CN115232817B

CN115232817B - Gene editing system for constructing triple-gene combined mutations in miniature pig nuclear transfer donor cells and its application

Info

Publication number: CN115232817B
Application number: CN202111019980.6A
Authority: CN
Inventors: 牛冬; 汪滔; 刘璐; 马翔; 段星; 曾为俊; 王磊; 程锐; 赵泽英; 陶裴裴; 黄彩云
Original assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Current assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2025-10-21
Anticipated expiration: 2041-09-01
Also published as: CN115232817A

Abstract

The invention discloses a gene editing system for constructing a miniature pig nuclear transfer donor cell with combined mutation of three genes (GHR gene, IGF1 gene and IGF2 gene) and application thereof. The invention provides a kit, which comprises GHR-E5-gRNA2 shown in SEQ ID NO. 36, GHR-E5-gRNA3 shown in SEQ ID NO. 37, IGF1-E4-gRNA1 shown in SEQ ID NO. 38, IGF1-E4-gRNA2 shown in SEQ ID NO. 39, IGF2-E4-gRNA2 shown in SEQ ID NO. 40, IGF2-E4-gRNA7 shown in SEQ ID NO. 41 and NCN proteins. The NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein. The invention adopts CRISPR/Cas9 technology and double gRNA editing to perform combined knockout of GHR gene, IGF1 gene and IGF2 gene, and obtains three single cell clones with combined knockout of genes, thereby laying a foundation for culturing small pigs and growth and development disorder or retardation model pigs through somatic cell nuclear transfer animal cloning technology in the later period.

Description

Gene editing system for constructing three-gene combined mutation miniature pig nuclear transplantation donor cells and application thereof

Technical Field

The invention belongs to the technical field of biology, in particular to the technical field of gene editing, and more particularly relates to a gene editing system for constructing a miniature pig nuclear transfer donor cell with combined mutation of three genes (GHR gene, IGF1 gene and IGF2 gene) and application thereof.

Background

Typically, animal growth is regulated by a growth axis. The growth axis is the neuroendocrine system consisting of a series of hormones and their receptors in the hypothalamus-pituitary-target organ of animals, and consists of growth hormone releasing factor (GRF), growth Hormone (GH) and insulin-like growth factor (IGF). The growth hormone receptor (Growth hormone receptor, GHR) is a transmembrane protein encoded by a single gene and is one of the members of the cytokine receptor superfamily. GHR plays an important role in the growth and development process and metabolism of animals, and the functional deficiency of GHR can lead to the growth and development retardation of animals. Insulin-like growth factor I (IGF 1) and insulin-like growth factor II (IGF 2) play an important role in bone development and growth. Both bone and cartilage cells are capable of producing IGF1 and IGF2, allowing proliferation of bone and cartilage cells and functional changes (e.g., collagen and glycosaminoglycans, respectively) to occur. Although the relative concentrations of IGF1 and IGF2 in bone vary from species to species and from embryonic to postnatal, both are essential for normal development. Studies have shown that the absence of either growth factor in IGF1 and IGF2 results in reduced size of animals at birth.

Pigs are domestic animals domesticated for a long time, have mild characters and are ideal companion animals for human beings, but the pigs are not widely used as pets in the market all the time because of the large general body types, so that the development of miniature pigs can increase the occupancy of the pigs in the pet market and have wide application prospects. In addition, the miniature pig produced by editing the genes related to the growth can simulate genetic diseases such as growth and development disorder or retardation of human beings, and an effective animal model is provided for the treatment and pathogenesis research of the genetic diseases related to the human beings.

Gene editing is a biotechnology that has been greatly developed in recent years, and includes editing technologies from homologous recombination-based gene editing to nuclease-based ZFN, TALEN, CRISPR/Cas9 and the like, wherein CRISPR/Cas9 technology is currently the most advanced gene editing technology.

Disclosure of Invention

The invention aims to provide a gene editing system for constructing miniature pig nuclear transfer donor cells with combined mutation of three genes (GHR gene, IGF1 gene and IGF2 gene) and application thereof.

The invention provides a kit comprising GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN proteins.

The invention also provides a kit comprising GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and PRONCN proteins.

The invention also provides a kit comprising GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and specific plasmids.

The invention also provides application of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein in preparation of a kit.

The invention also provides application of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and PRONCN proteins in preparation of a kit.

The invention also provides applications of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and specific plasmids in preparation of the kit.

Any of the above kits further comprises pig cells.

The invention also provides a method for preparing the recombinant cell, which comprises the following steps of co-transfecting GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein into a pig cell to obtain the recombinant cell.

The kit is used for preparing recombinant cells, (b) preparing miniature pigs, (c) cultivating pigs with reduced body size, (d) preparing model pigs with retarded growth, preparing cell models with retarded growth or tissue models with retarded growth or organ models with retarded growth, preparing model pigs with retarded growth, and preparing (g) preparing cell models with retarded growth or tissue models with retarded growth or organ models with retarded growth.

The cotransfection adopts a specific electric shock transfection mode.

The parameters for electric shock transfection can be 1450V, 10ms, 3 pulses.

The cotransfection can be specifically performed by using a mammalian nuclear transfection kit (Neon kit, thermofisher) and a Neon TM transfection system electrotransfection apparatus.

The proportion of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein is ：0.4-0.6μg GHR-E5-gRNA2：0.4-0.6μg GHR-E5-gRNA3：0.4-0.6μg IGF1-E4-gRNA1：0.4-0.6μg IGF1-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA7：5-7μg NCN protein in sequence.

The proportion of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein is ：0.5μg GHR-E5-gRNA2：0.5μg GHR-E5-gRNA3：0.5μg IGF1-E4-gRNA1：0.5μg IGF1-E4-gRNA2：0.5μg IGF2-E4-gRNA2：0.5μg IGF2-E4-gRNA7：6μg NCN protein in sequence.

The proportion of pig cells, GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein is 10 ten thousand pig cells ：0.4-0.6μg GHR-E5-gRNA2：0.4-0.6μg GHR-E5-gRNA3：0.4-0.6μg IGF1-E4-gRNA1：0.4-0.6μg IGF1-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA7：5-7μg NCN proteins in sequence.

The proportion of pig cells, GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein is 10 ten thousand pig cells ：0.5μg GHR-E5-gRNA2：0.5μg GHR-E5-gRNA3：0.5μg IGF1-E4-gRNA1：0.5μg IGF1-E4-gRNA2：0.5μg IGF2-E4-gRNA2：0.5μg IGF2-E4-gRNA7：6μg NCN proteins in sequence.

Any of the GHR-E5-gRNA2 is sgRNA, and the target sequence binding region is shown as nucleotide 3-22 in SEQ ID NO. 36.

Specifically, the GHR-E5-gRNA2 is shown as SEQ ID NO. 36.

Specifically, the GHR-E5-gRNA2 is shown as SEQ ID NO.11.

Any of the GHR-E5-gRNA3 is sgRNA, and the target sequence binding region is shown as nucleotide numbers 3-22 in SEQ ID NO. 37.

Specifically, the GHR-E5-gRNA3 is shown in SEQ ID NO. 37.

Specifically, the GHR-E5-gRNA3 is shown in SEQ ID NO. 12.

The IGF1-E4-gRNA1 is sgRNA, and the target sequence binding region is shown as nucleotide numbers 3-22 in SEQ ID NO. 38.

Specifically, IGF1-E4-gRNA1 is shown as SEQ ID NO. 38.

Specifically, IGF1-E4-gRNA1 is shown as SEQ ID NO. 16.

The IGF1-E4-gRNA2 is sgRNA, and the target sequence binding region is shown as nucleotide numbers 3-22 in SEQ ID NO. 39.

Specifically, IGF1-E4-gRNA2 is shown as SEQ ID NO: 39.

Specifically, IGF1-E4-gRNA2 is shown as SEQ ID NO. 17.

The IGF2-E4-gRNA2 is sgRNA, and the target sequence binding region is shown as nucleotide numbers 3-22 in SEQ ID NO. 40.

Specifically, IGF2-E4-gRNA2 is shown as SEQ ID NO. 40.

Specifically, IGF2-E4-gRNA2 is shown as SEQ ID NO. 23.

The IGF2-E4-gRNA7 is sgRNA, and the target sequence binding region is shown as nucleotide numbers 3-22 in SEQ ID NO. 41.

Specifically, IGF2-E4-gRNA7 is shown as SEQ ID NO. 41.

Specifically, IGF2-E4-gRNA7 is shown as SEQ ID NO. 28.

Any of the above NCN proteins is a Cas9 protein or a fusion protein with a Cas9 protein.

Specifically, the NCN protein is shown as SEQ ID NO. 3.

Any of the above-mentioned pig cells are pig somatic cells.

Any of the above-described porcine cells are porcine fibroblasts.

Any of the above described porcine cells are porcine primary fibroblasts.

The preparation method of the NCN protein comprises the following steps:

(1) Introducing plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain recombinant bacteria;

(2) Culturing the recombinant bacteria by adopting a liquid culture medium at 30 ℃, then adding IPTG, performing induction culture at 25 ℃, and then collecting the bacteria;

(3) Crushing the collected thalli, and collecting a crude protein solution;

(4) Purifying the His ₆ -tagged fusion protein from the crude protein solution using affinity chromatography;

(5) Cutting fusion protein with His ₆ label by enterokinase with His ₆ label, and removing protein with His ₆ label by Ni-NTA resin to obtain purified NCN protein;

the plasmid pKG-GE4 has the fusion gene shown in the 5209-9852 nucleotide in SEQ ID NO. 1.

The preparation method of the NCN protein specifically comprises the following steps:

(1) The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.

(2) Inoculating the recombinant bacteria obtained in the step (1) to a liquid LB culture medium containing ampicillin, and carrying out shake culture;

(3) Inoculating the bacterial liquid obtained in the step (2) to a liquid LB culture medium, carrying out shaking culture at 30 ℃ and 230rpm until the OD _600nm value=1.0, then adding IPTG to ensure that the concentration of the IPTG in the system is 0.5mM, carrying out shaking culture at 25 ℃ and 230rpm for 12 hours, and then centrifuging to collect bacterial cells;

(4) Washing the thalli obtained in the step (3) with PBS buffer solution;

(5) Adding the thalli obtained in the step (4) into a crude extraction buffer solution, suspending the thalli, crushing the thalli, centrifugally collecting supernatant, filtering by adopting a filter membrane with the aperture of 0.22 mu m, and collecting filtrate;

(6) Purifying the His ₆ -tagged fusion protein (fusion protein shown in SEQ ID NO: 2) from the filtrate obtained in step (5) by affinity chromatography;

(7) Taking the post-column solution collected in the step (6), concentrating the post-column solution by using an ultrafiltration tube, and diluting the post-column solution by using 25mM Tris-HCl (pH 8.0);

(8) Adding the recombinant bovine enterokinase with His ₆ tag into the solution obtained in the step (7), and performing enzyme digestion;

(9) Uniformly mixing the solution obtained in the step (8) with Ni-NTA resin, incubating, and centrifuging to collect supernatant;

(10) Concentrating the supernatant obtained in the step (9) by using an ultrafiltration tube, and then adding the concentrated supernatant into an enzyme stock solution to obtain the NCN protein solution.

The specific method for purifying the His ₆ -tagged fusion protein from the filtrate obtained in step (5) by affinity chromatography is as follows:

the method comprises the steps of firstly, balancing a Ni-NTA agarose column by using 5 column volumes of balancing solution (the flow rate is 1 ml/min), then loading 50ml of filtrate obtained in the step (5) (the flow rate is 0.5-1 ml/min), then washing the column by using 5 column volumes of balancing solution (the flow rate is 1 ml/min), then washing the column by using 5 column volumes of buffer solution (the flow rate is 1 ml/min) to remove the impurity proteins, then eluting by using 10 column volumes of eluent at the flow rate of 0.5-1ml/min, and collecting post-column solution (90-100 ml).

Any one of the PRONCN proteins comprises the following elements, in order from upstream to downstream, a signal peptide, a chaperone protein, a protein tag, a protease cleavage site, a nuclear localization signal, a Cas9 protein and a nuclear localization signal.

The function of the signal peptide is to promote secretory expression of the protein. The signal peptide may be selected from the group consisting of an E.coli alkaline phosphatase (phoA) signal peptide, a Staphylococcus aureus protein A signal peptide, an E.coli outer membrane protein (ompa) signal peptide, or a signal peptide of any other prokaryotic gene, preferably alkaline phosphatase signal peptide (phoA SIGNAL PEPTIDE). The alkaline phosphatase signal peptide is used for guiding the secretion and expression of the target protein into the periplasmic cavity of the bacterium so as to be separated from the intracellular protein of the bacterium, and the target protein secreted into the periplasmic cavity of the bacterium is expressed in a soluble way and can be cracked by the signal peptidase in the periplasmic cavity of the bacterium.

The chaperone protein functions to increase the solubility of the protein. The chaperone may be any protein that aids in disulfide bond formation, preferably a thioredoxin (TrxA protein). Thioredoxin, which can serve as a molecular chaperone to help the co-expressed target protein (e.g., cas9 protein) form disulfide bonds, improving the stability of the protein, the correctness of folding, and increasing the solubility and activity of the target protein.

The function of the protein tag is for protein purification. The Tag may be a His Tag (His-Tag, his ₆ protein Tag), GST Tag, flag Tag, HA Tag, c-Myc Tag or any other protein Tag, more preferably a His Tag. His tag can be combined with Ni column, and target protein can be purified by one-step Ni column affinity chromatography, so that the purification process of target protein can be greatly simplified.

The protease cleavage site functions to cleave off the nonfunctional segment after purification to release the native form of Cas9 protein. The protease may be selected from enterokinase (Enterokinase), factor Xa, thrombin (Thrombin), TEV protease (TEV protease), HRV 3C protease (HRV 3C protease), WELQut protease or any other endoprotease, further preferably enterokinase. EK is enterokinase cleavage site, which is convenient for cutting fused TrxA-His segment by enterokinase to obtain the natural form Cas9 protein. After the commercial enterokinase enzyme digestion fusion protein with the His tag is used, the TrxA-His segment and the enterokinase with the His tag can be removed through one-time affinity chromatography to obtain the natural form of the Cas9 protein, so that the damage and the loss of the target protein caused by repeated purification and dialysis are avoided.

The nuclear localization signal may be any nuclear localization signal, preferably an SV40 nuclear localization signal and/or nucleoplasmin nuclear localization signal. NLS is a nuclear localization signal, and an NLS site is designed at the N end and the C end of Cas9 respectively, so that Cas9 can enter the nucleus more effectively for gene editing.

The Cas9 protein may be saCas or spCas9, preferably spCas9 protein.

The PRONCN protein is specifically shown as SEQ ID NO. 2.

The specific plasmid comprises the following elements, namely a promoter, an operator, a ribosome binding site, a PRONCN protein coding gene and a terminator from upstream to downstream.

The promoter may specifically be a T7 promoter. The T7 promoter is a prokaryotic expression strong promoter and can efficiently drive the expression of exogenous genes.

The operon may specifically be the Lac operon. The Lac operon is a regulatory element for lactose induced expression, and can induce the expression of the target protein at low temperature after bacteria grow to a certain amount, thereby avoiding the influence of the premature expression of the target protein on the growth of host bacteria, and remarkably improving the solubility of the expressed target protein by the induced expression at low temperature.

The ribosome binding site is a ribosome binding site for protein translation, and is necessary for protein translation.

The terminator may specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the tail end of the target gene, and prevent other downstream sequences except the target gene from being transcribed and translated.

For the codon of the spCas9 protein, the codon is optimized, so that the codon completely adapts to the codon preference of the E.coli BL21 (DE 3) strain for efficiently expressing the escherichia coli selected by the application, and the expression level of the Cas9 protein is improved.

The T7 promoter is shown as 5121-5139 nucleotides in SEQ ID NO. 1.

The Lac operon is shown as 5140-5164 nucleotides in SEQ ID NO. 1.

The ribosome binding site is shown as nucleotide 5178-5201 in SEQ ID NO. 1.

The coding sequence of the alkaline phosphatase signal peptide is shown as 5209-5271 nucleotides in SEQ ID NO. 1.

The coding sequence of the TrxA protein is shown as 5272-5598 nucleotide in SEQ ID NO. 1.

The coding sequence of His-Tag is shown as 5620-5637 nucleotide in SEQ ID NO. 1.

The coding sequence of the enterokinase enzyme cutting site is shown as 5638-5652 nucleotides in SEQ ID NO. 1.

The coding sequence of the nuclear localization signal is shown as 5656-5670 nucleotides in SEQ ID NO. 1.

The coding sequence of the spCas9 protein is shown as 5701-9801 nucleotide in SEQ ID NO. 1.

The coding sequence of the nuclear localization signal is shown as 9802-9849 nucleotides in SEQ ID NO. 1.

T7 terminator is shown as 9902-9949 nucleotides in SEQ ID NO. 1.

Specifically, the specific plasmid is plasmid pKG-GE4.

The plasmid pKG-GE4 has the DNA molecule shown in the 5121-9949 nucleotide of SEQ ID NO. 1.

Specifically, any one of the plasmids pKG-GE4 is shown as SEQ ID NO. 1.

The invention also protects recombinant cells, which are cells in which GHR gene, IGF1 gene and IGF2 gene are mutated in combination.

The invention also protects recombinant cells, which are cells in which GHR genes, IGF1 genes and IGF2 genes are knocked out in a combined way.

The recombinant cell may be a recombinant cell prepared by any of the methods described above.

The recombinant cell may be one in which the genotype of the IGF1 gene is heterozygous and the genotype of the GHR gene is either a double allele identical mutant or a double allele different mutant and the genotype of the IGF2 gene is either a double allele identical mutant or a double allele different mutant.

The recombinant cell may be a single cell clone numbered 9, 35, 36, 54, 63, 65, 69, 75, 79 or 81 in table 1.

Any of the above mutations is a deletion and/or insertion and/or substitution of one or more nucleotides.

The invention also protects the application of the recombinant cells in the preparation of miniature pigs.

And (3) taking the recombinant cells as nuclear transfer donor cells to clone somatic cells, so that cloned pigs, namely miniature pigs, can be obtained.

The miniature pig may be used as a pet pig.

Miniature pigs refer to pigs that are smaller in size than the pig from which the pig cells were derived.

The invention also protects the application of the recombinant cells in preparing model pigs with retarded growth and development.

And (3) taking the recombinant cells as nuclear transfer donor cells to clone somatic cells, so that cloned pigs, namely model pigs with slow growth and development, can be obtained.

The invention also protects pig tissues of a model pig prepared by the recombinant cells, namely a tissue model with slow growth and development.

The invention also protects a pig organ, namely an organ model with slow growth and development, of a model pig prepared by utilizing the recombinant cells.

The invention also protects pig cells of a model pig prepared by using the recombinant cells, namely a cell model with slow growth and development.

The invention also protects the use of said recombinant cells, said tissue model of bradykinin, said organ model of bradykinin, said cell model of bradykinin or said model of bradykinin in pigs as (d 1) or (d 2) or (d 3) or (d 4) as follows:

(d1) Screening medicines for treating growth retardation;

(d2) Performing drug effect evaluation of the growth retardation drug;

(d3) Performing efficacy evaluation of gene therapy and/or cell therapy of growth retardation;

(d4) The pathogenesis of growth retardation is studied.

Any of the above may be congenital growth retardation.

Any of the above may be autosomal recessive inherited congenital growth developmental retardation.

The growth retardation described above is caused by a combined mutation of GHR gene, IGF1 gene and IGF2 gene.

The invention also protects the application of the recombinant cells in preparing model pigs with growth and development disorder.

And (3) taking the recombinant cells as nuclear transfer donor cells to clone somatic cells, so that cloned pigs, namely model pigs with dysgenesis and development, can be obtained.

The invention also protects pig tissues of a model pig prepared by the recombinant cells, namely a tissue model of growth and development disorder.

The invention also protects the pig organ of the model pig prepared by the recombinant cells, namely an organ model of growth and development disorder.

The invention also protects the pig cells of the model pig prepared by the recombinant cells, namely a cell model of growth and development disorder.

The invention also protects the use of said recombinant cells, said tissue model of a growth disorder, said organ model of a growth disorder, said cell model of a growth disorder or said model pig of a growth disorder as follows (d 5) or (d 6) or (d 7) or (d 8):

(d5) Screening medicines for treating growth and development disorder;

(d6) Performing drug effect evaluation of the growth and development disorder drug;

(d7) Performing efficacy evaluation of gene therapy and/or cell therapy of the growth and development disorder;

(d8) The pathogenesis of the growth and development disorder is studied.

Any of the above described growth and development disorders may be congenital growth and development disorders.

Any of the above described growth and development disorders may be autosomal recessive inherited congenital growth and development disorders.

The growth dysfunction described above is caused by combined mutations of the GHR gene, IGF1 gene and IGF2 gene.

Any of the above pigs may specifically be a from-river fragrant pig.

Pig GHR gene information, encoding growth hormone receptor (growth hormone receptor), chromosome 16, geneID 397488,Sus scrofa.

The amino acid sequence of the pig GHR gene is shown as SEQ ID NO. 8.

The pig GHR gene has a DNA segment shown in SEQ ID NO. 9.

Pig IGF1 gene information encoding insulin-like growth factor 1 (insulin like growth factor 1), chromosome 5, geneID 397491,Sus scrofa.

The amino acid sequence of pig IGF1 gene is shown in SEQ ID NO. 14.

The pig IGF1 gene has a DNA segment shown as SEQ ID NO. 15.

Pig IGF2 gene information encoding insulin-like growth factor 2 (insulin like growth factor 2), chromosome 2, geneID 396916,Sus scrofa.

The amino acid sequence of pig IGF2 gene is shown in SEQ ID NO. 20.

The pig IGF2 gene has a DNA segment shown as SEQ ID NO. 21.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The subject (pig) of the invention has better applicability than other animals (rats, mice, primates).

Rodents such as rats and mice have great differences from humans in terms of body type, organ size, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that more than 95% of drugs that are validated in mice are ineffective in human clinical trials. In the case of large animals, primates are animals with the closest relationship to humans, but are small in size, late in sexual maturity (mating begins at 6-7 years old), and single animals, the population expansion rate is extremely slow, and the raising cost is high. In addition, primate cloning is inefficient, difficult and costly.

The pig is an animal which has the closest relationship with human except primate, and has the similar body shape, weight, organ size and the like as human, and has the similar anatomical, physiological, immunological, nutritional metabolism, disease pathogenesis and the like as human. Meanwhile, the pigs are early in sexual maturity (4-6 months), have high fertility and have more piglets, and can form a larger group within 2-3 years. In addition, the cloning technology of pigs is very mature, and the cloning and feeding costs are much lower than those of primates. Pigs are thus very suitable animals as models of human diseases.

(2) The vector constructed by the invention uses a strong promoter T7-lac capable of efficiently expressing the target protein to express the target protein, and uses a signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) to guide the secretory expression of the target protein into a bacterial periplasmic cavity so as to separate from bacterial intracellular proteins, wherein the target protein secreted into the bacterial periplasmic cavity is expressed in a soluble way. Meanwhile, the fusion expression of the thioredoxin TrxA and the Cas9 protein is adopted, the TrxA can help the co-expressed target protein to form disulfide bonds, the stability and folding correctness of the protein are improved, and the solubility and activity of the target protein are increased. In order to facilitate purification of the target protein, a His tag is designed, and the target protein can be purified by one-step Ni column affinity chromatography, so that the purification process of the target protein is greatly simplified. Meanwhile, an enterokinase enzyme cutting site is designed behind the His tag, so that fused TrxA-His polypeptide fragments can be conveniently cut off, and the Cas9 protein in a natural form is obtained. After the fusion protein is digested by using the enterokinase with the His tag, the TrxA-His polypeptide fragment and the enterokinase with the His tag can be removed by one-time affinity chromatography to obtain the natural form of the Cas9 protein, thereby avoiding the damage and the loss of the target protein caused by multiple purification dialysis. Meanwhile, the N end and the C end of the Cas9 are respectively designed with an NLS site, so that the Cas9 can enter a cell nucleus more effectively for gene editing. In addition, the E.coli BL21 (DE 3) strain is selected as a target protein expression strain, and the strain can efficiently express and clone exogenous genes in an expression vector (such as pET-32 a) containing a phage T7 promoter. Meanwhile, the codon of the Cas9 protein is optimized, so that the codon is completely adapted to the codon preference of an expression strain, and the expression level of the target protein is improved. In addition, after bacteria grow to a certain quantity, the invention uses IPTG to induce the expression of the target protein at low temperature, thereby avoiding the influence of the premature expression of the target protein on the growth of host bacteria, and obviously improving the solubility of the expressed target protein by the induction expression at low temperature. Through the optimization design and experimental implementation, the activity of the obtained Cas9 protein is remarkably improved compared with that of commercial Cas9 protein.

(3) The Cas9 high-efficiency protein constructed and expressed by the invention is combined with the in vitro transcribed gRNA to carry out gene editing, and the optimal dosage ratio of Cas9 and gRNA is optimized.

(4) The invention adopts double gRNA combination to carry out mutation, and compared with single gRNA, the invention can effectively reduce the generation of non-frame shift mutation. If a single gRNA is used to mutate a target gene, in non-homologous end joining (NHEJ) random repair of DNA, there is a 1/3 probability that a non-frameshift mutation of the base will occur, and it is highly probable that the non-frameshift mutation will not disrupt the function of the target gene, failing to achieve the intended goal of inactivating the target gene. When the double gRNA is used for cutting and mutating the target gene, one fragment of the target gene can be removed, and the fragment deletion frame shift mutation of the target gene can be effectively generated by designing to remove base fragments which are not multiple of 3. In addition, the double gRNAs can cause the deletion of theoretical fragments, and the situation of independent cutting of single gRNAs exists, so that the gene mutation efficiency is greatly improved.

(5) The target gene knockout monoclonal strain obtained by the invention is used for cloning somatic cell nuclear transfer animals, so that the cloned pig with the target gene knockout can be directly obtained, and the gene variation can be inherited stably.

The method of microinjection of gene editing material into fertilized ovum and embryo transplantation adopted in mouse model production is not suitable for the production of large animals with long gestation period, such as pigs, because the probability of directly obtaining the offspring of gene mutation is low and the hybrid breeding of the offspring is needed. Therefore, the method for editing primary cells in vitro, cutting double gRNA and screening positive editing single cell clones with high technical difficulty and high challenges is adopted, and the miniature pig or model pig is directly obtained by somatic cell nuclear transfer animal cloning technology in the later stage, so that the manufacturing period of the miniature pig or model pig can be greatly shortened, and the labor, material resources and financial resources are saved.

The CRISPR/Cas9 technology is combined with double gRNA editing to perform combined knockout of GHR genes, IGF1 genes and IGF2 genes, and three single cell clones with the combined knockout genes are obtained, so that a foundation is laid for culturing small pigs or model pigs through a somatic cell nuclear transfer animal cloning technology in the later period.

The invention is helpful for researching and revealing pathogenesis of growth and development disorder/growth and development retardation caused by abnormal functions of GHR genes, IGF1 genes and IGF2 genes, can also be used for researching drug screening, drug effect evaluation, gene therapy, cell therapy and the like, can provide effective experimental data for further clinical application, and further provides a powerful experimental means for successfully treating the growth and development disorder/growth and development retardation of human beings. The invention has great application value for developing and revealing pathogenesis of the disease in the development of growth disorder/growth retardation medicine.

Drawings

FIG. 1 is an electrophoretogram of example 1 using ear tissue-extracted genome of pig designated 1 as a template for PCR amplification using different primer pairs.

FIG. 2 is an electrophoretogram of PCR amplification using 18 pig genomic DNAs as templates and primer sets consisting of GHR-E5-F132 and GHR-E5-R467 in example 1.

FIG. 3 is a graph of sequencing peaks comparing editing efficiency for different combinations of targets in example 1.

FIG. 4 is an electrophoretogram of example 2 using ear tissue extracted genome of pig designated 1 as a template for PCR amplification using different primer pairs.

FIG. 5 is an electrophoretogram of PCR amplification using 18 pig genomic DNAs as templates and primer sets composed of IGF1-E4-F114 and IGF1-E4-R575, respectively, in example 2.

FIG. 6 is a graph of sequencing peaks comparing editing efficiency for different combinations of targets in example 2.

FIG. 7 is an electrophoretogram of example 3 using ear tissue extracted genome of pig designated 1 as a template for PCR amplification using different primer pairs.

FIG. 8 is an electrophoretogram of PCR amplification using primer pairs composed of IGF2-E4-F136 and IGF2-E4-R728 using genomic DNAs of 18 pigs as templates, respectively, in example 3.

FIG. 9 is a graph of sequencing peaks comparing editing efficiency for different combinations of targets in example 3.

FIG. 10 is a schematic diagram of the structure of plasmid pET-32 a.

FIG. 11 is a schematic diagram of the structure of plasmid pKG-GE 4.

FIG. 12 is an electrophoretogram of the optimization of the ratio of gRNA to NCN protein in example 6.

Fig. 13 is an electrophoretogram of the comparison of gene editing efficiency of NCN protein and commercial Cas9 protein in example 6.

FIG. 14 shows the result of reverse sequencing of the single cell clone No. 63 (GHR gene) compared with the wild-type sequence.

FIG. 15 shows the result of reverse sequencing of the single cell clone No. 63 (IGF 1 gene) compared with the wild type sequence.

FIG. 16 shows the result of reverse sequencing of the single cell clone No. 63 (IGF 2 gene) compared with the wild type sequence.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The recombinant plasmids constructed in the examples were all subjected to sequencing verification. The commercial Cas9-A protein is a commercially available Cas9 protein with good effect. The commercial Cas9-B protein is a commercially available Cas9 protein with good effect. Complete medium (% by volume) 15% foetal calf serum (Gibco) +83% DMEM medium (Gibco) +1% Penicillin-Streptomycin (Gibco) +1% HEPES (Solarbio). Cell culture conditions were 37℃in a constant temperature incubator with 5% CO ₂、5％O₂.

The primary fibroblasts of pigs used in the examples were prepared from the tissue of the ear of a Jiang Xiang pig from a first time. A method for preparing primary fibroblast of pig comprises collecting pig ear tissue 0.5g, removing hair and bone tissue, soaking in 75% alcohol for 30-40s, washing with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) for 5 times, washing once with PBS buffer, shearing tissue with ② scissors, digesting with 5mL of 0.1% collagenase solution (Sigma) at 37deg.C for 1 hr, centrifuging for 5min, discarding supernatant, resuspending the precipitate with 1mL of complete culture solution ③, spreading into 10mL of complete culture solution, sealing with 0.2% gelatin (VWR) plate, culturing until the cell grows to about 60% of the bottom of the plate, and culturing with trypsin after ④. For carrying out subsequent electrotransformation experiments.

Plasmid pKG-GE3 is a circular plasmid, as shown in SEQ ID NO. 2 of patent application 202010084343.6. In SEQ ID NO. 2 of patent application 202010084343.6, nucleotide 395-680 constitutes the CMV enhancer, nucleotide 682-890 constitutes the EF1a promoter, nucleotide 986-1006 encodes the Nuclear Localization Signal (NLS), nucleotide 1016-1036 encodes the Nuclear Localization Signal (NLS), nucleotide 1037-5161 encodes the Cas9 protein, nucleotide 5162-5209 encodes the Nuclear Localization Signal (NLS), nucleotide 5219-5266 encodes the Nuclear Localization Signal (NLS), nucleotide 5276-5332 encodes the cleavage polypeptide P2A (the amino acid sequence of cleavage polypeptide P2A is "ATNFSLLKQAGDVEENPGP", the cleavage site at which cleavage occurs between the first amino acid residue and the second amino acid residue from the C-terminus, nucleotide 5333-6046 encodes the EGFP protein, nucleotide 6056-6109 encodes the cleavage polypeptide T2A (the amino acid sequence of cleavage polypeptide T2A is "EGRGSLLTCGDVEENPGP", the cleavage site at which cleavage site occurs between the first amino acid residue from the C-terminus is "377", the amino acid sequence of cleavage site at which cleavage site is the nucleotide position of the cleavage site is "677", the amino acid sequence of nucleotide No. 3-7643 is the amino acid sequence of the cleavage site is encoded between the first amino acid residue from the first amino acid residue of the cleavage site of the cleavage element, nucleotide position 2A is "3782", the amino acid sequence of the cleavage element No. 2A is encoded between nucleotide No. 10-7 and the amino acid sequence of the polypeptide 11B is encoded by the polypeptide B. In SEQ ID NO. 2 of patent application 202010084343.6, nucleotides 911 to 6706 form a fusion gene, expressing a fusion protein. Due to the presence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously forms three proteins, a protein with Cas9 protein, a protein with EGFP protein and a protein with Puro protein.

The pKG-U6gRNA vector, i.e., plasmid pKG-U6gRNA, is a circular plasmid, as shown in SEQ ID NO. 3 of patent application 202010084343.6. In SEQ ID NO. 3 of patent application 202010084343.6, nucleotides 2280 to 2539 constitute the hU6 promoter and nucleotides 2558 to 2637 are used for transcription to form the gRNA backbone. When in use, a DNA molecule of about 20bp (target sequence binding region for transcription to form gRNA) is inserted into plasmid pKG-U6gRNA to form a recombinant plasmid, and the recombinant plasmid is transcribed in cells to obtain gRNA.

Example 1 screening of GHR Gene efficient gRNA target combinations

Pig GHR gene information, encoding growth hormone receptor (growth hormone receptor), chromosome 16, geneID 397488,Sus scrofa. The amino acid sequence of the protein coded by the pig GHR gene is shown as SEQ ID NO. 8. In pig genomic DNA, the GHR gene shares 17 exons, wherein the coding exons are 10, and 1-6 coding exons code for extracellular region of GHR protein. In the invention, the 5 th coding exon of the GHR gene is taken as a target area for gene editing, and the 5 th coding exon and partial nucleotide sequences on the upstream and downstream of the 5 th coding exon are shown as SEQ ID NO. 9.

1. GHR gene preset target region and adjacent genome sequence conservation analysis

18 From Jiangxiang pigs, of which 10 females (designated 1,2,3, 4, 5, 6, 7, 8, 9, 10 respectively) and 8 males (designated A, B, C, D, E, F, G, H respectively) were bred.

GHR-E5-F132:CTGGAATTAAGGGCCGACAGA;

GHR-E5-R454:GTCAGAAAGGCATGAAGGCTGTA;

GHR-E5-F133:TGGAATTAAGGGCCGACAGA;

GHR-E5-R467:CATGGAGAGAAAAGTCAGAAAGGC。

The genome was extracted from ear tissue of swine designated as 1 as a template, and PCR amplification was performed using different primer pairs, followed by 1% agarose gel electrophoresis. The electrophoresis diagram is shown in fig. 1. In FIG. 1, the primer set consisting of GHR-E5-F132 and GHR-E5-R454 is used, the primer set consisting of GHR-E5-F132 and GHR-E5-R467 is used in set 2, the primer set consisting of GHR-E5-F133 and GHR-E5-R454 is used in set 3, and the primer set consisting of GHR-E5-F133 and GHR-E5-R467 is used in set 4. As a result, it was found that the target fragment was amplified preferably using a primer set composed of GHR-E5-F132 and GHR-E5-R467.

PCR amplification was performed using 18 pig genomic DNAs as templates, respectively, using primer pairs consisting of GHR-E5-F132 and GHR-E5-R467, followed by 1% agarose gel electrophoresis. The electrophoresis diagram is shown in fig. 2. The PCR amplified product was recovered and sequenced, and the sequencing result was compared with the GHR gene sequence of the porcine reference genome (susscrofa 11.1.1) for analysis. The conserved regions common to 18 pigs were selected for the design of the gRNA targets.

2. Screening target

A plurality of targets are initially screened by screening NGG (avoiding possible mutation sites), and 4 targets are further screened from the targets through preliminary experiments.

The 4 targets were as follows:

GHR-E5-g1:CAGGGCTCTGTAAACCGTGA;

GHR-E5-g2:GACGGACCCCATCTGTCCAG;

GHR-E5-g3:AAGTCTCTAGTTCAGGTGAA;

GHR-E5-g4:TGGACAGATGGGGTCCGTCA。

3. Preparation of gRNA

Plasmid pKG-U6gRNA was taken and digested with restriction enzyme BbsI, and the vector backbone (about 3kb linear fragment) was recovered.

GHR-E5-g1-S and GHR-E5-g1-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (GHR-E5-g 1). Plasmid pKG-U6gRNA (GHR-E5-g 1) expresses the sgRNA _GHR-E5-g1 shown in SEQ ID NO. 10.

sgRNA_GHR-E5-g1(SEQ ID NO:10):

CAGGGCUCUGUAAACCGUGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

GHR-E5-g2-S and GHR-E5-g2-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (GHR-E5-g 2). Plasmid pKG-U6gRNA (GHR-E5-g 2) expresses the sgRNA _GHR-E5-g2 shown in SEQ ID NO. 11.

sgRNA_GHR-E5-g2(SEQ ID NO:11):

GACGGACCCCAUCUGUCCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

GHR-E5-g3-S and GHR-E5-g3-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (GHR-E5-g 3). Plasmid pKG-U6gRNA (GHR-E5-g 3) expresses the sgRNA _GHR-E5-g3 shown in SEQ ID NO. 12.

sgRNA_GHR-E5-g3(SEQ ID NO:12):

AAGUCUCUAGUUCAGGUGAAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

GHR-E5-g4-S and GHR-E5-g4-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (GHR-E5-g 4). Plasmid pKG-U6gRNA (GHR-E5-g 4) expresses the sgRNA _GHR-E5-g4 shown in SEQ ID NO. 13.

sgRNA_GHR-E5-g4(SEQ ID NO:13):

UGGACAGAUGGGGUCCGUCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

GHR-E5-g1-S:caccgCAGGGCTCTGTAAACCGTGA;

GHR-E5-g1-A:aaacTCACGGTTTACAGAGCCCTGc;

GHR-E5-g2-S:caccGACGGACCCCATCTGTCCAG;

GHR-E5-g2-A:aaacCTGGACAGATGGGGTCCGTC;

GHR-E5-g3-S:caccgAAGTCTCTAGTTCAGGTGAA;

GHR-E5-g3-A:aaacTTCACCTGAACTAGAGACTTc;

GHR-E5-g4-S:caccgTGGACAGATGGGGTCCGTCA;

GHR-E5-g4-A:aaacTGACGGACCCCATCTGTCCAc。

GHR-E5-g1-S, GHR-E5-g1-A, GHR-E5-g2-S, GHR-E5-g2-A, GHR-E5-g3-S, GHR-E5-g3-A, GHR-E5-g4-S, GHR-E5-g4-A are all single-stranded DNA molecules.

4. Editing efficiency comparison of different target combinations

1. Co-transfection

The first group was to cotransfect plasmid pKG-U6gRNA (GHR-E5-g 1) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (GHR-E5-g 1), 1.08. Mu.g plasmid pKG-GE3.

The second group was to cotransfect plasmid pKG-U6gRNA (GHR-E5-g 2) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (GHR-E5-g 2) 1.08. Mu.g plasmid pKG-GE3.

The third group was to cotransfect plasmid pKG-U6gRNA (GHR-E5-g 3) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (GHR-E5-g 3), 1.08. Mu.g plasmid pKG-GE3.

The fourth group was to cotransfect plasmid pKG-U6gRNA (GHR-E5-g 4) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (GHR-E5-g 4) 1.08. Mu.g plasmid pKG-GE3.

Fifth group, pig primary fibroblast, electric transfer operation is carried out without adding plasmid with the same electric transfer parameters.

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofisher) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 12 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time after electrotransformation was 48 hours.

3. After step 2 was completed, cells were digested and collected with trypsin, lysed, genomic DNA was extracted, PCR amplified with primer pairs consisting of GHR-E5-F132 and GHR-E5-R467, and then subjected to 1% agarose gel electrophoresis.

And (3) cutting and recovering a target product, sending the target product to a sequencing company for sequencing (a sequencing peak diagram is shown in figure 3), and analyzing the sequencing peak diagram by using a webpage plate Synthego ICE tool to obtain the gene editing efficiency of different targets. The gene editing efficiencies of the first group to the fourth group were 24%, 51%, 25%, 16% in this order, and no gene editing occurred in the fifth group. The results show that the editing efficiency of GHR-E5-g2 and GHR-E5-g3 is higher.

Example 2 screening of highly efficient gRNA target combinations of IGF1 Gene

Pig IGF1 gene information encoding insulin-like growth factor 1 (insulin like growth factor 1), chromosome 5, geneID 397491,Sus scrofa. The amino acid sequence of the protein coded by the pig IGF1 gene is shown as SEQ ID NO. 14. In porcine genomic DNA, IGF1 gene shares 8 exons, with 7 encoding exons. In the invention, the 4 th coding exon of IGF1 gene is taken as a target area for gene editing, and the 4 th coding exon and partial nucleotide sequences on the upstream and downstream of the 4 th coding exon are shown as SEQ ID NO. 15.

1. IGF1 gene preset target region and adjacent genome sequence conservation analysis

IGF1-E4-F114:AACAGTGGAACCCAGAAGGAC;

IGF1-E4-R547:ACTGTTTGGCACACTCACACT;

IGF1-E4-F189:TGTGGGTTGACAAGAGGTGG;

IGF1-E4-R575:TCTCAGGGAGAGAACCAGGG。

The genome was extracted from ear tissue of swine designated as 1 as a template, and PCR amplification was performed using different primer pairs, followed by 1% agarose gel electrophoresis. The electrophoresis pattern is shown in FIG. 4. In FIG. 4, group 1 was the primer set using IGF1-E4-F114 and IGF1-E4-R547, group 2 was the primer set using IGF1-E4-F114 and IGF1-E4-R575, group 3 was the primer set using IGF1-E4-F189 and IGF1-E4-R547, and group 4 was the primer set using IGF1-E4-F189 and IGF 1-E4-R575. As a result, it was revealed that the target fragment was amplified preferably using a primer set composed of IGF1-E4-F114 and IGF 1-E4-R575.

PCR amplification was performed using 18 pig genomic DNAs as templates, respectively, using primer pairs composed of IGF1-E4-F114 and IGF1-E4-R575, followed by 1% agarose gel electrophoresis. The electrophoresis pattern is shown in FIG. 5. The PCR amplified product was recovered and sequenced, and the sequencing result was compared to IGF1 gene sequence of the porcine reference genome (susscrofa 11.1.1) for analysis. The conserved regions common to 18 pigs were selected for the design of the gRNA targets.

2. Screening target

The 4 targets were as follows:

IGF1-E4-g1:GAGCCTTGGGCATGTCCGTG;

IGF1-E4-g2:GCTTCCGGAGCTGTGATCTG;

IGF1-E4-g3:GGCGCCACAGACGGGCATCG;

IGF1-E4-g4:CGTCCGTGCCCAGCGCCACA。

3. Preparation of gRNA

IGF1-E4-g1-S and IGF1-E4-g1-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 1-E4-g 1). Plasmid pKG-U6gRNA (IGF 1-E4-g 1) expresses the sgRNA _IGF1-E4-g1 shown in SEQ ID NO. 16.

sgRNA_IGF1-E4-g1(SEQ ID NO:16):

GAGCCUUGGGCAUGUCCGUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-g2-S and IGF1-E4-g2-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 1-E4-g 2). Plasmid pKG-U6gRNA (IGF 1-E4-g 2) expresses the sgRNA _IGF1-E4-g2 shown in SEQ ID NO: 17.

sgRNA_IGF1-E4-g2(SEQ ID NO:17):

GCUUCCGGAGCUGUGAUCUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-g3-S and IGF1-E4-g3-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 1-E4-g 3). Plasmid pKG-U6gRNA (IGF 1-E4-g 3) expresses the sgRNA _IGF1-E4-g3 shown in SEQ ID NO. 18.

sgRNA_IGF1-E4-g3(SEQ ID NO:18):

GGCGCCACAGACGGGCAUCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-g4-S and IGF1-E4-g4-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 1-E4-g 4). Plasmid pKG-U6gRNA (IGF 1-E4-g 4) expresses the sgRNA _IGF1-E4-g4 shown in SEQ ID NO. 19.

sgRNA_IGF1-E4-g4(SEQ ID NO:19):

CGUCCGUGCCCAGCGCCACAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-g1-S:caccGAGCCTTGGGCATGTCCGTG;

IGF1-E4-g1-A:aaacCACGGACATGCCCAAGGCTC;

IGF1-E4-g2-S:caccGCTTCCGGAGCTGTGATCTG;

IGF1-E4-g2-A:aaacCAGATCACAGCTCCGGAAGC;

IGF1-E4-g3-S:caccGGCGCCACAGACGGGCATCG;

IGF1-E4-g3-A:aaacCGATGCCCGTCTGTGGCGCC;

IGF1-E4-g4-S:caccgCGTCCGTGCCCAGCGCCACA;

IGF1-E4-g4-A:aaacTGTGGCGCTGGGCACGGACGc。

IGF1-E4-g1-S、IGF1-E4-g1-A、IGF1-E4-g2-S、IGF1-E4-g2-A、IGF1-E4-g3-S、IGF1-E4-g3-A、IGF1-E4-g4-S、IGF1-E4-g4-A Are all single-stranded DNA molecules.

4. Editing efficiency comparison of different target combinations

1. Co-transfection

The first group was to cotransfect plasmid pKG-U6gRNA (IGF 1-E4-g 1) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand porcine primary fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 1-E4-g 1), 1.08. Mu.g plasmid pKG-GE3.

The second group was to cotransfect plasmid pKG-U6gRNA (IGF 1-E4-g 2) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand porcine primary fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 1-E4-g 2) 1.08. Mu.g plasmid pKG-GE3.

The third group was to cotransfect plasmid pKG-U6gRNA (IGF 1-E4-g 3) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 1-E4-g 3), 1.08. Mu.g plasmid pKG-GE3.

The fourth group was to cotransfect plasmid pKG-U6gRNA (IGF 1-E4-g 4) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand porcine primary fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 1-E4-g 4) 1.08. Mu.g plasmid pKG-GE3.

3. After step 2 was completed, cells were digested and collected with trypsin, lysed, genomic DNA was extracted, PCR amplified with primer pairs composed of IGF1-E4-F114 and IGF1-E4-R575, and then subjected to 1% agarose gel electrophoresis.

And (3) cutting and recycling a target product, sending the target product to a sequencing company for sequencing (a sequencing peak diagram is shown in fig. 6), and analyzing the sequencing peak diagram by using a webpage plate Synthego ICE tool to obtain the gene editing efficiency of different targets. The gene editing efficiency of the first group to the fourth group was 25%, 29%, 5%, 1% in this order, and no gene editing occurred in the fifth group. The results show that IGF1-E4-g1 and IGF1-E4-g2 have higher editing efficiency.

Example 3 screening of highly efficient gRNA target combinations of IGF2 Gene

Pig IGF2 gene information encoding insulin-like growth factor 2 (insulin like growth factor 2), chromosome 2, geneID 396916,Sus scrofa. The amino acid sequence of the protein coded by the pig IGF2 gene is shown as SEQ ID NO. 20. In porcine genomic DNA, the IGF2 gene shares 12 exons, with 5 encoding exons. In the invention, the 4 th coding exon of IGF2 gene is taken as a target area for gene editing, and the 4 th coding exon and partial nucleotide sequences on the upstream and downstream of the 4 th coding exon are shown as SEQ ID NO. 21.

1. IGF2 gene preset target region and adjacent genome sequence conservation analysis

IGF2-E4-F278:CCTTGGTCCTGTGGGACTTC;

IGF2-E4-R728:CGGGGTATCTGGGGAAGTTG;

IGF2-E4-F136:GCACCCCACTCTCACTTCTT;

IGF2-E4-R602:GAAGACTGCCCTGGCTTCTG。

The genome was extracted from ear tissue of swine designated as 1 as a template, and PCR amplification was performed using different primer pairs, followed by 1% agarose gel electrophoresis. The electrophoresis pattern is shown in FIG. 7. FIG. 7 shows a primer set of IGF2-E4-F136 and IGF2-E4-R602, a primer set of IGF2-E4-F278 and IGF2-E4-R602, a primer set of IGF2-E4-F136 and IGF2-E4-R728, and a primer set of IGF2-E4-F278 and IGF 2-E4-R728. As a result, it was revealed that the target fragment was amplified preferably using a primer set composed of IGF2-E4-F136 and IGF 2-E4-R728.

PCR amplification was performed using 18 pig genomic DNAs as templates, respectively, using primer pairs composed of IGF2-E4-F136 and IGF2-E4-R728, followed by 1% agarose gel electrophoresis. The electrophoresis pattern is shown in FIG. 8. The PCR amplified product was recovered and sequenced, and the sequencing result was compared to IGF2 gene sequence of the porcine reference genome (susscrofa 11.1.1) for analysis. The conserved regions common to 18 pigs were selected for the design of the gRNA targets.

2. Screening target

Several targets were initially screened by screening NGG (avoiding possible mutation sites), from which 8 targets were further screened by pre-experiments.

The 8 targets were as follows:

IGF2-E4-g1:AGCACTCTTCCACGATGCCA;

IGF2-E4-g2:GGCGCAGTAGGTCTCCAGCA;

IGF2-E4-g3:CCACCCCCGCCAAGTCCGAG;

IGF2-E4-g4:CTTCCACGATGCCACGGCTG;

IGF2-E4-g5:CCGCCGCAGCCGTGGCATCG;

IGF2-E4-g6:CACGGCTGCGGCGGTTCACG;

IGF2-E4-g7:CCACGATGCCACGGCTGCGG;

IGF2-E4-g8:AGTAGGTCTCCAGCAGGGCC。

3. Preparation of gRNA

IGF2-E4-g1-S and IGF2-E4-g1-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 1). Plasmid pKG-U6gRNA (IGF 2-E4-g 1) expresses the sgRNA _IGF2-E4-g1 shown in SEQ ID NO. 22.

sgRNA_IGF2-E4-g1(SEQ ID NO:22):

AGCACUCUUCCACGAUGCCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g2-S and IGF2-E4-g2-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 2). Plasmid pKG-U6gRNA (IGF 2-E4-g 2) expresses the sgRNA _IGF2-E4-g2 shown in SEQ ID NO. 23.

sgRNA_IGF2-E4-g2(SEQ ID NO:23):

GGCGCAGUAGGUCUCCAGCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g3-S and IGF2-E4-g3-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 3). Plasmid pKG-U6gRNA (IGF 2-E4-g 3) expresses the sgRNA _IGF2-E4-g3 shown in SEQ ID NO: 24.

sgRNA_IGF2-E4-g3(SEQ ID NO:24):

CCACCCCCGCCAAGUCCGAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g4-S and IGF2-E4-g4-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 4). Plasmid pKG-U6gRNA (IGF 2-E4-g 4) expresses the sgRNA _IGF2-E4-g4 shown in SEQ ID NO. 25.

sgRNA_IGF2-E4-g4(SEQ ID NO:25):

CUUCCACGAUGCCACGGCUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g5-S and IGF2-E4-g5-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 5). Plasmid pKG-U6gRNA (IGF 2-E4-g 5) expresses the sgRNA _IGF2-E4-g5 shown in SEQ ID NO. 26.

sgRNA_IGF2-E4-g5(SEQ ID NO:26):

CCGCCGCAGCCGUGGCAUCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g6-S and IGF2-E4-g6-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 6). Plasmid pKG-U6gRNA (IGF 2-E4-g 6) expresses the sgRNA _IGF2-E4-g6 shown in SEQ ID NO: 27.

sgRNA_IGF2-_E4-g6(SEQ ID NO:27):

CACGGCUGCGGCGGUUCACGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g7-S and IGF2-E4-g7-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 7). Plasmid pKG-U6gRNA (IGF 2-E4-g 7) expresses the sgRNA _IGF2-E4-g7 shown in SEQ ID NO. 28.

sgRNA_IGF2-E4-g7(SEQ ID NO:28):

CCACGAUGCCACGGCUGCGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g8-S and IGF2-E4-g8-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules having cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (IGF 2-E4-g 8). Plasmid pKG-U6gRNA (IGF 2-E4-g 8) expresses the sgRNA _IGF2-E4-g8 shown in SEQ ID NO. 29.

sgRNA_IGF2-E4-g8(SEQ ID NO:29):

AGUAGGUCUCCAGCAGGGCCguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-g1-S:caccgAGCACTCTTCCACGATGCCA;

IGF2-E4-g1-A:aaacTGGCATCGTGGAAGAGTGCTc;

IGF2-E4-g2-S:caccGGCGCAGTAGGTCTCCAGCA;

IGF2-E4-g2-A:aaacTGCTGGAGACCTACTGCGCC;

IGF2-E4-g3-S:caccgCCACCCCCGCCAAGTCCGAG;

IGF2-E4-g3-A:aaacCTCGGACTTGGCGGGGGTGGc;

IGF2-E4-g4-S:caccgCTTCCACGATGCCACGGCTG;

IGF2-E4-g4-A:aaacCAGCCGTGGCATCGTGGAAGc;

IGF2-E4-g5-S:caccgCCGCCGCAGCCGTGGCATCG;

IGF2-E4-g5-A:aaacCGATGCCACGGCTGCGGCGGc;

IGF2-E4-g6-S:caccgCACGGCTGCGGCGGTTCACG;

IGF2-E4-g6-A:aaacCGTGAACCGCCGCAGCCGTGc;

IGF2-E4-g7-S:caccgCCACGATGCCACGGCTGCGG;

IGF2-E4-g7-A:aaacCCGCAGCCGTGGCATCGTGGc;

IGF2-E4-g8-S:caccgAGTAGGTCTCCAGCAGGGCC;

IGF2-E4-g8-A:aaacGGCCCTGCTGGAGACCTACTc。

IGF2-E4-g1-S、IGF2-E4-g1-A、IGF2-E4-g2-S、IGF2-E4-g2-A、IGF2-E4-g3-S、IGF2-E4-g3-A、IGF2-E4-g4-S、IGF2-E4-g4-A、IGF2-E4-g5-S、IGF2-E4-g5-A、IGF2-E4-g6-S、IGF2-E4-g6-A、IGF2-E4-g7-S、IGF2-E4-g7-A、IGF2-E4-g8-S、IGF2-E4-g8-A Are all single-stranded DNA molecules.

4. Editing efficiency comparison of different target combinations

1. Co-transfection

The first group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 1) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 1), 1.08. Mu.g plasmid pKG-GE3.

The second group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 2) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 2) 1.08. Mu.g plasmid pKG-GE3.

The third group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 3) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 3), 1.08. Mu.g plasmid pKG-GE3.

The fourth group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 4) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 4) 1.08. Mu.g plasmid pKG-GE3.

The fifth group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 5) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 5) 1.08. Mu.g plasmid pKG-GE3.

The sixth group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 6) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 6) 1.08. Mu.g plasmid pKG-GE3.

The seventh group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 7) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand porcine primary fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 7), 1.08. Mu.g plasmid pKG-GE3.

The eighth group was to cotransfect plasmid pKG-U6gRNA (IGF 2-E4-g 8) and plasmid pKG-GE3 into porcine primary fibroblasts. The ratio was about 20 ten thousand primary swine fibroblasts, 0.92. Mu.g plasmid pKG-U6gRNA (IGF 2-E4-g 8), 1.08. Mu.g plasmid pKG-GE3.

And ninth group, pig primary fibroblast, and performing electrotransformation operation without adding plasmid according to the same electrotransformation parameters.

3. After completion of step 2, cells were digested and collected with trypsin, lysed, genomic DNA was extracted, PCR amplified with primer pairs composed of IGF2-E4-F136 and IGF2-E4-R728, and then subjected to 1% agarose gel electrophoresis.

And (3) cutting and recycling a target product, sending the target product to a sequencing company for sequencing (a sequencing peak diagram is shown in fig. 9), and analyzing the sequencing peak diagram by using a webpage plate Synthego ICE tool to obtain the gene editing efficiency of different targets. The gene editing efficiency of the first group to the eighth group was 9%, 14%, 1%, 11%, 5%, 18%, 6% in this order, and no gene editing occurred in the ninth group. The results show that IGF2-E4-g2 and IGF2-E4-g7 have higher editing efficiency.

Example 4 construction of prokaryotic Cas9 efficient expression vector

The schematic structure of plasmid pET-32a is shown in FIG. 10.

Plasmid pKG-GE4 was obtained by transformation with plasmid pET-32a as starting plasmid. The plasmid pET32a-T7lac-phoA is SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (called plasmid pKG-GE4 for short), is a circular plasmid as shown in SEQ ID NO. 1, and the structural schematic diagram is shown in FIG. 11.

In SEQ ID NO. 1, nucleotides 5121 to 5139 constitute the T7 promoter, nucleotides 5140 to 5164 encode the Lac operator (Lac operator), nucleotides 5178 to 5201 constitute the Ribosome Binding Site (RBS), nucleotides 5209 to 5271 encode the alkaline phosphatase signal peptide (phoA SIGNAL PEPTIDE), nucleotides 5272 to 5598 encode the TrxA protein, nucleotides 5620 to 5637 encode the His-Tag (also called His ₆ Tag), nucleotides 5638 to 5652 encode the enterokinase cleavage site (EK cleavage site), nucleotides 5656 to 5670 encode the nuclear localization signal, nucleotides 5701 to 9801 encode the Cas9 protein, nucleotides 9802 to 9849 encode the nuclear localization signal, and nucleotides 9902 to 9949 constitute the T7 terminator. The nucleotides encoding spCas9 protein have been codon optimized for the e.coli BL21 (DE 3) strain.

The plasmid pKG-GE4 is mainly modified by ① retaining the coding region of TrxA protein, which can help the expressed target protein form disulfide bond and increase the solubility and activity of the target protein, adding the coding sequence of alkaline phosphatase signal peptide before the coding region of TrxA protein, which can guide the expressed target protein to be secreted into the periplasm cavity of the bacterial membrane and be digested by prokaryotic periplasm signal peptide, ② adding the coding sequence of His-Tag after the coding sequence of TrxA protein, his-Tag can be used for enriching the expressed target protein, ③ adding the coding sequence of enterokinase enzyme cleavage site DDDDDDK (Asp-Asp-Asp-Lys) downstream of the coding sequence of His-Tag, the purified protein can remove His-Tag and the fused TrxA protein upstream under the action of enterokinase, ④ inserting the 9 genes expressed by the strain of escherichia coli BL21 (DE 3), and simultaneously adding the localization of the coding sequence of Cas-Tag at the upstream and downstream of the gene, and increasing the localization of the coding sequence.

The fusion gene in the plasmid pKG-GE4 is shown as 5209-9852 nucleotide in SEQ ID NO. 1, and encodes a fusion protein shown in SEQ ID NO. 2 (fusion protein TrxA-His-EK-NLS-spCas9-NLS, which is called PRONCN protein for short). Due to the presence of the alkaline phosphatase signal peptide and enterokinase cleavage site, the fusion protein is cleaved by enterokinase to form the protein shown in SEQ ID NO. 3, and the protein shown in SEQ ID NO. 3 is named NCN protein.

EXAMPLE 5 preparation and purification of NCN protein

1. Induction of expression

1. The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.

2. The recombinant strain obtained in step 1 was inoculated into a liquid LB medium containing 100. Mu.g/ml ampicillin, and cultured overnight at 37℃under shaking at 200 rpm.

3. Inoculating the bacterial liquid obtained in the step 2 to a liquid LB culture medium, culturing at 30 ℃ and 230rpm until the OD _600nm value=1.0, adding isopropyl thiogalactoside (IPTG) to ensure that the concentration of the isopropyl thiogalactoside in the system is 0.5mM, culturing at 25 ℃ and 230rpm for 12 hours, centrifuging at 4 ℃ and 10000g for 15 minutes, and collecting bacterial bodies.

4. And (3) washing the thalli obtained in the step (3) with PBS buffer solution.

2. Purification of fusion protein TrxA-His-EK-NLS-spCas9-NLS

1. And (3) adding the crude extraction buffer solution into the thalli obtained in the step one, suspending the thalli, crushing the thalli by using a homogenizer (1000 par circulation is performed three times), centrifuging at 4 ℃ and 15000g for 30min, collecting supernatant, filtering the supernatant by using a filter membrane with the aperture of 0.22 mu m, and collecting filtrate. In this step, 10ml of the crude extraction buffer was mixed per g of the wet cells.

The crude extraction buffer contained 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, 1mM PMSF, the balance ddH ₂ O.

2. The fusion protein was purified by affinity chromatography.

The method comprises the steps of firstly, balancing a Ni-NTA agarose column by using 5 column volumes of balancing solution (the flow rate is 1 ml/min), then loading 50ml of filtrate obtained in the step 1 (the flow rate is 0.5-1 ml/min), then washing the column by using 5 column volumes of balancing solution (the flow rate is 1 ml/min), then washing the column by using 5 column volumes of buffer solution (the flow rate is 1 ml/min) to remove the impurity proteins, then eluting by using 10 column volumes of eluent at the flow rate of 0.5-1ml/min, and collecting post-column solution (90-100 ml).

Ni-NTA agarose column, gold Style, L00250/L00250-C, packing 10ml.

The equilibration solution contained 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, the balance ddH ₂ O.

Buffer containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 50mM Imidazole, the balance ddH ₂ O.

The eluent contained 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 500mM Imidazole, the remainder ddH ₂ O.

3. Cleavage of fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein

1.15 Ml of the post-column solution collected in step two was concentrated to 200. Mu.l using an Amicon ultrafiltration tube (Sigma, UFC9100, capacity 15 ml) and then diluted to 1ml with 25mM Tris-HCl (pH 8.0). 6 ultrafiltration tubes were used to give a total of 6ml.

2. Commercial sources of recombinant bovine enterokinase (organisms, C620031, recombinant bovine enterokinase light chain, his ₆ tag, recombinant Bovine Enterokinase LIGHT CHAIN, his) with His ₆ tag were added to the solution (about 6 ml) obtained in step 1 and digested at 25 ℃ for 16 hours. 2 units of enterokinase were added in a ratio of 50. Mu.g protein.

3. The solution (about 6 ml) from step 2 was taken, mixed with 480. Mu.l of Ni-NTA resin (Kirsrui, L00250/L00250-C), spun at room temperature for 15min, centrifuged at 7000g for 3min, and the supernatant (4-5.5 ml) was collected.

4. The supernatant obtained in the step 3 was concentrated to 200. Mu.l using an Amicon ultrafiltration tube (Sigma, UFC9100, capacity: 15 ml), and then added to an enzyme stock solution to adjust the protein concentration to 5mg/ml, thereby obtaining an NCN protein solution.

The protein in NCN protein solution is sequenced, and 15 amino acid residues at the N end are shown in positions 1 to 15 of SEQ ID NO. 3, namely NCN protein.

The NCN proteins used in the subsequent examples were each provided by NCN protein solutions.

The enzyme stock solution (pH 7.4) contained 10mM Tris,300mM NaCl,0.1mM EDTA,1mM DTT,50% by volume of glycerol, the balance being ddH ₂ O.

Example 6 Performance of NCN protein

The selection of 2 gRNA targets targeting TTN genes was as follows:

TTN-gRNA1:AGAGCACAGTCAGCCTGGCG;

TTN-gRNA2:CTTCCAGAATTGGATCTCCG。

The primers used to identify the target fragment comprising the gRNA in the TTN gene were as follows:

TTN-F55:TACGGAATTGGGGAGCCAGCGGA;

TTN-R560:CAAAGTTAACTCTCTGTGTCT。

1. Preparation of gRNA

1. Preparation of TTN-T7-gRNA1 transcription template and TTN-T7-gRNA2 transcription template

The TTN-T7-gRNA1 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO. 4.

The TTN-T7-gRNA2 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO. 5.

2. In vitro transcription to obtain gRNA

TTN-T7-gRNA1 transcription template is adopted, TRANSCRIPT AID T7HIGH YIELD Transcription Kit (Fermentas, K0441) is adopted for in vitro transcription, and then MEGA CLEAR ^TM Transcription Clean-Up Kit (Thermo, AM 1908) is used for recovery and purification, so that TTN-gRNA1 is obtained. TTN-gRNA1 is single-stranded RNA, and is shown in SEQ ID NO. 6.

TTN-T7-gRNA2 transcription template is adopted, TRANSCRIPT AID T7HIGH YIELD Transcription Kit (Fermentas, K0441) is adopted for in vitro transcription, and then MEGA CLEAR ^TM Transcription Clean-Up Kit (Thermo, AM 1908) is used for recovery and purification, so that TTN-gRNA2 is obtained. TTN-gRNA2 is single-stranded RNA, as shown in SEQ ID NO. 7.

2. Optimization of dosage proportion of gRNA and NCN proteins

1. Co-transfected porcine primary fibroblasts

The first group was to co-transfect porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. The ratio was about 10 ten thousand porcine primary fibroblasts with 0.5. Mu.g TTN-gRNA1, 0.5. Mu.g TTN-gRNA2, 4. Mu.g NCN protein.

The second group is to co-transfect porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. The ratio was about 10 ten thousand porcine primary fibroblasts 0.75. Mu.g TTN-gRNA1 0.75. Mu.g TTN-gRNA2 4. Mu.g NCN protein.

Third group, porcine primary fibroblasts were co-transfected with TTN-gRNA1, TTN-gRNA2 and NCN proteins. The ratio is about 10 ten thousand porcine primary fibroblasts 1 μg TTN-gRNA 24 μg NCN protein.

Fourth group, porcine primary fibroblasts were co-transfected with TTN-gRNA1, TTN-gRNA2 and NCN proteins. The ratio was about 10 ten thousand porcine primary fibroblasts 1.25. Mu.g TTN-gRNA 1:1.25. Mu.g TTN-gRNA 2:4. Mu.g NCN protein.

And fifth group, co-transfecting the TTN-gRNA1 and the TTN-gRNA2 into the primary fibroblast of the pig. The ratio is about 10 ten thousand pig primary fibroblasts 1 mug TTN-gRNA1 and 1 mug TTN-gRNA2.

3. After the step 2 is completed, cells are digested and collected by trypsin, genomic DNA is extracted, PCR amplification is performed by using a primer pair consisting of TTN-F55 and TTN-R560, and then 1% agarose gel electrophoresis is performed.

The electrophoresis pattern is shown in FIG. 12. The 505bp band is wild type band (WT), and the 254bp band (about 251bp of the 505bp theoretical deletion of the wild type band) is deletion mutation band (MT).

Gene deletion mutation efficiency = (MT gray scale/MT band bp number)/(WT gray scale/WT band bp number + MT gray scale/MT band bp number) ×100%. The first group of gene deletion mutation efficiency was 19.9%, the second group of gene deletion mutation efficiency was 39.9%, the third group of gene deletion mutation efficiency was 79.9%, and the fourth group of gene deletion mutation efficiency was 44.3%. The fifth group had no mutation.

The results show that the gene editing efficiency is highest when the mass ratio of the two gRNAs to the NCN protein is 1:1:4, and the actual dosage is 1 mug to 4 mug. Thus, the optimum amount of the two gRNAs and NCN protein was determined to be 1. Mu.g/4. Mu.g.

3. Comparison of Gene editing efficiency of NCN protein and commercial Cas9 protein

1. Co-transfected porcine primary fibroblasts

Cas9-A group TTN-gRNA1, TTN-gRNA2 and commercial Cas9-A protein were co-transfected into porcine primary fibroblasts. The ratio is about 10 ten thousand pig primary fibroblasts with 1 mug TTN-gRNA 1to 1 mug TTN-gRNA2 to 4 mug Cas9-A protein.

The pKG-GE4 group was prepared by cotransfecting porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. The ratio is about 10 ten thousand porcine primary fibroblasts 1 μg TTN-gRNA 24 μg NCN protein.

Cas9-B group TTN-gRNA1, TTN-gRNA2 and commercial Cas9-B protein were co-transfected into porcine primary fibroblasts. The ratio is about 10 ten thousand pig primary fibroblasts with 1 mug TTN-gRNA 1to 1 mug TTN-gRNA2 to 4 mug Cas9-B protein.

Control group TTN-gRNA1, TTN-gRNA2 were co-transfected into porcine primary fibroblasts. The ratio is about 10 ten thousand pig primary fibroblasts 1 mug TTN-gRNA1 and 1 mug TTN-gRNA2.

The electrophoresis pattern is shown in FIG. 13. The gene deletion mutation efficiency of the commercial Cas9-A protein is 28.5%, the gene deletion mutation efficiency of the NCN protein is 85.6%, and the gene deletion mutation efficiency of the commercial Cas9-B protein is 16.6%.

The results show that compared with the commercial Cas9 protein, the NCN protein prepared by the method provided by the invention has the advantage that the gene editing efficiency is obviously improved.

EXAMPLE 7 preparation of GHR, IGF1, IGF2 Gene-Combined knockdown from Jiangxiang pig Single cell clones

The high-efficiency gRNA combinations of the GHR genes selected in example 1 (GHR-E5-g 2 and GHR-E5-g 3), the high-efficiency gRNA combinations of the IGF1 genes selected in example 2 (IGF 1-E4-g1 and IGF1-E4-g 2), and the high-efficiency gRNA combinations of the IGF2 genes selected in example 3 (IGF 2-E4-g2 and IGF2-E4-g 7) were selected.

1. Preparation of gRNA

1. Preparation of transcription templates

The GHR-E5-T7-g2 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO. 30.

The GHR-E5-T7-g3 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO. 31.

IGF1-E4-T7-g1 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO. 32.

IGF1-E4-T7-g2 transcription template is a double stranded DNA molecule as shown in SEQ ID NO. 33.

IGF2-E4-T7-g2 transcription template is a double stranded DNA molecule as shown in SEQ ID NO. 34.

IGF2-E4-T7-g7 transcription template is a double stranded DNA molecule as shown in SEQ ID NO. 35.

2. In vitro transcription to obtain gRNA

The transcription template was taken, subjected to in vitro transcription using TRANSCRIPT AID T7. 7 HIGH YIELD Transcription Kit (Fermentas, K0441), and then recovered and purified using MEGA CLEAR ^TM Transcription Clean-Up Kit (Thermo, AM 1908) to give the gRNA as single-stranded RNA.

The GHR-E5-T7-g2 transcription template yields gRNA as GHR-E5-gRNA2, as shown in SEQ ID NO. 36.

The GHR-E5-T7-g3 transcription template yields gRNA as GHR-E5-gRNA3, as shown in SEQ ID NO. 37.

IGF1-E4-T7-g1 transcription template to obtain gRNA which is IGF1-E4-gRNA1, as shown in SEQ ID NO. 38.

IGF1-E4-T7-g2 transcription template to obtain gRNA which is IGF1-E4-gRNA2, as shown in SEQ ID NO: 39.

IGF2-E4-T7-g2 transcription template to obtain gRNA which is IGF2-E4-gRNA2, as shown in SEQ ID NO. 40.

IGF2-E4-T7-g7 transcription template to obtain gRNA which is IGF2-E4-gRNA7, as shown in SEQ ID NO. 41.

GHR-E5-gRNA2(SEQ ID NO:36):

GGGACGGACCCCAUCUGUCCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

GHR-E5-gRNA3(SEQ ID NO:37):

GGAAGUCUCUAGUUCAGGUGAAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-gRNA1(SEQ ID NO:38):

GGGAGCCUUGGGCAUGUCCGUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF1-E4-gRNA2(SEQ ID NO:39):

GGGCUUCCGGAGCUGUGAUCUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-gRNA2(SEQ ID NO:40):

GGGGCGCAGUAGGUCUCCAGCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

IGF2-E4-gRNA7(SEQ ID NO:41):

GGCCACGAUGCCACGGCUGCGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

2. Transfection of porcine primary fibroblasts

1. The porcine primary fibroblasts were co-transfected with GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein. The proportion is about 10 ten thousand pig primary fibroblast ：0.5μg GHR-E5-gRNA2：0.5μg GHR-E5-gRNA3：0.5μg IGF1-E4-gRNA1：0.5μg IGF1-E4-gRNA2：0.5μg IGF2-E4-gRNA2：0.5μg IGF2-E4-gRNA7：6μg NCN proteins. Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofisher) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time after electrotransformation was 48 hours.

3. After completion of step 2, the cells were digested with trypsin and collected, then washed with complete medium, then resuspended with complete medium, and then each individual monoclonal was individually picked and transferred to 96-well plates (1 cell per well, 100 μl of complete medium per well) and cultured for 2 weeks (new complete medium was changed every 2-3 days).

4. After completion of step 3, cells were digested with trypsin and collected (about 2/3 of the resulting cells per well were inoculated into 6-well plates filled with complete culture medium, and the remaining 1/3 were collected in 1.5mL centrifuge tubes).

5. The 6-well plate of step 4 was used to culture until the cells grew to 80% confluence, the cells were digested with trypsin and collected, and the cells were frozen using cell frozen stock (90% complete medium+10% dmso, volume ratio).

6. Taking the centrifuge tube in the step 4, taking cells, performing cell lysis, extracting genome DNA, performing PCR amplification by adopting three primer pairs (a primer pair consisting of GHR-E5-F132 and GHR-E5-R467, a primer pair consisting of IGF1-E4-F114 and IGF1-E4-R575, and a primer pair consisting of IGF2-E4-F136 and IGF 2-E4-R728) respectively, and then performing electrophoresis. Porcine primary fibroblasts were used as wild-type control (WT).

7. After step 6 is completed, the PCR amplification product is recovered and sequenced.

The sequencing result of the primary fibroblast cells of pigs only has one, and the genotype of the primary fibroblast cells is wild type (also called homozygous wild type). If there are two types of single cell clones, one is identical to the sequencing result of the pig primary fibroblast, the other is mutated (mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the pig primary fibroblast, the genotype of the single cell clone is heterozygous, if the sequencing result of the single cell clone is two types, the genotype of the single cell clone is a double allele different mutant type if the mutation comprises deletion, insertion or substitution of one or more nucleotides compared with the sequencing result of the pig primary fibroblast, if the sequencing result of the single cell clone is one type and the mutation (mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the pig primary fibroblast, the genotype of the single cell clone is the same mutant type as the double allele, if the sequencing result of the single cell clone is one type and is identical with the sequencing result of the pig primary fibroblast, the genotype of the single cell clone is the wild type (can also be referred to as the wild type).

The homozygous knockout of IGF1 gene can greatly affect mice, which is easy to cause death of cloned mice, the homozygous knockout of GHR gene does not cause death of cloned mice, and the homozygous knockout of IGF2 gene does not cause death of cloned mice. Thus, the target single cell clone is in the form of a triple gene combination wherein the genotype of the IGF1 gene is heterozygous, the genotype of the GHR gene is a double allele identical mutant or a double allele different mutant, and the genotype of the IGF2 gene is a double allele identical mutant or a double allele different mutant.

A total of 93 single cell clones were obtained. The results are shown in Table 1. And screening according to genotypes identified by sequencing results, wherein 10 single cell clones with the numbers of 9, 35, 36, 54, 63, 65, 69, 75, 79 and 81 are target single cell clones, and the ratio of the single cell clones is 11%. Exemplary, three gene sequencing results for single cell clone number 63 are shown in figures 14 to 16 in comparison to Wild Type (WT) cells. The target single cell clone can be used for subsequent cloned pig production. And (3) taking the cells as nuclear transfer donor cells to clone somatic cells, so that cloned pigs can be obtained, namely miniature pigs.

TABLE 1 GHR genotyping results of IGF1, IGF2 Gene Joint editing Single cell clones

The single cell clone No. 9 shows the genotype of the GHR gene as the double allele identical mutant (deletion of 29bp in mutation form, deletion region corresponding to position 339 to 367 of sequence 9), the genotype of IGF1 gene as heterozygous (deletion of 16bp and 12bp in mutation form, deletion region corresponding to position 380 to 395 and 460 to 471 of sequence 15), and the genotype of IGF2 gene as the double allele identical mutant (deletion of 53bp in mutation form, deletion region corresponding to position 338 to 390 of sequence 21).

The single cell clone with the number 35 shows that the genotype of the GHR gene is a double allele different mutant (29 bp deletion on one chromosome, the deletion region corresponds to the position 339 to 367 of the sequence 9, 18bp deletion on the other chromosome, the deletion region corresponds to the position 353 to 370 of the sequence 9), the genotype of the IGF1 gene is a heterozygous type (77 bp deletion, 384 to 460 of the deletion region corresponds to the position 15), and the genotype of the IGF2 gene is a double allele identical mutant (53 bp deletion, 338 to 390 of the sequence 21).

The single cell clone with the number of 36 shows the genotype of the GHR gene as a double allele identical mutant (deletion of 1bp in mutation form, deletion region corresponding to position 368 of sequence 9), the genotype of the IGF1 gene as heterozygous (deletion of 77bp in mutation form, deletion region corresponding to position 384 to 460 of sequence 15), and the genotype of the IGF2 gene as a double allele identical mutant (deletion of 53bp in mutation form, deletion region corresponding to position 338 to 390 of sequence 21).

The single cell clone with the number 54 shows that the genotype of the GHR gene is a double allele different mutant (the mutation form on one chromosome is 29bp deletion, the deletion region corresponds to the position 339 to 367 of the sequence 9, the mutation form on the other chromosome is 1bp deletion, the deletion region corresponds to the position 368 of the sequence 9), the genotype of the IGF1 gene is heterozygous (the mutation form is 77bp deletion, the deletion region corresponds to the position 384 to 460 of the sequence 15), and the genotype of the IGF2 gene is the double allele same mutation form (the mutation form is 53bp deletion, the deletion region corresponds to the position 338 to 390 of the sequence 21).

The single cell clone with the number 63 shows the genotype of the GHR gene as the double allele identical mutant (deletion of 29bp in mutation form, deletion region corresponding to the position 339 to 367 of the sequence 9), the genotype of the IGF1 gene as the heterozygous type (deletion of 77bp in mutation form, deletion region corresponding to the position 384 to 460 of the sequence 15), and the genotype of the IGF2 gene as the double allele identical mutant (deletion of 53bp in mutation form, deletion region corresponding to the position 338 to 390 of the sequence 21).

The single cell clone with the number of 65 shows that the genotype of the GHR gene is a double allele different mutant (29 bp deletion on one chromosome, the deletion region corresponds to the position 339 to 367 of the sequence 9, 18bp deletion on the other chromosome, the deletion region corresponds to the position 353 to 370 of the sequence 9), the genotype of the IGF1 gene is a heterozygous type (77 bp deletion, 384 to 460 of the deletion region corresponds to the position 15), and the genotype of the IGF2 gene is a double allele identical mutant (53 bp deletion, 338 to 390 of the sequence 21).

The single cell clone with the number 69 shows that the genotype of the GHR gene is a double allele variant (29 bp deletion on one chromosome, deletion region corresponding to the positions 339 to 367 of the sequence 9, 18bp deletion on the other chromosome, deletion region corresponding to the positions 353 to 370 of the sequence 9), the genotype of the IGF1 gene is a heterozygous type (16 bp and 12bp deletion, deletion region corresponding to the positions 380 to 395 and 460 to 471 of the sequence 15), the genotype of the IGF2 gene is a double allele variant (53 bp deletion on one chromosome, deletion region corresponding to the positions 338 to 390 of the sequence 21, 3bp deletion and 1bp insertion on the other chromosome, deletion region corresponding to the positions 339 to 341 of the sequence 21, insertion corresponding to the positions 392 to 393 nucleotides).

The single cell clone numbered 75, the genotype of GHR gene was the same mutant as the double allele (18 bp deletion in the form of mutation, 353 to 370 positions in the region of deletion corresponding to sequence 9), the genotype of IGF1 gene was heterozygous (7 bp and 3bp deletions in the form of mutation, 380 to 386 positions and 459 to 461 positions in the region of deletion corresponding to sequence 15), and the genotype of IGF2 gene was the same mutant as the double allele (53 bp deletion in the form of mutation, 338 to 390 positions in the region of deletion corresponding to sequence 21).

The single cell clone No. 79 shows the genotype of the GHR gene as the double allele identical mutant (18 bp deletion, the deletion region corresponding to the position 353 to 370 of the sequence 9), the genotype of the IGF1 gene as the heterozygous gene (77 bp deletion, the deletion region corresponding to the position 384 to 460 of the sequence 15), and the genotype of the IGF2 gene as the double allele identical mutant (53 bp deletion, the deletion region corresponding to the position 338 to 390 of the sequence 21).

The single cell clone with the number of 81 shows the genotype of the GHR gene as the double allele identical mutant (deletion of 29bp in mutation form, deletion region corresponding to the position 339 to 367 of the sequence 9), the genotype of the IGF1 gene as the heterozygous type (deletion of 77bp in mutation form, deletion region corresponding to the position 384 to 460 of the sequence 15), and the genotype of the IGF2 gene as the double allele identical mutant (deletion of 53bp in mutation form, deletion region corresponding to the position 338 to 390 of the sequence 21).

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Nanjing Kidney Gene engineering Co., ltd

<120> Gene editing System for constructing Tri-Gene-combination-mutated miniature pig Nuclear transplantation donor cells and use thereof

<130> GNCYX212503

<160> 41

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9974

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900

tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100

cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160

ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gaaacaaagc 5220

actattgcac tggcactctt accgttactg tttacccctg tgacaaaagc catgagcgat 5280

aaaattattc acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg 5340

atcctcgtcg atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg 5400

gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa 5460

aaccctggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 5520

aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 5580

ctcgacgcta acctggccgg ttctggttct ggccatatgc accatcatca tcatcatgac 5640

gatgacgata agatgcccaa aaagaaacga aaggtgggta tccacggagt cccagcagcc 5700

gacaaaaaat atagcatcgg cctggacatc ggtaccaaca gcgttggctg ggcagtgatc 5760

actgatgaat acaaagttcc atccaaaaaa tttaaagtac tgggcaacac cgaccgtcac 5820

tctatcaaaa aaaacctgat tggtgctctg ctgtttgaca gcggcgaaac tgctgaggct 5880

acccgtctga aacgtacggc tcgccgtcgc tacactcgtc gtaaaaaccg catctgttat 5940

ctgcaggaaa ttttctctaa cgaaatggca aaagttgatg atagcttctt tcatcgtctg 6000

gaagagagct tcctggtgga agaagataaa aaacacgaac gtcacccgat tttcggtaac 6060

attgtggatg aggttgccta ccacgagaaa tatccgacca tctaccatct gcgtaaaaaa 6120

ctggttgata gcactgacaa agcggatctg cgtctgatct acctggctct ggcacacatg 6180

atcaaattcc gtggtcactt cctgatcgaa ggtgatctga accctgataa ctccgacgtg 6240

gacaaactgt tcattcagct ggttcagacc tataaccagc tgttcgaaga aaacccgatc 6300

aacgcgtccg gtgtagacgc taaggcaatt ctgtctgcgc gtctgtctaa gtctcgtcgt 6360

ctggaaaacc tgattgcgca actgccaggt gaaaagaaaa acggcctgtt cggcaatctg 6420

atcgccctgt ccctgggtct gactccgaac tttaaatcca actttgacct ggcggaagat 6480

gccaagctgc agctgagcaa agatacctat gacgatgacc tggataacct gctggcacag 6540

atcggtgatc agtatgccga tctgttcctg gccgcgaaaa acctgtctga tgcgattctg 6600

ctgtctgata tcctgcgcgt taacactgaa attactaaag cgccgctgag cgcatccatg 6660

attaaacgtt acgatgaaca ccaccaggat ctgaccctgc tgaaagcgct ggtgcgtcag 6720

cagctgccgg aaaaatacaa ggagatcttc ttcgaccaga gcaaaaacgg ttacgcgggc 6780

tacattgatg gtggtgcatc tcaggaggaa ttctacaaat tcattaaacc gatcctggaa 6840

aaaatggatg gtactgaaga gctgctggtt aaactgaatc gtgaagatct gctgcgcaaa 6900

cagcgtacct tcgataacgg ttccatcccg catcagattc atctgggcga actgcacgct 6960

atcctgcgcc gtcaggaaga cttttatccg ttcctgaaag acaaccgtga gaaaattgaa 7020

aaaatcctga ccttccgtat tccgtactat gtaggtccgc tggcgcgtgg taactcccgt 7080

ttcgcttgga tgacccgcaa aagcgaagaa accatcaccc cgtggaattt cgaagaagtc 7140

gttgacaaag gcgcgtccgc gcagtctttc atcgaacgca tgacgaactt cgacaaaaac 7200

ctgccgaacg agaaagtgct gccgaaacac tctctgctgt acgagtactt cactgtgtac 7260

aacgaactga ccaaagtgaa atacgtcacc gaaggtatgc gtaaaccggc attcctgtcc 7320

ggtgagcaaa aaaaagcaat cgtggatctg ctgttcaaaa ccaaccgtaa agtaaccgtg 7380

aaacagctga aggaagacta tttcaagaaa atcgaatgtt ttgattctgt tgaaatctcc 7440

ggcgtggaag atcgcttcaa tgcgtccctg ggtacgtatc acgacctgct gaaaattatc 7500

aaagacaaag attttctgga caacgaggaa aacgaagaca tcctggagga tattgtactg 7560

accctgaccc tgttcgaaga ccgtgagatg atcgaagaac gcctgaaaac ctacgcccac 7620

ctgttcgatg acaaggtaat gaagcagctg aaacgtcgtc gttataccgg ctggggtcgt 7680

ctgtcccgta aactgatcaa tggcatccgt gataaacagt ctggcaaaac catcctggac 7740

ttcctgaaat ccgacggttt cgcgaatcgt aacttcatgc aactgattca tgacgattct 7800

ctgactttca aagaagacat ccagaaagca caggtttccg gccagggtga ctctctgcac 7860

gagcacattg ccaatctggc tggttctccg gctattaaaa agggtattct gcagactgtg 7920

aaagtagttg atgagctggt caaagtaatg ggccgtcaca agccggaaaa cattgtgatc 7980

gaaatggcac gtgaaaacca gacgacccag aaaggtcaga aaaactctcg tgaacgcatg 8040

aaacgtatcg aagaaggcat caaagaactg ggctctcaga tcctgaagga acaccctgta 8100

gaaaataccc agctgcagaa cgaaaagctg tatctgtatt acctgcagaa cggccgcgat 8160

atgtatgtgg accaggaact ggatatcaac cgcctgtccg attacgatgt agatcacatc 8220

gtgccgcaaa gcttcctgaa agacgacagc attgacaaca aagtactgac ccgttctgat 8280

aagaaccgtg gcaaatccga taacgtcccg tctgaagaag ttgttaaaaa aatgaaaaac 8340

tattggcgtc agctgctgaa cgcgaaactg atcacccagc gtaagttcga caatctgact 8400

aaagctgagc gcggtggtct gtccgaactg gataaagcgg gttttatcaa acgccagctg 8460

gttgaaaccc gtcagatcac gaagcacgtt gcgcagattc tggactctcg tatgaacacc 8520

aaatacgacg aaaacgacaa actgatccgc gaggttaagg ttatcaccct gaaaagcaaa 8580

ctggtatccg attttcgtaa agactttcag ttctacaaag tgcgcgaaat taacaactat 8640

caccacgctc acgatgcata tctgaatgca gttgttggca cggcgctgat caaaaagtat 8700

ccgaaactgg aatctgaatt cgtatacggc gattacaaag tgtatgacgt tcgtaagatg 8760

atcgcaaaat ccgagcagga aattggtaag gcgacggcga aatacttctt ttattccaat 8820

attatgaact ttttcaaaac cgaaatcacc ctggcgaatg gtgaaattcg taaacgcccg 8880

ctgatcgaaa ccaacggtga aactggtgaa atcgtttggg acaaaggccg cgacttcgcg 8940

accgtgcgta aagttctgtc tatgccgcaa gtgaacatcg tcaagaagac cgaagtacaa 9000

accggcggtt ttagcaaaga gagcattctg ccaaaacgta actccgacaa actgatcgcg 9060

cgcaagaaag actgggatcc gaaaaaatac ggtggtttcg attctccaac cgttgcttat 9120

tccgttctgg tggtagccaa agttgagaaa ggtaaaagca aaaaactgaa atccgtaaag 9180

gaactgctgg gtattactat catggagcgt agctccttcg aaaaaaaccc gatcgatttt 9240

ctggaagcga aaggctataa agaagtcaaa aaggacctga tcatcaaact gccaaaatac 9300

agcctgttcg agctggaaaa cggccgtaaa cgtatgctgg catctgcggg cgaactgcag 9360

aaaggcaacg agctggctct gccgtccaaa tacgtgaact ttctgtacct ggcctctcac 9420

tacgaaaaac tgaaaggttc cccggaagac aacgaacaga aacagctgtt cgtagagcag 9480

cacaaacact acctggacga gatcatcgaa cagatttctg aattttctaa acgtgtgatt 9540

ctggctgatg cgaatctgga taaagttctg tctgcctata acaagcatcg tgacaaaccg 9600

atccgcgaac aggctgagaa catcatccac ctgttcactc tgactaacct gggcgcgcca 9660

gcggctttca agtactttga taccaccatt gaccgcaagc gttacacctc cactaaagaa 9720

gtgctggacg cgactctgat ccaccagtcc atcaccggtc tgtacgagac ccgtatcgat 9780

ctgagccagc tgggcggtga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 9840

aagaaaaagt gacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9900

actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9960

aactatatcc ggat 9974

<210> 3

<211> 1547

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Thr Lys Ala Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp

20 25 30

Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp

35 40 45

Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu

50 55 60

Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu

65 70 75 80

Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly

85 90 95

Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys

100 105 110

Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn

115 120 125

Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His Asp

130 135 140

Asp Asp Asp Lys Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly

145 150 155 160

Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr

165 170 175

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

180 185 190

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

195 200 205

Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala

210 215 220

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

225 230 235 240

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

245 250 255

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

260 265 270

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

275 280 285

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

290 295 300

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

305 310 315 320

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

325 330 335

Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val

340 345 350

Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly

355 360 365

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

370 375 380

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

385 390 395 400

Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys

405 410 415

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

420 425 430

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

435 440 445

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

450 455 460

Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu

465 470 475 480

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

485 490 495

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

500 505 510

Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly

515 520 525

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

530 535 540

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

545 550 555 560

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

565 570 575

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

580 585 590

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

595 600 605

Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg

610 615 620

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

625 630 635 640

Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu

645 650 655

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

660 665 670

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

675 680 685

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

690 695 700

Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg

705 710 715 720

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

725 730 735

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

740 745 750

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

755 760 765

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

770 775 780

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

785 790 795 800

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

805 810 815

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

820 825 830

Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser

835 840 845

Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser

850 855 860

Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly

865 870 875 880

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

885 890 895

Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys

900 905 910

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

915 920 925

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

930 935 940

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

945 950 955 960

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

965 970 975

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

980 985 990

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

995 1000 1005

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1010 1015 1020

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1025 1030 1035 1040

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1045 1050 1055

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1060 1065 1070

Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg

1075 1080 1085

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1090 1095 1100

Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr

1105 1110 1115 1120

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1125 1130 1135

Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu

1140 1145 1150

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1155 1160 1165

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1170 1175 1180

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1185 1190 1195 1200

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1205 1210 1215

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1220 1225 1230

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1235 1240 1245

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1250 1255 1260

Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1265 1270 1275 1280

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1285 1290 1295

Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1300 1305 1310

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1315 1320 1325

Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1330 1335 1340

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1345 1350 1355 1360

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1365 1370 1375

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1380 1385 1390

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1395 1400 1405

Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1410 1415 1420

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1425 1430 1435 1440

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1445 1450 1455

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1460 1465 1470

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys

1475 1480 1485

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu

1490 1495 1500

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1505 1510 1515 1520

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala

1525 1530 1535

Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1540 1545

<210> 4

<211> 1399

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala

1 5 10 15

Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

20 25 30

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

35 40 45

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

50 55 60

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

65 70 75 80

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

85 90 95

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

100 105 110

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

115 120 125

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

130 135 140

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

145 150 155 160

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

165 170 175

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

180 185 190

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

195 200 205

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

210 215 220

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

225 230 235 240

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

245 250 255

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

260 265 270

Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

275 280 285

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

290 295 300

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

305 310 315 320

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

325 330 335

Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

340 345 350

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

355 360 365

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

370 375 380

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

385 390 395 400

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

405 410 415

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

420 425 430

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

435 440 445

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

450 455 460

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

465 470 475 480

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

485 490 495

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

500 505 510

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

515 520 525

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

530 535 540

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

545 550 555 560

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

565 570 575

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

580 585 590

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

595 600 605

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

610 615 620

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

625 630 635 640

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

645 650 655

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

660 665 670

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

675 680 685

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

690 695 700

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

705 710 715 720

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

725 730 735

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

740 745 750

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

755 760 765

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

770 775 780

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

785 790 795 800

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

805 810 815

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

820 825 830

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

835 840 845

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

850 855 860

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

865 870 875 880

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

885 890 895

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

900 905 910

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

915 920 925

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

930 935 940

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

945 950 955 960

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

965 970 975

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

980 985 990

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

995 1000 1005

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

1010 1015 1020

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1025 1030 1035 1040

Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn

1045 1050 1055

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1060 1065 1070

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val

1075 1080 1085

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1090 1095 1100

Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1105 1110 1115 1120

Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1125 1130 1135

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1140 1145 1150

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1155 1160 1165

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1170 1175 1180

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys

1185 1190 1195 1200

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1205 1210 1215

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1235 1240 1245

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1250 1255 1260

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1265 1270 1275 1280

Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile

1285 1290 1295

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1300 1305 1310

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1315 1320 1325

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr

1330 1335 1340

Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala

1345 1350 1355 1360

Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1365 1370 1375

Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala

1380 1385 1390

Gly Gln Ala Lys Lys Lys Lys

1395

<210> 4

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggagagc acagtcagcc tggcggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 5

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggcttcc agaattggat ctccggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 6

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

ggagagcaca gucagccugg cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 7

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

ggcuuccaga auuggaucuc cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 8

<211> 638

<212> PRT

<213> Sus scrofa

<400> 8

Met Asp Leu Trp Gln Leu Leu Leu Thr Leu Ala Val Ala Gly Ser Ser

1 5 10 15

Asp Ala Phe Ser Gly Ser Glu Ala Thr Pro Ala Val Leu Val Arg Ala

20 25 30

Ser Gln Ser Leu Gln Arg Val His Pro Gly Leu Glu Thr Asn Ser Ser

35 40 45

Gly Lys Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Leu Glu Thr Phe

50 55 60

Ser Cys His Trp Thr Asp Gly Val Arg His Gly Leu Gln Ser Pro Gly

65 70 75 80

Ser Ile Gln Leu Phe Tyr Ile Arg Arg Ser Thr Gln Glu Trp Thr Gln

85 90 95

Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys

100 105 110

Tyr Phe Asn Ser Ser Tyr Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys

115 120 125

Leu Thr Ser Asn Gly Gly Thr Val Asp Gln Lys Cys Phe Ser Val Glu

130 135 140

Glu Ile Val Gln Pro Asp Pro Pro Ile Gly Leu Asn Trp Thr Leu Leu

145 150 155 160

Asn Ile Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu

165 170 175

Pro Pro Pro Asn Ala Asp Val Gln Lys Gly Trp Ile Val Leu Glu Tyr

180 185 190

Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr Gln Trp Lys Met Met Asp

195 200 205

Pro Val Leu Ser Thr Ser Val Pro Val Tyr Ser Leu Arg Leu Asp Lys

210 215 220

Glu Tyr Glu Val Arg Val Arg Ser Arg Gln Arg Asn Ser Glu Lys Tyr

225 230 235 240

Gly Glu Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Pro

245 250 255

Phe Ala Cys Glu Glu Asp Phe Arg Phe Pro Trp Phe Leu Ile Ile Ile

260 265 270

Phe Gly Ile Phe Gly Leu Thr Val Ile Leu Phe Leu Leu Ile Phe Ser

275 280 285

Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro

290 295 300

Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu

305 310 315 320

Glu Val Asn Thr Ile Leu Ala Ile His Asp Asn Tyr Lys His Glu Phe

325 330 335

Tyr Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Asp

340 345 350

Pro Asp Glu Lys Thr Glu Gly Ser Asp Thr Asp Arg Leu Leu Asn Asn

355 360 365

Asp His Glu Lys Ser Leu Thr Ile Leu Gly Ala Lys Asp Asp Asp Ser

370 375 380

Gly Arg Thr Ser Cys Tyr Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn

385 390 395 400

Ala Asn Asp Val Cys Asp Gly Thr Ala Glu Val Ala Gln Pro Gln Arg

405 410 415

Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn

420 425 430

Asn Ser Pro Ser Asn Asp Ala Ala Pro Ala Thr Gln Gln Pro Ser Val

435 440 445

Ile Leu Ala Glu Glu Asn Lys Pro Arg Pro Leu Ile Ile Ser Gly Thr

450 455 460

Asp Ser Thr His Gln Thr Ala His Thr Gln Leu Ser Asn Pro Ser Ser

465 470 475 480

Leu Ala Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala

485 490 495

Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Ile Ser

500 505 510

Gln Cys Asp Met His Leu Glu Val Val Ser Pro Cys Pro Ala Asn Phe

515 520 525

Ile Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile

530 535 540

Ala Met Ala Pro His Val Glu Val Glu Ser Arg Val Ala Pro Ser Phe

545 550 555 560

Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Thr Ala

565 570 575

Gly Arg Ser Gly Thr Ala Glu Cys Ala Pro Ser Ser Glu Met Pro Val

580 585 590

Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Val

595 600 605

Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser

610 615 620

Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro

625 630 635

<210> 9

<211> 730

<212> DNA

<213> Sus scrofa

<400> 9

tagctgttct ctctatccat cccgctcacc ctccaaataa actgcctgta cccaaatcct 60

catctctagt actggtttct taaataagcc ctaagaaata atgttgggaa taaaaacaca 120

atggtttgtc cctggaatta agggccgaca gaggaatgat tgacaagaac cgctctgaag 180

ctgtgaccca ggaaaacatt tctagaagtg gttgttcttc accactttaa atatgtgttt 240

cattaggacc atccatcacc ctcctgatct catgccttgc cttttctttt tattcggcag 300

attcttctgg aaagcctaaa ttcaccaagt gccgttcacc tgaactagag actttttcat 360

gccactggac agatggggtc cgtcacggtt tacagagccc tggatccata cagctgttct 420

atattagaag gtacagcctt catgcctttc tgacttttct ctccatgaat tttctgatta 480

aaatgtactg agtcatatgc aatagtagga acggaaatga tttattttga tgatctaaat 540

gtattcattc atttattcaa aaaatattaa tgaagccctt attgtctgtt gcacactatt 600

ttgggcactg gagatacagg aatgattaca aaaagataag gtctctggtc tcctggagat 660

ttgttcccag ctggtgaaga cagataacaa aaaaattttt ttaattaaat gtcagctggt 720

aatatgggtt 730

<210> 10

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

cagggcucug uaaaccguga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 11

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

gacggacccc aucuguccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 12

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

aagucucuag uucaggugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 13

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

uggacagaug ggguccguca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 14

<211> 130

<212> PRT

<213> Sus scrofa

<400> 14

Met His Ile Thr Ser Ser Ser His Leu Phe Tyr Leu Ala Leu Cys Leu

1 5 10 15

Leu Ser Phe Thr Ser Ser Ala Thr Ala Gly Pro Glu Thr Leu Cys Gly

20 25 30

Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe

35 40 45

Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro

50 55 60

Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg

65 70 75 80

Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala Arg

85 90 95

Ser Val Arg Ala Gln Arg His Thr Asp Met Pro Lys Ala Gln Lys Glu

100 105 110

Val His Leu Lys Asn Thr Ser Arg Gly Ser Ser Gly Asn Lys Asn Tyr

115 120 125

Arg Met

130

<210> 15

<211> 782

<212> DNA

<213> Sus scrofa

<400> 15

tgggcataga caagatcctt gactacaggt gattaagaac ctaaggagaa ttagcacaaa 60

taaatgcatg atgagtgagg tctgccaacg aatgtggcac tgactgcagg agaaacagtg 120

gaacccagaa ggacctacag ggtcaggaat tgttggagaa gcttcaagaa gaggttgact 180

tacaactgtg tgggttgaca agaggtggga agaggagagt ctgcaggggc caggtggaac 240

tgtgacgata gtgtattatt ccactctaaa gccaggcccc tctgcatttg atttgaacag 300

acaagcccac agggtacggc tccagcagtc ggagggcgcc acagacgggc atcgtggatg 360

agtgctgctt ccggagctgt gatctgagga ggctggagat gtactgtgca cccctcaagc 420

ctgccaagtc ggcccgctcc gtccgtgccc agcgccacac ggacatgccc aaggctcaga 480

aggtaagcca gcctgggcgg ggtcagccat cctcaagaga cttatcagtg tgagtgtgcc 540

aaacagttat tgtacccctg gttctctccc tgagaggtcc aactcttcca tcactccaca 600

ttgcaaatcc tcccttccac tgctctggac ctctgatcac caaaagatgg tggagaagag 660

tgactaaacc tgggctttgg tatcagacaa aactgaaggt ttaccttcac ccatcaccag 720

ctgacagccc ttggccaaat aatgtatcct tccaagcctt agtttcatca gtaaagatgg 780

ga 782

<210> 16

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

gagccuuggg cauguccgug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 17

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

gcuuccggag cugugaucug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 18

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

ggcgccacag acgggcaucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 19

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

cguccgugcc cagcgccaca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 20

<211> 181

<212> PRT

<213> Sus scrofa

<400> 20

Met Gly Ile Pro Met Arg Lys Pro Leu Leu Val Leu Leu Val Phe Leu

1 5 10 15

Ala Leu Ala Ser Cys Cys Tyr Ala Ala Tyr Arg Pro Ser Glu Thr Leu

20 25 30

Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg

35 40 45

Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Asn Arg Arg Ser Arg

50 55 60

Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu

65 70 75 80

Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr

85 90 95

Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys

100 105 110

Phe Phe Arg Tyr Asp Thr Trp Lys Gln Ser Ala Gln Arg Leu Arg Arg

115 120 125

Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly Arg Thr Leu Ala Lys

130 135 140

Glu Leu Glu Ala Val Arg Glu Ala Lys Arg His Arg Pro Leu Thr Ala

145 150 155 160

Arg Pro Thr Arg Asp Pro Ala Ala His Gly Gly Ala Ser Pro Glu Ala

165 170 175

Ser Gly His Arg Lys

180

<210> 21

<211> 758

<212> DNA

<213> Sus scrofa

<400> 21

ccaaacagcc ttgggtcgag gcccaagagg ctgggcccgg tttaaggacg gggagggagg 60

cgccaagagg ccaggggctg gtcccgagca cgcccgcacc cgctcacccc cgctgtcccc 120

tctccttccc cggggggccc ctgtgcaccc cactctcact tcttctgctc gaggccacga 180

ggctggctgt ccccgcaagg tgaccgggcg tcctgtctgg agggcggggg ccggggcggc 240

tgggggcacc gtccgtgccc ggggcccctg tgctgacgtg ccctcccctt ggtcctgtgg 300

gacttccagg caggccggca agccgcgtga accgccgcag ccgtggcatc gtggaagagt 360

gctgcttccg tagctgcgac ctggccctgc tggagaccta ctgcgccacc cccgccaagt 420

ccgagaggga cgtgtcgacc cctccgaccg tgcttccggt aaggcagccc ctctctcggc 480

agcgcccccc ccccgggggg ggctgtctcc tctgagccgg gggaccgggg cgcagccggc 540

tcttgggctt caagtgctgc cagaggggcc ttccccgctg gggaccctgg ccagaagcca 600

gggcagtctt cgctctgtcg cagggcaggc aggcaggagg accccgcaga ggttgttgtt 660

ctgggacagg ggctgggggg ccaggccccc ccctgacggg cccttcccct ctcaggacaa 720

cttccccaga taccccgtgg gcaagttctt ccgctatg 758

<210> 22

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

agcacucuuc cacgaugcca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 23

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

ggcgcaguag gucuccagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 24

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

ccacccccgc caaguccgag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 25

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

cuuccacgau gccacggcug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 26

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

ccgccgcagc cguggcaucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 27

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 27

cacggcugcg gcgguucacg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 28

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 28

ccacgaugcc acggcugcgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 29

<211> 100

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 29

aguaggucuc cagcagggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 30

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 30

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

atagggacgg accccatctg tccaggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 31

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 31

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggaagtc tctagttcag gtgaagtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 32

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 32

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

atagggagcc ttgggcatgt ccgtggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 33

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 33

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

atagggcttc cggagctgtg atctggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 34

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 34

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggggcgc agtaggtctc cagcagtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 35

<211> 225

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 35

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggccacg atgccacggc tgcgggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 36

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 36

gggacggacc ccaucugucc agguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 37

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 37

ggaagucucu aguucaggug aaguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 38

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 38

gggagccuug ggcauguccg ugguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 39

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 39

gggcuuccgg agcugugauc ugguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 40

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 40

ggggcgcagu aggucuccag caguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 41

<211> 102

<212> RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 41

ggccacgaug ccacggcugc ggguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

Claims

1. A kit comprising GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7, and NCN protein;

The GHR-E5-gRNA2 is the sgRNA, the target sequence binding region of the GHR-E5-gRNA2 is shown as 3-22 nucleotides in SEQ ID NO:36, the target sequence binding region of the GHR-E5-gRNA3 is shown as 3-22 nucleotides in SEQ ID NO:37, the IGF1-E4-gRNA1 is the sgRNA, the target sequence binding region of the IGF1-E4-gRNA2 is shown as 3-22 nucleotides in SEQ ID NO:38, the IGF1-E4-gRNA2 is the sgRNA, the target sequence binding region of the IGF2-E4-gRNA2 is shown as 3-22 nucleotides in SEQ ID NO:39, the target sequence binding region of the IGF2-E4-gRNA7 is shown as 3-22 nucleotides in SEQ ID NO:40, and the NCN protein is shown as 3-NCID NO: 3;

The preparation method of the NCN protein comprises the following steps:

(2) Culturing the recombinant bacteria by adopting a liquid culture medium at 30 ℃, then adding IPTG and performing induction culture at 25 ℃, and then collecting thalli;

(3) Crushing the collected thalli, and collecting a crude protein solution;

The plasmid pKG-GE4 is shown as SEQ ID NO. 1;

The kit is used for preparing recombinant pig fibroblasts, (b) small pigs, (c) growing pigs with reduced body types, (d) preparing model pigs with retarded growth, preparing cell models with retarded growth or tissue models with retarded growth or organ models with retarded growth, preparing model pigs with retarded growth, and preparing (f) preparing model pigs with retarded growth or tissue models with retarded growth or organ models with retarded growth.

Application of GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein in preparation of kit;

GHR-E5-gRNA2 is the GHR-E5-gRNA2 of claim 1, GHR-E5-gRNA3 is the GHR-E5-gRNA3 of claim 1, IGF1-E4-gRNA1 is the IGF1-E4-gRNA1 of claim 1, IGF1-E4-gRNA2 is the IGF1-E4-gRNA2 of claim 1, IGF2-E4-gRNA2 is the IGF2-E4-gRNA2 of claim 1, IGF2-E4-gRNA7 is the IGF2-E4-gRNA7 of claim 1, and NCN protein is the NCN protein of claim 1;

3. A method for producing a recombinant cell, comprising the steps of co-transfecting a pig fibroblast with GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein to obtain a recombinant cell, wherein GHR-E5-gRNA2 is GHR-E5-gRNA2 as defined in claim 1, GHR-E5-gRNA3 is GHR-E5-gRNA3 as defined in claim 1, IGF1-E4-gRNA1 is IGF1-E4-gRNA1 as defined in claim 1, IGF1-E4-gRNA2 is IGF1-E4-gRNA2 as defined in claim 1, IGF2-E4-gRNA2 is 2-E4-gRNA2 as defined in claim 1, IGF2-E4-gRNA2 is defined in claim 1, and IGF2-E4-gRNA2 is NCN protein as defined in claim 1-4-gRNA.

4. The method of claim 3, wherein the ratio of pig fibroblasts, GHR-E5-gRNA2, GHR-E5-gRNA3, IGF1-E4-gRNA1, IGF1-E4-gRNA2, IGF2-E4-gRNA7 and NCN protein is 10 ten thousand pig cell ：0.4-0.6μg GHR-E5-gRNA2：0.4-0.6μg GHR-E5-gRNA3：0.4-0.6μg IGF1-E4-gRNA1：0.4-0.6μg IGF1-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA2：0.4-0.6μg IGF2-E4-gRNA7：5-7μg NCN proteins.