CN109182380B - Preparation method and application of baculovirus-expressed classical swine fever E2 subunit vaccine - Google Patents

Preparation method and application of baculovirus-expressed classical swine fever E2 subunit vaccine Download PDF

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CN109182380B
CN109182380B CN201810920169.7A CN201810920169A CN109182380B CN 109182380 B CN109182380 B CN 109182380B CN 201810920169 A CN201810920169 A CN 201810920169A CN 109182380 B CN109182380 B CN 109182380B
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CN109182380A (en
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徐慧玲
何放
方维焕
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Zhejiang University ZJU
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Abstract

The invention relates to the technical field of molecular biology and biological products for livestock, and aims to provide a swine fever E2 subunit vaccine expressed by baculovirus, which is swine fever SP-E2 protein containing signal peptide expressed by a baculovirus expression system, wherein the sequence of the signal peptide SP is shown as SEQ ID No.1, and the nucleic acid sequence expressing the E2 protein is shown as SEQ ID No. 2. After the SP-E2 subunit vaccine developed by the invention is used for immunizing mice, specific antibodies for producing swine fever can be generated in preference to QZ14-E2 and HZ08-E2 proteins, and the level of neutralizing antibodies is far higher than that of QZ14-E2 and HZ08-E2 proteins; after the pig is immunized, specific antibodies can be induced. The vaccine of the invention has high yield, strong immunogenicity and good safety, can distinguish infected animals from immunized animals through differential diagnosis, and can fundamentally purify the classical swine fever virus.

Description

Preparation method and application of baculovirus-expressed classical swine fever E2 subunit vaccine
Technical Field
The invention belongs to the technical field of molecular biology and biological products for livestock, and particularly relates to a preparation method and application of a baculovirus expressed classical swine fever E2 subunit vaccine.
Background
Classical Swine Fever (CSF) is a highly-contact and lethal infectious disease of pigs caused by Classical Swine Fever Virus (CSFV), is clinically characterized by acute onset, high fever retention, generalized punctate bleeding and spleen infarction, mainly harms domestic pigs and wild pigs, and causes great economic loss to the world pig industry. The prevalence trend of swine fever is greatly changed, the coexistence of typical swine fever and atypical swine fever, the concurrent recessive infection and persistent infection and the phenomenon of immune failure occur at present. This new epidemic presents new challenges to the swine industry worldwide.
At present, vaccination is still an important measure for preventing swine fever, but the traditional vaccine is an attenuated vaccine, and cannot distinguish Infected and immunized animals (differential Infected from vaccine animals, DIVA), so that the requirement for preventing swine fever cannot be met.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a preparation method and application of a baculovirus expressed classical swine fever E2 subunit vaccine.
In order to solve the technical problem, the solution of the invention is as follows:
provides a swine fever E2 subunit vaccine expressed by baculovirus, which is swine fever SP-E2 protein containing signal peptide expressed by a baculovirus expression system, wherein the sequence of the signal peptide SP is shown as SEQ ID NO.1, and the nucleic acid sequence for expressing the E2 protein is shown as SEQ ID NO. 2.
The amino acid sequence of the swine fever E2 subunit vaccine is shown in SEQ ID NO. 3.
The invention further provides application of the classical swine fever E2 subunit vaccine in preventing and treating classical swine fever virus infection.
The preparation method of the swine fever SP-E2 subunit vaccine comprises the following steps:
1) constructing shuttle plasmids containing E2 and signal peptide target genes thereof, transforming the shuttle plasmids into DH10Bac competent cells to enable the cells to generate transposition, and obtaining recombinant bacmids through blue-white spot screening;
2) transfecting the recombinant bacmid into an insect cell to obtain a baculovirus, and carrying out passage on the baculovirus on the insect cell;
3) infecting a large number of insect cells with the P3 generation virus liquid, purifying and obtaining E2 protein;
4) the SP-E2 protein and seppic 206 water adjuvant are fully emulsified to prepare the swine fever SP-E2 subunit vaccine.
Further, the specific operation steps of the method are as follows:
(1) amplification of target Gene
Primers SP-F, SP-R, E2-F and E2-R were designed according to Genbank;
firstly, obtaining SP signal peptide by amplification of a primer SP-F, SP-R, obtaining an E2 fragment by amplification of a primer E2-F, E2-R, and obtaining a target gene SP-E2 by amplification of a primer SP-F, E2-R by taking the SP signal peptide and E2 protein as templates;
the sequences of the primers SP-F, SP-R, E2-F and E2-R are shown as SEQ ID NO. 4-7;
(2) construction of shuttle plasmid
The pFastBacHTB plasmid is digested for 2h by EcoRI and XholI restriction endonuclease at 37 ℃, then recovered, the digested vector and the target gene are recombined for 30min by homologous recombinase at 37 ℃, Ecoli competent cells are transformed, the spots are picked and identified, and positive bacteria are sequenced;
(3) obtaining recombinant bacmids
Transforming a positive plasmid into a DH10Bac competent cell, selecting a white spot after 48 hours, performing PCR identification by using primers M13-F and M13-R, and extracting bacmid;
the sequences of the primers M13-F and M13-R are shown as SEQ ID NO. 8-9;
(4) transfection and baculovirus harvesting
Transfection of reagents with liposomes
Figure BDA0001764007840000021
II, transfecting SF9 cells by using the Reagent according to the Invitrogen instruction, culturing for 96h at 27 ℃, generating pathological changes in the cells, collecting P1 generation virus solution, re-infecting High Five cells with the P1 generation virus solution, culturing for 96h at 27 ℃, then collecting P2 generation virus solution, and obtaining P3 generation virus solution by using the same method;
(5) western blot identification of SP-E2
Adding P1-generation venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 96h at 27 ℃, and then carrying out Western blot identification, wherein primary antibodies are positive serum, negative serum and monoclonal antibodies of CSFV, and secondary antibodies are rabbit anti-pig secondary antibodies marked by HRP and goat anti-mouse secondary antibodies marked by FITC;
(6) IFA identification of SP-E2
Adding the P1-substituted venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 48h at 27 ℃, and then performing indirect immunofluorescence experiment, wherein primary antibody is positive serum and negative serum of CSFV and monoclonal antibody, and secondary antibody is FITC-labeled rabbit anti-pig secondary antibody and FITC-labeled goat anti-mouse secondary antibody;
(7) expression and purification of proteins
Culturing High Five cells in 250ml shake flask with 50ml culture medium until the cell density reaches 1.5X 106At each ml, cells were infected at MOI of 1, cultured at 27 ℃ at 115rpm/h for 96 hours, centrifuged at 4000rpm for 10min, the supernatant was collected, the protein was purified by a nickel column, and the purified protein was quantified and identified by Western Blot.
Description of the inventive principles:
hog cholera virus is a member of the genus pestivirus of the family flaviviridae, and comprises a large Open Reading Frame (ORF) encoding a polyprotein of 3898 amino acids that is processed by the host cell and viral autoproteases to form 4 structural proteins (C, Erns, E1, and E2) and 8 non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B). Wherein, the envelope glycoprotein E2 is a main protective antigen protein and can induce protective immunity against swine fever.
Baculovirus expression systems have been rapidly developed in recent years, and have been widely used because of their advantages such as simple operation, high safety, ability to express proteins on a suspension culture scale, post-translational modification, immunogenicity of proteins, and the like, which are similar to those of natural proteins. The classical swine fever E2 subunit vaccine prepared by using the baculovirus expression system has strong immunogenicity and good safety, and can distinguish infected animals from immunized animals through differential diagnosis and fundamentally purify classical swine fever viruses.
Because the baculovirus expression system expresses different hog cholera E2 proteins, the expression yield, the immune effect and the like of the baculovirus expression system are greatly different, so that the hog cholera E2 subunit vaccine expressed by the baculovirus expression system is not popularized yet
Compared with the prior art, the invention has the beneficial effects that:
1. after the SP-E2 subunit vaccine developed by the invention is used for immunizing mice, specific antibodies for producing swine fever can be generated in preference to QZ14-E2 and HZ08-E2 proteins, and the level of neutralizing antibodies is far higher than that of QZ14-E2 and HZ08-E2 proteins; after the pig is immunized, specific antibodies can be induced.
2. The vaccine of the invention has high yield, strong immunogenicity and good safety, can distinguish infected animals from immunized animals through differential diagnosis, and can fundamentally purify the classical swine fever virus.
Drawings
FIG. 1 is a schematic diagram of the construction of SP-E2 recombinant baculovirus.
FIG. 2 shows PCR amplification of a target gene and PCR identification of a recombinant bacmid (left panel shows amplification of a target gene, and right panel shows PCR identification of a recombinant bacmid).
FIG. 3 shows the expression results of SP-E2 protein in insect cells (SDS-PAGE and Western blot on the left).
FIG. 4 shows the result of IFA identification of SP-E2 protein in insect cells (Sf9 cells infected with Bac-SP-E248 h, swine fever standard positive serum and standard negative serum, monoclonal antibody IFA identification of SP-E2 expression in insect cells).
FIG. 5 shows the results of the purification and identification of SP-E2, E2, CSP-CE2, SP-CE2 and CSP-E2 proteins (the left graph shows the 10-fold concentration of the protein, and the right graph shows the 2-fold concentration of the protein).
FIG. 6 shows the results of neutralization titer, ELISA and IFA of serum from mice immunized with SP-E2 subunit vaccine for 0, 7, 14, 21 and 28 days. (in the figure, A represents the result of ELISA, B represents the neutralizing titer of serum, C represents the result of IFA of serum immunized for 7 days, and D represents the result of IFA of serum immunized for 14 days.)
FIG. 7 shows serum blocking ELISA results of SP-E2 subunit vaccine for swine immunization at days 0, 7, 14, 21, and 28.
Detailed Description
The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
The sequences of the primers used in the expression of the protein of the invention are shown in the following table:
Figure BDA0001764007840000041
example 1 expression of hog cholera SP-E2 protein
1) Amplification of target Gene
A primer SP-F, SP-R, E2-F, E2-R is designed according to Genbank, SP-F, SP-R is used for amplification to obtain an SP signal peptide, E2-F, E2-R is used for amplification to obtain an E2 fragment, and then SP and E2 are used as templates, and SP-F, E2-R primer is used for amplification to obtain a target gene SP-E2 (shown in figure 2).
2) Construction of shuttle plasmid
The pFastBacHTB plasmid is digested for 2 hours by EcoRI and XholI restriction enzymes at 37 ℃, and then recovered, the digested vector and the target gene are recombined for 30 minutes by homologous recombinase at 37 ℃, Ecoli competent cells are transformed, spots are picked and identified, and positive bacteria are sequenced.
3) Obtaining recombinant bacmids
The positive plasmid is transformed into DH10Bac competent cells, white spots are selected after 48h, and bacmid is extracted after PCR identification is carried out by using M13 primer (shown in figure 2).
4) Transfection and baculovirus harvesting
Transfection of reagents with liposomes
Figure BDA0001764007840000042
II Reagent transfects SF9 cells according to the Invitrogen instruction, cultures for 96h at 27 ℃ until the cells are diseased, collects virus liquid of P1 generation, re-infects High Five cells with virus liquid of P1 generation, cultures for 96h at 27 ℃ to obtain virus liquid of P2 generation, and obtains virus liquid of P3 generation by the same method.
5) Western blot identification of SP-E2
Adding the P1 generation venom into a 96-well plate of High Five cells with the density of 70-80%, infecting at 27 ℃ for 96h, and then carrying out Western blot identification, wherein primary antibodies are positive serum and negative serum of CSFV and monoclonal antibodies, secondary antibodies are rabbit anti-pig secondary antibodies marked by HRP and goat anti-mouse secondary antibodies marked by FITC, and the results are shown in figure 3.
6) IFA identification of SP-E2
Adding P1-substituted venom into 96-well plate of High Five cells with 70-80% density, and infecting at 27 deg.C for 48h
Then, an indirect immunofluorescence experiment is carried out, wherein the primary antibody is positive serum, negative serum and monoclonal antibody of CSFV, the secondary antibody is FITC-labeled rabbit-anti-pig secondary antibody and FITC-labeled goat-anti-mouse secondary antibody, and the result is shown in figure 4.
7) Expression and purification of proteins
Culturing with 50mlMedium High Five cells were cultured in 250ml shake flasks until the cell density reached 1.5X 106At each ml, cells were infected at MOI of 1, cultured at 27 ℃ at 115rpm/h for 96 hours, centrifuged at 4000rpm for 10min, the supernatant was collected, the protein was purified by a nickel column, and the purified protein was quantified and identified by Western Blot.
As shown in FIG. 5, the SP signal peptide can promote the secretory expression of the E2 protein and can increase the protein expression level of CE2 and E2.
Example 2 preparation of the subunit vaccine of swine fever SP-E2 and animal immunization experiment.
The SP-E2 protein expressed in example 1 was mixed with Seppic 206 water adjuvant at a ratio of 1: 1 at a ratio of 350rpm for 10min, and storing at 4 ℃ in a sealed and dark place.
Test example 1
16 female BALB/c mice of 6 weeks old were randomly divided into four groups, each group consisting of 4 mice, the first group subcutaneously injected with SP-E2 protein 50 μ g, the second group subcutaneously injected with HZ08-E2 protein 50 μ g, the third group subcutaneously injected with QZ14-E2 protein 50 μ g, the fourth group subcutaneously injected with PBS, and the second immunization was performed at day 14 of prime, and blood was collected at 0d, 7d, 14d, 21, and 28d after prime, respectively, by indirect ELISA, indirect immunofluorescence, and neutralization experiments of serum, as shown in FIG. 6, which indicated that the SP-E2 subunit vaccine immunized mice were able to generate swine fever specific antibodies in preference to their QZ14-E2, HZ08-E2 proteins, and that the level of neutralizing antibodies was much higher than those of QZ14-E2, HZ08-E2 proteins as shown in FIG. 6.
Test example 2
12 piglets are randomly divided into 4 groups, 3 piglets in an immunization group are divided into 3 piglets in an immunization group, the immunization doses are respectively 5 mug, 15 mug and 30 mug, 1 piglet in a control group is immunized with DMEM culture medium, blood is collected respectively in 0d, 7d, 14d, 21 and 28d of immunization, and detection is carried out by a blocking ELISA kit, as shown in figure 7, an antibody of a swine immunized with 5 mug is positive when the swine is immunized for 21d, an antibody of a swine immunized with 15 mug and 30 mug is partially positive when the swine is immunized for 14d, and all antibodies are positive when the swine is immunized for 21d, so that the swine fever SP-E2 protein subunit vaccine has good immunization effect.
The above description is only an embodiment of the present invention.
It should be noted that, for those skilled in the art, various modifications can be made without departing from the technical principle of the present invention, and these modifications are also considered to be within the scope of the present invention.
Sequence listing
<110> Zhejiang university
Preparation method and application of baculovirus-expressed classical swine fever E2 subunit vaccine
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<213> Artificial Sequence (Artificial Sequence)
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atgataaaag tattaagagg gcaggtcgtg caaggtataa tatggctgct gctggtgacc 60
ggggcacaag gg 72
<210> 2
<211> 993
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cggctgtcct gcaaggaaga ctacaggtat gcgatatcat caaccaatga gatagggccg 60
ctaggggctg aaggcctcac caccacctgg agagagtata gccatggttt gcagctggat 120
gacgggactg tcagggccat ctgcactgcg gggtccttca aagttatagc acttaatgtg 180
gtcagtagga ggtacctggc atcattacac aagagggctt tgcccacctc agtaacattt 240
gaactcctat ttgatgggac tagcccagca attgaggaga tgggagatga ctttggattt 300
gggctgtgcc cttttgacac aaccccagtg gtcaaaggga agtacaatac cactctatta 360
aacggcagtg ctttctacct agtctgccca ataggatgga cgggtgtcat agagtgcacg 420
gcagtgagcc ccacaacctt aagaacagaa gtggtgaaga ccttcaagag agagaagcct 480
ttcccacaca gagtggattg cgtgaccact atagtagaaa aagaagacct gttctactgc 540
aagtgggggg gtaattggac atgtgtgaaa ggcaacccgg tgacctacat gggggggcaa 600
gtaaaacaat gcaggtggtg cggttttgac ttcaaggagc ccgatgggct cccacactac 660
cccataggca agtgcatcct agcaaatgag acgggttaca gggtagtgga ttccacagac 720
tgcaatagag atggcgtcgt tatcagcact gaaggagaac acgagtgctt gattggtaac 780
accaccgtca aggtgcacgc gttggatgga agactgggcc ctatgccgtg cagacccaaa 840
gaaatcgtct ctagcgcggg acctgtaagg aaaacttcct gtactttcaa ttacacaaag 900
acactaagaa acaagtacta tgagcccagg gacagctatt ttcagcaata tatgcttaag 960
ggcgagtacc aatactggtt tgacctggac gtg 993
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<213> amino acid Sequence encoding hog cholera E2-SP (Artificial Sequence)
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Met Ile Lys Val Leu Arg Gly Gln Val Val Gln Gly Ile Ile Trp Leu
1 5 10 15
Leu Leu Val Thr Gly Ala Gln Gly Arg Leu Ser Cys Lys Glu Asp Tyr
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Arg Tyr Ala Ile Ser Ser Thr Asn Glu Ile Gly Pro Leu Gly Ala Glu
35 40 45
Gly Leu Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu Asp
50 55 60
Asp Gly Thr Val Arg Ala Ile Cys Thr Ala Gly Ser Phe Lys Val Ile
65 70 75 80
Ala Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Arg
85 90 95
Ala Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Ser
100 105 110
Pro Ala Ile Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro
115 120 125
Phe Asp Thr Thr Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu
130 135 140
Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val
145 150 155 160
Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val
165 170 175
Lys Thr Phe Lys Arg Glu Lys Pro Phe Pro His Arg Val Asp Cys Val
180 185 190
Thr Thr Ile Val Glu Lys Glu Asp Leu Phe Tyr Cys Lys Trp Gly Gly
195 200 205
Asn Trp Thr Cys Val Lys Gly Asn Pro Val Thr Tyr Met Gly Gly Gln
210 215 220
Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Lys Glu Pro Asp Gly
225 230 235 240
Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Gly
245 250 255
Tyr Arg Val Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile
260 265 270
Ser Thr Glu Gly Glu His Glu Cys Leu Ile Gly Asn Thr Thr Val Lys
275 280 285
Val His Ala Leu Asp Gly Arg Leu Gly Pro Met Pro Cys Arg Pro Lys
290 295 300
Glu Ile Val Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr Phe
305 310 315 320
Asn Tyr Thr Lys Thr Leu Arg Asn Lys Tyr Tyr Glu Pro Arg Asp Ser
325 330 335
Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp
340 345 350
Leu Asp Val
355
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aagcgcgcgg aattcatgat aaaagtatta agagggc 37
<210> 5
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<213> Artificial Sequence (Artificial Sequence)
<400> 5
cccttgtgcc ccggtcacca gc 22
<210> 6
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
accggggcac aagggcggct gtcctgtaag gaagact 37
<210> 7
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctcgacaagc ttctacacgt ccaggtcaaa ccagtatt 38
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tgtaaaacga cggccagt 18
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggaaacag ctatgacc 18

Claims (4)

1. A swine fever E2 subunit vaccine expressed by baculovirus is characterized in that the vaccine is swine fever SP-E2 protein containing signal peptide expressed by a baculovirus expression system, wherein the nucleotide sequence for coding the signal peptide SP is shown as SEQ ID NO.1, and the nucleotide sequence for coding the E2 protein is shown as SEQ ID NO. 2.
2. The classical swine fever E2 subunit vaccine of claim 1, wherein the amino acid sequence of the classical swine fever E2 subunit vaccine is shown in SEQ ID No. 3.
3. The method for preparing the baculovirus-expressed classical swine fever E2 subunit vaccine of claim 1, comprising the steps of:
1) constructing shuttle plasmids containing E2 and signal peptide target genes thereof, transforming the shuttle plasmids into DH10Bac competent cells to enable the cells to generate transposition, and obtaining recombinant bacmids through blue-white spot screening;
2) transfecting the recombinant bacmid into an insect cell to obtain a baculovirus, and carrying out passage on the baculovirus on the insect cell;
3) infecting a large number of insect cells with the P3 generation virus liquid, purifying and obtaining E2 protein;
4) the SP-E2 protein and seppic 206 water adjuvant are fully emulsified to prepare the swine fever SP-E2 subunit vaccine.
4. The preparation method according to claim 3, wherein the specific operation steps of the preparation method are as follows:
(1) amplification of target Gene
Primers SP-F, SP-R, E2-F and E2-R were designed according to Genbank;
firstly, obtaining SP signal peptide by amplification of a primer SP-F, SP-R, obtaining an E2 fragment by amplification of a primer E2-F, E2-R, and obtaining a target gene SP-E2 by amplification of a primer SP-F, E2-R by taking the SP signal peptide and E2 protein as templates;
the sequences of the primers SP-F, SP-R, E2-F and E2-R are shown as SEQ ID NO. 4-7;
(2) construction of shuttle plasmid
The pFastBacHTB plasmid is digested for 2h by EcoRI and XholI restriction endonuclease at 37 ℃, then recovered, the digested vector and the target gene are recombined for 30min by homologous recombinase at 37 ℃, Ecoli competent cells are transformed, the spots are picked and identified, and positive bacteria are sequenced;
(3) obtaining recombinant bacmids
Transforming the positive plasmid into DH10Bac competent cells, selecting white spots after 48h, performing PCR identification by using primers M13-F and M13-R, and extracting bacmids;
the sequences of the primers M13-F and M13-R are shown as SEQ ID NO. 8-9;
(4) transfection and baculovirus acquisition
Transfecting SF9 cells by using a liposome transfection Reagent Cellffectin II Reagent according to Invitrogen specifications, culturing the cells at 27 ℃ for 96 hours until the cells are diseased, collecting P1 generation virus liquid, re-infecting P1 generation virus liquid into High Five cells, culturing the cells at 27 ℃ for 96 hours, then harvesting P2 generation virus liquid, and obtaining P3 generation virus liquid by the same method;
(5) western blot identification of SP-E2
Adding P1-generation venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 96h at 27 ℃, and then carrying out Western blot identification, wherein primary antibodies are positive serum, negative serum and monoclonal antibodies of CSFV, and secondary antibodies are rabbit anti-pig secondary antibodies marked by HRP and goat anti-mouse secondary antibodies marked by FITC;
(6) IFA identification of SP-E2
Adding the P1-substituted venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 48h at 27 ℃, and then performing indirect immunofluorescence experiment, wherein primary antibody is positive serum and negative serum of CSFV and monoclonal antibody, and secondary antibody is FITC-labeled rabbit anti-pig secondary antibody and FITC-labeled goat anti-mouse secondary antibody;
(7) expression and purification of proteins
Culturing High Five cells in 250ml shake flask with 50ml culture medium until the cell density reaches 1.5X 106At each ml, cells were infected at MOI =1, cultured at 27 ℃ at 115rpm/h for 96h, centrifuged at 4000rpm for 10min, the supernatant was collected, the protein was purified by a nickel column, and the purified protein was quantified and identified by Western Blot.
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