CN107337718B - Gene for coding porcine circovirus type 2Cap protein and application thereof - Google Patents
Gene for coding porcine circovirus type 2Cap protein and application thereof Download PDFInfo
- Publication number
- CN107337718B CN107337718B CN201710466551.0A CN201710466551A CN107337718B CN 107337718 B CN107337718 B CN 107337718B CN 201710466551 A CN201710466551 A CN 201710466551A CN 107337718 B CN107337718 B CN 107337718B
- Authority
- CN
- China
- Prior art keywords
- porcine circovirus
- protein
- circovirus type
- recombinant
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10051—Methods of production or purification of viral material
Abstract
The invention provides a gene which is easy to express in escherichia coli and codes porcine circovirus type 2Cap protein, and the gene contains a sequence shown in SEQ ID NO. 1; a recombinant expression vector containing the gene and a recombinant bacterium containing the recombinant vector; the invention also provides a method for producing the porcine circovirus type 2Cap protein by inducing the recombinant bacteria and application of the protein in producing porcine circovirus type 2 vaccines. The invention realizes the high-efficiency expression of the full-length Cap protein in the Escherichia coli, and has the advantages of simple operation, low cost and easy large-scale production; the recombinant bacterium and the vaccine have wide application prospect in the aspect of preventing porcine circovirus type 2 diseases.
Description
Technical Field
The invention relates to a gene for coding porcine circovirus type 2Cap protein and application thereof, belonging to the field of molecular biology.
Background
Porcine circovirus type 2 (PCV-2) is one of the more serious viruses which have been newly discovered in recent years and endanger the swine industry, and the viruses are closely related to various disease syndromes of pigs, including Post-weaning multisystemic wasting syndrome (PMWS) of pigs, reproductive failure (SAMS) of sows, and the like. Since PMWS was first reported in Canada in 1991, porcine circovirus infection has now spread around the world in the 2010 International Pig Veterinary Society (IPVS), and is one of the most important infectious diseases recognized globally as harming the swine industry. In recent years, domestic porcine circovirus infection is on the rise, and the domestic porcine circovirus infection is very popular in domestic swinery, thereby causing great economic loss to the domestic pig industry.
PCV-2 genome is 1767bp or 1768bp in total length and comprises 11 Open Reading Frames (ORFs), wherein the ORFs 1 and 2 are the two largest open reading frames and respectively code replication-related proteins (Rep proteins) and capsid proteins (Cap proteins). The Cap protein is a main structural protein, and researches show that the N end of the Cap protein contains a Nuclear Localization Signal (NLS) consisting of 41 amino acid residues and contains a large number of rare codons of Escherichia coli, which seriously influences the expression of foreign proteins. Related studies have demonstrated that animals immunized with Cap protein can produce neutralizing antibodies to the virus. The ORF2 gene has been the focus of developing genetically engineered vaccines. However, in the prior art, the yield of Cap protein adopting a eukaryotic expression system is low, and the separation and purification are difficult; however, the N-terminal of ORF2 encoded protein has a large number of rare codons of Escherichia coli connected in series or in a single connection, which seriously affects the expression in Escherichia coli, so most of the currently reported prokaryotic expression is truncated Cap protein, which is not complete Cap protein though the protein expression amount is high, and cannot form complete virus-like particles, and some functional signal regions and conserved sequences cannot be expressed.
Disclosure of Invention
Aiming at the problems that the existing complete Cap protein is difficult to use escherichia coli for large-scale production and the like, the invention provides a gene for coding porcine circovirus type 2Cap protein, which is easy to be expressed in a soluble manner.
Another object of the present invention is to provide a recombinant expression vector containing the above gene.
It is still another object of the present invention to provide a recombinant bacterium containing the above gene, which has a high expression level of a target protein and whose expression product can be easily purified.
The invention also aims to provide application of the porcine circovirus type 2Cap protein expressed by the recombinant strain in preparation of porcine circovirus type 2 vaccines.
In order to achieve the purpose, the invention adopts the following technical scheme.
A gene for coding porcine circovirus type 2Cap protein contains a sequence shown in SEQ ID NO. 1.
The gene contains BamH I and Xho I restriction enzyme cutting sites.
The gene sequence of the coded porcine circovirus type 2Cap protein is shown in SEQ ID NO. 2.
The recombinant expression vector containing the gene is obtained by inserting the gene between BamH I and Xho I enzyme cutting sites of a prokaryotic expression vector pET30 a.
The recombinant bacterium containing the gene is obtained by transforming the recombinant expression vector into escherichia coli BL 21.
The recombinant strain expresses the porcine circovirus type 2Cap protein in an inducible expression mode.
The sequence of the recombinant strain expressing the porcine circovirus type 2Cap protein is shown in SEQ ID NO. 3.
An application of the recombinant bacterium in preparing porcine circovirus type 2 vaccine.
A method for preparing porcine circovirus type 2 vaccine by using the recombinant strain comprises the following steps:
(1) inducing the recombinant bacteria to express, crushing, cracking and collecting the supernatant, adding formaldehyde to inactivate to prepare an inactivated protein solution, wherein the protein solution qualified in inactivation detection is the vaccine antigen;
(2) mixing and stirring the vaccine antigen and the adjuvant to obtain the porcine circovirus type 2 vaccine.
The concentration of formaldehyde is 0.2% w.t; the inactivation time was 24 hours.
The antigen titer of the supernatant is not less than 1: 8; the content of PCV2Cap protein is not less than 150 mu g/mL.
The volume ratio of the protein inactivation liquid antigen to the adjuvant is 85: 15.
The adjuvant is ISA series adjuvant, preferably ISA 15 AVG.
The vaccine is of the oil-in-water type.
A preparation method of porcine circovirus type 2Cap protein comprises the following steps: and (3) carrying out induced culture on the recombinant strain, collecting the strain, crushing to obtain a lysate supernatant, and purifying to obtain the porcine circovirus type 2Cap protein.
The culture medium is a liquid LB culture medium containing kanamycin.
The inoculation amount of the recombinant bacteria is 1-5%, and preferably 2-3%.
The inducer for induction culture is isopropyl-beta-D-thiogalactoside (IPTG), the concentration is 0.2-1.2mmol/L, the induction time is 1-8h, and the induction initial amount is bacterial liquid OD600The value is 0.6-1.0.
Preferably, the concentration of IPTG is 0.4-0.8mmol/L, and the induction time is 2-5 h.
The invention has the following advantages:
the preparation of recombinant protein antigen by prokaryotic expression system makes the production of antigen easy, and has low cost and high yield. If the expression is high, the method has great advantages. However, since prokaryotes and eukaryotes have bias in codon usage and there are a large number of rare codons of Escherichia coli in tandem or in a single unit at the N-terminus of the protein encoded by ORF2, these factors will seriously affect expression in E.coli. In order to improve the soluble expression quantity of the ORF2 gene in Escherichia coli, the gene is optimized on the basis of not changing the amino acid sequence of the protein encoded by the gene, and the ORF2 gene after codon optimization can express the target protein more efficiently, and the expression rate is more than 90%.
ORF2 encoded protein does not need modification processing such as glycosylation after translation, and the protein exists in the form of peptide chain, and prokaryotic expression product has the same primary structure and the same antigenic determinant as natural Cap protein. The invention optimizes the codon of ORF2 gene, changes rare codon which is difficult to express in colibacillus, greatly improves the expression quantity of protein, simultaneously, the expressed complete Cap protein amino acid sequence is completely consistent with the parent sequence, does not change the immunogenicity, and lays a good foundation for ensuring the immune effect of the prepared genetic engineering vaccine. The invention realizes the high-efficiency expression of the full-length Cap protein in the Escherichia coli, and carries out codon optimization on the porcine circovirus type 2Cap protein coding gene, so that the recombinant porcine circovirus type 2Cap protein can be expressed with high yield, high efficiency and solubility, and the problem of low expression quantity of the Cap protein in prokaryotic cells is solved. As the prokaryotic expression system of the escherichia coli is mature, the method is simple to operate, low in cost and easy for large-scale production.
The recombinant porcine circovirus type 2Cap protein has good activity, and the compound containing the recombinant protein can generate high-level porcine circovirus type 2 specific antibody after immunizing piglets. Therefore, the method has wide application prospect in the aspect of preventing porcine circovirus type 2.
Drawings
FIG. 1 shows PCR detection results of PCV2 ORF2 gene; wherein, M is DL2000 DNA Marker, 1 is negative control, and 2 is positive plasmid;
FIG. 2 shows the SDS-PAGE result of Cap protein; wherein, M is a protein Marker, 1 is a bacterial liquid before induction, and 2 is a cracking supernatant after induction;
FIG. 3 shows the detection result of Cap protein Western-blot electrophoresis.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 construction and identification of recombinant expression vector pET30a-ORF 2.
Acquisition and codon optimization of ORF2 Gene
PCV2 SD strain (CGMCC NO.5774, preservation date: 2, 8 days 2012) is inoculated to PK-15 cells without PCV2 pollution, viruses are harvested after 72h to serve as templates, and the porcine circovirus type 2 whole genome is amplified by PCR, wherein the primer sequences are as follows:
the sequence of the upstream primer is as follows:
5’-GAACCGCGGGCTGGCTGAACTTTTGAAAGT-3’(SEQ ID NO.4)；
the sequence of the downstream primer is as follows:
5’-GCACCGCGGAAATTTCTGACAAACGTTACA-3’(SEQ ID NO.5)；
the PCV2 SD strain genome sequence is shown as SEQ ID NO.6 (GenBank No. DQ 346683), the ORF2 gene of the virus shown as SEQ ID NO.7 is optimized and modified into escherichia coli preference codons, and the sequence is shown as SEQ ID NO. 1. And (3) adding the 5 'end and the 3' end of the ORF2 gene subjected to optimization and modification into BamH I and Xho I restriction enzyme sites respectively, wherein the sequences are shown as SEQ ID No.2, and carrying out artificial synthesis.
2. Construction and identification of recombinant expression vector pET30a-ORF2
2.1 construction of recombinant expression vector pET30a-ORF2
The synthesized recombinant ORF2 gene is subjected to double enzyme digestion and then is connected with the corresponding enzyme digestion site of the pET30a prokaryotic expression vector to construct a pET30a-ORF2 expression vector. The ligation product was transformed into competent cells of E.coli BL21 by heat stress, plated on LB plate containing kanamycin, and cultured at 37 ℃. Selecting a single colony, culturing in an LB culture medium containing kanamycin, extracting plasmids by an alkaline lysis method, carrying out PCR amplification and BamH I and XhoI double enzyme digestion identification, obtaining a target strip from positive plasmids, sequencing the positive plasmids, and proving that the optimized ORF2 gene is successfully inserted into pET30a plasmid without point mutation by a sequencing result, wherein the prokaryotic expression vector is successfully constructed, and the recombinant plasmid with correct sequencing is named as pET30a-ORF 2.
2.2 PCR of the recombinant expression vector pET30a-ORF2
According to the modified ORF2 gene sequence, a pair of detection primers is designed to amplify the ORF2 gene, and the primer sequences are shown as follows: the sequence of the upstream primer is as follows:
5’-ATGGATCCATGACCTACCCGCGTCGTC-3’(SEQ ID NO.8)；
the sequence of the downstream primer is as follows:
5’-GTCTCGAGCTTAGGGTTGAGCGGCGGG-3’(SEQ ID NO.9)。
using the ligation product as a template, the following reagents were added to establish a 50 μ LPCR system:
the following amplification conditions were used: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 61 ℃ for 30 s; extension at 72 ℃ for 10 min.
The PCR product was recovered and stored at-20 ℃ until use. The PCR product was identified by 1% agarose gel electrophoresis, as shown in FIG. 1, where M is a standard DNA Marker (DL2000), 1 is a negative control, and 2 is a positive plasmid amplification result, containing a target band of about 700 bp.
2.3 double restriction enzyme identification of recombinant expression vector pET30a-ORF2
The purified pET30a-ORF2 prokaryotic expression vector plasmid is subjected to double enzyme digestion by BamH I and Xho I, and 5 mu L of the digestion product is subjected to electrophoresis on 2% agarose gel at a concentration of 5V/cm for 40 min. The enzyme digestion system is as follows:
the recombinant plasmid identified as positive by double enzyme digestion is re-sequenced, and the result shows that no point mutation exists in the ORF2 gene.
Example 2 construction and inducible expression of recombinant expression strain BL21/pET30a-ORF 2.
1. Recombinant plasmid transformation competent Escherichia coli BL21
Coli BL21(NEB) was transformed with the plasmid for the prokaryotic expression vector pET30a-ORF2, which was sequenced correctly in example 1, to obtain a recombinant expression strain BL21/pET30a-ORF 2.
2. Inducible expression of recombinant expression strain BL21/pET30a-ORF2
Selecting single colony of recombinant bacteria, inoculating into 10mL LB culture medium containing kanamycin, and shake culturing at 37 deg.C to OD600Reaching 0.6-1.0, adding IPTG to the final concentration of 1mmol/L, and continuing culturing for 5 hours. Centrifuging to collect thallus, discarding supernatant, washing with PBS (0.015mol/L, pH7.2), adding 3mL lysate, ultrasonic lysing for 3min, centrifuging, collecting supernatant, adding appropriate amount of 5 xSDS loading buffer solution, performing SDS-PAGE detection with 12% polyacrylamide gel, freezing the rest sample at-20 deg.C, and setting culture without IPTG induction as control.
3. Identification of expression product of recombinant expression strain BL21/pET30a-ORF2
BL21/pET30a-ORF2 cells were collected, subjected to SDS-PAGE, and visualized by Coomassie blue staining after electrophoresis, and the results are shown in FIG. 2: the recombinant expression plasmids respectively generate an approximately 37kDa protein band, the size of the protein band is basically consistent with the molecular weight of the expected fusion protein, the expression rate reaches more than 90 percent, and the control has no protein band. Western-blot detection is carried out on the recombinant protein subjected to induced expression by using the PCV2 monoclonal antibody, and the result shows that a specific target band is presented at 37KDa, as shown in figure 3.
Example 3 soluble expression of recombinant expression Strain BL21/pET30a-ORF2
Inoculating 2% of the recombinant strain into liquid LB culture medium containing kanamycin, and shake culturing at 37 deg.C for 2 hr to OD600When the concentration is 0.82, adding IPTG with the final concentration of 0.8mmol/L for induction for 3 hours, centrifuging at 4 ℃ and 5000rpm for 10 minutes, washing the precipitate with PBS (0.015mol/L, pH7.2), then adding 10mL of lysate, ultrasonically cracking for 20 minutes, centrifuging at 4 ℃ and 10000rpm for 15 minutes, and collecting the supernatant; purifying and concentrating to obtain the porcine circovirus type 2Cap protein.
Example 4 preparation of porcine circovirus type 2 vaccine.
A porcine circovirus type 2 vaccine was prepared as follows:
(1) crushing, cracking and collecting supernate of the recombinant bacteria after induction expression, purifying and concentrating the supernate, and measuring the antigen titer to be 1:16 by adopting an agar diffusion method; total protein content was determined by the BCA method, followed by SDS-PEGA electrophoresis and then by scanning on a gel imager, and PCV2Cap protein content was calculated to be 155. mu.g/mL. Adding 0.2% formaldehyde into the supernatant, shaking at 37 ℃, inactivating for 24 hours to prepare a protein inactivation solution, and obtaining a vaccine antigen after the detection is qualified;
(2) mixing antigen and oil phase adjuvant ISA 15AVG at a ratio of 85:15, stirring, and emulsifying to obtain oil-in-water (O/W) vaccine.
Example 5 efficacy and biological testing of porcine circovirus type 2 vaccine.
1. Antibody detection
10 pigs of 14-21-day-old PCV2 negative healthy susceptible piglets are divided into 2 groups, each group has 5 pigs, the 1 st group is respectively injected with 2.0mL of gene engineering vaccine through muscle, the 2 nd group is not inoculated and is used as a blank control for isolated feeding and observation. Blood is collected 28 days after immunization, PCV2 antibody titer is determined by an ELISA method, and PCV2 serum ELISA antibody titer is not less than 1: 1600, PCV2 serum ELISA antibody titers in control pigs were not higher than 1: 400. the specific results are as follows:
as can be seen from Table 1, the PCV2 serum ELISA antibody titer of the genetically engineered vaccine immunization group pigs is not lower than 1: 1600, PCV2 serum ELISA antibody titers in the blank control pigs were not higher than 1: 400.
table 1 serum antibody titer test results of vaccine immunoassay pigs.
2. Immunological method of counteracting toxic pathogen
15 pigs which are 14-21 days old and are PCV2 negative healthy susceptible piglets are divided into 3 groups, each group comprises 5 pigs, the 1 st group is an immune challenge group, 2.0mL of vaccine is injected into muscles respectively, the 2 nd group is a non-immune challenge group, the 3 rd group is a blank control group (non-immune and non-challenge), and the 3 groups are separately fed in isolation. The piglets in groups 1 and 2 are respectively injected with 5.0mL of keyhole limpet hemocyanin (KLH/ICFA,0.5mg/mL) emulsified by Freund's incomplete adjuvant and 10mL of thioglycollic acid culture medium through intramuscular injection on 28 days after immunization and 3 days before challenge. When attacking toxinRespectively inoculating SD strain virus culture solution (not less than 10) to piglets in groups 1 and 25.5TCID50Per nasal drip 2.0 mL), intramuscular injection of 3.0mL, 3 and 6 days after challenge, intramuscular injection of 5.0mL of keyhole limpet hemocyanin (KLH/ICFA,0.5mg/mL) emulsified in Freund's incomplete adjuvant to each piglet in groups 1 and 2, and intraperitoneal injection of 10mL of thioglycolic acid medium. Weighing all piglets on the current day of infection and 28 th day after challenge, measuring body temperature of each group of piglets every day on the first 7 days after challenge, finishing 28 days after challenge, performing autopsy, taking inguinal lymph nodes of the piglets to perform immunohistochemical detection, and judging according to body temperature, relative daily gain and immunohistochemical detection, wherein test results are detailed in tables 2-5. The non-immune attacking group should have at least 4 attacks of disease, and the immune attacking group should have at least 4 protections.
TABLE 2 thermometer of post-challenge test pigs
As can be seen from Table 2, the virus solution of porcine circovirus type 2 SD strain (10) was used after immunization with the genetically engineered vaccine5.5TCID50mL), the body temperature of the test pig is not obviously changed; the body temperature of 5 test pigs in the non-immune challenge group is increased (more than or equal to 40 ℃) and lasts for more than 3 days.
TABLE 3 weight change after challenge
As can be seen from Table 3, the swine used the virus liquid of porcine circovirus type 2 SD strain (10) in the immune challenge group and the non-immune challenge group5.5TCID50mL), the statistical software SPSS 20.0 is used for significance analysis, the average relative daily gain of the immune challenge group is more than 0.05 compared with the average relative daily gain of a blank control group, the difference is not significant, and all pigs in the group have no clinical symptoms caused by porcine circovirus type 2. Compared with the average relative daily gain of a blank control group, the average relative daily gain of the non-immune challenge group is less than 0.01, the difference is very obvious, and all pigs in the group have the average relative daily gainClinical symptoms caused by porcine circovirus type 2.
TABLE 4 immunohistochemical detection results of post-challenge test pigs
Note: "+" indicates positive; "-" indicates negative.
As can be seen from Table 4, antigens were detected by immunohistochemistry 28 days after challenge in the non-immunized challenge group and the virus solution of porcine circovirus type 2 SD strain (10) was administered after immunization with the vaccine in the test pigs5.5TCID50/mL) no antigen could be detected by immunohistochemistry at 28 days of challenge.
TABLE 5 vaccine immunoprotective efficacy test results
Note: "+" indicates positive; "-" indicates negative.
By combining the above, after immune challenge, the immune group can achieve 5/5 protection, and the non-immune challenge group 5/5 has diseases, which shows that the vaccine has good immune effect.
<110> institute of zootechnics of academy of agricultural sciences of Shandong province
<120> gene for coding porcine circovirus type 2Cap protein and application thereof
<130> 20170614
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 705
<212> DNA
<213> Artificial sequence
<400> 1
atgacctacc cgcgtcgtcg ttaccgtcgt cgtcgtcacc gtccgcgttc tcacctgggt 60
caaatcctgc gtcgtcgtcc gtggctggtt cacccgcgtc tgcgttaccg ttggcgtcgt 120
aaaaacggca tcttcaacac ccgtctgtct cgtaccatcg gttacaccgt taaagcgacc 180
accgttcgca ctccaagctg ggcggtcgat atgatgcgct ttaacatcaa cgacttcctg 240
ccgccaggtg gcggctctaa cccgctgacc gttccgttcg aatactaccg tatccgtaaa 300
gtgaaagttg aattctggcc gtgcagcccg atcacccagg gcgatcgcgg cgtcggttcc 360
accgcagtga tcctggatga taacttcgtg accaaagcga acgcgctgac ctacgatccg 420
tacgttaact attctagccg ccacaccatc ccgcagccat tctcctacca cagccgttat 480
ttcaccccga agccggtgct ggatcgtacc atcgactact tccagccgaa caacaaacgt 540
aaccagctgt ggctgcgtct gcagaccacc ggtaacgtgg atcacgttgg cctgggtact 600
gcgttcgaaa actctaaata cgaccaggac tacaacattc gtatcaccat gtacgttcag 660
ttccgtgagt tcaacctgaa agacccgccg ctcaacccta agtaa 705
<210> 2
<211> 717
<212> DNA
<213> Artificial sequence
<400> 2
ggatccatga cctacccgcg tcgtcgttac cgtcgtcgtc gtcaccgtcc gcgttctcac 60
ctgggtcaga tcctgcgtcg tcgtccgtgg ctggttcacc cgcgtctgcg ttaccgttgg 120
cgtcgtaaaa acggcatctt caacacccgt ctgtctcgta ccatcggtta caccgttaaa 180
gcgaccaccg ttcgcactcc aagctgggcg gtcgatatga tgcgctttaa catcaacgac 240
ttcctgccgc caggtggcgg ctctaacccg ctgaccgttc cgttcgaata ctaccgtatc 300
cgtaaagtga aagttgaatt ctggccgtgc agcccgatca cccagggcga tcgcggcgtc 360
ggttccaccg cagtgatcct ggatgataac ttcgtgacca aagcgaacgc gctgacctac 420
gatccgtacg ttaactattc tagccgccac accatcccgc agccattctc ctaccacagc 480
cgttatttca ccccgaagcc ggtgctggat cgtaccatcg actacttcca gccgaacaac 540
aaacgtaacc agctgtggct gcgtctgcag accaccggta acgtggatca cgttggcctg 600
ggtactgcgt tcgaaaactc taaatacgac caggactaca acattcgtat caccatgtac 660
gttcagttcc gtgagttcaa cctgaaagac ccgccgctca accctaagta actcgag 717
<210> 3
<211> 234
<212> PRT
<213> Porcine circovirus
<400> 3
Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg
1 5 10 15
Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro
20 25 30
Arg Leu Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg
35 40 45
Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Ala Thr Thr Val Arg Thr
50 55 60
Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu
65 70 75 80
Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr
85 90 95
Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr
100 105 110
Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn
115 120 125
Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr
130 135 140
Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr
145 150 155 160
Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro
165 170 175
Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn
180 185 190
Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp
195 200 205
Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe
210 215 220
Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys
225 230
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence
<400> 4
gaaccgcggg ctggctgaac ttttgaaagt 30
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence
<400> 5
gcaccgcgga aatttctgac aaacgttaca 30
<210> 6
<211> 1767
<212> DNA
<213> Porcine circovirus
<400> 6
accagcgcac ttcggcagcg gcagcacctc ggcagcacct cagcagcaac atgcccagca 60
aaaagaatgg aagaagcgga ccccaaccac acaaaaggtg ggtgttcacg ctgaataatc 120
cttccgagga cgagcgcaag aaaatacggg agcttccaat ctcccttttt gattatttta 180
ttgttggcga ggagggtaat gaggaaggac gaacacccca cctccagggg ttcgctaatt 240
ttgtgaagaa gcaaacattt aataaagtga aatggtattt cggtgcccgc tgccacatcg 300
agaaagcgaa aggaactgat cagcagaata aagaatattg cagtaaagaa ggcaacttac 360
tgattgaatg tggagctcct agatctcaag gacaacgcag tgacctgtct actgctgtga 420
gtaccttgct ggagagcggg agtctggtga ccgttgcaga gcagcaccct gtaacgtttg 480
tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg cagaagcgtg 540
attggaagac taatgtacac gtcattgtgg ggccacctgg gtgtggtaaa agcaaatggg 600
ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac aagtggtggg 660
atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg ctgccctggg 720
atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaa ggtggaactg 780
tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttg gaatggtact 840
cctcaactgc tgtcccagct gtagaagctc tttatcggag gattacttcc ttggtatttt 900
ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc ctttcccccc 960
catgccctga atttccatat gaaataaatt actgagtctt ttttatcatt tcgtaatggt 1020
ttttattatt cattaagggt taagtggggg gtctttaaga ttaaattctc tgaattgtac 1080
atacatggtt acacggatat tgtattcctg gtcgtatata ctgttttcga acgcagtgcc 1140
gaggcctacg tggtctacat ttccagcagt ttgtagtctc agccacagct gatttctttt 1200
gttgtttggt tggaagtaat caatagtgga atctaggaca ggtttggggg taaagtagcg 1260
ggagtggtag gagaagggct gggttatggt atggcgggag gagtagttta cataggggtc 1320
ataggtgagg gctgtggcct ttgttacaaa gttatcatct agaataacag cactggagcc 1380
cactcccctg tcaccctggg tgatcgggga gcagggccag aattcaacct taacctttct 1440
tattctgtag tattcaaagg gcacagagcg ggggtttgag ccccctcctg ggggaagaaa 1500
gtcattaata ttgaatctca tcatgtccac cgcccaggag ggcgttctga ctgtggttcg 1560
cttgatagta tatccgaagg tgcgggagag gcgggtgttg aagatgccat ttttccttct 1620
ccagcggtaa cggtggcggg ggtggacgag ccaggggcgg cggcggagga tctggccaag 1680
atggctgcgg gggcggtgtc ttcttctccg gtaacgcctc cttggatacg tcatatctga 1740
aaacgaaaga agtgcgctgt aagtatt 1767
<210> 7
<211> 723
<212> DNA
<213> Porcine circovirus
<400> 7
atgacgtatc caaggaggcg ttaccgaaga cgaagacacc gctactgcat aggttcctcc 60
ccccgcagcc atcttggcca aatcctccgc cgccgcccct ggctcgtcca cccccgcctc 120
cgttaccgct ggagaaggaa aaatggcatc ttcaacaccc gcctctcccg caccatcggt 180
tatactgtca aggctaccac agtcagaacg ccctcctggg cggtggacat gatgagattt 240
aatattaatg attttcttcc cccaggaggg ggctcaaacc ccctcactgt gccctttgaa 300
tactacagaa taagaaaggt taaggttgaa ttctggccct gctccccaat cacccagggt 360
gacaggggag tgggctccac tgctgttatt ctagatgata actttgtaac aaaggccaat 420
gccctaacct atgaccccta tgtaaactac tcctcccgcc ataccatacc ccagcccttc 480
tcctaccact cccgctattt cacccccaaa cctgtccttg ataggacaat cgattacttc 540
caacccaata acaaaagaaa tcaactctgg ctgagactac aaactactgg aaatgtagac 600
catgtaggcc tcggcactgc gttcgaaaac agtaaatacg accaggacta caatatccgt 660
ataaccatgt atgtacaatt cagagaattt aatcttaaag accccccact taaccctaag 720
tga 723
<210> 8
<211> 27
<212> DNA
<213> Artificial sequence
<400> 8
atggatccat gacctacccg cgtcgtc 27
<210> 9
<211> 27
<212> DNA
<213> Artificial sequence
<400> 9
gtctcgagct tagggttgag cggcggg 27
Claims (13)
1. A gene for coding porcine circovirus type 2Cap protein is characterized in that the sequence is shown as SEQ ID number 1.
2. The gene according to claim 1, further comprisingBamH I andXhoi restriction enzyme cutting site.
3. The gene of claim 2, wherein the gene sequence encoding the porcine circovirus type 2Cap protein is represented by SEQ ID number 2.
4. A recombinant expression vector containing the gene of claim 2 or 3, wherein the recombinant expression vector is obtained by inserting the gene into a prokaryotic expression vector pET30aBamH I andXhoi is obtained between enzyme cutting sites.
5. A recombinant bacterium containing the recombinant expression vector of claim 4, wherein the recombinant bacterium is obtained by transforming Escherichia coli BL21 with the recombinant expression vector.
6. The recombinant strain of claim 5, wherein the expression of the porcine circovirus type 2Cap protein is inducible expression; the porcine circovirus type 2Cap protein sequence is shown as SEQ ID number 3.
7. Use of the recombinant bacterium of claim 5 in the preparation of a porcine circovirus type 2 vaccine.
8. A method for preparing the porcine circovirus type 2 vaccine by using the recombinant strain of claim 5, which is characterized by comprising the following steps:
(1) inducing the recombinant bacteria to express, crushing, cracking and collecting the supernatant, adding formaldehyde to inactivate to prepare an inactivated protein solution, wherein the protein solution qualified in inactivation detection is the vaccine antigen;
(2) mixing and stirring the vaccine antigen and the adjuvant to obtain the porcine circovirus type 2 vaccine.
9. The method according to claim 8, wherein the supernatant antigenic titer is not less than 1:8, and the PCV2Cap protein content is not less than 150 μ g/mL; the concentration of formaldehyde is 0.2% w.t; the inactivation time is 24 hours; the volume ratio of the vaccine antigen to the adjuvant is 85: 15; the vaccine is of the oil-in-water type.
10. The method according to any one of claims 8 or 9, wherein the adjuvant is selected from the ISA series adjuvants.
11. A preparation method of porcine circovirus type 2Cap protein is characterized by comprising the following steps: inducing and culturing recombinant bacteria, collecting thalli, crushing to obtain lysate supernatant, and purifying to obtain porcine circovirus type 2Cap protein;
the sequence of the coding gene of the porcine circovirus type 2Cap protein is shown as SEQ ID number 1.
12. The production method according to claim 11, wherein the medium is a liquid LB medium containing kanamycin; the inoculation amount of the recombinant bacteria is 1-5%; the inducer for induction culture is isopropyl-beta-D-thiogalactoside, the concentration is 0.2-1.2mmol/L, the induction time is 1-8h, and the induction initial amount is bacterial liquid OD600The value is 0.6-1.0.
13. The method according to claim 11 or 12, wherein the amount of the recombinant strain is 2-3%, the concentration of isopropyl- β -D-thiogalactoside is 0.4-0.8mmol/L, and the induction time is 2-5 h.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710466551.0A CN107337718B (en) | 2017-06-20 | 2017-06-20 | Gene for coding porcine circovirus type 2Cap protein and application thereof |
| AU2017101605A AU2017101605A4 (en) | 2017-06-20 | 2017-11-15 | A gene encoding capsid protein of porcine circovirus type 2 (PCV2) and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710466551.0A CN107337718B (en) | 2017-06-20 | 2017-06-20 | Gene for coding porcine circovirus type 2Cap protein and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107337718A CN107337718A (en) | 2017-11-10 |
| CN107337718B true CN107337718B (en) | 2021-06-01 |
Family
ID=60220688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710466551.0A Active CN107337718B (en) | 2017-06-20 | 2017-06-20 | Gene for coding porcine circovirus type 2Cap protein and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN107337718B (en) |
| AU (1) | AU2017101605A4 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182603A (en) * | 2018-09-30 | 2019-01-11 | 广西壮族自治区兽医研究所 | A kind of the multiple PCR detection primer group and kit of quick differentiation PCV2, PCV3 and Mhp |
| CN109810179B (en) * | 2019-02-21 | 2021-08-17 | 成都天邦生物制品有限公司 | Separated nucleic acid molecule and method for preparing porcine circovirus type 2 Cap protein |
| PE20211141A1 (en) * | 2019-12-19 | 2021-06-25 | Farm Veterinarios S A C | SALMONELLA ENTERITIDIS RECOMBINANTE AND ITS USE AS A SWINE VACCINE |
| CN110974951B (en) * | 2019-12-31 | 2022-12-30 | 山东省农业科学院畜牧兽医研究所 | Bivalent inactivated vaccine and preparation method thereof |
| CN111560386A (en) * | 2020-03-13 | 2020-08-21 | 山东省农业科学院畜牧兽医研究所 | Soluble porcine circovirus type 2 Cap protein and application thereof |
| CN112011556B (en) * | 2020-08-06 | 2022-03-08 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine circovirus 2b and 2d type bivalent virus-like particle vaccine as well as preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103205A (en) * | 2013-03-01 | 2013-05-15 | 江苏省农业科学院 | Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene |
| CN105087624A (en) * | 2015-08-18 | 2015-11-25 | 肇庆大华农生物药品有限公司 | Expression vector of Cap protein of porcine circovirus (PCV) 2 as well as construction method and application thereof |
| CN105087605A (en) * | 2015-09-08 | 2015-11-25 | 南京农业大学 | Gene encoding recombinant porcine circovirus type 2 Cap protein and application thereof |
-
2017
- 2017-06-20 CN CN201710466551.0A patent/CN107337718B/en active Active
- 2017-11-15 AU AU2017101605A patent/AU2017101605A4/en not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103205A (en) * | 2013-03-01 | 2013-05-15 | 江苏省农业科学院 | Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene |
| CN105087624A (en) * | 2015-08-18 | 2015-11-25 | 肇庆大华农生物药品有限公司 | Expression vector of Cap protein of porcine circovirus (PCV) 2 as well as construction method and application thereof |
| CN105087605A (en) * | 2015-09-08 | 2015-11-25 | 南京农业大学 | Gene encoding recombinant porcine circovirus type 2 Cap protein and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells;Trundova M等;《Virus Genes》;20061201;第34卷(第2期);第200页、第204页左栏最后一段 * |
| Nucleotide Sequence of Porcine Circovirus Associated with Postweaning Multisystemic Wasting Syndrome in Pigs;Hamel A L等;《Journal of Virology》;19980601;第72卷(第6期);第5262-5267页 * |
| 猪圆环病毒2型Cap蛋白原核可溶性表达及分析;陈稳等;《西北农业学报》;20130731;第22卷(第7期);第1-6页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107337718A (en) | 2017-11-10 |
| AU2017101605A4 (en) | 2017-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107337718B (en) | Gene for coding porcine circovirus type 2Cap protein and application thereof | |
| CN103739717B (en) | Recombinant protein subunit vaccine for resisting porcine circovirus serotype 2 | |
| Andrianova et al. | Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs | |
| CN108273054A (en) | Swine foot-and-mouth disease virus is O-shaped, A type Fc polypeptide bivalent vaccines and its preparation method and application | |
| CN102813918B (en) | Porcine circovirus 2 type recombinant subunit vaccine and preparation method thereof | |
| CN104004068A (en) | Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof | |
| KR102200773B1 (en) | A antigen fused with porcine Fc fragment and vaccine composition comprising the same | |
| CN114437236B (en) | Recombinant African swine fever virus multi-epitope fusion protein, preparation and application thereof | |
| CN109021115B (en) | Porcine circovirus trivalent subunit vaccine | |
| CN108503697B (en) | Zika virus subunit vaccine expressed by drosophila cells | |
| CN110974951B (en) | Bivalent inactivated vaccine and preparation method thereof | |
| JP2023526770A (en) | Recombinant spike protein of novel coronavirus (COVID-19, coronavirus infectious disease 2019) forming a trimer, method for mass production of said recombinant spike protein in plants, and method for producing vaccine composition based on this (Method for production of novel coronavirus trimeric spike protein in plants and use for vaccination) | |
| CN108610424B (en) | Recombinant protein and application thereof in preparation of porcine circovirus and porcine reproductive and respiratory syndrome virus vaccine | |
| CN108715607B (en) | Recombinant cholera toxin B subunit protein, PEDV inactivated vaccine, preparation and application | |
| CN111560386A (en) | Soluble porcine circovirus type 2 Cap protein and application thereof | |
| CN105622762B (en) | Chemotactic factor-mediated O-type foot-and-mouth disease targeting compound epitope protein for cattle and vaccine | |
| CN104497148B (en) | A kind of preparation for recombinating ubiquitination blue-ear disease vaccine | |
| CN112480270B (en) | Pseudorabies virus gB-gC epitope concatemer and application thereof | |
| CN116496362B (en) | Antigen combination of porcine rotavirus and application thereof | |
| CN114891120B (en) | Bivalent avian adenovirus specific antigen fusion protein | |
| CN101564533B (en) | Hepatitis E protein-nucleic acid composite vaccine and preparation method thereof | |
| CN112592410B (en) | Canine adenovirus gene engineering subunit vaccine, preparation method and application thereof | |
| KR102571164B1 (en) | Design for recombinant protein for influenza virus surface protein hemagglutinin, a method of mass-production of its in plants, and pharmaceutical composition for treating and preventing influenza virus containing its | |
| CN108586617B (en) | Method for improving immune response of PCV2VLPs vaccine by displaying TFlg at C terminal | |
| WO2006062266A1 (en) | Genes of a korean isolate rbiv and a vaccine against rbiv |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |