CN114480439A - Porcine parvovirus VP2 protein gene and application thereof - Google Patents

Porcine parvovirus VP2 protein gene and application thereof Download PDF

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CN114480439A
CN114480439A CN202210170184.0A CN202210170184A CN114480439A CN 114480439 A CN114480439 A CN 114480439A CN 202210170184 A CN202210170184 A CN 202210170184A CN 114480439 A CN114480439 A CN 114480439A
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recombinant
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porcine parvovirus
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张冬冬
岳丰雄
刘汉平
李姝蕊
林艳
李刚
项聪英
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Chengdu Shiji Biopharmaceutical Co ltd
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Abstract

The invention relates to a porcine parvovirus VP2 protein gene and application thereof. The vaccine prepared by emulsifying the recombinant expressed porcine parvovirus structural protein and an adjuvant has the advantages of good safety, high antibody titer, high immune efficiency and long immune duration, can achieve an ideal immune effect only by single immunization, and has practical application value.

Description

Porcine parvovirus VP2 protein gene and application thereof
Technical Field
The invention belongs to the technical field of gene recombinant vaccines, and particularly relates to a porcine parvovirus VP2 protein gene
Background
Porcine Parvovirus (PPV) is extremely resistant and highly contagious, and is one of the pathogens that cause reproductive disorders in sows, and is clinically characterized mainly by abortion, fetal death, and mummification in pregnant sows. The disease is distributed globally at present, is particularly harmful to pregnant sows and piglets, obstructs the industrialization process of pig breeding and causes huge economic loss. PPV is a small non-enveloped virus, the virus particle is hexagonal or circular in appearance, icosahedral three-dimensional symmetry is realized, the diameter is 18-26nm, the genome is single-strand negative strand DNA, and the total length is about 5000 bp. The VP2 protein is the main capsid protein of PPV, accounts for about 80% of total capsid protein of PPV, is composed of 579 amino acids, has a molecular mass of 64KU, carries main antigenic determinant, has the ability of self-assembling into virus-like particles (VLP), has good reactogenicity, and can induce animal organism to generate immune response.
The past development of PPV vaccines goes through the development process from attenuated vaccines to inactivated vaccines, although the use of the vaccines effectively controls the clinical PPV infection, the attenuated vaccines have the risk of virulence reversion or recombination, and meanwhile, the inactivated vaccines also have the risk of virus dispersion caused by incomplete virus inactivation, and the development of efficient and safe novel PPV vaccines under the condition has important significance for effectively preventing and controlling the PPV. VLPs vaccines are formed by self-assembly of viral structural proteins, do not contain viral nucleic acids, can mediate efficient humoral and cellular immune responses, and are currently considered safe and effective vaccine candidates. However, no research report on the generation of recombinant porcine parvovirus VP2 protein vaccine with sufficient protective efficacy for immunized animals exists at present.
Disclosure of Invention
The invention aims to provide a porcine parvovirus VP2 protein gene fragment, and the nucleotide sequence of the porcine parvovirus VP2 protein gene fragment is shown as SEQ ID NO. 1.
The invention also provides a recombinant vector which comprises a nucleotide sequence shown as SEQ ID NO. 1.
Further, it is a plasmid vector connected with the nucleotide sequence shown as SEQ ID NO. 1; the plasmid vector is a pFastBacDual plasmid.
The invention also provides a recombinant cell for expressing the porcine parvovirus VP2 protein gene, which is any one of Escherichia coli, yeast, insect, plant or mammalian cells comprising the recombinant vector.
The invention also provides a recombinant porcine parvovirus VP2 protein, which is expressed by the recombinant cell and has an amino acid sequence shown in SEQ ID NO. 2.
The invention also provides a preparation method of the recombinant porcine parvovirus VP2 protein, which comprises the following steps:
1) connecting the porcine parvovirus VP2 protein gene fragment with an expression vector subjected to double enzyme digestion, introducing into escherichia coli, and extracting a recombinant baculovirus gene-rod particle with a nucleotide sequence shown as SEQ ID NO. 1;
2) transfecting the recombinant baculovirus gene-bacmid obtained in the step 1) into insect cells for virus packaging and recombinant protein expression, and collecting supernatant to obtain the recombinant baculovirus gene-bacmid.
Further, the expression vector in the step 1) is a pFastBacDual plasmid vector; the introduced escherichia coli is introduced escherichia coli TOP10 competent cells and/or escherichia coli DH10Bac competent cells; and/or, the insect cells transfected in the step 2) are Sf9 cells and/or High Five cells.
Furthermore, the introduced escherichia coli is introduced into an escherichia coli TOP10 competent cell, an expression donor plasmid is extracted, and then the expression donor plasmid is introduced into an escherichia coli DH10Bac competent cell; the insect cells are transfected into Sf9 cells firstly, virus packaging and virus reproduction are carried out, and then recombinant baculovirus is transfected into High Five cells for recombinant protein expression.
The invention relates to a recombinant porcine parvovirus VP2 protein vaccine, which is prepared by inactivating the recombinant porcine parvovirus VP2 protein with diethylene imine and then adding 201 adjuvant for emulsification.
Further, the concentration of the diethylene imine is 0.2% w/w, the inactivation temperature is 32 ℃, the time is 96 hours, and 0.02% w/w sodium thiosulfate is added after inactivation is finished.
Further, the mass ratio of the recombinant porcine parvovirus VP2 protein to the adjuvant is 1: 1; the stirring speed of the emulsification is 500rpm/min, and the time is 10 minutes.
The invention provides a porcine parvovirus VP2 protein gene, and a vaccine prepared by emulsifying a recombinant porcine parvovirus structural protein expressed by the gene with an adjuvant has the advantages of high safety, high antibody titer, high immune effect and long immune duration, can achieve an ideal immune effect by only one-time immunization, and has practical application value.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples.
Drawings
FIG. 1VP2 gene fragment
FIG. 2 construction of parvovirus VP donor plasmid pFastPV-VP 2
FIG. 3 construction principle of recombinant baculovirus
FIG. 4P1 recombinant baculovirus inoculation sf9 day 4 cell status
FIG. 5 recombinant baculovirus inoculation HighFive supernatant on day 6 HA Titers assay
FIG. 6 SDS-PAGE gel electrophoresis of PPV-VP2
FIG. 7 SEM photograph of PPV-VP2 VLP
Detailed Description
Example 1 recombinant porcine parvovirus VP2 protein vaccine study
1. Gene association
1) Chemically synthesized porcine parvovirus VP2 protein sequence
Synthesizing a corresponding codon-optimized gene sequence according to a GeneBank parvovirus VP2 protein sequence (AY686602.1), wherein the codon-optimized gene sequence is shown as SEQ ID NO. 1:
ATGTCAGAAAACGTAGAACAGCATAACCCAATTAATGCCGGTACGGAGTTGTCGGCTACCGGAAACGAATCTGGAGGGGGTGGTGGCGGCGGCGGCGGTAGAGGAGCGGGCGGCGTAGGAGTCTCTACAGGAAGTTTTAACAATCAGACTGAGTTCCAGTATCTCGGCGAGGGTCTAGTACGAATTACAGCGCACGCGAGTCGGCTGATTCATTTAAATATGCCGGAGCACGAGACGTATAAGAGGATACACGTGCTTAATTCTGAAAGTGGAGTGGCAGGCCAAATGGTACAGGACGATGCACATACGCAAATGGTTACACCCTGGTCACTAATTGACGCAAACGCGTGGGGGGTCTGGTTTAATCCTGCAGATTGGCAATTGATATCGAACAACATGACAGAAATAAATCTTGTCTCGTTCGAACAGGAGATTTTTAACGTTGTCCTTAAGACTATAACAGAATCAGCTACCTCGCCCCCCACGAAAATATACAATAATGATTTAACGGCCAGTCTGATGGTGGCCTTAGACACGAACAATACTTTACCCTACACCCCTGCTGCGCCGCGGAGCGAAACCCTCGGTTTTTATCCGTGGCTGCCGACGAAACCAACGCAGTACCGCTATTATCTATCATGTACCCGGAACCTAAATCCACCTACTTACACTGGGCAGAGTCAACAGATTACAGATTCTATACAAACCGGCCTCCACAGCGACATCATGTTCTATACAATTGAGAATGCCGTGCCAATACATCTCCTGCGTACGGGGGATGAGTTTAGTACCGGAATTTACCACTTCGATACGAAGCCTCTTAAATTGACGCATTCCTGGCAGACCAACAGATCCCTCGGTCTTCCGCCCAAATTGCTGACTGAGCCCACGACAGAGGGGGATCAACACCCAGGGACGCTACCAGCTGCAAACACACGGAAGGGCTATCATCAAACTATCAATAACTCTTATACAGAAGCAACAGCTATACGTCCCGCCCAAGTTGGCTACAATACACCGAACATGAACTTTGAATACTCCAATGGTGGACCTTTCCTGACGCCGATCGTTCCCACTGCAGATACACAGTACAACGATGATGAACCAAATGGAGCCATCAGGTTCACTATGGATTATCAGCACGGACATTTAACTACAAGCAGCCAAGAGCTAGAGCGCTATACTTTTAACCCCCAATCGAAATGTGGAAGAGCTCCCAAACAGCAATTCAACCAACAAGCGCCTTTAAACTTGGAAAATACTAATAATGGTACCTTGCTTCCGTCCGACCCGATCGGAGGTAAACCTAATATGCATTTCATGAACACCTTAAATACGTACGGGCCGTTGACTGCGCTTAACAATACGGCCCCTGTTTTTCCCAACGGTCAGATCTGGGACAAAGAGCTGGACACCGACCTAAAGCCACGACTCCATGTCACGGCTCCTTTCGTGTGCAAAAACAATCCACCAGGGCAACTCTTCGTAAAGATTGCGCCTAACCTCACCGACGATTTTAACGCCGACTCCCCGCAACAACCGCGTATCATCACTTACAGTAATTTCTGGTGGAAGGGGACGTTGACCTTTACCGCTAAGATGCGCAGCTCCAATATGTGGAATCCCATCCAGCAGCACACTACTACAGCA GAAAACATTG GTAATTACATCCCTACCAACATAGGGGGTATTAAAATGTTTCCAGAATATTCTCAGCTTATTCCTAGGAAGTTATATTGA
2) PCR amplification of the PPV VP2 Gene
PPV-VP2 gene sequence was amplified by PCR, electrophoresed in 1% agarose, and DNA sequence was purified by gel recovery kit. The amplification primer sequences are as follows:
F(SEQ ID NO.3)：
CACCATCGGGCGCGGATCCATGTCAGAAAACGTAGAACAGC
R(SEQ ID NO.4):
TAGTACTTCTCGACAAGCTTTCAATATAACTTCCTAGGAATAA
sample addition system (50 μ l):
Figure BDA0003517122130000041
PCR amplification procedure:
Figure BDA0003517122130000042
and (3) purifying and recovering PCR products:
1. preparing 1% agarose gel (containing dye), adding PCR product sample into the sample adding hole, and performing electrophoresis at constant voltage of 100V for 15 min; the results are shown in figure 1 of the drawings,
2. cutting the target DNA strip on a gel cutting instrument, putting the cut DNA strip into a clean EP tube, and purifying and recovering DNA fragments according to a DNA gel recovery box of Tiangen biochemical technology;
3) ligation of PCR products with Donor plasmid vectors
The original pFastBacDual plasmid vector is cut by BamHI and HindIII, and the cutting system is as follows:
50. mu.l of digestion system:
40μl pFastBacDual(100ng/μl)
2.5μl BamHⅠ
2.5μl HindⅢ
5μl CutSmart buffer
enzyme digestion is carried out for 3 hours at 37 ℃, then 6X DNA sample loading buffer solution is added for carrying out 1 percent gel electrophoresis, electrophoresis is carried out for 15min at constant pressure of 100V, then DNA fragments are purified and recovered, the method is the same as the above,
the ligation is carried out by an easy Geno rapid recombinant cloning kit, and the ligation system is as follows:
Figure BDA0003517122130000051
incubation was carried out at 50 ℃ for 15min, then placed on ice and transformed into E.coli TOP10 competent cells. The conversion steps are as follows:
taking 50 μ L of Escherichia coli competent cells, adding appropriate amount of plasmid (volume not more than 4 μ L), ice-cooling for 30min, heat-shocking at 42 deg.C for 90s, immediately returning to ice, and ice-cooling for 2 min; adding 400 μ L LB culture medium, and culturing at 37 deg.C with shaking table for 45-60 min; 50-100. mu.L of the culture broth was applied to LB solid medium containing ampicillin (100. mu.g/mL) and cultured overnight at 37 ℃ in an inverted state.
4) Screening, sequencing and confirmation
Picking up a plurality of single colonies from a culture dish, culturing the single colonies in 5mL LB culture medium overnight, extracting donor plasmids through a plasmid extraction kit, carrying out enzyme digestion for identification, and finally sending the donor plasmids to a sequencing company for sequencing to confirm that the sequence is correct. The constructed protein sequence is shown as SEQ ID NO. 2:
MSENVEQHNPINAGTELSATGNESGGGGGGGGGRGAGGVGVSTGSFNNQTEFQYLGEGLVRITAHASRLIHLNMPEHETYKRIHVLNSESGVAGQMVQDDAHTQMVTPWSLIDANAWGVWFNPADWQLISNNMTEINLVSFEQEIFNVVLKTITESATSPPTKIYNNDLTASLMVALDTNNTLPYTPAAPRSETLGFYPWLPTKPTQYRYYLSCTRNLNPPTYTGQSQQITDSIQTGLHSDIMFYTIENAVPIHLLRTGDEFSTGIYHFDTKPLKLTHSWQTNRSLGLPPKLLTEPTTEGDQHPGTLPAANTRKGYHQTINNSYTEATAIRPAQVGYNTPNMNFEYSNGGPFLTPIVPTADTQYNDDEPNGAIRFTMDYQHGHLTTSSQELERYTFNPQSKCGRAPKQQFNQQAPLNLENTNNGTLLPSDPIGGKPNMHFMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPFVCKNNPPGQLFVKIAPNLTDDFNADSPQQPRIITYSNFWWKGTLTFTAKMRSSNMWNPIQQHTTTA ENIGNYIPTN IGGIKMFPEY SQLIPRKLY
5) transferred to DH10Bac competent cells
The expression donor plasmid pFastPV-VP 2 (figure 2) with correct sequencing is transformed into DH10Bac competent cells, white plaque screening is carried out, a PCR method is used for identifying positive colonies, and a genome is extracted, namely the recombinant baculovirus gene-bacmid.
The constructed donor plasmid pFastPPV-VP2 was transformed into TOP10 competent cells with the aim of using TOP10 as a cloning host strain for plasmid pFastPPV-VP2 for plasmid production (in small or large quantities).
Plasmid pFastPV-VP 2 is extracted from TOP10 clone strain and transformed into DH10Bac competent cells, and foreign genes (VP2) are inserted into a shuttle vector Bacmid capable of being proliferated in escherichia coli by utilizing site-specific transposition, so that recombinant Bacmid (namely recombinant virus genome) transformants are constructed. The construction principle is shown in the following (fig. 3) by culturing single bacterial plaque (positive transformant), extracting to obtain recombinant bacmid (recombinant virus genome), and then transfecting insect cells to carry out recombinant virus packaging.
6) Transfection of cells for viral packaging
Recombinant bacmid was transferred into sf9 cells using the transfection reagent cellfectin (invitrogen) to obtain P1 recombinant baculovirus, the harvested virus was inoculated with sf9, a clear change in sf9 cells was observed after 96h, demonstrating successful packaging of the baculovirus, designated BacPPV-VP 2. sf9 cytopathic effect is shown in FIG. 4
2. Antigen expression and identification
1) HA and SDS-PAGE identification
High Five cells are commonly used for recombinant protein expression using baculovirus expression vector systems, and secreted protein expression levels can be much higher than Sf9 cells. The VP2 optimized sequence is optimized based on codon preference of High Five cells, and is more suitable for secreting and expressing the target VP2 protein in the High Five cells. Therefore, the prepared recombinant baculovirus is inoculated to the HighFive cell for target antigen expression.
The cell density of the HighFive is 2X 106Cells/ml, MOI 0.2 PFU/cell, 125ml shake flask placed 27 deg.C (120 rpm) environment for antigen expression. After 6 days, the cell viability rate is reduced to below 10%, the supernatant is harvested, and the HA titer of VP2 is 1: 217-18The expression level of the antigen was satisfactory (FIG. 5), and the objective band of SDS-PAGE was evident (FIG. 6).
2) Electron microscope detection and identification
The prepared protein sample solution was pipetted 10. mu.l onto a copper mesh with a support membrane, left for 2 minutes, and then excess liquid was pipetted off the edge of the bead with filter paper, followed by addition of 2% uranium acetate, staining for 1 minute, and then staining solution was pipetted off with filter paper, dried, and then observed in an environmental transmission electron microscope H-9500Hitachi (FIG. 7).
3. Preparation of PPV-VP2 subunit inactivated vaccine
The supernatant harvested on the 6 th day of the inoculation of the recombinant baculovirus on the HighFive is inactivated by adding 0.2% (w/w) of diethylene imine (BEI) at 32 ℃ for 96 hours, and the inactivation is stopped by adding 0.02% (w/w) of sodium thiosulfate. And then, a commercial ISA201 adjuvant is adopted, and the porcine parvovirus VP2 subunit inactivated vaccine is obtained through emulsification. The specific emulsification method comprises the following steps: respectively heating the two phases to 31 ℃ by using a water bath kettle, placing a container containing the adjuvant under a stirrer, and slowly adding the antigen liquid into the container containing the adjuvant, wherein the mass ratio of the adjuvant to the antigen is 1: stirring speed of 500rpm/min for 10 min.
4. Safety and efficacy test of porcine parvovirus subunit inactivated vaccine
1) Safety inspection
10 mice weighing 18-22g were injected with 0.2ml of vaccine (containing 0.1 head) subcutaneously, and the animals were continuously observed for 14 days, and the animals in the immunized group had no abnormal appetite and smooth back hair, which was consistent with the animals in the control group.
2) Efficacy test
A weight of 350-. After 28 days, serum was collected and separated together with 3 control guinea pigs under the same conditions, and HI antibody titer was measured. The test results show that the HI antibody titer of the control guinea pigs is not higher than 1: 8, the HI antibody titers of the immunized guinea pigs were not lower than 1: 64 (animals were fully protected against virulent porcine parvovirus challenge) (Table 1).
According to the standard regulation of the efficacy test of the porcine parvovirus inactivated vaccine in the third part of the 2020 edition animal pharmacopoeia: the immunized guinea pig HI titer should be no less than 1: 64. (HI titer is more than or equal to 1: 64 can completely protect animals against the virulent attack of porcine parvovirus, which means that the serum can inhibit the antigen 100% when diluted 64 times). The HI antibody titer of the vaccine disclosed by the invention can reach 1: 4096 (the antigen can still be inhibited by 100% after being diluted by 4096 times in serum), which is far higher than a qualified line and has good immunogenicity.
TABLE 1 porcine parvovirus subunit inactivated vaccine efficacy test results
Figure BDA0003517122130000071
Figure BDA0003517122130000081
3) Minimum immune dose test
The harvested antigen solution was diluted with sterile PBS to HA titer 1: 212(64-fold dilution), 1: 214(32-fold dilution) followed by inactivation of the emulsion-vaccinated guinea pigs. The results show that the HI antibody titer is not lower than 1 after the guinea pig is immunized by the diluted vaccine for 28 days: 64 (table 2). The invention sets the vaccine preparation standard of the porcine parvovirus VP2 subunit inactivated vaccine antigen liquid as HA titer 1: 212。
TABLE 2 porcine parvovirus subunit inactivated vaccine minimal immunization dose results
Figure BDA0003517122130000082
Figure BDA0003517122130000091
4) Duration of immunization test
The porcine parvovirus VP2 subunit inactivated vaccine qualified in safety test is used for immunizing healthy guinea pigs, and antibody detection is carried out on the immunized guinea pigs at different time, and test results show that 28 days after single immunization, the serum HI antibody level reaches the peak value, the antibody peak is within 2-6 months, the antibody level gradually decreases within 6-9 months, and the HI antibody level at 9 th month is 1: around 64, thus the duration of the immunization was defined as 6 months.
In conclusion, the invention provides the recombinant porcine parvovirus structural protein expressed by the porcine parvovirus VP2 protein gene with the nucleotide sequence shown as SEQ ID NO.1, and the vaccine prepared by emulsifying the recombinant porcine parvovirus structural protein with the adjuvant has the advantages of good safety, high antibody titer, high immune efficiency and long immune duration, can achieve ideal immune effect only by single immunization, and has practical application value.
SEQUENCE LISTING
<110> Chengdu Smith biopharmaceutical Limited
<120> porcine parvovirus VP2 protein gene and application thereof
<130> GY768-2021P0114482CCR3
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1740
<212> DNA
<213> Artificial sequence
<400> 1
atgtcagaaa acgtagaaca gcataaccca attaatgccg gtacggagtt gtcggctacc 60
ggaaacgaat ctggaggggg tggtggcggc ggcggcggta gaggagcggg cggcgtagga 120
gtctctacag gaagttttaa caatcagact gagttccagt atctcggcga gggtctagta 180
cgaattacag cgcacgcgag tcggctgatt catttaaata tgccggagca cgagacgtat 240
aagaggatac acgtgcttaa ttctgaaagt ggagtggcag gccaaatggt acaggacgat 300
gcacatacgc aaatggttac accctggtca ctaattgacg caaacgcgtg gggggtctgg 360
tttaatcctg cagattggca attgatatcg aacaacatga cagaaataaa tcttgtctcg 420
ttcgaacagg agatttttaa cgttgtcctt aagactataa cagaatcagc tacctcgccc 480
cccacgaaaa tatacaataa tgatttaacg gccagtctga tggtggcctt agacacgaac 540
aatactttac cctacacccc tgctgcgccg cggagcgaaa ccctcggttt ttatccgtgg 600
ctgccgacga aaccaacgca gtaccgctat tatctatcat gtacccggaa cctaaatcca 660
cctacttaca ctgggcagag tcaacagatt acagattcta tacaaaccgg cctccacagc 720
gacatcatgt tctatacaat tgagaatgcc gtgccaatac atctcctgcg tacgggggat 780
gagtttagta ccggaattta ccacttcgat acgaagcctc ttaaattgac gcattcctgg 840
cagaccaaca gatccctcgg tcttccgccc aaattgctga ctgagcccac gacagagggg 900
gatcaacacc cagggacgct accagctgca aacacacgga agggctatca tcaaactatc 960
aataactctt atacagaagc aacagctata cgtcccgccc aagttggcta caatacaccg 1020
aacatgaact ttgaatactc caatggtgga cctttcctga cgccgatcgt tcccactgca 1080
gatacacagt acaacgatga tgaaccaaat ggagccatca ggttcactat ggattatcag 1140
cacggacatt taactacaag cagccaagag ctagagcgct atacttttaa cccccaatcg 1200
aaatgtggaa gagctcccaa acagcaattc aaccaacaag cgcctttaaa cttggaaaat 1260
actaataatg gtaccttgct tccgtccgac ccgatcggag gtaaacctaa tatgcatttc 1320
atgaacacct taaatacgta cgggccgttg actgcgctta acaatacggc ccctgttttt 1380
cccaacggtc agatctggga caaagagctg gacaccgacc taaagccacg actccatgtc 1440
acggctcctt tcgtgtgcaa aaacaatcca ccagggcaac tcttcgtaaa gattgcgcct 1500
aacctcaccg acgattttaa cgccgactcc ccgcaacaac cgcgtatcat cacttacagt 1560
aatttctggt ggaaggggac gttgaccttt accgctaaga tgcgcagctc caatatgtgg 1620
aatcccatcc agcagcacac tactacagca gaaaacattg gtaattacat ccctaccaac 1680
atagggggta ttaaaatgtt tccagaatat tctcagctta ttcctaggaa gttatattga 1740
<210> 2
<211> 579
<212> PRT
<213> Artificial sequence
<400> 2
Met Ser Glu Asn Val Glu Gln His Asn Pro Ile Asn Ala Gly Thr Glu
1 5 10 15
Leu Ser Ala Thr Gly Asn Glu Ser Gly Gly Gly Gly Gly Gly Gly Gly
20 25 30
Gly Arg Gly Ala Gly Gly Val Gly Val Ser Thr Gly Ser Phe Asn Asn
35 40 45
Gln Thr Glu Phe Gln Tyr Leu Gly Glu Gly Leu Val Arg Ile Thr Ala
50 55 60
His Ala Ser Arg Leu Ile His Leu Asn Met Pro Glu His Glu Thr Tyr
65 70 75 80
Lys Arg Ile His Val Leu Asn Ser Glu Ser Gly Val Ala Gly Gln Met
85 90 95
Val Gln Asp Asp Ala His Thr Gln Met Val Thr Pro Trp Ser Leu Ile
100 105 110
Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Ala Asp Trp Gln Leu
115 120 125
Ile Ser Asn Asn Met Thr Glu Ile Asn Leu Val Ser Phe Glu Gln Glu
130 135 140
Ile Phe Asn Val Val Leu Lys Thr Ile Thr Glu Ser Ala Thr Ser Pro
145 150 155 160
Pro Thr Lys Ile Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
165 170 175
Leu Asp Thr Asn Asn Thr Leu Pro Tyr Thr Pro Ala Ala Pro Arg Ser
180 185 190
Glu Thr Leu Gly Phe Tyr Pro Trp Leu Pro Thr Lys Pro Thr Gln Tyr
195 200 205
Arg Tyr Tyr Leu Ser Cys Thr Arg Asn Leu Asn Pro Pro Thr Tyr Thr
210 215 220
Gly Gln Ser Gln Gln Ile Thr Asp Ser Ile Gln Thr Gly Leu His Ser
225 230 235 240
Asp Ile Met Phe Tyr Thr Ile Glu Asn Ala Val Pro Ile His Leu Leu
245 250 255
Arg Thr Gly Asp Glu Phe Ser Thr Gly Ile Tyr His Phe Asp Thr Lys
260 265 270
Pro Leu Lys Leu Thr His Ser Trp Gln Thr Asn Arg Ser Leu Gly Leu
275 280 285
Pro Pro Lys Leu Leu Thr Glu Pro Thr Thr Glu Gly Asp Gln His Pro
290 295 300
Gly Thr Leu Pro Ala Ala Asn Thr Arg Lys Gly Tyr His Gln Thr Ile
305 310 315 320
Asn Asn Ser Tyr Thr Glu Ala Thr Ala Ile Arg Pro Ala Gln Val Gly
325 330 335
Tyr Asn Thr Pro Asn Met Asn Phe Glu Tyr Ser Asn Gly Gly Pro Phe
340 345 350
Leu Thr Pro Ile Val Pro Thr Ala Asp Thr Gln Tyr Asn Asp Asp Glu
355 360 365
Pro Asn Gly Ala Ile Arg Phe Thr Met Asp Tyr Gln His Gly His Leu
370 375 380
Thr Thr Ser Ser Gln Glu Leu Glu Arg Tyr Thr Phe Asn Pro Gln Ser
385 390 395 400
Lys Cys Gly Arg Ala Pro Lys Gln Gln Phe Asn Gln Gln Ala Pro Leu
405 410 415
Asn Leu Glu Asn Thr Asn Asn Gly Thr Leu Leu Pro Ser Asp Pro Ile
420 425 430
Gly Gly Lys Pro Asn Met His Phe Met Asn Thr Leu Asn Thr Tyr Gly
435 440 445
Pro Leu Thr Ala Leu Asn Asn Thr Ala Pro Val Phe Pro Asn Gly Gln
450 455 460
Ile Trp Asp Lys Glu Leu Asp Thr Asp Leu Lys Pro Arg Leu His Val
465 470 475 480
Thr Ala Pro Phe Val Cys Lys Asn Asn Pro Pro Gly Gln Leu Phe Val
485 490 495
Lys Ile Ala Pro Asn Leu Thr Asp Asp Phe Asn Ala Asp Ser Pro Gln
500 505 510
Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp Trp Lys Gly Thr Leu
515 520 525
Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met Trp Asn Pro Ile Gln
530 535 540
Gln His Thr Thr Thr Ala Glu Asn Ile Gly Asn Tyr Ile Pro Thr Asn
545 550 555 560
Ile Gly Gly Ile Lys Met Phe Pro Glu Tyr Ser Gln Leu Ile Pro Arg
565 570 575
Lys Leu Tyr
<210> 3
<211> 41
<212> DNA
<213> Artificial sequence
<400> 3
caccatcggg cgcggatcca tgtcagaaaa cgtagaacag c 41
<210> 4
<211> 43
<212> DNA
<213> Artificial sequence
<400> 4
tagtacttct cgacaagctt tcaatataac ttcctaggaa taa 43

Claims (10)

1. A porcine parvovirus VP2 protein gene fragment is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1.
2. A recombinant vector characterized by: it comprises a nucleotide sequence shown as SEQ ID NO. 1.
3. The recombinant vector according to claim 2, wherein: it is a plasmid vector connected with a nucleotide sequence shown as SEQ ID NO. 1; the plasmid vector is a pFastBacDual plasmid.
4. A recombinant cell for expressing a porcine parvovirus VP2 protein gene, which is characterized in that: it is any of E.coli, yeast, insect, plant or mammalian cells comprising the recombinant vector of claims 2 and 3.
5. A recombinant porcine parvovirus VP2 protein, which is characterized in that: it is expressed by the recombinant cell of claim 4, and the amino acid sequence of the recombinant cell is shown as SEQ ID NO. 2.
6. A method for preparing the recombinant porcine parvovirus VP2 protein of claim 5, which comprises the following steps:
1) connecting the porcine parvovirus VP2 protein gene fragment of claim 1 with an expression vector after double enzyme digestion, then introducing into escherichia coli, and extracting recombinant baculovirus gene-bacmid with a nucleotide sequence shown as SEQ ID NO. 1;
2) transfecting the recombinant baculovirus gene-bacmid obtained in the step 1) into insect cells for virus packaging and recombinant protein expression, and collecting supernatant to obtain the recombinant baculovirus gene-bacmid.
7. The method of claim 6, wherein: the expression vector in the step 1) is a pFastBacDual plasmid vector; the introduced escherichia coli is introduced escherichia coli TOP10 competent cells and/or escherichia coli DH10Bac competent cells; and/or, the insect cells transfected in the step 2) are Sf9 cells and/or High Five cells.
8. The method of claim 7, wherein: the introduced escherichia coli is introduced into an escherichia coli TOP10 competent cell, an expression donor plasmid is extracted, and then the expression donor plasmid is introduced into an escherichia coli DH10Bac competent cell; the insect cells are transfected into Sf9 cells firstly, virus packaging and virus reproduction are carried out, and then recombinant baculovirus is transfected into High Five cells for recombinant protein expression.
9. A recombinant porcine parvovirus VP2 protein vaccine is characterized in that: the vaccine is prepared by inactivating the recombinant porcine parvovirus VP2 protein of claim 5 by using diethylene imine and then adding 201 adjuvant for emulsification.
10. The recombinant porcine parvovirus VP2 protein vaccine of claim 9, wherein: the concentration of the diethylene imine is 0.2% w/w, the inactivation temperature is 32 ℃, the time is 96h, and 0.02% w/w sodium thiosulfate is added after the inactivation is finished; the mass ratio of the recombinant porcine parvovirus VP2 protein to the adjuvant is 1: 1; the stirring speed of the emulsification was 500rpm/min for 10 minutes.
CN202210170184.0A 2022-02-23 2022-02-23 Porcine parvovirus VP2 protein gene and application thereof Pending CN114480439A (en)

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