CN114908056A - Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof - Google Patents

Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof Download PDF

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CN114908056A
CN114908056A CN202210547481.2A CN202210547481A CN114908056A CN 114908056 A CN114908056 A CN 114908056A CN 202210547481 A CN202210547481 A CN 202210547481A CN 114908056 A CN114908056 A CN 114908056A
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钱平
任旭皎
李祥敏
荣振香
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Huazhong Agricultural University
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Abstract

The invention provides a recombinant CHO cell line for expressing porcine pseudorabies virus gDFc or gBFc fusion protein, a construction method and application thereof, and belongs to the technical field of vaccines. The recombinant CHO cell line provided by the invention expresses PRVgDFc or gBFc and is used for preparing subunit vaccine, and after a mouse is immunized, a stronger specific immune response aiming at PRV can be generated. The recombinant CHO cell line prepared by the invention can obtain the gDFc and/or the gBFc protein antigen in a dimer form, and the prepared subunit vaccine can stimulate organisms to generate strong humoral immunity and cellular immunity reaction, has good protection effect on mice, and can be used as a promising candidate genetic engineering vaccine to prevent porcine pseudorabies.

Description

Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof
Technical Field
The invention belongs to the technical field of vaccines, and particularly relates to a recombinant CHO cell line for expressing porcine pseudorabies virus (PRV) gDFc or gBFc fusion protein, and a construction method and application thereof.
Background
Pseudorabies (PR), also known as Aujeszky's disease, is an acute febrile infectious disease in pigs caused by Pseudorabies virus (PRV) [1,2] . The main clinical features of PR in pigs are reproductive disorders, neurological and respiratory diseases [3–5] . The disease can result in significant economic losses due to reduced reproductive capacity of sows and weight loss of adult pigs following PRV infection [2] . Currently, several strategies based on DIVA (differentiating between infected and vaccinated) vaccination programs are applied to eradicate PR in commercial herds [6] . Some developed countries, e.g. the United states [3] Several European countries [7] And New Zealand [8] PR has been eradicated. Since 2011, the increased frequency and severity of infections in Chinese herds and other animals has again led to an epidemic of PR [9] . Therefore, more effective vaccines are considered as essential tools for the prevention of PRV.
PRV is a member of the Herpesviridae (Herpesviridae), sub-family α -Herpesviridae (Alphaherpesvirinae), and consists of a double stranded DNA genome of approximately 150kb, divided into a unique long region (UL) and a unique short region (US). Comprising 70 open reading frames encoding 70-100 viral proteins: 16 membrane proteins, 11 glycoproteins of gB, gC, gD, gE, gG, gH, gI, gK, gL, gM and gN [3,10] . To date, all live PRV vaccine strains have been reported to contain one or more gene deletions [11,12] . Several PRV variant-based vaccines have been reported, such as gE/gI gene-deleted PRV based on Tianjin strain, gE/gI gene-deleted PRV based on ZJ01 strain, and TK/gE/gI gene-deleted vaccine based on PR HN1201 strain [13–15] . Because PRV attenuated live vaccines still may present safety risks such as virulence reversion. Therefore, the development of a safe and effective PRV vaccine is important for the prevention and control of porcine pseudorabies.
Reference to the literature
[1]Li,X.；Zhang,W.；Liu,Y.；Xie,J.；Hu,C.；Wang,X.,Role of p53 in pseudorabies virus replication,pathogenicity,and host immune responses. Veterinary research 2019,50,(1),9.
[2]Pomeranz,L.E.；Reynolds,A.E.；Hengartner,C.J.,Molecular biology of pseudorabies virus:impact on neurovirology and veterinary medicine. Microbiology and molecularbiology reviews:MMBR 2005,69,(3),462-500.
[3]Klupp,B.G.；Hengartner,C.J.；Mettenleiter,T.C.；Enquist,L.W., Complete,annotated sequence of the pseudorabies virus genome.Journal of virology 2004,78,(1),424-40.
[4]An,T.Q.；Peng,J.M.；Tian,Z.J.；Zhao,H.Y.；Li,N.；Liu,Y.M.；Chen, J.Z.；Leng,C.L.；Sun,Y.；Chang,D.；Tong,G.Z.,Pseudorabies virus variant in Bartha-K61-vaccinated pigs,China,2012.Emerging infectious diseases 2013,19, (11),1749-55.
[5]Wu,R.；Bai,C.；Sun,J.；Chang,S.；Zhang,X.,Emergence of virulent pseudorabies virus infection in northern China.Journal of veterinary science 2013,14,(3),363-5.
[6]van Oirschot,J.T.,Diva vaccines that reduce virus transmission.Journal of biotechnology 1999,73,(2-3),195-205.
[7]Moynagh,J.,Aujeszky's disease and the European Community. Veterinary microbiology 1997,55,(1-4),159-66.
[8]Davidson,R.M.,Control and eradication of animal diseases in New Zealand.New Zealand veterinaryjournal 2002,50,(3 Suppl),6-12.
[9]Sun,Y.；Luo,Y.；Wang,C.H.；Yuan,J.；Li,N.；Song,K.；Qiu,H.J., Control of swine pseudorabies in China:Opportunities and limitations. Veterinary microbiology 2016,183,119-24.
[10]Mettenleiter,T.C.,Immunobiology of pseudorabies(Aujeszky's disease).Veterinary immunology and immunopathology 1996,54,(1-4),221-9.
[11]van Oirschot,J.T.；Kaashoek,M.J.；Rijsewijk,F.A.；Stegeman,J.A., The use of marker vaccines in eradication of herpesviruses.Journal of biotechnology 1996,44,(1-3),75-81.
[12]Vilnis,A.；Sussman,M.D.；Thacker,B.J.；Senn,M.；Maes,R.K., Vaccine genotype and route of administration affect pseudorabies field virus latency load after challenge.Veterinarymicrobiology 1998,62,(2),81-96.
[13]Wang,C.H.；Yuan,J.；Qin,H.Y.；Luo,Y.；Cong,X.；Li,Y.；Chen,J.； Li,S.；Sun,Y.；Qiu,H.J.,A novel gE-deleted pseudorabies virus(PRV)provides rapid and complete protection from lethal challenge with the PRV variant emerging in Bartha-K61-vaccinated swine population in China.Vaccine 2014,32, (27),3379-85.
[14]Zhang,C.；Guo,L.；Jia,X.；Wang,T.；Wang,J.；Sun,Z.；Wang,L.；Li, X.；Tan,F.；Tian,K.,Construction ofa triple gene-deleted Chinese Pseudorabies virus variant and its efficacy study as a vaccine candidate on suckling piglets. Vaccine 2015,33,(21),2432-7.
[15]Gu,Z.；Dong,J.；Wang,J.；Hou,C.；Sun,H.；Yang,W.；Bai,J.；Jiang, P.,A novel inactivated gE/gI deleted pseudorabies virus(PRV)vaccine completely protects pigs from an emerged variant PRV challenge.Virus research 2015,195,57-63.
[16]Li,J.；Li,X.；Ma,H.；Ren,X.；Hao,G.；Zhang,H.；Zhao,Z.；Fang,K.； Li,X.；Rong,Z.；Sun,S.；Chen,H.；Qian,P.,Efficient mucosal vaccination of a novel classical swine fever virus E2-Fc fusion protein mediated by neonatal Fc receptor.Vaccine 2020,38,(29),4574-4583.
[17]Mackness,B.C.；Jaworski,J.A.；Boudanova,E.；Park,A.；Valente,D.； Mauriac,C.；Pasquier,O.；Schmidt,T.；Kabiri,M.；Kandira,A.；
Figure BDA0003649638130000031
K.； Qiu,H.,Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulationhalf-life.mAbs 2019,11,(7),1276-1288.
Disclosure of Invention
In view of the above, the present invention aims to provide a recombinant CHO cell line expressing PRV gDFc or gfbc fusion protein and a construction method thereof, so that the expressed fusion protein prevents the virulence from returning to the original level, and the prepared subunit vaccine can stimulate the organism to generate strong humoral immunity and cellular immune response, and generate a good immune protection effect on mice, can be used as a promising candidate genetic engineering vaccine to prevent porcine pseudorabies, and has a high clinical application value.
The invention provides a construction method of a recombinant CHO cell line for expressing porcine pseudorabies virus gDFc or gBFc fusion protein, which comprises the following steps:
1) artificially synthesizing sequences of PRV gD protein and porcine IgG3Fc protein with optimized codons, and respectively cloning the gD sequence and the IgG3Fc sequence into a pUC57 vector to obtain pUC57-gD and pUC57-IgG3 Fc;
2) artificially synthesizing a codon-optimized sequence for encoding PRV gB protein, and cloning the codon-optimized sequence into a pUC57 vector to obtain pUC 57-gB;
3) performing enzyme digestion on the pUC57-gD obtained in the step 1), recovering a gD gene, and cloning the gD gene to pEE12.4 subjected to the same enzyme digestion treatment to obtain pEE12.4-gD;
4) subjecting pUC57-IgG3Fc obtained in the step 1) to enzyme digestion, recovering an IgG3Fc gene, and cloning to pEE12.4-gD subjected to the same enzyme digestion treatment to obtain pEE12.4-gDFc;
5) subjecting the pUC57-gB obtained in the step 2) to enzyme digestion, recovering a gB gene, and cloning to pEE12.4-gDFc subjected to the same enzyme digestion treatment to obtain pEE12.4-gBFc;
6) transfecting pEE12.4-gDFc in the step 4) or pEE12.4-gBFc in the step 5) to CHO-S cells, and obtaining a recombinant CHO cell line CHO-S-gDFc or CHO-S-gBFc after screening and cell cloning;
wherein there is no chronological restriction between step 1) and step 2).
Preferably, the nucleotide sequence of the codon-optimized PRV gD protein-encoding sequence in step 1) is shown as SEQ ID NO. 1;
preferably, the nucleotide sequence of the codon-optimized sequence encoding the IgG3Fc protein in step 1) is shown as SEQ ID NO. 2;
preferably, the nucleotide sequence of the codon-optimized sequence encoding PRV gB protein in step 2) is shown as SEQ ID NO. 3;
preferably, the 5 'end of the codon-optimized sequence encoding PRV gD protein of step 1) further comprises a sequence encoding an IL-2 signal peptide, and the 3' end of the codon-optimized sequence encoding IgG3Fc protein further comprises a sequence encoding 8 × His;
the nucleotide sequence of the sequence for coding the IL-2 signal peptide is shown as SEQ ID NO. 4;
the nucleotide sequence of the sequence for coding 8 XHis is shown in SEQ ID NO. 5.
Preferably, the codon-optimized PRV gB protein-encoding sequence of step 2) comprises a sequence encoding an Ig κ signal peptide;
the nucleotide sequence of the sequence for encoding the Ig kappa signal peptide is shown as SEQ ID NO. 6.
The invention provides a PRV gDFc or gBFc expressing recombinant CHO cell line prepared by the preparation method.
The invention provides a fusion protein with porcine pseudorabies virus immunogenicity, which is a gDFc fusion protein and/or a gBFc fusion protein obtained by the expression of a recombinant CHO cell line;
the amino acid sequence of the gDFc fusion protein is shown in SEQ ID NO. 7;
the amino acid sequence of the gBFc fusion protein is shown as SEQ ID NO. 8.
The invention provides an application of the recombinant CHO cell line or the fusion protein in preparation of a vaccine for preventing porcine pseudorabies.
The invention provides a vaccine for preventing porcine pseudorabies, which comprises the fusion protein and an adjuvant.
The CHO cell is one of the most important recombinant protein expression systems, the expressed protein has correct processing and modification, and the biological activity of the expressed protein is closer to that of a natural protein; after the exogenous gene is integrated, the cell is stable, and the recombinant gene can be efficiently amplified and expressed; the expressed target protein can be secreted into supernatant, and the protein can be easily purified.
The PRV gDFc or gBFc expression recombinant CHO cell line CHO-S-gDFc or CHO-S-gBFc provided by the invention can be applied to the preparation of subunit vaccine of porcine pseudorabies virus. After the mouse is immunized, the invention can generate stronger specific immune response aiming at the porcine pseudorabies virus. The fusion proteins gDFc and gBFc prepared by the method can form a stable dimer form, and the prepared subunit vaccine can stimulate organisms to generate strong humoral immunity and cellular immune response, has a good protection effect on mice, can be used as a promising candidate genetic engineering vaccine for preventing porcine pseudorabies, and has a high clinical application value.
Drawings
FIG. 1 is a schematic structural diagram of a PRVgDFc and gBFc fusion protein;
FIG. 2 is a diagram showing the double restriction enzyme identification of the transfer vectors pEE12.4-gDFc and pEE12.4-gBFc; wherein, FIG. 2A is a diagram of the result of double restriction enzyme identification of the transfer vector pEE12.4-gDFc; m: DL15000 DNA Marker, 1-3: BamHI/Sma I double enzyme digestion product; FIG. 2B is a diagram showing the results of double restriction enzyme identification of the transfer vector pEE12.4-gBFc; m: DL15000 DNAmarker, 1-3: BamHI/Sma I double enzyme digestion product;
FIG. 3 is a diagram showing Western blotting identification results after transfection of CHO-S with transfer vectors pEE12.4-gDFc and pEE12.4-gBFc; wherein, FIG. 3A is a Western blotting identification result diagram after pEE12.4-gDFc transfects CHO-S; m: protein molecular mass standard; FIG. 3B is a western blotting identification result diagram after transfection of CHO-S with pEE12.4-gBFc; m: protein molecular mass standard;
FIG. 4 shows the results of drug screening in CHO-S-gDFc and CHO-S-gBFc cell lines; wherein, FIG. 4A shows the result of drug-adding screening in CHO-S-gDFc cell line; m: protein molecular mass standard; 1-24: samples from different wells in a 24-well plate; FIG. 4B shows the results of drug-adding screening in CHO-S-gBFc cell line; m: protein molecular mass standard; 1-24: samples from different wells in a 24-well plate;
FIG. 5 is a graph showing the results of immunofluorescence assays in CHO-S-gDFc and CHO-S-gBFc cell lines;
FIG. 6 is a diagram showing the result of Western blotting identification of a CHO cell line stably expressing PRV gDFc or gBFc obtained by screening; wherein, FIG. 6A is a Western blotting identification result chart of the CHO-S-gDFc stable cell line; m: protein molecular mass standard; FIG. 6B is a Westernblotting identification result chart for the CHO-S-gBFc stable cell line; m: protein molecular mass standard;
FIG. 7 is a graph showing the results of PRV gDFc and gBFc protein purification; wherein, FIG. 7A is a SDS-PAGE identification result of purified gDFc protein; m: protein molecular mass standard; FIG. 7B is a SDS-PAGE identification of purified gBFc protein; m: protein molecular mass standard;
FIG. 8 is a representation of the PRV gDFc and gBFc fusion protein dimers; wherein, FIG. 8A is a diagram showing the identification results of denatured and non-denatured SDS-PAGE and Westernblotting of PRVgDFc fusion protein; m: protein molecular mass standard; FIG. 8B is a diagram showing the results of denatured and non-denatured SDS-PAGE and Westernblotting identification of PRV gBFc fusion proteins; m: protein molecular mass standard;
FIG. 9 shows the results of detecting the levels of PRV-specific antibodies at different time points after immunization of mice; figure 9A is gD-specific antibody levels; figure 9B is gB-specific antibody levels;
FIG. 10 shows the results of detection of PRV neutralizing antibody levels;
FIG. 11 shows the results of detecting the levels of cytokines induced by mice immunized with the vaccine;
FIG. 12 shows the survival rate of PRV GX virulent strain after challenge and immunization of mice.
Detailed Description
The invention provides a construction method of a recombinant CHO cell line for expressing porcine pseudorabies virus gDFc and/or gBFc fusion protein, which comprises the following steps:
1) artificially synthesizing sequences of PRV gD protein and porcine IgG3Fc protein with optimized codons, and respectively cloning the gD sequence and the IgG3Fc sequence into a pUC57 vector to obtain pUC57-gD and pUC57-IgG3 Fc;
2) artificially synthesizing a codon-optimized sequence for encoding PRV gB protein, and cloning the codon-optimized sequence into a pUC57 vector to obtain pUC 57-gB;
3) performing enzyme digestion on the pUC57-gD obtained in the step 1), recovering a gD gene, and cloning the gD gene to pEE12.4 subjected to the same enzyme digestion treatment to obtain pEE12.4-gD;
4) subjecting pUC57-IgG3Fc obtained in the step 1) to enzyme digestion, recovering an IgG3Fc gene, and cloning to pEE12.4-gD subjected to the same enzyme digestion treatment to obtain pEE12.4-gDFc;
5) subjecting the pUC57-gB obtained in the step 2) to enzyme digestion, recovering a gB gene, and cloning to pEE12.4-gDFc subjected to the same enzyme digestion treatment to obtain pEE12.4-gBFc;
6) transfecting pEE12.4-gDFc in the step 4) or pEE12.4-gBFc in the step 5) into CHO-S cells, and obtaining a recombinant CHO cell line CHO-S-gDFc or CHO-S-gBFc after screening and cell cloning.
The invention artificially synthesizes sequences of PRV gD protein and porcine IgG3Fc protein which are encoded by codon optimization, and clones the gD sequence and the IgG3Fc sequence into a pUC57 vector respectively to obtain pUC57-gD and pUC57-IgG3 Fc; a codon-optimized sequence encoding PRV gB protein was synthesized and ligated into pUC57 vector to obtain pUC 57-gB.
In the present invention, the nucleotide sequence of the codon-optimized PRV gD protein-encoding sequence is preferably as shown in SEQ ID NO. 1. The 5' end of the codon-optimized PRV gD protein-encoding sequence also comprises a sequence encoding an IL-2 signal peptide. The nucleotide sequence of the sequence for coding the IL-2 signal peptide is shown as SEQ ID NO. 4. The nucleotide sequence of the codon-optimized IgG3Fc protein-encoding sequence is preferably shown as SEQ ID NO. 2. The 3' end of the codon optimized sequence encoding IgG3Fc protein also contained a sequence encoding 8 × His. The nucleotide sequence of the sequence for coding 8 XHis is shown in SEQ ID NO. 5. The nucleotide sequence of the codon-optimized PRV gB protein-encoding sequence is preferably shown as SEQ ID NO. 3. The codon-optimized PRV gB protein-encoding sequence contains a sequence encoding an Ig kappa signal peptide. The nucleotide sequence of the sequence for encoding the Ig kappa signal peptide is shown as SEQ ID NO. 6. The method for artificially synthesizing the gene sequence is not particularly limited in the present invention, and a method for artificially synthesizing a gene fragment known in the art may be used.
After pUC57-gD is obtained, the invention carries out enzyme digestion treatment on pUC57-gD, recovers the gD gene and clones the gD gene to pEE12.4 which is treated by the same enzyme digestion treatment, and obtains pEE12.4-gD.
In the present invention, the pUC57-gD is preferably subjected to double digestion with BamHI and Mlu I restriction enzymes. The double digestion conditions are preferably 37 ℃ for 3 h. After the enzyme digestion, the recovered gD gene is connected with pEE12.4 after the same enzyme digestion treatment. The present invention is not particularly limited in kind of the ligase, and a ligase known in the art, for example, T4 ligase may be used. The ligation reaction conditions are preferably 16 ℃ for 6 h.
After pEE12.4-gD is obtained, the invention carries out enzyme digestion treatment on pUC57-IgG3Fc, recovers IgG3Fc gene, clones pEE12.4-gD after the same enzyme digestion treatment, and obtains pEE12.4-gDFc.
In the invention, the enzyme digestion treatment method is preferably double enzyme digestion by using Mlu I and Sma I restriction enzymes. The cloning method is preferably to link the recovered IgG3Fc gene with linear pEE12.4-gD.
After obtaining pEE12.4-gDFc, the invention carries out enzyme digestion treatment on pUC57-gB, recovers gB gene, clones pEE12.4-gDFc after the same enzyme digestion treatment, and obtains pEE12.4-gBFc.
In the present invention, the enzyme digestion treatment is preferably performed by double digestion with Mlu I and Sma I restriction enzymes. The cloning method is preferably to replace gD in pEE12.4-gDFc by recovered gB gene to obtain pEE12.4-gBFc.
After pEE12.4-gDFc or pEE12.4-gBFc is obtained, the pEE12.4-gDFc or pEE12.4-gBFc is transfected into CHO-S cells, and after screening and cell cloning, a recombinant CHO cell line CHO-S-gDFc or CHO-S-gBFc is obtained.
The method of transfection is not particularly limited in the present invention, and any transfection method known in the art may be used. In the present example, recombinant vectors were transfected into CHO-S cells in the presence of Nano Trans CHO transfection reagent.
The invention provides a PRV gDFc or gBFc expressing recombinant CHO cell line prepared by the preparation method.
The invention provides a fusion protein with porcine pseudorabies virus immunogenicity, which is a gDFc fusion protein and/or a gBFc fusion protein obtained by the expression of a recombinant CHO cell line;
the amino acid sequence of the gDFc fusion protein is shown as SEQ ID NO. 7;
the amino acid sequence of the gBFc fusion protein is shown as SEQ ID NO. 8.
The invention provides an application of the recombinant CHO cell line or the fusion protein in preparation of a vaccine for preventing porcine pseudorabies.
The invention provides a vaccine for preventing porcine pseudorabies, which comprises the fusion protein and an adjuvant.
In the vaccine, the final concentration of the fusion protein is preferably 38-42 mug/mL, and more preferably 40 mug/mL. The present invention is not particularly limited in the kind of the adjuvant, and any kind of adjuvants known in the art may be used. In the embodiment of the invention, the adjuvant takes ISA 201VG as an example to prepare the vaccine. The volume ratio of the fusion protein to the adjuvant is preferably 1:1 (omega/omicron/omega), and the mixture is emulsified.
In the invention, the neutralizing antibody against PRV can be generated by vaccine immunized animals prepared by pseudorabies virus gDFc and gBFc fusion protein alone or in combination, and the neutralizing antibody against PRV can be generated by mice more effectively improved by the vaccine prepared by gDFc or gBFc + gDFc. The survival rates of mice in the gDFc group, the gfbc + gDFc group and the inactivated vaccine group were all 100%, while the survival rate in the bfc immunization group was 80%.
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Eukaryotic transfer vectors pEE12.4, CHO-S cells, E.coli DH5 alpha competence, etc., in the following examples were maintained and provided by the national emphasis on laboratory neuropathology, the university of agriculture university, Huazhong, and various molecular biological reagents were purchased from Bioreagents Inc.
Example 1
Construction method of transfer vector pEE12.4-gDFc and pEE12.4-gBFc for expressing fusion protein of pseudorabies virus gDFc and gBFc
1. Construction method of transfer vector pEE12.4-gDFc for expressing pseudorabies virus gDFc fusion protein
(1) Synthesis of PRV gD Gene and porcine IgG3Fc sequence
Nucleotide sequences encoding pseudorabies virus gD protein (GenBank accession No.: MF940935.1) and porcine IgG3Fc protein (GenBank accession No.: AK405781.1) were artificially synthesized and codon-optimized, a sequence encoding IL-2 signal peptide was added to the 5 'end of the gD sequence (SEQ ID NO:1), a sequence encoding 8 XHis was added to the 3' end of the IgG3Fc sequence (SEQ ID NO:2), and the artificially synthesized gD sequence and the IgG3Fc sequence were ligated to pUC57 vector, respectively, to obtain 57-gD and pUC57-IgG3 Fc.
(2) Enzyme digestion ligation assay
Performing double enzyme digestion on the pUC57-gD obtained in the step (1) and the eukaryotic transfer vector pEE12.4 by using BamH I restriction enzymes and Mlu I restriction enzymes respectively, wherein the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3h, and the cleavage system is shown in Table 1.
TABLE 1 double enzyme digestion System
Figure BDA0003649638130000101
The enzyme digestion product is recovered by using a DNA gel recovery kit of Tiangen Biochemical technology (Beijing) Co., Ltd, and then the recovered product is subjected to ligation reaction. The ligation reaction conditions were: the ligation was carried out at 16 ℃ for 6h, and the ligation reaction system is shown in Table 2.
TABLE 2 ligation reaction System
Figure BDA0003649638130000102
And (3) transforming the products of the ligation reaction to DH5 alpha competent cells, culturing for 12h on an LB solid culture medium containing ampicillin, selecting positive colonies, inoculating the positive colonies in an LB liquid culture medium containing ampicillin, culturing for 12-14 h at 37 ℃ by a shaking table 180r/min, extracting plasmids, and performing double enzyme digestion identification. The plasmid was sent to Biotech limited of Ongjingkidaceae for sequencing. The correctly sequenced recombinant plasmid was designated: pEE12.4-gD.
Carrying out double digestion on pUC57-IgG3Fc obtained in the step (1) and the recombinant plasmid pEE12.4-gD by using Mlu I and Sma I restriction enzymes under the conditions that: the enzyme was cleaved at 37 ℃ for 3h, and the cleavage system is shown in Table 3.
TABLE 3 double enzyme digestion System
Figure BDA0003649638130000111
The enzyme digestion product is recovered by using a DNA gel recovery kit of Tiangen Biochemical technology (Beijing) Co., Ltd, and then the recovered product is subjected to ligation reaction. The ligation reaction conditions were: ligation was carried out at 16 ℃ for 6h, and the ligation reaction system is shown in Table 4.
TABLE 4 ligation reaction System
Figure BDA0003649638130000112
And (3) transforming the products of the ligation reaction to DH5 alpha competent cells, culturing for 12h on an LB solid culture medium containing ampicillin, selecting positive colonies, inoculating the positive colonies to an LB liquid culture medium containing ampicillin, culturing for 12-14 h at 37 ℃ by a shaker at 180r/min, extracting plasmids, and performing double enzyme digestion identification.
The result is shown in FIG. 2A, and the plasmid was sequenced by Biotech, Inc., of Okagaku, Beijing. The correctly sequenced recombinant plasmid was designated: pEE12.4-gDFc.
2. Construction method of transfer vector pEE12.4-gBFc for expressing pseudorabies virus gBFc fusion protein
(1) Synthesis of PRV gB Gene sequence
The nucleotide sequence of the codon-optimized PRV gB protein (GenBank accession No.: MK618718.1) was synthesized and ligated into pUC57 vector to obtain pUC 57-gB. The sequence of the mouse Ig kappa signal peptide is contained in the polypeptide.
(2) Enzyme digestion ligation test
Carrying out double enzyme digestion on pUC57-gB obtained in the step (1) and the recombinant transfer vector pEE12.4-gDFc obtained in the step 1 by using BamHI/Mlu I restriction enzyme under the conditions that: the enzyme was cleaved at 37 ℃ for 3h, and the cleavage system is shown in Table 5.
TABLE 5 double enzyme digestion System
Figure BDA0003649638130000121
The enzyme digestion product is recovered by using a DNA gel recovery kit of Tiangen Biochemical technology (Beijing) Co., Ltd, and then the recovered product is subjected to ligation reaction. The ligation reaction conditions were: the ligation was carried out at 16 ℃ for 6h, and the ligation reaction system is shown in Table 6.
TABLE 6 ligation reaction System
Figure BDA0003649638130000122
And (3) transforming the products of the ligation reaction to DH5 alpha competent cells, culturing for 12h on an LB solid culture medium containing ampicillin, selecting positive colonies, inoculating the positive colonies to an LB liquid culture medium containing ampicillin, culturing for 12-14 h at 37 ℃ by a shaker at 180r/min, extracting plasmids, and performing double enzyme digestion identification.
The result is shown in FIG. 2B, and the plasmid was sequenced by Biotech, Inc., of Okagaku, Beijing. The correctly sequenced recombinant plasmid was designated: pEE12.4-gBFc.
Example 2
Construction method of recombinant CHO cell line for expressing fusion protein of pseudorabies virus gDFc and gBFc
At 37 deg.C, 5% CO 2 CHO-S cells were cultured in suspension on a shaker at 140r/min using serum-free medium MCHOS1(Sino Biological Inc) containing 6mM L-Glutamine. When the cell density reaches 2X 10 6 Transfection assays were performed at cells/mL. 2mL of cells were seeded in 6-well plates using NanoTrans CHO transfection reagents were used to transfect endotoxin-depleted pEE12.4-gDFc and pEE12.4-gBFc into CHO-S cells, respectively. And after 48h, taking 40 mu L of supernatant sample for Western blotting detection.
As a result, as shown in FIG. 3, the expression of the gDFc or gBFc protein was detected in the supernatant.
After the western blotting test confirmed the expression of the target protein, recombinant cell lines were screened. The cells that were verified to be correct after transfection were seeded into 24-well plates at a cell density of 2X 10 4 cells/mL. Meanwhile, methionine sulfoxide imine (MSX) with different concentrations (0 muM, 25 muM and 50 muM) is added into a culture medium for screening positive cells, and after 72 hours, 40 muL of supernatant samples are taken for Westernblotting detection.
The results are shown in FIG. 4. The pressure screening was continued for 10 days, during which the growth state of the cells was observed. When all cells in the negative control wells died, the wells growing into cell clusters were resuspended in CHO medium containing the corresponding concentration of MSX, and then monoclonal cell selection was performed by limiting dilution in 96-well plates. After at least 1 subcloning, the cell line is gradually expanded and cultured, and then frozen in liquid nitrogen for preservation after identification. The CHO cell lines stably expressing either the gDFc or gBFc proteins were designated CHO-S-gDFc and CHO-S-gBFc, respectively.
Example 3
Identification of CHO cell line expressing fusion proteins of pseudorabies virus gDFc and gBFc
1. Immunofluorescence assay for detecting expression of gDFc and gBFc proteins
Inoculating the CHO cell line stably expressing the gDFc or gBFc protein obtained by screening into a 24-well plate, wherein the inoculation density is 2 multiplied by 10 4 cells/mL, 5% CO at 37 ℃ 2 In the incubator, the growth is allowed to adhere to the wall. When the cells grew to 30-50%, the medium was discarded and washed 3 times with PBS (pH7.4) for 5min each. Cells were fixed with pre-cooled 1:1 ═ acetone: methanol fixative for 30min at-20 ℃ and then washed 3 times with PBS for 5min each. Blocking was performed for 1h at room temperature using 2% BSA in PBS. The goat anti-mouse IgG-Fc monoclonal antibody labeled with FITC (1: 1000-fold dilution) (Abcam Co.) was incubated at 37 ℃ for 1 hour in the absence of light. PBS wash 3 times, each time for 5 min. The DAPI was added at the working concentration,the reaction was carried out at 37 ℃ for 10min, followed by 3 washes with PBS for 5min each. Observed under an inverted fluorescence microscope and photographed.
As a result, as shown in FIG. 5, green fluorescence labeled with FITC was observed in the positive cells.
2. Westernblotting detection of gDFc and gBFc protein expression
The CHO cell line which is obtained by screening and stably expresses the gDFc or gBFc protein is enlarged and cultured into a 500mL shake flask until the cell density grows to 4 multiplied by 10 6 At cells/mL, 40. mu.L of supernatant sample was subjected to Westernblotting assay. After running SDS-PAGE, the cells were transferred to a PVDF membrane, and PRV gDFc or gBFc protein expression was detected by ECL display using His-tagged murine monoclonal antibody as a primary antibody (MBL Co.), and HRP-tagged goat anti-mouse IgG (Wuhan Boston Biond) as a secondary antibody.
As shown in FIG. 6, the constructed CHO cell line secreted gdFc or gBFc to the culture supernatant.
3. SDS-PAGE detection of purified gDFc and gBFc proteins
The CHO cell line which is obtained by screening and stably expresses the gDFc or gBFc protein is enlarged and cultured into a 500mL shake flask until the cell density grows to 4 multiplied by 10 6 cell/mL, 4 ℃, 10000r/min centrifugation for 10min to collect cell supernatant, 0.22 μm filter supernatant, and nickel medium 4 ℃ combined overnight. The gDFc or gfbc protein bound to nickel media was then eluted and purified using NGC Quest 10 chromatography system (BIO-RAD, USA). After the eluted proteins were concentrated using a 30kDa size ultrafiltration tube, a 40. mu.L sample was selected for SDS-PAGE detection.
The results are shown in FIG. 7, where a specific band appears at the target size.
4. Identification of PRV gDFc and gBFc protein dimers
Taking two 40 mu L recombinant CHO cell line expression samples, adding beta-Mercaptoethanol denaturant into one part, and not adding beta-Mercaptoethanol into the other part. SDS-PAGE and Westernblotting assays were then performed with reference to the above procedure.
As shown in FIG. 8, the size of the undenatured bands of the gDFc and gBFc proteins was about 2 times that of the bands with the denaturant, indicating that proteins expressed by fusion of gD or gB with IgG3Fc form a stable dimer form by the action of disulfide bonds.
Example 4
Preparation method of PRV gDFc and gBFc subunit vaccine
The PRV gDFc and gBFc proteins were expressed and purified in large quantities as described in example 3, the proteins were quantified by BCA method, and then emulsified with ISA 201VG adjuvant at a ratio of 1:1(ω/omicron/ω) at a concentration of 40 μ g/mL. The prepared subunit vaccine is stored at 4 ℃ for later use after aseptic detection.
Example 5
Mouse immunization and challenge protection test for PRV gDFc and gBFc subunit vaccines
1. Immunization and challenge procedures
40 7-week-old BALB/c female mice were selected and randomly divided into 4 vaccine immunization groups (8 mice/group) and 1 PBS negative control group (8 mice). The different subunit vaccines are adopted for combined immunization. The hind legs were vaccinated intramuscularly at an immunization dose of 40 μ g/mouse. Group a (PBS); group B (inactivated vaccine); group C (gDFc + ISA 201 VG); group D (gBFc + ISA 201 VG); group E (gDFc + gBFc + ISA 201 VG). 2 weeks after the initial immunization, booster immunizations were performed once with the same dose of vaccine. Blood samples (anticoagulated blood and serum) are collected at 0 day, 14 days, 21 days, 28 days and 35 days after immunization respectively, ELISA is used for detecting the antibody level of the gD or gB protein in the serum, the expression conditions of relevant cytokines (IFN-gamma, IL-2, IL-4 and IL-10) are detected, and the immunogenicity of each subunit vaccine is compared. 3 weeks after the second immunization, PRV-GX virulent strain (100 LD) 50 ) Mice immunized were infected intranasally. The survival rate of the mice is recorded every day 1-14 days after the challenge.
2. Indirect ELISA detection of mouse serum antibody level based on gD or gB protein
PRV specific antibody titers in sera of mice at different time points after immunization are detected by an indirect ELISA method to evaluate whether the gDFc and gBFc subunit vaccines can effectively promote humoral immune responses of organisms. ELISA plates were coated with 2. mu.g/mL purified gD or gB protein and coated overnight at 4 ℃. 5% BSA was blocked at 37 ℃ for 1h, PBST was washed 3 times (5 min/time), and diluted serum to be tested was added to each wellmu.L (1:3200), incubated at 37 ℃ for 1h, washed 3 times with PBST, and added to each well with 100. mu.L (1:10000) of a diluted HRP-labeled goat anti-mouse IgG secondary antibody (Wuhan Antriety Biotech, Ltd.). PBST was washed 3 times, and 100. mu.L of TMB substrate developing solution (Beijing Soilebao Tech., Ltd.) was added thereto to develop color at room temperature for 20 min. 2mol/L H added into each hole 2 SO 4 The reaction was stopped with 50. mu.L of a stop solution, and OD was measured 450 The value is obtained.
The results are shown in fig. 9, with a significant increase in serum antibody levels against gD and gB proteins 28 days after immunization. And the specific antibody levels of the gDFc or gfbc immunised group alone were significantly higher than those of the other vaccine immunised groups (p < 0.05). There was no significant change in serum antibody levels 35 days after immunization. Whereas no corresponding antibody was detected in the PBS control group. The results show that the subunit vaccine prepared by the recombinant CHO cell line expression protein can effectively induce organisms to generate PRV specific antibodies. And the gDFc or the gfbc can more effectively promote the humoral immune response of the mouse compared with the inactivated vaccine.
3. PRV neutralizing antibody level detection
(1) The serum was inactivated at 56 ℃ for 30 min. Diluting the inactivated serum by 2 times with serum-free DMEM (modified eagle Medium), wherein the serum is diluted to 1:4096, and each dilution is repeated for 2 times;
(2) add 50. mu.L of virus fluid (100 TCID) per well 50 ) Uniformly mixing, incubating in an incubator at 37 ℃ for 90min, and setting virus control and cell control at the same time;
(3) after 90min, 100. mu.L of PK-15 cell suspension was added to each well, and the 96-well plate was placed at 37 ℃ with 5% CO 2 Culturing for 72h in an incubator;
(4) after 72h, the cytopathic effect was observed under a microscope, and then the neutralization titer of the serum was calculated according to the Reed-Muench method, and the maximum dilution of the serum that could completely inhibit the cytopathic effect was taken as the neutralization titer of the serum.
The results are shown in fig. 10, and all vaccine immunization groups showed neutralizing antibodies against PRV 28 days after immunization. At 35 days post-immunization, neutralizing antibody levels reached a maximum and the gDFc and gfbc + gDFc immunization groups were significantly higher than the other immunization groups (p < 0.05). Whereas no neutralizing antibodies against PRV were detected in the PBS group. The above results indicate that gDFc or gfbc + gDFc can more effectively enhance the production of neutralizing antibodies against PRV in mice.
4. Cytokine level detection
The peripheral blood lymphocytes obtained by the isolation were resuspended in RPMI 1640 medium containing 10% FBS. Adjusting cell density to 4X 10 6 cells/mL were inoculated into 24-well plates containing 10. mu.g/mL of purified gD or gB protein. After culturing for 48h at 37 ℃, cell culture supernatant is taken, the cell factors are detected by a mouse interferon ELISA kit (IFN-gamma, IL-2, IL-4 and IL-10) (Xinbo Sheng Biotech), and the concentration of different cell factors is calculated according to a standard curve.
The results are shown in FIG. 11, where all vaccine immunization groups produced higher levels of IL-2(Th1 type cytokine). The results show that PRV gDFc and gBFc subunit vaccines can induce stronger cellular immune response and are Th1 type dominant cellular immune response.
5. Survival rate of mice
To evaluate the immunoprotective effects of PRV gDFc and gBFc subunit vaccines on mice, all immunized mice were vaccinated intranasally with a virulent PRV-GX strain (100 LD) 50 ). The survival rate of the mice within 1-14 days after challenge is shown in fig. 12, and the survival rates of the gDFc, the gfbc + gDFc and the inactivated vaccine group are all 100%, while the survival rate of the gfbc immune group is 80%. The results show that the PRV gDFc and gBFc subunit vaccines are used for immunization in a single and combined immunization mode, and the PRV gDFc and gBFc subunit vaccines can generate strong protection effect on mice.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof
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gtgaataata aggacctgct gtctcctatc accaggacaa tctctaaggc cacaggccct 420
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Ala Ala Thr Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser
1 5 10 15
Leu Ala Leu Val Thr Asn Ser Val Pro Ala Pro Thr Phe Pro Pro Pro
20 25 30
Ala Tyr Pro Tyr Thr Glu Ser Cys Gln Leu Thr Leu Thr Thr Val Pro
35 40 45
Ser Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu
50 55 60
Asp Pro Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg
65 70 75 80
Leu Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His
85 90 95
Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe
100 105 110
Ile Glu Tyr Thr Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg
115 120 125
Arg Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe
130 135 140
Pro Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe
145 150 155 160
Asn Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile
165 170 175
Leu Thr Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe
180 185 190
Ala Arg Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser
195 200 205
Asp Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro
210 215 220
Phe Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg
225 230 235 240
Lys Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Ala Pro Gly
245 250 255
Val Ser Arg His Arg Gly Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly
260 265 270
Gly Gly Ser Gly Ser Asp Ile Glu Pro Pro Thr Pro Ile Cys Pro Glu
275 280 285
Ile Cys Ser Cys Pro Ala Ala Glu Val Leu Gly Ala Pro Ser Val Phe
290 295 300
Leu Phe Pro Pro Lys Pro Lys Asp Ile Leu Met Ile Ser Arg Thr Pro
305 310 315 320
Lys Val Thr Cys Val Val Val Asp Val Ser Gln Glu Glu Ala Glu Val
325 330 335
Gln Phe Ser Trp Tyr Val Asp Gly Val Gln Leu Tyr Thr Ala Gln Thr
340 345 350
Arg Pro Met Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
355 360 365
Leu Pro Ile Gln His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys
370 375 380
Lys Val Asn Asn Lys Asp Leu Leu Ser Pro Ile Thr Arg Thr Ile Ser
385 390 395 400
Lys Ala Thr Gly Pro Ser Arg Val Pro Gln Val Tyr Thr Leu Pro Pro
405 410 415
Ala Trp Glu Glu Leu Ser Lys Ser Lys Val Ser Ile Thr Cys Leu Val
420 425 430
Thr Gly Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly
435 440 445
Gln Gln Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln Gln Asp
450 455 460
Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val Asp Lys Val
465 470 475 480
Arg Trp Gln Arg Gly Asp Leu Phe Gln Cys Ala Val Met His Glu Ala
485 490 495
Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Lys Thr Gln Gly Lys
500 505 510
Gly Ser Gly Ser His His His His His His His His
515 520
<210> 8
<211> 546
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ala Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu
1 5 10 15
Trp Val Pro Gly Ser Thr Gly His His His His His His Ala Ala Val
20 25 30
Thr Arg Ala Ala Ser Ala Ser Pro Ala Pro Gly Thr Gly Ala Thr Pro
35 40 45
Asp Gly Phe Ser Ala Glu Glu Ser Leu Glu Glu Ile Asp Gly Ala Val
50 55 60
Ser Pro Gly Pro Ser Asp Ala Pro Asp Gly Glu Tyr Gly Asp Leu Asp
65 70 75 80
Ala Arg Thr Ala Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr
85 90 95
Val Cys Pro Pro Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu
100 105 110
Gln Ala Cys Pro Glu Tyr Ser Gln Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asn Asp Met Leu Ser Arg Ile Ala Ala Ala Trp Cys Glu Leu
130 135 140
Gln Asn Lys Asp Arg Thr Leu Trp Gly Glu Met Ser Arg Leu Asn Pro
145 150 155 160
Ser Ala Val Ala Thr Ala Ala Leu Gly Gln Arg Val Ser Ala Arg Met
165 170 175
Leu Gly Asp Val Met Ala Ile Ser Arg Cys Val Glu Val Arg Gly Gly
180 185 190
Val Tyr Val Gln Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys
195 200 205
Tyr Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile
210 215 220
Glu Gly Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Ser Arg Asp Leu
225 230 235 240
Ile Glu Pro Cys Thr Gly Asn His Arg Arg Tyr Phe Lys Leu Gly Gly
245 250 255
Gly Tyr Val Tyr Tyr Glu Asp Tyr Ser Tyr Val Arg Met Val Glu Val
260 265 270
Pro Glu Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Thr Pro Gly Gly
275 280 285
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Asp Ile Glu Pro Pro
290 295 300
Thr Pro Ile Cys Pro Glu Ile Cys Ser Cys Pro Ala Ala Glu Val Leu
305 310 315 320
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Ile Leu
325 330 335
Met Ile Ser Arg Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser
340 345 350
Gln Glu Glu Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Gln
355 360 365
Leu Tyr Thr Ala Gln Thr Arg Pro Met Glu Glu Gln Phe Asn Ser Thr
370 375 380
Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys
385 390 395 400
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Leu Ser Pro
405 410 415
Ile Thr Arg Thr Ile Ser Lys Ala Thr Gly Pro Ser Arg Val Pro Gln
420 425 430
Val Tyr Thr Leu Pro Pro Ala Trp Glu Glu Leu Ser Lys Ser Lys Val
435 440 445
Ser Ile Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro Asp Ile Asp Val
450 455 460
Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Gly Asn Tyr Arg Thr
465 470 475 480
Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys
485 490 495
Leu Ala Val Asp Lys Val Arg Trp Gln Arg Gly Asp Leu Phe Gln Cys
500 505 510
Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile
515 520 525
Ser Lys Thr Gln Gly Lys Gly Ser Gly Ser His His His His His His
530 535 540
His His
545

Claims (10)

1. A construction method of a recombinant CHO cell line for expressing porcine pseudorabies virus gDFc or gBFc fusion protein is characterized by comprising the following steps:
1) artificially synthesizing sequences of PRV gD protein and porcine IgG3Fc protein with optimized codons, and respectively connecting the gD sequence and the IgG3Fc sequence to a pUC57 vector to obtain pUC57-gD and pUC57-IgG3 Fc;
2) artificially synthesizing a codon-optimized sequence for encoding PRV gB protein, and connecting the codon-optimized sequence to a pUC57 vector to obtain pUC 57-gB;
3) performing enzyme digestion on the pUC57-gD obtained in the step 1), recovering a gD gene, and cloning the gD gene to pEE12.4 subjected to the same enzyme digestion treatment to obtain pEE12.4-gD;
4) subjecting the pUC57-IgG3Fc obtained in the step 1) to enzyme digestion, recovering an IgG3Fc gene, and cloning to pEE12.4-gD subjected to the same enzyme digestion treatment to obtain pEE12.4-gDFc;
5) subjecting the pUC57-gB obtained in the step 2) to enzyme digestion, recovering a gB gene, and cloning to pEE12.4-gDFc subjected to the same enzyme digestion treatment to obtain pEE12.4-gBFc;
6) transfecting pEE12.4-gDFc in the step 4) or pEE12.4-gBFc in the step 5) to CHO-S cells, and obtaining a recombinant CHO cell line CHO-S-gDFc or CHO-S-gBFc after screening and cell cloning;
wherein, there is no time sequence limit between step 1) and step 2).
2. The method for constructing according to claim 1, wherein the nucleotide sequence of the codon-optimized PRV gD protein-encoding sequence in step 1) is shown as SEQ ID NO. 1.
3. The method for constructing a recombinant human immunodeficiency virus (IgG) protein according to claim 1, wherein the nucleotide sequence of the codon-optimized IgG3Fc protein encoding sequence in step 1) is shown as SEQ ID NO. 2.
4. The method for constructing according to claim 1, wherein the nucleotide sequence of the codon-optimized PRV gB protein-encoding sequence in step 2) is shown as SEQ ID NO. 3.
5. The method of claim 1, wherein the codon-optimized sequence encoding PRV gD protein of step 1) further comprises, at its 5' end, a sequence encoding an IL-2 signal peptide,
the 3' end of the codon-optimized sequence encoding IgG3Fc protein further comprises a sequence encoding 8 × His;
the nucleotide sequence of the sequence for coding the IL-2 signal peptide is shown as SEQ ID NO. 4;
the nucleotide sequence of the sequence for coding 8 XHis is shown in SEQ ID NO. 5.
6. The method of claim 1, wherein the codon-optimized PRV gB protein-encoding sequence of step 2) comprises a sequence encoding an Ig κ signal peptide;
the nucleotide sequence of the sequence for encoding the Ig kappa signal peptide is shown as SEQ ID NO. 6.
7. A PRV gDFc-or gBFc-expressing recombinant CHO cell line produced by the production method according to any one of claims 1 to 6.
8. A fusion protein with porcine pseudorabies virus immunogenicity is characterized in that the gDFc fusion protein and/or the gBFc fusion protein is expressed by the recombinant CHO cell line of claim 7;
the amino acid sequence of the gDFc fusion protein is shown as SEQ ID NO. 7;
the amino acid sequence of the gBFc fusion protein is shown as SEQ ID NO. 8.
9. Use of the recombinant CHO cell line according to claim 7 or the fusion protein according to claim 8 for the preparation of a vaccine for the prevention of porcine pseudorabies.
10. A vaccine for preventing porcine pseudorabies, comprising the fusion protein of claim 8 and an adjuvant.
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