CN112921042A - Prokaryotic expression method for amplifying buffalo IL-2 gene coding region primer and IL-2 protein - Google Patents
Prokaryotic expression method for amplifying buffalo IL-2 gene coding region primer and IL-2 protein Download PDFInfo
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Abstract
The primer for amplifying the buffalo IL-2 gene coding region provided by the invention comprises a nucleotide sequence shown by an upstream primer SEQ ID NO. 1 and a downstream primer SEQ ID NO. 2. Provides the application of the primer in PCR amplification; the recombinant expression plasmid Pet-32a-IL-2 is constructed by adding the expression vector pET-32a, and the IPTG is used for inducing the expression of the target protein and the purification method thereof, and the expressed IL-2 protein can react with the His tag antibody and the goat IL-2 polyclonal body.
Description
Technical Field
The invention belongs to the field of veterinary biotechnology, and particularly relates to a procaryon expression method for amplifying buffalo IL-2 gene coding region primers and IL _2 protein.
Background
Buffalo interleukin-2 protein (IL-2, hereinafter referred to as "IL-2"), also known as T cell growth factor (TCRF), consists of 155 amino acids and has a molecular weight of 17.5 KDa. IL-2 protein is synthesized and secreted mainly by T lymphocytes after being stimulated by antigenic substances or mitogens; in addition, B lymphocytes, natural killer cells (NK cells) and monocyte-macrophages are also capable of producing IL-2.
Research shows that the buffalo IL-2 protein can promote the increase of CD4+ and CD8+ of T lymphocytes and enhance the killing activity of natural killer cells (NK cells), monocytes and the like. The buffalo IL-2 protein can be used as an immunologic adjuvant, can improve the immunologic function of the vaccine when being used together with the vaccine, for example, the buffalo IL-2 protein has good function in the immunization of bovine bronchitis virus and bovine herpes virus vaccines, can effectively prevent and treat bovine mastitis, can enhance the resistance to the herpes virus by using the recombinant IL-2 protein in mice and cows, and improves the antiviral function.
At present, the research on cattle IL-2 in China is reported more [13-19], while the research on buffalo IL-2 protein is limited on the clone sequence analysis of genes [20-21 ]. The buffalo is important livestock in the south, has the characteristics of heat resistance, coarse feeding resistance and the like, and has the advantages that the milk of the buffalo is rich in nutrition, the economic value of the buffalo is further improved, the feeding scale is increased, and epidemic diseases are still prevalent, wherein the bovine viral diarrhea viruses are distributed in the world, and after infection, the cows have symptoms of fever, diarrhea, abortion, stillbirth, malformed fetuses and the like, so that the buffalo is an important factor influencing the development of the cow industry, and the research and development of new vaccine medicaments are an important means for preventing and controlling diseases.
Disclosure of Invention
The invention aims to provide a buffalo IL-2 protein and a prokaryotic expression method thereof, and the specific scheme is as follows:
the primer for amplifying the coding region of the buffalo IL-2 gene comprises an upstream primer and a downstream primer which respectively have nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Furthermore, the upstream primer has a BamH I cleavage site, and the downstream primer has a HindIII cleavage site.
The application of the primer for amplifying the buffalo IL-2 gene coding region in PCR amplification.
Further, the reaction procedure of the PCR amplification is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and 34 cycles; and finally, carrying out extension for 5min at 72 ℃ for the first time to obtain an amplification product, carrying out agarose gel electrophoresis separation on the amplification product, connecting the amplification product with a pMD18-T vector to form a cloning vector, transforming DH5 alpha competent cells, screening positive clones, and carrying out double enzyme digestion identification.
A recombinant expression plasmid Pet-32a-IL-2 of buffalo IL-2 protein.
Further, the expression vector pET-32a is included, the expression vector pET-32a and the T-IL-2 plasmid are subjected to double enzyme digestion for 6h at 37 ℃ through HindIII and BamHI restriction enzymes, the digestion product is recovered by cutting gel, and the expression vector pET-32a and the buffalo IL-2 protein coding region gene fragment are connected at 16 ℃ overnight.
A recombinant gene engineering bacterium containing the recombinant expression plasmid Pet-32a-IL-2, wherein the host cell of the recombinant gene engineering bacterium is escherichia coli.
The buffalo IL-2 protein prokaryotic expression method comprises the following steps:
(1) primer synthesis: according to the sequence of the coding region of the buffalo IL-2 protein gene, adopting Oligo7.37 to carry out primer design, respectively adding Hind III and BamHI restriction endonuclease sites in the nucleotide sequences shown by an upstream primer SEQ ID NO 1 and a downstream primer SEQ ID NO 2, extracting the total RNA of lymphocyte stimulated by ConA for amplifying the coding region of the IL-2 gene, connecting the amplified product with a pMD18-T vector to form a cloning vector, transforming DH5 alpha competent cells to obtain a T-IL-2 plasmid, and storing at-20 ℃ for later use;
(2) construction of recombinant expression plasmid Pet-32 a-IL-2: carrying out double enzyme digestion on an expression vector Pet-32a and the T-IL-2 plasmid in the step (1) at 37 ℃ for 6h by virtue of Hind III and BamH I restriction enzymes, cutting glue to recover a digestion product, connecting a buffalo IL-2 coding region gene fragment and the expression vector pET-32a at 16 ℃ overnight, transferring the obtained product into BL21 competent cells, and carrying out PCR (polymerase chain reaction) and sequencing verification, screening and inserting positive bacteria to obtain a recombinant expression plasmid Pet-32 a-IL-2;
(3) inducing expression: and (3) selecting the positive bacteria in the step (2) to perform shaking table overnight culture at 37 ℃ to serve as seed liquid, inoculating according to the inoculation amount of 1% the next day, adding IPTG (isopropyl-beta-thiogalactoside) after culturing for 4 hours until the final concentration is 1.0mmol/L for induction expression, continuing induction culture at 37 ℃ for 6 hours, and centrifugally collecting the bacteria.
A purification method of recombinant expression IL-2 protein adopts His tag protein purification reagent for purification, and the elution steps are as follows: eluting hetero protein with 50mmol/L imidazole, eluting target protein with 220mmol/L imidazole, ultrafiltering molecular sieve to concentrate target protein, and replacing the original buffer with PBS.
The primer of the buffalo IL-2 gene coding region or the application of the recombinant expression plasmid Pet-32a-IL-2 in preparing bovine viral diarrhea virus medicines or vaccines.
THE ADVANTAGES OF THE PRESENT INVENTION
The invention constructs a recombinant expression plasmid pET-32a-IL-2 based on the gene fragment of the buffalo IL-2 coding region, the recombinant buffalo IL-2 protein is efficiently expressed in the solubility of escherichia coli, can react with His labeled antibody and goat IL-2 polyclonal body after purification, can effectively inhibit cytopathic effect caused by bovine viral diarrhea virus on bovine kidney cells, and has good antiviral effect. The invention lays a foundation for researching and developing recombinant buffalo IL-2 protein vaccines and therapeutic biological agents, and has important practical significance for the healthy development of the buffalo breeding industry.
Drawings
FIG. 1 shows the result of electrophoresis of the PCR amplification product of buffalo IL-2 protein gene of the present invention. In the figure, the m.dna standard DL 1000 is used; 1: an IL-2 gene amplification product; 2: negative control m.dnamarkerdl 1000.
FIG. 2 shows the PCR identification result of the recombinant plasmid pET-32a-IL-2 colony of the present invention. In the figure, the m.dna standard DL2000 is used; 1-4: PCR was performed for each of the selected different pET-32a-IL-2 colonies.
FIG. 3 shows SDS-PAGE analysis and soluble expression analysis of the recombinant protein of the present invention. In the figure, M. protein molecular weight standards (15-130ku) are used; 1: pET-32a empty vector; 2: pET-32a-IL-2 vector (Total protein); 3: a soluble protein; 4: insoluble proteins.
FIG. 4 shows the purification analysis of the recombinant IL-2 protein of the present invention. In the figure, M. protein Marker (11-245ku) is adopted; 1: pET-32a empty vector; 2: pET-32a-IL-2 vector; 3: purified IL-2.
FIG. 5 shows the Western blot identification results of the recombinant IL-2 protein of the present invention. In panel a, the His-tag antibody in panel a was used; protein Marker (11-245 ku); 1: pET-32a empty vector; 2.: pET-32a-IL-2 vector; in panel B, a polyclonal IL-2 antibody is used; protein Marker (15-130 ku); 1: pET-32a empty vector; 2: pET-32a-IL-2 vector.
FIG. 6 shows the antiviral activity assay of recombinant IL-2 protein of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in detail and fully with reference to the accompanying drawings and specific embodiments, which should be noted that the present invention is not limited by the scope of the claims.
The various sources of reagents and materials in the examples of the invention are as follows:
1. test materials, viruses and fresh spleens of buffalos were collected from a certain slaughterhouse of south-of-Guangxi; bovine Viral Diarrhea Virus (BVDV), bovine kidney cells (MDBK cells) were provided by cantonese veterinary biotechnology focus laboratory preservation.
2. The reagent and instrument nucleic acid extraction reagent TRIzol kit and the IL-2 goat-derived polyclonal antibody are both products of Invitrogen corporation of America; pMD-18T vector, T4 DNA ligase, Hind III and BamH I restriction enzymes are all products of Bao bioengineering (Dalian) company; the bovine spleen lymphocyte separation fluid kit, the pET-32a (+) vector, the sword bean protein A (Concanavanin A, ConA), the porin electrophoresis preformed gel (15%) and the DAB color development fluid are all products of Beijing Solebao scientific and technological Limited company; coli DH5 alpha and BL21 competent cells were all products of Beijing Quanjin Biotech company; the His-labeled mouse-derived monoclonal antibody, the HRP-labeled goat anti-mouse IgG (H + L) and the HRP-labeled donkey anti-goat IgG (H + L) are all products of Wuhan Sanying technology company; the His label protein purification kit (soluble protein) is a product of century company Beijing kang. The micro nucleic acid analyzer NanoDrop2000 is a product of Saimer Feishel.
Example 1: primer synthesis
(1) Referring to the published buffalo IL-2 coding region sequence (accession number AF363786.1) on GenBank, the coding region sequence of buffalo IL-2 protein is shown as SEQ ID NO:3, Oligo7.37 is used for primer design, the upstream primer and the downstream primer are respectively added with BamHI and HindIII cleavage sites and protective bases, and the upstream primer is:
ataggatcctcaactcctgccacaatgtac (underlined BamH I cleavage site), and the downstream primers are:
ataaagcttagtcattgttgagtagatgctt (underlined Hind III sites), amplified 500bp in length, synthesized by Guangzhou Rui Bo, for amplification of the IL-2 gene coding region.
(2) Spleen lymphocyte culture
Extracting Buffalo spleen lymphocyte with reference to bovine spleen lymphocyte kit, adjusting cell density of obtained lymphocyte suspension to 2 × 10 with DMEM medium6One cell/mL of the cells were inoculated into 6-well cell culture plates and cultured for 6 hours, and 10. mu.g/. mu.L of ConA 40. mu.L was added to 20mL of MEM mediumAnd incubating the cells in a CO2 incubator at 37 ℃ for 18h, and collecting lymphocytes.
(3) Extraction of RNA and amplification of buffalo IL-2 protein coding region
Total RNA was extracted from the cells as described above with reference to TRIzol kit instructions, and the concentration of nucleic acid was measured at 300 ng/. mu.L using NanoDrop2000, and cDNA was synthesized with reference to Takara reverse transcription kit instructions.
Taking cDNA as a template, amplifying the gene segment of the IL-2 coding region by PCR, wherein the total reaction system is 50 mu L: mu.L of 2 XTransTaq-TPCRSuperMix 25, 1. mu.L of each of the upstream and downstream primers (primer concentration 10. mu. mol/L), 4. mu.L of template, and 50. mu.L of nuclease-free water. Reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and 34 cycles; the final extension at 72 ℃ was 5 min. Separating the amplified product by agarose gel electrophoresis, connecting with a pMD18-T vector, constructing a cloning vector, transforming DH5 alpha competent cells, screening positive clones, carrying out double enzyme digestion identification, sending correct positive recombinant plasmids to Huada company for sequencing, comparing by using a Blast program of NCBI, predicting an amino acid sequence encoded by an IL-2 gene by DNAMAN software, and storing the obtained T-IL-2 plasmid at-20 ℃ for later use. The amino acid sequence is shown in SED ID NO. 4.
Using cDNA obtained by reverse transcription as a template, a 500bp band was observed by agarose gel separation of the PCR amplification product, which was consistent with the expected size, as shown in FIG. 1.
Example 2: construction of recombinant expression vector Pet-32a-IL-2
The pET-32a vector and the T-IL-2 plasmid are subjected to double enzyme digestion for 6h at 37 ℃ by HindIII and BamHI restriction enzyme, the enzyme digestion product is recovered by cutting gel, the pET-32a vector and the T-IL-2 plasmid are connected at 16 ℃ overnight, the obtained product is transferred into BL21 competent cells, and positive bacteria with completely correct insertion are screened through PCR and sequencing verification.
A single colony PCR product of the recombinant expression plasmid pET-32a-IL-2 is separated by agarose to show a band of about 1200 bp (the target fragment is 500bp, and the vector sequencing primer amplification product is 700bp), which is consistent with the expected result, and is shown in figure 2.
Example 3: inducible expression and solubility assays
Selecting positive bacteria, culturing overnight in a shaker at 37 deg.C as seed solution, inoculating with 1% inoculum size the next day, culturing for 4 hr, adding IPTG to final concentration of 1.0mmol/L, and performing induced culture at 37 deg.C for 6 hr. Collecting induced bacterial liquid of 50mL, adding precooled bacterial lysate and PMSF to resuspend the thalli, crushing the thalli by ultrasonic until the liquid is clear, respectively taking mixed liquid (total protein), centrifuging supernatant (soluble protein), carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on a sediment (insoluble protein) sample, staining by Coomassie brilliant blue, and carrying out photographing analysis by a protein gel imaging system after decoloration.
The buffalo IL-2 protein gene coding region sequence totally encodes 155 amino acids, the molecular weight is about 17ku, and the buffalo IL-2 protein gene coding region sequence is connected to an expression vector pET-32a to be induced and expressed by IPTG, and SDS-PAGE analysis shows that: the recombinant IL-2 protein is expressed in E.coli well at about 36ku (including the tag protein), and the recombinant protein is mainly in the supernatant, i.e., is a soluble protein, as shown in FIG. 3.
Example 4: purification of IL-2 proteins
Purification was performed using a His-tag protein purification kit. After sample loading and balancing, eluting by using eluent with imidazole concentration of 50, 80, 100, 120, 150, 180, 200, 220, 250, 300 and 500mmol/L respectively, collecting eluent flowing out of each group, determining protein concentration by using NanoDrop2000 and selecting eluent with the next 9 concentrations for SDS-PAGE electrophoretic analysis. The formal elution steps are: eluting hetero protein with 50mmol/L imidazole, eluting target protein with 220mmol/L imidazole, ultrafiltering molecular sieve to concentrate target protein, and replacing the original buffer with PBS.
After the recombinant IL-2 protein is purified, a single band can be seen through SDS-PAGE analysis, and the IL-2 recombinant protein with higher purity can be seen, as shown in figure 4.
Experiment 1: western-blot identification of recombinant protein
Performing SDS-PAGE electrophoresis on a protein sample, transferring the protein to a PVDF membrane, sealing skim milk at room temperature for 4h, respectively adding 1:5000 diluted His-labeled mouse monoclonal antibody and 0.1 mu g/mL IL-2 goat-derived polyclonal antibody, incubating overnight at 4 ℃, washing the membrane for 4 times by PBST (basic-matrix-dependent test) the next day, respectively adding 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody and 1:5000 diluted HRP-labeled donkey anti-sheep secondary antibody, incubating at room temperature for 1h, washing the membrane for 4 times by PBST, and taking a DAB enhanced reagent by color development and photographing. Western-blot identification is carried out by respectively using a monoclonal antibody with a His tag and an IL-2 polyclonal antibody, a specific band is formed at the 36ku position, and a corresponding band does not appear in a control empty vector, so that the recombinant buffalo IL-2 protein has good reactogenicity and can specifically react with the His tag protein antibody and the IL-2 polyclonal antibody (fig. 5A and B).
Experiment 2: IL-2 antiviral Activity assay
Whether recombinant IL-2 protein has antiviral activity was determined using cytopathy inhibition. After the MDBK cells of the 24-well cell culture plate grow to be a single layer, adding DMEM containing recombinant protein with the final concentration of 3mg/ml into the IL-2 treatment group for co-incubation for 24 hours, abandoning the culture solution the next day, and adding BVDV solution with 100TCID50 into each well; the virus treatment group was added with 100TCID50 BVDV solution only; the untreated group was normal MDBK cells, without IL-2 and BVDV, and the cytopathic condition was examined under the microscope after 72 h.
BVDV/MDBK cytopathic inhibition is adopted to evaluate the antiviral activity of the purified IL-2, and the result shows that after 72 hours, typical cytopathic atrophy, plaque and desquamation appear in the cells of a treatment group; the cells in the IL-2 treated group had decreased cell density, less lesions, and smooth and clean edges of the surviving cells, compared to the untreated group, and it can be seen that the purified IL-2 protein had better antiviral activity, as shown in FIG. 6.
According to the invention, at a cellular level, the IL-2 protein expressed by a pronucleus is purified, concentrated, the buffer solution is replaced, and the mixture is uniformly mixed with a cell culture solution to act on MDBK cells infected with BVDV, and the result shows that the IL-2 protein has the effect of resisting BVDV on MDBK.
Therefore, the IL-2 disclosed by the invention has a better application prospect in bovine infectious diseases. Quiorga et al injected recombinant bovine IL-2 protein into bovine udders significantly reduced the incidence of bacterial mastitis. Meanwhile, the secretion level of IL-2 in the body of the cattle reflects different states of the cattle infected with mycobacterium. In addition, the use effect of the vaccine can be enhanced by taking IL-2 as an immunologic adjuvant, after the inactivated vaccine of the foot-and-mouth disease is injected into the water cattle by Mingala and the like, the dynamic change of the in-vivo cell factor is monitored, the IL-2 is found to reach the peak value in the second week, and the influence of the interaction of the IL-2 and the vaccine on the antiviral process is suggested; wyckoff and the like use recombinant bovine IL-2 protein and vaccine when infecting Brucella, so that the immune response of a bovine body is enhanced, and the resistance to Brucella is improved.
Description of technical terms in the present invention:
1. interleukins are a cytokine of the chemokine family. It is a cytokine derived from multiple cells (mainly produced by activated T cells) and having a pleiotropic effect (mainly promoting the growth, proliferation, differentiation of lymphocytes); has important effects on immune response of organisms, virus infection resistance and the like, and can stimulate the proliferation of T cells which are started by specific antigens or mitogenic factors; can activate T cells and promote cytokine production; stimulating NK cell proliferation, enhancing NK killing activity, generating cytokines, and inducing LAK cell generation; promoting B cell proliferation and secretion of antibodies; macrophages are activated.
2. Amino acid sequence: the type and arrangement of amino acid residues constituting a protein or polypeptide are usually indicated by three-letter method, which is conventionally used in the art, or one-letter method, which is conventionally used in the art, and those skilled in the art should be able to convert the three-letter amino acid sequence into the one-letter amino acid sequence. It should be clear that a person skilled in the art can understand and convert it no matter what way the application is presented. For example: alanine, a single letter is a, and a three letter is Ala; arginine, wherein one letter is R, and the three letters are Arg; aspartic acid, wherein the single letter is D, and the three letters are Asp; cysteine, with the single letter being C and the three letters being Cys; glutamine, one letter is Q, and the three letters are Gln; glutamic acid, wherein one letter is E, and the three letters are Glu; histidine, H in single letter and His in three letters; isoleucine, I in single letter and Ile in three letters; glycine, G is a single letter, and Gly is a three letter; asparagine, with N as the single letter and Asn as the three letters; leucine with the single letter of L and the three letters of Leu; lysine, wherein one letter is K, and the three letters are Lys; methionine, with a single letter of M and a three letter of Met; phenylalanine, with one letter F and three letters Phe; proline, P in one letter and Pro in three letters; serine, wherein the single letter is S, and the three letters are Ser; threonine, wherein one letter is T, and the three letters are Thr; tryptophan with W as a single letter and Trp as a three-letter; tyrosine, with one letter being Y and the three letters being Tyr; valine, V in single letter and Val in three letters.
Sequence listing
<110> Guangxi Zhuang nationality autonomous region veterinary research institute
<120> prokaryotic expression method for amplifying buffalo IL-2 gene coding region primer and IL _2 protein
<130> LWY2021015
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> buffalo (Bubalus bubalis)
<400> 1
ataggatcct caactcctgc cacaatgtac 30
<210> 2
<211> 31
<212> DNA
<213> buffalo (Bubalus bubalis)
<400> 2
ataaagctta gtcattgttg agtagatgct t 31
<210> 3
<211> 468
<212> DNA
<213> buffalo (Bubalus bubalis)
<400> 3
atgtacaaga tacaactctt gtcttgcatt gcactaactc ttgcactcgt tgcaaacggt 60
gcacctactt caagctctac ggggaacaca atgaaagaag tgaagtcatt gctgctggat 120
ttacagttgc ttttggagaa agttaaaaat cccgagaacc tcaagctctc caggatgcat 180
acatttaact tttacgtgcc caaggttaac gctacagaat tgaagcatct taagtgttta 240
ctagaagaac tcaaacttct agaggaagtg ctaaatttag ctccaagcaa aaacctgaac 300
cccagagaga tcaaggattc aatggacaat atcaagagaa tagttttgga actacaggga 360
tctgaaacag gattcacatg tgaatatgat gatgcgacag taaaggctgt agaatttctg 420
aacaaatgga ttaccttttg tcaaagcatc tactcaacaa tgacttga 468
<210> 4
<211> 155
<212> PRT
<213> buffalo (Bubalus bubalis)
<400> 4
Met Tyr Lys Ile Gln Leu Leu Ser Cys Ile Ala Leu Thr Leu Ala Leu
1 5 10 15
Val Ala Asn Gly Ala Pro Thr Ser Ser Ser Thr Gly Asn Thr Met Lys
20 25 30
Glu Val Lys Ser Leu Leu Leu Asp Leu Gln Leu Leu Leu Glu Lys Val
35 40 45
Lys Asn Pro Glu Asn Leu Lys Leu Ser Arg Met His Thr Phe Asn Phe
50 55 60
Tyr Val Pro Lys Val Asn Ala Thr Glu Leu Lys His Leu Lys Cys Leu
65 70 75 80
Leu Glu Glu Leu Lys Leu Leu Glu Glu Val Leu Asn Leu Ala Pro Ser
85 90 95
Lys Asn Leu Asn Pro Arg Glu Ile Lys Asp Ser Met Asp Asn Ile Lys
100 105 110
Arg Ile Val Leu Glu Leu Gln Gly Ser Glu Thr Gly Phe Thr Cys Glu
115 120 125
Tyr Asp Asp Ala Thr Val Lys Ala Val Glu Phe Leu Asn Lys Trp Ile
130 135 140
Thr Phe Cys Gln Ser Ile Tyr Ser Thr Met Thr
145 150 155
Claims (10)
1. The primer for amplifying the coding region of the buffalo IL-2 gene is characterized by comprising an upstream primer and a downstream primer which respectively have nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
2. The primer for amplifying the coding region of the buffalo IL-2 gene as claimed in claim 1, wherein the upstream primer has a BamH I cleavage site and the downstream primer has a HindIII cleavage site.
3. Use of the primer for amplifying the coding region of buffalo IL-2 gene according to claim 1 in PCR amplification.
4. The use according to claim 3, wherein the reaction procedure of the PCR amplification is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and 34 cycles; and finally, carrying out extension for 5min at 72 ℃ for the first time to obtain an amplification product, carrying out agarose gel electrophoresis separation on the amplification product, connecting the amplification product with a pMD18-T vector to form a cloning vector, transforming DH5 alpha competent cells, screening positive clones, and carrying out double enzyme digestion identification.
5. A recombinant expression plasmid Pet-32a-IL-2 of buffalo IL-2 protein.
6. The recombinant expression plasmid Pet-32a-IL-2 as set forth in claim 5, including expression vector pET-32a, which is digested with HindIII and BamHI restriction enzymes at 37 deg.c for 6 hr, and digested with glue to recover the product, and the expression vector pET-32a and buffalo IL-2 coding region gene fragment are ligated at 16 deg.c overnight.
7. A recombinant genetically engineered bacterium containing the recombinant expression plasmid Pet-32a-IL-2 of claim 5, wherein the host cell of the recombinant genetically engineered bacterium is Escherichia coli.
8. A prokaryotic expression method of buffalo IL-2 protein according to claim 5, characterized in that it comprises the following steps:
(1) primer synthesis: according to the sequence of the coding region of the buffalo IL-2 protein gene, adopting Oligo7.37 to carry out primer design, respectively adding Hind III and BamHI restriction endonuclease in the nucleotide sequences shown by an upstream primer SEQ ID NO:1 and a downstream primer SEQ ID NO:2 to amplify the coding region of the IL-2 gene, extracting the total RNA of lymphocytes stimulated by ConA for amplifying the coding region of the IL-2 gene, connecting the amplified product with a pMD18-T vector to form a cloning vector, transforming DH5 alpha competent cells to obtain a T-IL-2 plasmid, and storing the T-IL-2 plasmid at the temperature of minus 20 ℃ for later use;
(2) construction of recombinant expression plasmid Pet-32 a-IL-2: carrying out double enzyme digestion on an expression vector Pet-32a and the T-IL-2 plasmid in the step (1) at 37 ℃ for 6h by virtue of Hind III and BamH I restriction enzymes, cutting glue to recover a digestion product, connecting a coding region of buffalo IL-2 protein and the expression vector pET-32a at 16 ℃ overnight, transferring the obtained product into BL21 competent cells, and carrying out PCR (polymerase chain reaction) and sequencing verification, screening and inserting positive bacteria to obtain a recombinant expression plasmid Pet-32 a-IL-2;
(3) inducing expression: and (3) selecting the positive bacteria in the step (2) to perform shaking table overnight culture at 37 ℃ to serve as seed liquid, inoculating according to the inoculation amount of 1% the next day, adding IPTG (isopropyl-beta-thiogalactoside) after culturing for 4 hours until the final concentration is 1.0mmol/L for induction expression, continuing induction culture at 37 ℃ for 6 hours, and centrifugally collecting the bacteria.
9. A purification method of recombinant buffalo IL-2 protein is characterized in that a His label protein purification kit is adopted for purification, and the elution steps are as follows: eluting hetero protein with 50mmol/L imidazole, eluting target protein with 220mmol/L imidazole, ultrafiltering molecular sieve to concentrate target protein, and replacing the original buffer with PBS.
10. The use of the primer of buffalo IL-2 gene coding region according to claim 1 or the recombinant expression plasmid Pet-32a-IL-2 according to claim 5 in the preparation of bovine viral diarrhea virus drugs or vaccines.
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