CN107164457A - A kind of high-throughput screening method of New-type wide-spectrum lysozyme - Google Patents

Abstract

The invention relates to a high-throughput screening method for a novel broad-spectrum lysozyme in the technical field of molecular biology. The method comprises the following steps: A, secreting and expressing the gene mutation library of the target lysozyme in eukaryotic cells to obtain fermentation broths containing target lysozymes with different activities; B, adding the fermentation broths containing target lysozymes with different activities respectively React in the reaction system to screen out lysozymes with high bactericidal activity against Gram-negative bacteria; wherein, the reaction system includes: a target expressing "donor fluorescent protein-protease recognition site-acceptor fluorescent protein" in the cell Lambert-negative bacteria, protease and protease reaction buffer. The screening method of the present invention significantly improves the sensitivity of detection through the cascade of FRET fluorescent protein pairs and site-specific proteases; at the same time, the method can utilize a fluorescent microplate reader and a 96 microwell plate to realize high-throughput detection of lysozyme .

Description

A high-throughput screening method for a novel broad-spectrum lysozyme

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a high-throughput screening method for novel broad-spectrum lysozyme.

Background technique

Lysozyme (lysozyme, EC3.2.1.17), also known as muramidase, can specifically hydrolyze peptidoglycan, the main component in the cell wall of prokaryotic bacteria, decompose the cell wall of microorganisms, and make the bacteria lose the protection of the cell wall and regenerate in the cell wall. Under the action of internal high osmotic pressure, it ruptures and dies, so as to achieve the purpose of sterilization. In 1921, the famous British bacteriologist Alexander Fleming discovered lysozyme in human nasal fluid, and later confirmed that lysozyme widely exists in the egg whites of birds and birds, various organs and tissues and body fluids of mammals, plants, molluscs and inside the insect.

As one of the most powerful antibacterial agents in higher organism tissues and body fluids, lysozyme is an important defense factor for biological organisms against the invasion of exogenous pathogenic bacteria. For example, human lysozyme is a small molecule basic globulin secreted by epithelial cells and monocyte-macrophages, which can recognize and destroy the cell structure of pathogenic bacteria, and attract leukocytes to the infection site through a signal cascade reaction, eventually Eliminate pathogenic bacteria that infect the human body. In addition, lysozyme can also directly combine with negatively charged viral proteins and form double salts with DNA-RNA apoproteins to inactivate the virus. In plants, such as the fresh juice of figs, lysozyme is rich in lysozyme, which is speculated to be closely related to the antiviral effect of plants. On the other hand, lysozyme also widely exists in microorganisms, such as various bacteria and phages. Phage lysozymes are involved in the breakdown of bacterial cell walls during phage infection. The main function of bacterial lysozyme is to participate in cell wall-related metabolic processes such as cell growth, division, and morphological changes. Therefore, bacteria will not develop true resistance to lysozyme, an autolysin widely contained in bacteria, which is of great significance for solving the increasingly serious problem of bacterial drug resistance and reducing the abuse of antibiotics.

Although lysozyme is powerful, it is not very effective at lysing Gram-negative bacteria. This is due to the different composition of the bacterial cell wall. The cell wall of Gram-positive bacteria is mainly composed of peptidoglycan, which has many and thick layers and contains a small amount of teichoic acid, while the outer layer of Gram-negative bacteria is mainly composed of lipopolysaccharide (LPS). Contains only a small amount of peptidoglycan. The main action site of lysozyme is the peptidoglycan in the cell wall, which hydrolyzes the β-1,4 glycosidic bond, so its effect on Gram-negative bacteria is not ideal.

In daily life, many pathogenic bacteria that people come into contact with, such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella and Clostridium perfringens, are all Gram-negative bacteria. In order to obtain lysozyme with high bactericidal activity against Gram-negative bacteria, lysozyme can be subjected to directed evolution to construct a random mutation library of lysozyme gene, and then screen out lysozyme with strong bactericidal effect on Gram-negative bacteria.

In the process of directed transformation, on the one hand, the required mutant library is huge, and a high-throughput method is required to improve the screening efficiency; on the other hand, lysozyme has a poor lysis effect on Gram-negative bacteria, and a sensitive indicator needs to be used. , A screening method that can effectively reflect the cracking effect. However, the existing screening methods, such as plate inhibition zone method and Oxford cup method, cannot satisfy the above two requirements at the same time. Therefore, it is urgent to construct a sensitive, reliable, and easy-to-operate high-throughput screening method for the screening of novel broad-spectrum lysozyme mutants.

Contents of the invention

The technical problem to be solved by the present invention is to provide a high-throughput screening method for a novel broad-spectrum lysozyme for the deficiencies in the prior art. This method improves the detection efficiency by cascading FRET fluorescent protein pairs with site-specific proteases. Sensitivity, and high-throughput detection of lysozyme can be achieved by using a fluorescent microplate reader and a 96-well plate.

For this reason, the invention provides a kind of high-throughput screening method of novel broad-spectrum lysozyme, which comprises the following steps:

A, the gene mutation library of the target lysozyme is secreted and expressed in eukaryotic cells, and the fermentation broth containing the target lysozyme with different activities is obtained;

B, adding fermented liquids containing lysozymes with different active targets to the reaction system for reaction, and screening out lysozymes with high bactericidal activity against Gram-negative bacteria;

Wherein, the reaction system includes: Gram-negative bacteria expressing "donor fluorescent protein-protease recognition site-acceptor fluorescent protein" in cells, protease and protease reaction buffer.

In the present invention, according to the signal intensity growth rate of the donor fluorescent protein in the reaction process, the bactericidal activity of lysozyme in the fermentation broth to Gram-negative bacteria is judged; specifically, the faster the signal intensity of the donor fluorescent protein increases in the reaction process Faster, the higher the bactericidal activity of lysozyme in the fermentation broth to Gram-negative bacteria.

In some embodiments of the present invention, the protease is a protease with recognition sequence specificity; in some specific embodiments of the present invention, the protease is TEV protease, enterokinase or coagulation factor Xa; in the present invention In some preferred embodiments, the protease is TEV protease.

In other embodiments of the present invention, the amino acid sequence of the recognition site of the TEV protease is: Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

In some embodiments of the present invention, the construction method of the target lysozyme gene mutation library is error-prone PCR and/or DNA shuffling.

In the present invention, since the emission spectrum of the donor fluorescent protein overlaps with the absorption spectrum of the acceptor fluorescent protein, when the distance between the donor fluorescent protein and the acceptor fluorescent protein is within 10 nm, an energy transfer phenomenon occurs, and the donor fluorescent protein The energy of the bulk fluorescent protein is transferred to the acceptor fluorescent protein, so that the fluorescence intensity of the donor protein is much lower than when it exists alone, while the fluorescence intensity of the acceptor protein is greatly enhanced.

In some specific embodiments of the present invention, the donor fluorescent protein is cyan fluorescent protein, and the acceptor fluorescent protein is yellow fluorescent protein.

In some embodiments of the invention, the eukaryotic cell is Pichia pastoris.

In other specific embodiments of the present invention, the method also includes the following steps:

C, re-screening the screened lysozyme with high bactericidal activity against Gram-negative bacteria, and verifying the activity of lysozyme according to the growth rate of the signal intensity of the donor fluorescent protein during the reaction.

The beneficial effects of the present invention are: the screening method of the present invention significantly improves the sensitivity of detection through the cascade of FRET fluorescent protein pairs and site-specific proteases by means of detection of fluorescent signals, and can effectively find positive mutations and excellent mutations gene; at the same time, the method can realize high-throughput detection of lysozyme by using a fluorescent microplate reader and a 96-well plate.

detailed description

In order to make the present invention easy to understand, the present invention will be described in detail below.

The screening method of the present invention is based on fluorescence resonance energy transfer technology (FRET), that is, when two fluorescent protein molecules are extremely close (within 10nm), if the emission spectrum of the donor fluorescent protein overlaps with the absorption spectrum of the acceptor fluorescent protein, then The phenomenon of energy transfer will occur, the energy of the donor fluorescent protein is transferred to the acceptor fluorescent protein, so that the fluorescence intensity of the donor protein is much lower than when it exists alone, and the fluorescence intensity of the acceptor protein is greatly enhanced. Taking cyan fluorescent protein and yellow fluorescent protein as an example, cyan fluorescent protein and yellow fluorescent protein are a pair of proteins that can undergo FRET effect, wherein the emission spectrum of cyan protein overlaps with the absorption spectrum of yellow protein.

Use the protease recognition site as the linker sequence, for example, use the TEV protease recognition site as the linker sequence to connect the cyan fluorescent protein and the yellow fluorescent protein, so that the cyan fluorescent protein and the yellow fluorescent protein form a FRET fluorescent protein pair with a very close distance, That is, "cyan fluorescent protein-TEV protease recognition site-yellow fluorescent protein". The recognition site of TEV protease is composed of seven amino acids Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

Before the reaction, due to the linker sequence (recognition site of TEV protease), the distance between the cyan fluorescent protein and the yellow fluorescent protein is very close, and the energy of the cyan fluorescent protein is transferred to the yellow fluorescent protein. At this time, the detection signal shows a weak cyan fluorescent signal, The yellow fluorescent signal is strong; when the fermentation broth containing lysozyme is added to the reaction solution, if the lysozyme has good bactericidal activity on the substrate bacteria (Gram-negative bacteria), the bacteria will be lysed and the internal FRET fluorescence will be released protein pair. The recognition site of the TEV protease of the protein pair is specifically recognized and decomposed by the TEV protease in the reaction solution to obtain independent cyan fluorescent protein and yellow fluorescent protein, and the FRET phenomenon disappears, which enhances the cyan fluorescent signal. Therefore, the bactericidal activity of lysozyme against Gram-negative bacteria can be determined by quantitatively measuring the growth rate of the signal intensity of cyan fluorescent protein.

The specific operations of the high-throughput screening method of the novel broad-spectrum lysozyme involved in the present invention are as follows:

(1) Construction of eukaryotic cells expressing the target lysozyme gene mutation library:

The specific type of target lysozyme can be selected according to actual needs. The gene sequence encoding the target lysozyme is searched through the nucleic acid database and the whole gene is synthesized to obtain the original target lysozyme gene sequence. The construction of the random mutation library of the target lysozyme gene is mainly realized by error-prone PCR and DNA shuffling (also called DNA shuffling).

1) The specific operation steps of error-prone PCR are as follows:

Design primers containing restriction sites for the original target lysozyme gene sequence, and perform error-prone PCR amplification by adjusting the ratio and reaction conditions of the PCR reaction system, such as changing the concentration of metal ions in the reaction system, increasing the number of PCR reaction cycles, etc. Amplification, which causes point mutations in the gene sequence in error-prone PCR products, resulting in amino acid changes.

2) The specific steps of DNA shuffling are as follows:

Select other lysozyme gene sequences that have homology with the original target lysozyme gene, and the number can be selected according to actual needs. Take 20 μl of each gene sequence and mix, add 0.2U DNaseI, digest at 16°C for 15 minutes, immediately add 6 μl EDTA, transfer to a 75°C water bath, and inactivate the enzyme for 10 minutes. Perform agarose gel electrophoresis on the mixed system after enzyme digestion, and select a suitable method to recover the fragments of the required size from the gel. If the template gene is about 2000bp, cut the gel block at 100-200bp, and use the gel recovery kit for gel recovery; if the template gene is about 1000bp, cut the gel block at about 50bp, because the fragment length is small, choose a low melting point during electrophoresis Agarose was electrophoresed, and the cut gel pieces were dissolved in 3 times the volume of TE solution, extracted with phenol-chloroform, and then ethanol-precipitated to recover small fragments of DNA.

Use the recovered fragmented DNA as a primer-free PCR template for primerless PCR. Using the principle of genetic recombination, the fragmented DNA can be used as primers for PCR. The system mix is as follows: 1 μl PFU enzyme, 5 μl buffer, 10 μl DNTP, 5 μl fragmented DNA , 29 μl ultrapure water, a total of 50 μl. The PCR program was: 94°C for 5 min, 94°C for 30 s, 46°C for 1 min, 72°C for 30 s, 50 cycles, 72°C for 10 min. The elongation time at 72°C is selected according to actual needs. Agarose gel electrophoresis was performed on the primer-free PCR product, and the fragment of the size of the original target lysozyme gene was recovered from the gel.

According to several homologous genes, primers containing the same restriction site were designed, and the designed primers were added, and the PCR products without primers in the previous step were used as templates to perform PCR with primers. According to the ratio of conventional PCR system, the PCR reaction was carried out according to the normal PCR reaction conditions. The PCR product with primers was subjected to agarose gel electrophoresis again, and the fragment of the size of the original target lysozyme gene was recovered from the gel.

The product after error-prone PCR or DNA shuffling was subjected to double enzyme digestion, and the expression vector was also subjected to double enzyme digestion with the same enzyme, and the required size fragment was recovered by agarose gel electrophoresis, and 5 μl of the recovered product was subjected to electrophoresis again, according to Determine the ratio of the target gene and the expression vector according to the brightness of the band, prepare the ligation system, connect overnight at 16°C, and construct the recombinant plasmid.

Transfer the recombinant plasmid into Top10 competent cells, and then apply all the competent cell suspension transferred to the recombinant plasmid on the plate, and apply 100-200 μl of the competent cell suspension transferred to the recombinant plasmid on each plate with a diameter of 90 mm. solution, cultured at 37°C for about 16 hours. Wash the grown strains off the plate with sterile water to form a bacterial suspension, recover the bacterial suspension, and use a plasmid extraction kit to extract the recombinant plasmid in the bacterial suspension.

Transfer the recombinant plasmid into eukaryotic competent cells, such as Pichia pastoris competent cells, spread all the eukaryotic competent cell suspensions transferred into the recombinant plasmid on the plate, and culture it statically to obtain the target lysate Enzyme Gene Mutation Libraries for Eukaryotic Cells.

(2) Construct a gram-negative bacterium that expresses "donor fluorescent protein-protease recognition site-acceptor fluorescent protein" in the cell:

The donor fluorescent protein used is cyan fluorescent protein, the acceptor fluorescent protein is photochromic fluorescent protein, and the protease is TEV protease;

The gene sequences of cyan fluorescent protein and yellow fluorescent protein were found through the NCBI database, and the recognition site gene of TEV protease was added as the intermediate linker sequence, and the "cyan fluorescent protein-TEV protease gene" with restriction enzyme sites at both ends was designed. The recognition site-yellow fluorescent protein "FRET fluorescent protein pair gene sequence is artificially synthesized. Both the FRET fluorescent protein pair gene and the intracellular expression vector of Gram-negative bacteria were digested with double enzymes, and the fragments recovered after electrophoresis were ligated to construct a recombinant plasmid.

The recombinant plasmid was introduced into Gram-negative bacterial competent cells, and the fermentation expression was induced, and the FRET fluorescent protein pair was successfully expressed in the strain identified by SDS-PAGE. Collect the bacteria by centrifugation, discard the supernatant, add sterile water to resuspend the bacteria, and then add sterile water after centrifugation to form a bacterial suspension with a specific concentration.

(3) Screening for lysozyme with high bactericidal activity against Gram-negative bacteria:

The yeast system that successfully secretes and expresses the target lysozyme gene mutation library is transferred into a 96 deep-well plate filled with yeast medium one by one, and induced fermentation is carried out in the deep-well plate to obtain a fermentation broth containing the target lysozyme.

Take 100 μl of fermentation broth from each well, and transfer it into a transparent microplate plate correspondingly, add 100 μl of pure water to each well, mix well and measure the OD ₆₀₀ value of the fermentation broth in each well in a microplate reader.

Add TEV protease and TEV protease buffer to the bacterial suspension of Gram-negative bacteria expressing "cyan fluorescent protein-TEV protease recognition site-yellow fluorescent protein", and the amount added is calculated according to the detection amount ( For detection, add 150 μl of bacterial suspension, 0.8 μl of TEV protease buffer and 0.5 μl of TEV protease) to each well, mix well to form a substrate mixture, and add 151 μl of substrate mixture to each well of the fluorescent microplate.

Then add 50 μl of fermentation broth to each well of the fluorescent microplate (no fermentation broth is added to the last two wells, only the substrate mixture is added as a negative control), pipette 1-2 times immediately, and then put it into a microplate reader for detection , the excitation wavelength is 433nm, and the emission wavelength is 475nm. The detection is performed every 1 minute for a total of 45 minutes.

The processing and analysis of screening indicators are as follows:

45 minutes before the reaction, take the time (min) as the abscissa and the cyan fluorescence signal intensity value as the ordinate to draw a graph to detect the stability at the beginning of the reaction. If the reaction is stable, that is, the reaction trend rises gently, the average value of the fluorescence signal intensity in the first 10 minutes can be calculated, and the average value within 10 minutes after 4 hours of reaction can be calculated at the same time. The difference between the two average values was divided by the OD ₆₀₀ value of each well of the fermentation broth, that is, the difference in fluorescence signal intensity/OD ₆₀₀ (the signal intensity growth rate of cyan fluorescent protein), and the activity of the mutant lysozyme was calculated based on this data. Sorting and comparing the data of each well with the data of the negative control well. Among them, the faster the cyan fluorescence signal increases and is higher than the value of the control well, it means that the lysozyme in the fermentation broth has a higher bactericidal activity on Gram-negative bacteria.

The screened positive yeast containing lysozyme with high bactericidal activity against Gram-negative bacteria was amplified and re-screened, and the activity of lysozyme was verified according to the growth rate of the signal intensity of cyan fluorescent protein during the reaction. The whole genome of the finally obtained positive yeast is extracted, the target gene is amplified and sequenced to obtain the gene sequence of a novel lysozyme with high bactericidal activity against Gram-negative bacteria.

The "tussah lysozyme gene" described in the present invention is the codon-optimized tussah lysozyme gene.

Example

In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, and these examples are for illustrative purposes only, and do not limit the scope of application of the present invention. The raw materials or components used in the present invention can be prepared by commercial channels or conventional methods unless otherwise specified.

Example 1: Construction of tussah silkworm lysozyme gene mutation library

Tussah silkworm is an economic insect in northern China. Tussah silkworm lysozyme (Aplyz) has the characteristics of wide temperature range and low optimum temperature, and has good application value. The tussah silkworm lysozyme gene sequence was found from the NCBI database. The mature peptide sequence is 363bp in total. The company carried out the whole gene synthesis. The synthesized gene sequence is as follows:

AAGTGGTTTACCAAATGTGGTCTAGTGCACGAGCTGAGGAGACAAGGCTTCGACGAGAGCCTAATGAGAGACTGGGTCTGTTTGGTTGAGAACGAAAGCAGCAGATATACTAATAAAATCGGTAAAGTGAATAAGAATGGTTCTCAAGACTACGGTTTGTTCCAGATCAATGACAAATATTGGTGTAGTAAGACCTCCACCCCCGGAAAGGATTGCAATGTGACTTGTAATCAATTGTTGACTGACGATATTACAGTTGCTGCTACCTGTGCGAAGAAGATTTACAAGAGACATAAGTTTAACGCTTGGTACGGATGGTTAAACCACTGTCAACACTCTCTTCCAGACATTAGCGACTGTTAA；

According to the expression vector pPICZαA to be connected and the above-mentioned tussah silkworm lysozyme gene sequence, two restriction enzymes, NotI and XhoI, were selected, and upstream and downstream primers containing restriction sites were designed. The primer sequences are as follows:

Aplyz-F: TCTACTCGAGAAAAAGAAAGTGGTTTACCA

Aplyz-R: TCGCTGACAATTCGCCGGCGTATAT

Firstly, normal PCR was carried out to amplify the gene of tussah lysozyme, and the strain was retained after being connected with T carrier, and then two methods of error-prone PCR and DNA shuffling were used to construct the gene mutation library of tussah silkworm lysozyme.

The specific operation steps of error-prone PCR are as follows:

Using the tussah silkworm lysozyme gene as a template, add the designed upstream and downstream primers, adjust the system ratio and reaction conditions, and perform error-prone PCR. The reaction system ratio is as follows:

The PCR reaction conditions were pre-denaturation at 94°C for 3 min, denaturation at 94°C for 1 min, annealing at 56°C for 30 s, and extension at 72°C for 30 s, a total of 50 cycles. 1% agarose gel electrophoresis for 45min, the gel imager was used to detect and record the results.

The specific steps of DNA shuffling are as follows:

The genes of human lysozyme gene 4 (LYZL4) and human lysozyme gene 6 (LYZL6) which have homology with tussah silkworm lysozyme gene sequence were selected, and tussah silkworm lysozyme gene was shuffled. Wherein, the gene sequence of human lysozyme gene 4 is as follows:

GAATTCCTCGAGTACATCTTAGGTAGATGTACTGTCGCAAAGAAACTGCATGACGGAGGTCTGGATTACTTCGAAGGATACTCTCTTGAGAATTGGGTGTGCTTGGCCTATTTTGAGTCTAAGTTCAATCCAATGGCCATATATGAAAATACTAGAGAGGGTTATACCGGATTTGGATTGTTTCAGATGAGAGGTAGTGATTGGTGCGGTGACCATGGTAGAAACAGATGTCATATGTCATGTTCCGCATTATTGAACCCAAACCTTGAAAAAACTATTAAGTGCGCTAAAACTATTGTTAAGGGTAAAGAAGGTATGGGTGCTTGGCCTACCTGGTCTAGATATTGTCAATACAGTGATACATTGGCTAGATGGCTAGACGGATGTAAGCTTTAAGCGGCCGC

The gene sequence of human lysozyme gene 6 is as follows:

GAATTCCTCGAGTCTTTGATTTCTAGATGCGATTTGGCTCAAGTTTTGCAGTTGGAGGACTTGGACGGTTTCGAGGGTTACTCTTTGTCTGACTGGTTGTGCTTGGCCTTCGTCGAGTCTAAGTTCAACATCTCTAAGATCAACGAGAACGCCGACGGATCTTTCGACTACGGATTGTTCCAGATCAACTCTCACTACTGGTGCAACGACTACAAATCTTACTCTGAGAACTTGTGCCATGTCGATTGCCAGGACTTGTTGAACCCAAACTTGTTGGCTGGAATCCATTGCGCCAAGAGAATCGTCTCTGGAGCCAGAGGAATGAACAACTGGGTCGAGTGGAGATTGCACTGCTCTGGTAGACCTTTGTTCTATTGGTTGACCGGTTGCAGATTGAGATGAGCGGCCGC

Primers containing the same restriction sites were designed according to the tussah silkworm lysozyme gene sequence, human lysozyme gene 4 gene sequence and human lysozyme gene 6 gene sequence. Primers were designed as follows:

Take 20 μl of tussah silkworm lysozyme gene, human lysozyme gene 4, and human lysozyme gene 6, mix them, add 6 μl of DNaseI buffer, add about 0.2 U of DNaseI, digest for 15 minutes, add 10 μl of EDTA, and inactivate the enzyme at 75°C 10min. The mixed system after enzyme digestion was electrophoresed on 1.5% low-melting point agarose gel for 45 minutes, and the gel imager was used to detect and record the results.

Select the band at about 50bp, cut off the gel block under ultraviolet light, add 3 times the volume of TE solution, put it in a 70°C water bath until the gel melts, add an equal volume of balanced phenol to extract once, and centrifuge at 12000rpm for 10min to take it Cleared into another PE tube, remove excess agar. Use an equal volume of phenol:(chloroform:isoamyl alcohol) (1:1) and chloroform:isoamyl alcohol (24:1) to extract once each, and take the supernatant.

Add 1/10 of the total volume of sodium acetate (pH 5.2), then add three times the volume of pre-cooled absolute ethanol, mix well and freeze overnight in a -20°C refrigerator. Take out the centrifuge tube, put it into a pre-cooled 4°C centrifuge, centrifuge at 13000rpm for 15min at high speed, suck up the supernatant as much as possible, add 1ml of 75% frozen ethanol to each of the two centrifuge tubes, centrifuge at 13000rpm for 10min. Desalt again by repeatedly adding 1 ml of 75% ice-cold ethanol. Naturally air-dry at room temperature, add 13 μL deionized water to each of the two centrifuge tubes to dissolve the DNA, and then take 3 μL of the DNA solution from each centrifuge tube to run electrophoresis to check the recovery effect after ethanol precipitation.

Using the recovered small fragment DNA product as a template, without adding primers, using homologous recombination, the small fragments serve as templates for each other, and perform primer-free PCR. The system is as follows:

The PCR reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 46°C for 1 min, extension at 72°C for 30 s, a total of 50 cycles, and a final extension at 72°C for 10 min. Use 1% agarose gel electrophoresis for 45min on the PCR products without primers, detect and record the results with a gel imager, and recover the DNA at about 400bp using the gel recovery kit. In order to increase the richness of the fragments, DNaseI can be used again for enzyme digestion and PCR without primers, and the fragments can be recovered again.

Use the PCR product without primers as a template, add the above six primers, and perform PCR with primers. The system is as follows:

The PCR reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55.5°C for 30 s, extension at 72°C for 30 s, a total of 33 cycles, and finally extension at 72°C for 10 min. The amplified product was electrophoresed on a 1% agarose gel for 45 minutes, detected by a gel imager and recorded, and the DNA at about 400 bp was recovered using a gel recovery kit.

NotI and XhoI restriction endonucleases were selected to perform double enzyme digestion on the error-prone PCR product, the DNA shuffling product and the expression plasmid pPICZαA. After double digestion, electrophoresis was performed on the system to recover fragments of the required size, and the gel-recovered product was electrophoresed again, and the concentration ratio was judged according to the brightness of the bands, and the target gene and expression plasmid were connected overnight to form a recombinant expression plasmid.

Transfer the recombinant plasmid into Top10 competent cells, and then apply all the competent cell suspension transferred to the recombinant plasmid on the plate, and apply 100-200 μl of the competent cell suspension transferred to the recombinant plasmid on each plate with a diameter of 90 mm. solution, cultured at 37°C for about 16 hours. Wash the grown strains off the plate with sterile water to form a bacterial suspension, recover the bacterial suspension, and use a plasmid extraction kit to extract the recombinant plasmid in the bacterial suspension.

Example 2: Transferring recombinant expression plasmids into Pichia pastoris cells

Pichia pastoris expression system is a good eukaryotic expression system, combined with pPICZαA expression plasmid can achieve good extracellular expression.

The specific operation steps for the preparation of Pichia pastoris competent cells are as follows:

(1) Pick a single clone of Pichia pastoris GS115 on the YPDS plate and inoculate it in 10 ml of YPD liquid medium. To ensure ventilation, seal it with 6 layers of gauze and culture overnight at 30°C and 250rpm.

(2) Transfer to a 250ml Erlenmeyer flask with baffles containing 50ml YPD liquid medium according to the inoculum size of 1%. Cultivate at 30°C with shaking at 250rpm for about 20h until the OD ₆₀₀ is 1.3-1.5.

(3) Transfer the bacterial solution into a pre-cooled 50ml centrifuge tube, place in an ice bath for at least 30 minutes to fully cool the cells, and collect the cells by centrifugation at 4°C and 4000 rpm for 5 minutes.

(4) Carefully discard the supernatant, resuspend the bacteria in 50ml ice-cold sterile deionized water, centrifuge at 4000rpm at 4°C for 5min.

(5) Resuspend the bacteria again with 25ml of ice-cold sterile deionized water.

(6) Carefully discard the supernatant, resuspend the cells with 5ml ice-cold 1mol/L sorbitol solution, centrifuge at 4000rpm at 4°C for 5min.

(7) Carefully discard the supernatant, resuspend the cells with 1ml sorbitol, and dispense 80μL per tube for use.

The specific steps for the electrotransformation of recombinant plasmids into Pichia pastoris GS115 competent cells are as follows:

(1) Add 10 μL of the linearized recombinant plasmid pPICZαA-ApLyz to 80 μL of Pichia pastoris GS115 competent cells, and pre-cool on ice for 10 min.

(2) Adjust the electrotransfer voltage of the electrotransfer instrument to 1.5kV.

(3) Add the ice-bathed mixture into the 0.2cm pre-cooled electric cup, tap it a few times to make the mixture sink to the bottom of the electric cup, and gently wipe off the water outside the electric cup.

(4) Put the electroporation cup into the electroporation apparatus, and the electroporation time is about 5.0 ms. Immediately after electroporation, 1 ml of pre-cooled 1 mol/L sorbitol solution is added.

(5) Transfer the electroshocked bacterial solution into a sterile centrifuge tube, incubate at 30°C for 1 hour, then add 500 μL of YPD liquid medium and mix gently, and continue to recover for 1 hour.

(6) The resuscitation solution was spread on the YPDS screening plate containing a lower concentration of Zeocin (100 μg/ml) resistance, 200 μL was applied to each plate, and all the resuscitation solution was coated.

(7) Static culture at 30°C for 48 hours.

Example 3: Construction of Escherichia coli expressing "cyan fluorescent protein-TEV protease recognition site-yellow fluorescent protein" in the cell

Escherichia coli is a typical Gram-negative bacterium, which is convenient to obtain, easy to operate and cultivate, and has a lipopolysaccharide outer membrane on the cell wall, so lysozyme has a poor cleavage effect on it.

According to the fluorescent protein gene and TEV protease recognition site gene sequences found in the NCBI database, the "cyan fluorescent protein-TEV protease recognition site-yellow fluorescent protein" gene was synthesized with XhoI and NocI restriction sites at both ends. sequence. The cloning plasmid and expression plasmid pET28a containing the protein pair gene were double-digested with two restriction endonucleases respectively, and the desired fragment was recovered by electrophoresis. Prepare the connection system, connect the fluorescent protein pair gene and the expression plasmid pET28a overnight, and construct the recombinant expression plasmid.

Take out BL21(DE3) competent cells and thaw on ice for 1-2min, add all the overnight ligation system to the competent cells, and then transfer to 42°C water bath for 90s in ice bath for 30min, then immediately transfer to ice for 2-3min, each tube Add 900 μl LB medium, recover at 200 rpm at 37°C for 1 hour. Aspirate 100 μl of the recovered bacterial suspension and spread it on the LB plate containing kanamycin.

Use the pET28a universal primer to perform colony PCR amplification on the colonies grown on the plate, verify the Escherichia coli successfully transformed into the plasmid, and then carry out fermentation. The specific steps of fermentation are as follows:

1) Transfer the Escherichia coli successfully transformed into the recombinant expression plasmid into 50ml LB liquid medium (conical flask, 250ml specification) containing an appropriate amount of kanamycin, and culture it on a shaker at 37°C until the _OD600 is between 0.4-1, The best is 0.6, about 3h.

2) Take 0.5ml of the prepared IPTG with a concentration of 100mM and add it to a group of medium to make the working concentration 1mM (because pET28a contains T7lac promoter, if other plasmids contain T7 promoter, dilute the working concentration of IPTG to 0.4mM ); another group of culture medium without IPTG was used as a control, and the two groups were cultured on a shaker for 3 h.

3) Place the Erlenmeyer flask on ice for 5 minutes, centrifuge at 5000×g at 4°C for 5 minutes, discard the supernatant, resuspend the bacteria in 1/4 container volume of pre-cooled sterile water, centrifuge again, and set at 4°C Centrifuge at 5000×g for 5 minutes.

4) Resuspend the strains in sterile water.

Example 4: Screening for novel tussah lysozyme with high bactericidal activity against Escherichia coli

Pick the Pichia pastoris with the lysozyme gene grown on the YPD plate into a 96-deep well plate filled with 1ml of BMGY medium, pick one strain into one well, and after culturing for 3 days, centrifuge at 4000rpm for 20min, discard For the supernatant, add 1ml of BMMY medium to each well, add 20μl of methanol, add 20μl of methanol to each well every 24h to induce fermentation expression, and stop the fermentation after 72h of fermentation.

Pipette 100 μl of the expressed fermentation broth into each well, and transfer the corresponding wells into a transparent normal 96-well plate, then add 100 μl of water to each well, mix well by pipetting, and put it into a microplate reader to measure the OD ₆₀₀ value.

Add TEV protease and TEV protease buffer to the Escherichia coli suspension expressing fluorescent protein pairs in the cells, and the amount added is calculated according to the detection amount (when testing, add 150 μl of bacterial suspension and 0.8 μl of TEV protease buffer to each well) and 0.5 μl TEV protease), mix well to form a substrate mixture, and add 151 μl substrate mixture to each well of a black 96-well plate.

Add 50 μl of fermentation broth to each well of the black 96-well plate (no fermentation broth is added to the last two wells, only the substrate mixture is added as a negative control), pipette 1-2 times immediately, put into a microplate reader for detection, and stimulate The wavelength is 433nm, and the emission wavelength is 475nm. It is detected once every 1 minute for a total of 45 minutes. After 4 hours of reaction, it is detected again for 10 minutes, and it is detected every 1 minute.

After the detection, the data were analyzed and processed by the method mentioned above to obtain yeast strains containing lysozyme with high bactericidal activity against Escherichia coli. The strains were expanded and cultivated, induced to ferment, and re-screened, and the data were analyzed by the method mentioned above processing to verify its activity. The whole genome of the finally obtained positive yeast is extracted, the target gene is amplified and sequenced to obtain the gene sequence of a novel lysozyme with high bactericidal activity against Gram-negative bacteria.

It should be noted that the above-mentioned embodiments are only used to explain the present invention, and do not constitute any limitation to the present invention. The invention has been described with reference to typical embodiments, but the words which have been used therein are words of description and explanation rather than words of limitation. The present invention can be modified within the scope of the claims of the present invention as prescribed, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the invention described therein refers to specific methods, materials and examples, it is not intended that the invention be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

SEQUENCE LISTING

<110> Beijing Technology and Business University

<120> A high-throughput screening method for a novel broad-spectrum lysozyme

<130> 2017

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 29

<212>DNA

<213> Aplyz-F

<400> 1

tctactcgag aaaagaaagt ggtttacca 29

<210> 2

<211> 25

<212>DNA

<213> Aplyz-R

<400> 2

tcgctgacaa ttcgccggcg tatat 25

<210> 3

<211> 29

<212>DNA

<213> Tussah lysozyme upstream primer

<400> 3

tctactcgag aaaagaaagt ggtttacca 29

<210> 4

<211> 25

<212>DNA

<213> Tussah lysozyme downstream primer

<400> 4

tatatgcggc cgcttaacag tcgct 25

<210> 5

<211> 31

<212>DNA

<213> human lysozyme 4 upstream primer

<400> 5

tctactcgag tacatcttag gtagatgtac t 31

<210> 6

<211> 23

<212>DNA

<213> human lysozyme 4 downstream primer

<400> 6

tatatgcggc cgcttaaagc tta 23

<210> 7

<211> 27

<212>DNA

<213> Human lysozyme gene 6 upstream primer

<400> 7

tctactcgag tctttgattt ctagatg 27

<210> 8

<211> 23

<212>DNA

<213> Downstream primer of human lysozyme gene 6

<400> 8

tatatgcggc cgctcatctc aat 23

<210> 9

<211> 363

<212>DNA

<213> Tussah lysozyme gene

<400> 9

aagtggttta ccaaatgtgg tctagtgcac gagctgagga gacaaggctt cgacgagagc 60

ctaatgagag actgggtctg tttggttgag aacgaaagca gcagatatac taataaaatc 120

ggtaaagtga ataagaatgg ttctcaagac tacggtttgt tccagatcaa tgacaaatat 180

tggtgtagta agacctccac ccccggaaag gattgcaatg tgacttgtaa tcaattgttg 240

actgacgata ttacagttgc tgctacctgt gcgaagaaga ttacaagag acataagttt 300

aacgcttggt acggatggtt aaaccactgt caacactctc ttccagacat tagcgactgt 360

taa 363

<210> 10

<211> 404

<212>DNA

<213> Human lysozyme gene 4

<400> 10

gaattcctcg agtacatctt aggtagatgt actgtcgcaa agaaactgca tgacggaggt 60

ctggattact tcgaaggata ctctcttgag aattgggtgt gcttggccta ttttgagtct 120

aagttcaatc caatggccat atatgaaaat actagagagg gttataccgg atttggattg 180

tttcagatga gaggtagtga ttggtgcggt gaccatggta gaaacagatg tcatatgtca 240

tgttccgcat tattgaaccc aaaccttgaa aaaactatta agtgcgctaa aactattgtt 300

aagggtaaag aaggtatggg tgcttggcct acctggtcta gatattgtca atacagtgat 360

acattggcta gatggctaga cggatgtaag ctttaagcgg ccgc 404

<210> 11

<211> 410

<212>DNA

<213> Human lysozyme gene 6

<400> 11

gaattcctcg agtctttgat ttctagatgc gatttggctc aagttttgca gttggaggac 60

ttggacggtt tcgagggtta ctctttgtct gactggttgt gcttggcctt cgtcgagtct 120

aagttcaaca tctctaagat caacgagaac gccgacggat ctttcgacta cggattgttc 180

cagatcaact ctcactactg gtgcaacgac tacaaatctt actctgagaa cttgtgccat 240

gtcgattgcc aggacttgtt gaacccaaac ttgttggctg gaatccattg cgccaagaga 300

atcgtctctg gagccagagg aatgaacaac tgggtcgagt ggagattgca ctgctctggt 360

agacctttgt tctattggtt gaccggttgc agattgagat gagcggccgc 410

<170> manual

<210> 12

<211> 7

<212> PRT

<213> TEV protease recognition site

<400> 12

Glu Asn Leu Tyr Phe Gln Gly

1 5

Claims (10)

1. a kind of high-throughput screening method of New-type wide-spectrum lysozyme, it comprises the following steps：

A, secreting, expressing is carried out by the gene mutation library of target lysozyme in eukaryotic, obtains target containing different activities molten The zymotic fluid of bacterium enzyme；

B, the zymotic fluid of the lysozyme of target containing different activities is added separately to be reacted in reaction system, is filtered out blue to leather The high lysozyme of family name's negative bacterium bactericidal activity；

Wherein, the reaction system includes：Intracellular expression " the recognition site of donor fluorescent protein-protein enzyme-acceptor fluorescence egg Gram-negative bacteria, protease and mmp reaction buffer solution in vain ".

2. according to the method described in claim 1, it is characterised in that according to the signal intensity of donor fluorescent albumen in course of reaction Growth rate, judges the lysozyme in zymotic fluid to Gram-negative bacteria bactericidal activity.

3. method according to claim 1 or 2, it is characterised in that described protease is with recognition sequence specificity Protease.

4. the method according to any one of claim 1-3, it is characterised in that described protease is TEV protease, intestines Kinases or factor Xa；Preferably, described protease is TEV protease.

5. method according to claim 4, it is characterised in that the amino acid sequence of the recognition site of the TEV protease For：Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

6. the method according to any one of claim 1-5, it is characterised in that the target lysozyme gene mutated library Construction method be fallibility PCR and/or DNA reorganization.

7. the method according to any one of claim 1-6, it is characterised in that the donor fluorescent albumen and acceptor fluorescence The distance between albumen is when within 10nm, on energy transfer to the acceptor fluorescent protein of donor fluorescent albumen.

8. method according to claim 7, it is characterised in that described donor fluorescent albumen is cyan fluorescent protein, institute The acceptor fluorescent protein stated is yellow fluorescence protein.

9. the method according to any one of claim 1-8, it is characterised in that the eukaryotic is Pichia pastoris.

10. the method according to any one of claim 1-9, it is characterised in that methods described is further comprising the steps of：

C, secondary screening is carried out to the lysozyme high to Gram-negative bacteria bactericidal activity filtered out, glimmering according to donor in course of reaction The signal intensity growth rate of photoprotein, verifies the activity of lysozyme.