CN106519038B

CN106519038B - Fusion protein, production method thereof and production method of transgenic rice

Info

Publication number: CN106519038B
Application number: CN201510585574.4A
Authority: CN
Inventors: 于为常; 陈磊; 黄彪; 李相鲁
Original assignee: Shenzhen Research Institute of CUHK
Current assignee: Shenzhen Research Institute of CUHK
Priority date: 2015-09-15
Filing date: 2015-09-15
Publication date: 2019-12-27
Anticipated expiration: 2035-09-15
Also published as: CN106519038A

Abstract

The invention relates to a fusion protein, a production method thereof and a production method of transgenic rice, wherein the fusion protein is a plant seed endosperm expression Ex4-TF fusion protein, and the gene sequence thereof comprises: the plant endosperm specific expression promoter pGluB-5, a plant endosperm storage protein signal peptide sp, an exenatide polypeptide Exendin-4, a linker polypeptide (GGGGS) x3linker connecting the Exendin-4 and a human iron transporter hTF, the human iron transporter hTF, a histone tag 6XHis used for protein purification, and a plant GluB-5 gene transcription terminator GluB-5-3' -UTR. The invention can realize the mass expression of the fusion protein in the whole endosperm by utilizing the GluB-5 promoter. Meanwhile, the continuous production of the Ex4-TF fusion protein medicine can be realized by utilizing the advantages of high yield, easy planting and management, rice seed storage resistance, good extraction and the like of the rice, the problems of waste and shortage can not be caused, and the production cost can be reduced.

Description

Fusion protein, production method thereof and production method of transgenic rice

Technical Field

The invention relates to a fusion protein and a production method thereof, in particular to a plant seed expression system of a recombinant exenatide fusion protein and a production method of transgenic rice.

Background

Diabetes is a chronic, multiple disease condition of the world. Type one diabetes is caused by insufficient secretion of insulin to regulate blood glucose, while type two diabetes is caused by poor utilization of insulin or insulin resistance. Insulin is a hormone secreted by pancreatic beta cells for regulating blood sugar concentration, and the loss or ineffective use of insulin often raises the blood sugar level of patients, and if not effectively treated, can cause serious complications (such as heart disease, stroke, diabetic retinopathy, renal failure, etc.) and even death.

Diabetes causes the death of millions of people each year, either directly or indirectly. World health organization data shows that there are 3.47 million diabetics worldwide, 90% of which are type 2 diabetes (http:// www.who.int/media/videos/fs 312/en /). China is the first major country of diabetes, the incidence of the disease is 6.7 percent at present, which is 6.4 percent higher than the international average level, and the high-risk population of diabetes in China is expanded to about 1.5 hundred million. More than 90 percent of diabetes in China is type II diabetes, and the diabetes prevention and treatment has important significance for improving the national quality of China.

The native exenatide (Exendin-4), a lizard salivary secretion, is a 39 peptide (Eng et al, 1992) that is 53% homologous to human glucagon-like peptide-1 (GLP-1), a incretin hormone, and also functions similarly to GLP-1 (Alarcon et al, 2006), and has a biological effect that is almost identical to GLP-1. The main difference from GLP-1 is that the 2 nd glycine (Gly) at the N terminal makes the N terminal not easy to be decomposed by DPP-IV (dipeptidyl peptidase-IV) in blood, while the same position of GLP-1 is alanine (Ala) which is easily cut by DPP-IV, so that the half-life of Exendin-4 after being absorbed into blood is longer (t1/2 is 9.7 h). Exendin-4 is a potent GLP-1 receptor agonist, and can simulate the sugar regulation effect of GLP-1, an endogenous polypeptide, and reduce fasting and postprandial blood glucose. The activity of Exenatide (Exenatide) is mediated primarily by binding to the human pancreatic GLP-1 receptor, initiating glucose-dependent insulin synthesis and secretion by cyclic adenosine monophosphate (cAMP) dependent and beta cell differentiation mechanisms. Exendin-4 is also commonly regarded as one of incretins because of its amino acid structure and biological activity related to GLP-1. Because the blood sugar reducing action time of Exendin-4 is longer, the beneficial effects of delaying gastric emptying and inhibiting ingestion impulse on the body weight of an obese patient are different from the beneficial effects of the traditional blood sugar reducing medicine, the potential of the Exendin-4 as a medicine for treating type 2 diabetes is huge.

The research finds that the protein can reduce the blood sugar concentration of a diabetic patient and improve the function marker of beta cells, and the excessive blood sugar reduction can not cause the blood sugar concentration of the patient to be too low, and the weight of the patient is reduced in the blood sugar reduction process, so that the pain of the patient is further relieved.

GLP-1 is secreted by small intestine endocrine L cells, and is secreted by diet stimulation. GLP-1 is secreted into the pancreas with the blood and binds to a receptor on the beta cells of the islets of Langerhans (GLP-1R), stimulating insulin synthesis and beta cell proliferation via the cAMP second messenger (Buteau et al, 1999; Stoffers et al, 2000; Furman et al, 2010). GLP-1 simultaneously inhibits blood glucose secretion (Orskov et al, 1988k Komatsu et al, 1989), delays gastric emptying (Wettergrenet et al, 1993; Willms et al, 1996; Nauck et al, 1997), suppresses appetite (Turton et al, 1996; Flint et al, 1998), and prevents beta cell death (Farilla et al, 2002; Wang et al, 2004). However, the short survival period of GLP-1 in vivo (half-life of about 2 minutes) is rapidly degraded by dipeptidyl peptidase IV (DPP-IV) and other proteases in vivo (Mentlein et al, 1993; Kieffer et al, 1995; Hupe-Sodmannet al, 1995), thus greatly limiting the potential for GLP-1 to develop drugs for the treatment of diabetes.

The discovery of the exenatide enables the treatment of the type II diabetes to have a specific therapeutic drug. Like GLP-1, exenatide also binds to the GLP-1 receptor (GLP-1R) (GLP-1R)et al, 1993; mann et al, 2010), stimulates the synthesis of insulin (Thorens et al, 1993) but, unlike GLP-1, exenatide has no cleavage site for DPP-IV and is therefore more stable in vivo (Thum et al, 2002; Hupe-Sodmann et al, 1995). Exenatide has a half-life in vivo of about 9.7 hours. Chemically synthesized Exenatide (Exenatide) was approved by the U.S. medical office FDA in 2005 to be marketed as a specific drug for type II diabetes (Furman,2012)The American Lily/Amylin pharmaceutical company developed artificially synthesized exenatide as a drug for treating type II diabetes in the U.S. market and obtained FDA approval, and was marketed in the U.S. market in 2005 under the trade name Bai Mida (Byetta).

However, chemically synthesized exenatide is very expensive in the market, which many patients cannot afford. In order to produce an inexpensive exenatide, many studies have been made on the production of exenatide by microbial fermentation or transgenic plants using genetic recombination (Yi et al 2006, Yin et al 2005; Kim et al 2009; Zhou et al 2008; Kwon et al 2013; Choi et al 2014), and good effects have been obtained.

However, one of the main disadvantages of these exenatides is that they require injection and are metabolized rapidly in vivo (half-life 9.7 hours), so frequent injections are required to maintain blood glucose level, causing pain and inconvenience to patients. The development of oral long-acting exenatide is urgently needed.

Kwon et al (2013) produce fusion proteins through the fusion of exenatide with cholera toxin beta subunit (CTB) and the high expression of chloroplasts (Kwon et al 2013), which can be absorbed through the binding of CTB to GM1 receptor on small intestine epidermal cells. Similarly, Choi et al (2014) produced a fusion protein Ex4-Tf that was not easily decomposed in the digestive tract by coupling with iron transporter (Tf), and absorbed into the blood through a membrane channel system due to the receptor of iron transporter (Tf) on the intestinal epithelial cell membrane, thereby achieving an effect similar to that of injecting exenatide into the blood, facilitating administration, and alleviating the pain of the patient. Experiments prove that the fusion protein can: 1. promoting beta cells of mouse pancreas to secrete insulin; 2. promoting beta cell proliferation; 3. promote the differentiation of rat beta cells (Choi J1, Diao H, Feng ZC, Lau A, Wang R, Jevnikar AM, Ma S.2014.A fusion protein derivative from plants promoting potential as an organic therapy for type 2 diabetes. plant Biotechnol J.12(4):425-35.doi: 10.1111/pbi.12149.).

In these methods, fusion proteins are produced by chloroplast transformation (Kwon et al 2013), leaf expression or transient expression of transgenic tobacco (Choi et al 2014), and one significant disadvantage is that fresh plant tissues such as leaves cannot be stored, sometimes decay due to non-timely extraction, and waste is caused, and sometimes required plant materials cannot be produced in time when needed, and drug production is affected. Although plant tissues can be maintained for up to 15 months by freeze-drying of the tissues (Kwon et al 2013), these treatments incur additional costs and increase costs.

One effective solution is to produce the fusion protein by means of a plant seed bioreactor. The fusion protein is expressed by utilizing the seed endosperm specificity expression promoter, and can be stably stored in a seed endosperm protein body. Since the crop seeds are easy to store and can be stored at room temperature for many years, and can be stored in a refrigerated warehouse for decades, the continuous production of the protein medicine can be realized, the problems of waste and shortage can not be caused, and the production cost can be reduced.

However, the prior art has not been able to achieve high expression of fusion proteins in the whole endosperm, and the previous expression was only in the aleurone layer or a part of cells.

Disclosure of Invention

The present invention aims to provide a fusion protein, a method for producing the same, and a method for producing transgenic rice, which allow exenatide fusion protein to be expressed in a large amount in the whole endosperm, and thus can be produced from plant seeds.

Therefore, the invention provides a fusion protein, which is a fusion protein expressed by the endosperm of plant seeds and containing Ex4-TF, wherein the gene sequence comprises: the plant endosperm specific expression promoter pGluB-5, a plant endosperm storage protein signal peptide sp, an exenatide polypeptide Exendin-4, a linker polypeptide (GGGGS) x3linker connecting the Exendin-4 and a human iron transporter hTF, the human iron transporter hTF, a histone tag 6XHis used for protein purification, and a plant GluB-5 gene transcription terminator GluB-5-3' -UTR.

The invention also provides a production method of the fusion protein, which comprises the following steps: 1) plant codon optimization and synthesis of the plant seed endosperm expression Ex4-TF fusion protein gene of claim 1; 2) constructing a plant binary expression vector for endosperm specific expression of the Ex4-TF fusion protein; 3) the Ex4-TF fusion protein gene is introduced into plant callus cells, and genetically transformed plant plants are obtained through screening.

Preferably, the plant is rice.

The invention also provides a production method of the transgenic rice, which comprises the following steps: 1) designing codon preference favorable for rice expression; 2) designing a DNA sequence of the plant seed endosperm expression Ex4-TF fusion protein gene according to claim 1, and obtaining the plant seed endosperm expression Ex4-TF fusion protein gene through gene synthesis; 3) cloning a rice seed endosperm specific expression promoter and a rice storage protein 3' -UTR expression terminator to clone the plant seed endosperm expression Ex4-TF fusion protein gene into a rice gene expression frame; 4) transgenic rice is obtained through gene transformation.

The invention can realize the mass expression of the fusion protein in the whole endosperm by utilizing the GluB-5 promoter. The GluB-5 promoter has strong expression in rice seeds, and the 3' -UTR of the GluB-5 gene not only plays a role in terminating expression, but also can promote the expression of the gene. Therefore, the expression of the fusion protein in rice seeds can be increased by using the GluB-5 promoter and the 3' terminator.

Meanwhile, the rice has the advantages of high yield, easy planting and management, storage resistance of rice seeds, good extraction and the like.

Drawings

FIG. 1 is a schematic diagram of rice seed endosperm expression Ex4-TF fusion protein, wherein FIGS. 1A and 1B show that exenatide in the fusion protein is located at the N-terminal and C-terminal of the fusion protein, respectively.

FIG. 2A is a flow chart of a scheme for expressing exenatide fusion protein in rice seeds according to an embodiment of the present invention.

FIG. 2B is a flow chart of the experimental detection of the present invention.

FIGS. 3A and 3B are the T-DNA structural diagrams of the vector pCAMBIA1301-Ex4-TF for expressing the Ex4-TF fusion protein from rice, respectively, of the Ex4-TFa fusion protein and the Ex4-TFb fusion protein.

FIGS. 4A and 4B are schematic diagrams of the regeneration on the differentiation induction medium of transgenic rice plants and the regenerated plants in culture flasks, respectively.

FIG. 5 is a result chart of GUS staining for identification of transgenic rice. WT is a wild-type rice callus, and 1-6 are different transgenic resistant calli.

FIG. 6 is a result diagram of PCR identification of transgenic plants. Vector is a plant binary expression Vector as a positive control, and WT is a wild type rice DNA as a negative control.

FIG. 7 is a graph showing the result of Southern Blot identifying the band obtained by hybridization using transgenic plant exenatide gene as a probe. Wherein 0 is wild type rice DNA as negative control, 1, 8 is single copy transgene event; 3. 7 is a two-copy transgene event; others are multicopy transgenic events.

FIG. 8 is a diagram showing the results of Northern blot analysis of the expression of transgenes in rice seeds and hybridization analysis of the fusion gene using the synthetic exenatide gene as a probe. WT was a wild-type rice RNA as a negative control, and #1-5 was a transgenic rice RNA.

FIG. 9 shows the results of Western Blot analysis of the expression of the fusion protein gene in transgenic rice seeds and detection of the fusion protein using exenatide antibody, WT being a wild-type rice protein as a negative control.

FIG. 10 is a result chart showing the purification of the fusion protein expressed in the transgenic rice seeds by Western Blot analysis and the detection of the fusion protein using exenatide antibody, #1-7 is the content of the fusion protein in each elution fraction during the elution.

Detailed Description

In the embodiment, the DNA sequence of the fusion protein gene is designed by designing the codon preference favorable for rice expression, and the fusion protein gene is obtained by gene synthesis. The Ex4-TF fusion protein gene is cloned to a rice gene expression frame by cloning a rice seed endosperm specific expression promoter and a rice storage protein 3' -UTR expression terminator, transgenic rice is obtained by gene conversion, the fusion protein in the transgenic rice seed can be detected by Western analysis, and the fusion protein can be stably stored in a seed endosperm protein body. Since the crop seeds are easy to store and can be stored at room temperature for many years, and can be stored in a refrigerated warehouse for decades, the continuous production of the protein medicine can be realized, the problems of waste and shortage can not be caused, and the production cost can be reduced.

As shown in FIGS. 1A and 1B, rice seed endosperm expression Ex4-TF fusion protein, pGluB-5 is a rice endosperm specific expression promoter, sp is a rice endosperm storage protein signal peptide, Exendin-4 is an exenatide peptide polypeptide, (GGGGS) x3linker is a connecting polypeptide (the DNA sequence and the amino acid sequence of which are respectively SEQ ID NO.11 and SEQ ID NO.12) for connecting Exendin-4 and human iron transport protein (hTF), hTF is human iron transport protein, 6XHis is a histone label for protein purification (the DNA sequence and the amino acid sequence of which are respectively SEQ ID NO.9 and SEQ ID NO.10), and GluB-5-3' -UTR is a rice GluB-5 gene transcription terminator. In the fusion proteins of FIGS. 1A and 1B, exenatide is located at the N-terminus and C-terminus of the fusion protein, respectively.

As shown in FIG. 2A, the rice seed-specific expression of exenatide fusion protein comprises the following steps: 1. optimizing and synthesizing a fusion protein gene by using rice codons; 2. constructing a plant binary expression vector for expressing the endosperm specificity of the fusion protein; 3. the fusion protein gene is introduced into the rice callus cells by adopting an agrobacterium-mediated method, and a genetically transformed rice plant is obtained by screening hygromycin.

As shown in FIG. 2B, this example examined the expression of the fusion protein in rice by GUS staining, PCR, Southern Blot, Northern Blot and Western Blot.

The above steps are explained in detail below:

rice codon optimization and synthesis of exenatide fusion protein gene:

DNA sequences of genes (Ex4-TF fusion protein A and Ex4-TF fusion protein B) of the rice fusion protein are generated on a website (http:// www.idtdna.com/CodonOpt) by rice codons (http:// www.kazusa.or.jp/codon/cgi-bin/showcocodon. cgi? species ═ 311553) according to amino acid sequences of the fusion protein genes, and GenScript is submitted to synthesize gene fragments, wherein the DNA sequence and the amino acid sequence of the Ex4-TF fusion protein A gene are SEQ ID No.1 and SEQ ID No.2, respectively, and the DNA sequence and the amino acid sequence of the Ex4-TF fusion protein B gene are SEQ ID No.3 and SEQ ID No.4, respectively.

Cloning of rice endosperm expression promoter and transcription terminator:

designing a primer according to a promoter (containing a gene signal peptide) and a transcription terminator sequence of the rice GluB-5 gene in a rice genome, wherein a 5' primer (SEQ ID NO.13) of the rice GluB-5 promoter: 5'-GAGAAAAGAAGATTTGCTGACCCCA-3', respectively; 3' primer of rice GluB-5 promoter (SEQ ID NO. 14): 5'-AGCCATAGAACCATGGCAAAGGAG-3' are provided. 5' primer of rice GluB-5 transcription terminator (SEQ ID NO. 15): 5'-TAAACCCAAGGCATTATATACTAAA-3', respectively; 3' primer of rice GluB-5 transcription terminator (SEQ ID NO. 16): 5'-CATGACATGATCCGCTCCTCTCCCT-3' are provided. The sequence is amplified and cloned from the Nipponbare genome DNA of rice by PCR and verified by a gene sequencing method, wherein the DNA sequence of a promoter of the GluB-5 gene of the rice is SEQ ID NO.5, the DNA sequence and the amino acid sequence of a signal peptide of the GluB-5 gene of the rice are respectively SEQ ID NO.6 and SEQ ID NO.7, and the DNA sequence of a transcription terminator of the GluB-5 gene of the rice is SEQ ID NO. 8. (Nipponbare is a common variety for rice gene transformation, has high transformation efficiency, and can be used for other rice varieties, but the preferred variety is Nipponbare.)

Construction of binary expression vectors:

the promoter (containing the gene signal peptide) of the GluB-5 gene, the fusion protein gene and the transcription terminator are connected in vitro, the plasmid CAMBIA1301 is used as a framework (a vector is a common vector for gene transformation and is available in most laboratories), and the fusion protein gene expression frame is cloned between the left boundary and the right boundary (TL is the left boundary and TR is the right boundary) of T-DNA. And named pCAMBIA1301-Ex4-TRa and pCAMBIA1301-Ex4-TRb, and the T-DNA structures are shown in FIG. 3. It contains HptII gene, which can make transformed rice cell resist hygromycin, and the gene is expressed by P35S (CaMV35S promoter) and T35S (CaMV35S terminator). The Light chain gene BLC (Bevacizumab Light chain) and the Heavy chain gene BHC (Bevacizumab Heavy chain) of the bevacizumab are expressed and translated respectively to generate the Light chain and the Heavy chain of the bevacizumab, the expression of the two genes is started by PUbi (maize ubiquitin promoter), and the expression of the two genes is stopped by TNos (nopaline synthase gene terminator). In addition, the T-DNA region also contains the GUS gene (β -glucuronidase gene), which is blue-colored after histochemical staining, and is expressed by P35S (CaMV35S promoter) and by TNos (nopaline synthase gene terminator).

FIGS. 3A and 3B are the T-DNA structure diagrams of the vector pCAMBIA1301-Ex4-TF for expressing the Ex4-TF fusion protein in rice. LB and RB are respectively the left and right boundaries of Agrobacterium T-DNA, GluB-5-3' -UTR and pGluB-5 are respectively the transcription terminator and promoter of rice seed storage protein GluB-5, Ex4-TFa and Ex4-TFb are respectively exenatide fusion protein genes a and b, SP is the signal peptide of rice seed storage protein GluB-5, 35S Pro is the 35S promoter of cauliflower mosaic virus (CaMV), GUS is a reporter gene and Tnos is the transcription terminator of Agrobacterium genes. Ex4-TFa fusion protein (SP) - (Exendin-4) - (GGGGGGS) x3- (hTf) -6 XHis; ex4-TFb fusion protein SP-6xHis-hTf- (GGGGGGS) x 3-Exendin-4.

Agrobacterium-mediated transformation of rice:

inducing rice callus, namely removing shells of mature nipponica japonica seeds, disinfecting the seeds for 30 seconds by using 75% alcohol, disinfecting the seeds for 25 minutes by using 5% sodium hypochlorite solution, rinsing the seeds for 3 to 5 times by using sterile water, placing the seeds on sterile paper, drying the seeds on an ultra-clean workbench, inoculating the seeds on an N6D2 callus induction culture medium, inoculating 12 to 15 seeds on each culture dish, carrying out dark culture at the temperature of 28 ℃ for 4 weeks, cutting off radicles during the dark culture, and carrying out subculture once every two weeks.

Activation of Agrobacterium tumefaciens 1) Agrobacterium containing the fusion protein gene plasmid (EHA105) was inoculated in LB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, shake-cultured at 28 ℃ at 200 rpm for 18 hours; 2) 1ml of cultured agrobacterium is put into a 2ml sterilized centrifuge tube, the rotating speed is 6000 rpm, the bacteria are collected by centrifugation for 5 minutes, 200 mul of AAM liquid culture medium is used for resuspending the bacteria, 10 mul of bacteria liquid is inoculated into 50ml of AAM liquid culture medium containing 200 mul of MAS (acetosyringone), and the vibration culture is carried out for 2 hours at the rotating speed of 160 rpm at the temperature of 28 ℃ for preparation of transformation.

Agrobacterium transformation of rice embryogenic callus: 1) dip-dyeing, namely selecting compact callus particles with the diameter of 3-5 mm, and dip-dyeing in the prepared AAM bacterial liquid for 10 min; 2) co-culturing, namely placing the impregnated callus on sterile filter paper to absorb bacterial liquid on the tissue surface, transferring the tissue to a Co-culture medium N6D2-Co, and performing dark culture for 3 days at 26 ℃; 3) selecting and culturing, namely rinsing the callus cultured for 3 days in total with sterile water containing 0.01 percent of Tween for 5 times, then rinsing the callus in sterile water containing 500mg/L of cefamycin for 10 minutes, placing the callus on sterile filter paper, airing the water on the callus on an ultra-clean workbench, transferring the callus to a selective culture medium N6D-Se, and performing dark culture at 28 ℃ for four weeks; 4) differentiation culture, transferring the resistant callus to a differentiation culture medium MS-Re, illuminating for 16 hours, culturing for 8 hours in the dark at 28 ℃ until a green seedling is differentiated (figure 4). Transferring the plantlet to rooting culture medium MS-Hf in sterilized bottle, culturing for 2 weeks, washing the culture medium on the plantlet root, culturing in water for 3 days, and transplanting to field.

FIGS. 4A and 4B are schematic diagrams of the differentiation of transgenic rice plants, and FIG. 4A is the regeneration on the differentiation induction medium; 4B is the regenerated plant in the culture flask.

The culture medium required by the rice tissue culture is described as follows:

(1) N6D2 medium: 3.9g/L Chu's N6(Chu,1975) (CHP01-50LT, 10402809, Caisson, USA), N6 vitamins, (2mg/L glycine, 0.5mg/L niacin, 1.0mg/L vitamin B1, 0.5mg/L vitamin B6), 0.1g/L inositol, 1.0g/L casein hydrolysate, 0.5g/L proline, 0.5g/L glutamine, 2 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D), 30g/L sucrose, 1M KOH adjusted pH5.8, 3.0g/L plant gel, 121 ℃, 220KPa, sterilized for 20 minutes.

(2) N6D2-Co medium: 10g/L glucose, 1M KOH adjusted pH5.5, 3.0g/L plant gel, 121 ℃, 220KPa, sterilized for 10 minutes, added to N6D2, 200uM Acetosyringone (AS) after the medium cooled to 50 ℃.

(3) N6D2-Se medium: the N6D2 medium was cooled to 50 ℃ at 121 ℃ at 220KPa, sterilized for 20 minutes, and added with 50mg/L hygromycin B and 500mg/L cephamycin.

(4) MS-Re medium: 4.6g/L MS (M10400-50.0, P06968, rpi, USA), 2.0 mg/L6-benzylamino adenine (6-BA), 0.5mg/L NAA naphthalene acetic acid, 1.0mg/L Kinetin (KT), 30g/L sucrose, 30g/L sorbitol, 1M KOH regulation pH5.8, 3.0g/L Gelrite, 121 ℃, 220KPa, sterilization for 20 minutes, cooling to 50 ℃, adding 50mg/L hygromycin B and 500mg/L cephamycin.

(5) MS-HF medium: 2.3g/L MS (M10400-50.0, P06968, rpi, USA), 30g/L sucrose, 1M KOH pH5.8, 3.0g/L Gelrite, 121 ℃, 220KPa, sterilized for 20 minutes, cooled to 50 ℃, and added 50mg/L hygromycin B.

(6) AAM medium: 0.5g/L hydrolyzed casein, 68.5g/L sucrose, 36g/L glucose, 0.9g/L glutamine, 0.3g/L aspartic acid, 3g/L potassium chloride, 10mg/L manganese sulfate pentahydrate, 3.0mg/L boric acid, 2.0mg/L zinc sulfate heptahydrate, 0.25mg/L sodium molybdate dihydrate, 0.025mg/L copper sulfate pentahydrate, 0.025mg/L cobalt chloride hexahydrate, 0.75mg/L potassium iodide, 15mg/L calcium chloride dihydrate, 25mg/L magnesium sulfate heptahydrate, 4mg/L ethylenediaminetetraacetic acid (EDTA), 15mg/L sodium dihydrogen phosphate dihydrate, 1mg/L nicotinic acid, 1mg/L vitamin B6, 10mg/L vitamin B1, 100mg/L inositol, 176mg/L arginine, 75mg/L glycine

The pH was adjusted to 5.2 with 1M KOH and sterilized by filtration through 0.22uM filter.

Identification of transgenic Rice

GUS staining identification of transgenic rice: cutting the transgenic plantlet leaves into 2cm small sections, and placing the small sections in a 2mL centrifuge tube; the resistant callus was divided into small pieces with forceps, immersed in GUS staining solution (100mM sodium phosphate buffer (pH 7.0) containing 0.5mg/ml X-GluC, 1% Triton X-100, 1% DMSO and 10mM EDTA), evacuated for 15min, incubated at 37 ℃ for 12h, decolorized with 85% ethanol several times, photographed, and as shown in FIG. 5, the results of the test on transgenic rice were identified for GUS staining, where WT was wild-type rice callus and 1-6 were different transgenic resistant calli.

PCR identification of transgenic Rice: the PCR reaction solution was prepared by mixing 10. mu.l of Premix Ex Taq Hot Start Version (the reagent is a product of TAKARA, which contains polymerase, deoxynucleotide mixture, and buffer solution required for PCR reaction, only DNA template and primer were added during PCR reaction, other PCR products were also available), 500ng of genomic DNA, 1. mu.l of each of 10. mu.M of forward and reverse primers of exenatide synthetic gene, and a volume of 20. mu.l was determined by double distilled water. The PCR procedure was: 5 minutes at 95 ℃,20 seconds at 60 ℃, 1.5 minutes at 72 ℃, 30 cycles, 5 minutes at 72 ℃. After the PCR reaction, 5. mu.L of the reaction solution was subjected to electrophoresis detection.

The PCR primer sequence is as follows: 5' primer for synthesizing Exenatide gene (SEQ ID NO. 17): 5'-CACGGAGAGGGCACTTTCACGTCCG-3', respectively; 3' primer for synthesizing Exenatide gene (SEQ ID NO. 18): 5'-ACTGGGGGGAGGAGCACCGCTTGAC-3' are provided.

FIG. 6 shows the result of PCR identification of transgenic plants. Vector is a plant binary expression Vector as a positive control, WT is a wild type rice DNA as a negative control, and 1-10 are different transgenic rice lines (all lines contain transgenic Nipponbare, and the difference lies in the position of the transgene inserted into the genome). The PCR amplified full-length DNA was 117 bp.

Southern blot identification of transgenic rice:

1) preparation of digoxigenin-labeled probe: the DNACR product of the exenatide synthetic gene is used as a template, and a digoxin labeled probe is prepared by amplification of primers (5 'primer: 5'-CACGGAGAGGGCACTTTCACGTCCG-3'of the synthetic exenatide gene; 3' primer 5'-ACTGGGGGGAGGAGCACCGCTTGAC-3' of the synthetic exenatide gene). The PCR reaction solution was prepared by using template DNA10ng, forward and reverse primers 1. mu.L each, a perfect reaction protocol for Premix Ex Taq Hot Start Version 15. mu.L amplification, 1. mu.L DIG-dUTP, and double distilled water to a volume of 30. mu.L. The PCR amplification reaction program is as follows: 5 minutes at 95 ℃,20 seconds at 60 ℃, 1.5 minutes at 72 ℃, 30 cycles, 5 minutes at 72 ℃. After the PCR reaction, 3. mu.L of the reaction solution was taken for electrophoresis detection.

2) Enzymolysis of total DNA of transgenic rice: transgenic rice leaf total DNA15ug, enzyme digested with EcoR I, reacted at 37 ℃ overnight. The enzymatic reaction system of each sample is as follows: total DNA15ug, CutSmart buffer 10. mu.L, 100U EcoRI, double distilled water to 100. mu.L. Wherein, wild rice genome DNA is used as a negative control, and plasmid DNA is used as a positive control. After the enzymolysis reaction is finished, loading 5 mu L of the mixture to carry out electrophoresis detection, purifying, dissolving in 15 mu L of double distilled water, and storing at 4 ℃ or-20 ℃.

3) Electrophoresis of the enzymatic products: loading the purified enzymolysis product on 1.0% agarose gel, and performing electrophoresis at 5V/cm voltage for 3 hours; photographing and marking the position of the molecular weight; treating the gel in 0.2N hydrochloric acid for 10min, and rinsing with double distilled water; putting the mixture into a denaturation buffer solution for treatment for 30 minutes; the mixture is placed in a neutralization buffer for 30 minutes.

4) DNA imprinting to hybridization membrane: shearing a hybridization membrane with proper size according to the size of the agarose gel, and wetting the hybridization membrane with double distilled water; the glass plate was placed on a tray, 1 piece of 3M filter paper was soaked with transfer buffer, and placed on the glass plate to remove air bubbles from the filter paper and the glass plate. Adding a proper amount of transfer buffer solution into the tray; placing the gel on filter paper, and removing air bubbles between the gel and the blotting surface; placing the hybridization membrane on the gel and discharging bubbles again; putting 2 pieces of 3M filter paper (slightly larger than the hybridization membrane) on the hybridization membrane after the transfer buffer solution is thoroughly wetted; a safety film is placed around the gel to prevent the liquid in the liquid pool from flowing directly to the tissue layer above the gel (i.e. the "short circuit" phenomenon). Putting a stack of absorbent paper on the filter paper, pressing an object weighing about 300g, and transferring overnight; the hybridization membrane was rinsed once in 10-fold diluted transfer buffer, dried on a clean bench, and oven-dried at 80 ℃ for 2 hours.

5) And (3) hybridization: wetting and rolling the baked membrane with sterilized water, putting the membrane into a hybridization tube, adding 30ml of hybridization solution, and pre-hybridizing for 2 hours at 42 ℃; pouring out the pre-hybridization solution, and adding 10ml of new hybridization solution to preheat at 42 ℃; the prepared probe was removed, thawed on ice, added to the pre-heated hybridization solution, and hybridized overnight at 42 ℃.

6) And (3) post-hybridization treatment: the hybridization solution was poured out, and 100ml of elution buffer W1 was poured into the hybridization tube, and the solution was eluted at room temperature for 5 minutes, and this was repeated once. Pouring off the elution buffer W1, adding 100ml of elution buffer W2, eluting at 65 ℃ for 15 minutes, and repeating once; taking out the film, putting the film into a small flat disc paved with a preservative film, adding elution buffer W3, and eluting for 5 minutes; pouring off the elution buffer solution W3, adding 50ml of blocking solution S1, and incubating for 15 minutes at room temperature; pouring off the blocking solution S1, adding 10ml of digoxin antibody reaction solution S2, and incubating for 15 minutes; taking out the membrane and putting the membrane into another box, adding 100ml of elution buffer solution W3, eluting for 15 minutes at room temperature, and repeatedly eluting once; adding 20mL of detection buffer S3, and incubating for 5 minutes; pouring out the detection buffer S3, adding 2ml CSPD and incubating for 5 minutes; the film was taken out and spread on a cling film, wrapped up, and incubated at 37 ℃ for 5 minutes.

7) Developing and fixing: placing the wrapped film in a cassette, placing an X-film on the wrapped film in a darkroom, closing the darkclamp, and standing at 37 deg.C for 30 minutes; the film was taken out of the dark room, immersed in the developing solution for 2 minutes, washed in water, immersed in the fixative for 2 minutes, washed in running water, and then dried (fig. 7).

8) Reagents required for Southern blot:

denaturation buffer: 20g/L NaOH, 87.75g/L NaCl; neutralization buffer: 60.05g/L Tris (Tris-hydroxymethyl-aminomethane), 87.75g/L NaCl, and 37% concentrated hydrochloric acid for adjusting the pH value to 7.2; transfer buffer (20 × SSC): 175.3g/L NaCl, 88.2g/L trisodium citrate 4M hydrochloric acid adjusted pH 7.0; elution buffer W1: 2 XSSC; 0.1% SDS (sodium dodecyl sulfate) (M/V); elution buffer W2: 0.5 XSSC; 0.1% SDS; elution buffer W3: 11.607g/L maleic acid, 8.775g/L NaCl, solid NaOH is used for adjusting the pH value to 7.5, and 0.3% Tween 20 is added before use; detection buffer S3: adjusting the pH value to 9.5 with 12.1g/L Tris and 5.85g/L NaCl; hybridization solution: 70g/L SDS, 50% formamide, 25% 20XSSC, 2% skimmed milk powder (Roche), 50ml of 1M sodium phosphate buffer (pH7.2), 1.0g/L sodium dodecylsarcosinate; sealing liquid S1: dissolving 1% skimmed milk powder (M/V) in elution buffer W3; digoxin antibody reaction solution S2: Anti-Digoxigenin-AP was centrifuged at 10000 rpm for 5 minutes, and the supernatant was pipetted at 1: 10000 dissolved in blocking solution S1.

FIG. 7 is a diagram showing the result of Southern Blot for identifying transgenic plants, which is a band obtained by hybridization using exenatide gene as a probe. Wherein 0 is wild type rice DNA as negative control, 1, 8 is single copy transgene event; 3. 7 is a two-copy transgene event; others are multicopy transgenic events.

Northern blot analysis of transgene expression in seeds:

1) extraction of total RNA of rice: total RNA of rice seeds was extracted using QIAGEN RNeasy Mini Kit RNA extraction Kit.

2) Detection and quantification of total RNA quality of rice: a sample of 1. mu.L was applied to 0.8% agarose gel and observed under an ultraviolet lamp after about 5min at 120V. For quantification, sample 1. mu.L + 99. mu. LDEPC H₂And O, after mixing uniformly, measuring the absorption values at 260nm and 280nm on an Eppendorfs nucleic acid quantitative detector (Eppendorfs AG22331, Hamburg), and calculating the yield and the concentration of the RNA.

3) Denaturation treatment of RNA samples: taking 1 mu g of plasmid DNA for enzymolysis, and taking a positive control; adding equal amount of Denation Buffer into 15 mu gRNA; meanwhile, 3 mu g RNA Marker and 40-80 pg enzyme-cut plasmid DNA are taken, and equivalent renaturation Buffer is also added respectively. Incubate at 60 ℃ for 20min, and cool on ice.

4) Formaldehyde gel denaturing electrophoresis of RNA: sealing two ends of the rubber plate by using a transparent adhesive tape, and inserting the rubber comb; compounding glue (1.5%), adding 110ml EPC-H into a triangle flask₂O, 15mL of 10 XMOPS electrophoresis buffer solution and 2.25g of agarose, heating to melt, cooling to 65 ℃, adding 25mL of 37% formaldehyde solution, pouring into a gel plate, and waiting for the gel plate to solidify; adding 1 XMOPS electrophoresis buffer solution into an electrophoresis tank, and putting the gel into the electrophoresis tank; taking the denatured sample, adding 0.2 times of BPB-Solution and 1 mu LEB, uniformly mixing, applying the sample, and performing 70V voltage electrophoresis for 3.5 h.

5) Film transfer: placing the glue in DEPC-H₂Oscillating in O for 15 min; observing under an ultraviolet lamp, photographing, marking the position of molecular weight, and cutting glue; the remaining steps are the same as Southern Blot.

6) Northern blot analysis: the hybridization temperature was 50 ℃ and the elution buffer W3 was used in place of the W2 of Southern Blot, and the procedure was the same as that of Southern Blot (FIG. 8).

7) Preparation of buffers and reagents:

10 × MOPS electrophoresis buffer: 41.8g of propanesulfonic acid (MOPS) are dissolved in 700mL of water and the pH is adjusted to 7.0 with 2 mol/LNaOH. 20mL of 1mol/L sodium acetate and 20mL of 0.5mol/L EDTA (pH8.0) were added. The volume is adjusted to 1L by water. After treatment overnight with 1ml of EPC, filter sterilized.

RNA denaturation buffer: 0.8mL of 10 XMOPS electrophoresis buffer was added with 2mL of formamide, 1.3mL of 37% formaldehyde, and 0.04mL of 0.5M EDTA to a constant volume of 4mL and stored at-20 ℃ for further use.

5 × BPB-Solution: bromophenol blue (0.2%); glycerol (50%); 1 × MOPS.

Elution buffer W3 solution: 0.1 XSSC; 0.1% SDS.

FIG. 8 is a diagram showing the expression result of the transgenic rice seeds by Northern blot analysis, in which the expression of the fusion gene is analyzed by hybridization using the synthetic exenatide gene as a probe. WT is a wild-type rice RNA as a negative control, and #1-5 is a transgenic rice RNA, wherein the expression level of a transgenic line #5 is the highest, and the expression level of #1 is the lowest (the expression level of the transgene is influenced by the gene position and copy number, and the difference between the expression levels may reflect the difference between the expression levels, but the specific situation is not the key point of the invention, and a line with a high expression level is generally selected).

Western Blot analysis of the expression of the fusion protein in transgenic rice:

1) sample preparation: transgenic rice seeds are taken as materials and ground in liquid nitrogen. 30mg of the ground tissue was weighed, added to 30ul of 2XSDS buffer, vortexed, incubated at 70 ℃ for 10 minutes, centrifuged at 13000 rpm for 10 minutes, and the supernatant was removed to a new centrifuge tube.

2) Electrophoretic separation: washing the glass plate, sample comb, Spacer with detergent, and washing with ddH₂And washing with O for several times, wiping with ethanol, and air drying. Spacer was added between the two cleaned glass plates and the plates were loaded as suggested by Bio-Rad Mini II/III. 8.0ml of 10% separation gel is prepared according to the following volume and mixed evenly: 3.0ml ddH₂O; 2.1ml of 1.0mol/LTris-HCl pH 8.8; 2.8ml of 30% Acr-Bis; 80ul 10% SDS; 56ul 10% AP; 6ul TEMED. Pouring separation glue between the glass plates, immediately covering a layer of redistilled water, and polymerizing the glue after about 20 minutes. 3.0ml of 6% concentrated gel is prepared according to the following volume and mixed evenly: 2.0ml ddH₂O; 400ul 1.0mol/LTris-HCl pH 6.8; 600ul 30% Acr-Bis; 36ul 10% SDS; 24ul 10% AP; 4ul TEMED. The upper layer of redistilled water is poured off, the filter paper is sucked dry, the concentrated gel is poured, and the sample comb is inserted. After the gel is collected, the sample comb is slowly pulled out. The electrophoresis system is assembled, electrode buffer is added, 10 mu l of sample is loaded, and 100V electrophoresis is carried out for 2 hours.

3) Film transfer: the plate was removed, the gel peeled off, and the gel was equilibrated in transfer buffer for 10 min. 6 pieces of membrane and filter paper are cut according to the size of the glue, and the PVDF membrane is put into methanol for soaking and saturation for 3-5 seconds and then is put into transfer buffer solution for balancing for 10 min. Assembling and transferring the sandwich: from bottom to top are 3 layers of filter paper, glue, PVDF membrane, 3 layers of filter paper in proper order, and after putting each layer, the bubble is driven away with the test tube. Insert electrode, 20V, 45 min. After the membrane transfer is completed, the power supply is cut off, and the hybrid membrane is taken out.

4) Immunological hybridization and color development: the membrane was soaked in double distilled water for 10 minutes, and then taken out and placed in 20ml of a sealing solution of 5% skimmed milk powder to incubate for 1 hour at room temperature. Add 1000-fold diluted exenatide antibody and incubate slowly with shaking overnight at 4 ℃.15 ml TBS-T3 times for 5min each. A2000-fold diluted secondary horseradish peroxidase (HRP) -labeled antibody was added and incubated for 1 hour at room temperature with slow shaking. 15ml TBS-T was washed 3 times for 5min each. Protein detection was performed according to the Thermo Pierce ECL western blotting Substrate kit.

FIG. 9 is a diagram showing the result of Western Blot analysis of the expression of the fusion protein gene in transgenic rice seeds, in which exenatide antibody is used for detecting the fusion protein, WT is a negative control of wild-type rice protein, #1-5 is transgenic rice, and the expression level of the transgenic line #2 is the highest and the expression level of #1 is the lowest. (protein expression is not necessarily consistent with RNA expression, and for this study, protein expression is more important, since we ultimately wanted protein.)

FIG. 9 shows that the gene optimized by rice codon can be expressed in rice seeds

Purification of fusion proteins in transgenic rice seeds

7.1 seed crushing: rice seeds were ground into powder, dissolved with Phosphate Buffer (PB) (25mM PB 50mM NaCl, pH7.5) at room temperature for 1 hour, then precipitated with acetic acid at pH5 for 2 hours, and centrifuged at 12000g for 10min to take the supernatant.

7.2 filling of the chromatographic column: loading the chromatographic column with Ni-NTA agarose as affinity chromatographic medium for chelating Ni ions with 5-10 times of buffer A (10mM Na)₂HPO₄，1.8mM KH₂PO₄140mM NaCl, 2.7mM KCl, pH adjusted to 8.0 with high concentration NaOHL).

7.3 column passing: an equal volume of 2 × histone-tagged protein binding buffer (5mM imidazole, 500mM NaCl, 20mM Tris-HCl, pH 7.9) was added to the supernatant in 7.1 and loaded onto the column.

7.4 column washing: using 5-10 column volumes of column wash (10mM imidazole, 20mM Na)₂HPO₄500mM NaCl) to remove unbound contaminating proteins.

7.5 elution: with 10 volumes of eluent (500mM imidazole, 20mM Na)₂HPO₄500mM NaCl) was eluted, and the eluates of each fraction were collected separately and analyzed by western. The fusion protein was completely eluted in 7 volumes of eluent. (if the volume of the affinity column is 1ml, then 1 column volume of eluent is 1ml, "7 volume" is 7 column volumes)

FIG. 10 shows the results of Western Blot analysis of the purification of the fusion protein expressed in transgenic rice seeds, using exenatide antibody to detect the fusion protein, and #1-7 as the content of the fusion protein in each elution fraction during the elution. The fusion protein product was eluted between volumes 1-7. (laboratory purification using 1-5 grams of plant material extraction, 1ml of affinity column, 1-7 volumes 1-7ml of eluent.)

FIG. 10 shows that the gene optimized by rice codon can be not only expressed in rice seeds, but also purified by affinity chromatography. Thus, the present invention provides a fully implementable method for the production of exenatide fusion protein by plant seeds.

The embodiment of the invention can generate stable fusion protein through the fusion of the exenatide and the iron transport protein, and can be orally taken, and the medicine is identified by the receptor of the iron transport protein in intestinal cells and is transported to blood, thereby playing a role and playing a similar therapeutic effect of the injection of the exenatide.

The present invention is to produce fusion proteins by means of a plant seed bioreactor. The characteristic of specific expression of the rice storage protein gene in the seed endosperm is utilized, the rice storage protein promoter is utilized to drive the fusion protein gene to perform specific expression in the rice seeds by a transgenic method, the DNA sequence of the fusion protein gene is designed by designing the codon preference favorable for rice expression, and the fusion protein gene is obtained by gene synthesis. By cloning a rice seed endosperm specific expression promoter and a rice storage protein 3' -UTR expression terminator, an Ex4-TF fusion protein gene is cloned into a rice gene expression frame, transgenic rice is obtained through gene transformation, the fusion protein in the transgenic rice seed is detected, and the fusion protein can be stably stored in a seed endosperm protein body. Since the crop seeds are easy to produce and store, and can be stored at room temperature for many years, and can be stored in a refrigerated warehouse for decades, the continuous production of the protein medicine can be realized, the problems of waste and shortage can not be caused, and the production cost can be reduced.

Claims

1. A method for producing a fusion protein, comprising the steps of:

1) rice seed endosperm expression Ex4-TF fusion protein gene is optimized by rice codon and synthesized, and the gene sequence comprises: the rice endosperm specific expression promoter pGluB-5, rice endosperm storage protein signal peptide sp, exenatide polypeptide Exendin-4, a connecting polypeptide (GGGGS) x3linker connecting the Exendin-4 and human iron transporter hTF, histone tag 6XHis for protein purification, and rice GluB-5 gene transcription terminator GluB-5-3' -UTR; the Ex4-TF fusion protein genes are two, exenatide is respectively positioned at the N end and the C end of the fusion protein and respectively called an Ex4-TF fusion protein A gene and an Ex4-TF fusion protein B gene, wherein the DNA sequence of the Ex4-TF fusion protein A gene is SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 2; the DNA sequence of the Ex4-TF fusion protein B gene is SEQ ID NO.3, and the coded amino acid sequence is SEQ ID NO. 4;

the cloning method of the rice endosperm specific expression promoter and the GluB-5 gene transcription terminator comprises the following steps: designing a primer according to a promoter and a transcription terminator sequence of a rice GluB-5 gene in a rice genome, and amplifying and cloning the sequence from a rice Nipponbare genome DNA through PCR;

wherein, the 5 'primer of the rice GluB-5 promoter is shown as SEQ ID NO.13, the 3' primer of the rice GluB-5 promoter is shown as SEQ ID NO.14, the 5 'primer of the rice GluB-5 gene transcription terminator is shown as SEQ ID NO.15, and the 3' primer of the rice GluB-5 transcription terminator is shown as SEQ ID NO. 16;

2) constructing the rice binary expression vector for expressing the Ex4-TF fusion protein endosperm specificity, which comprises the following steps: connecting a promoter of a GluB-5 gene, an Ex4-TF fusion protein gene and a transcription terminator in vitro, taking a plasmid CAMBIA1301 as a framework, cloning an Ex4-TF fusion protein gene expression frame between the left and right boundaries of T-DNA, and forming a vector which comprises an Ex4-TF fusion protein gene, a GUS reporter gene for transgene detection and an HptII gene for enabling transformed rice cells to resist hygromycin;

3) the Ex4-TF fusion protein gene is introduced into rice callus cells, and genetically transformed rice plants are obtained through screening.

2. The method for producing a fusion protein according to claim 1, wherein: the Ex4-TF fusion protein gene is introduced into rice callus cells by adopting an agrobacterium-mediated method.

3. The method for producing a fusion protein according to claim 1, wherein: the genetic transformation rice plant obtained by screening is screened by hygromycin.

4.A fusion protein characterized by: obtained by the production method according to any one of claims 1 to 3.