A recombinant pseudorabies virus strain expressing classical swine fever virus e2 protein and application thereof

Abstract

The application discloses a recombinant pseudorabies virus strain expressing classical swine fever virus E2 protein and application, and relates to the technical field of biology.The recombinant E2 protein provided by the application successfully realizes high-level expression of the E2 protein by removing the transmembrane region of the E2 protein and using the 18aa signal peptide of the E2 protein itself and a pig albumin signal peptide.The recombinant PRV expressing the classical swine fever virus (CSFV) E2 protein can induce E2 antibodies and cellular immunity after immunization of mice.The recombinant pseudorabies virus strain provided by the application can be used as a bivalent vaccine for preventing CSFV and PRV infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a recombinant pseudorabies virus strain expressing E2 protein of porcine fever virus and application. BACKGROUND

[0002] E2 protein is located on the surface of porcine fever virus (CSFV) envelope, participates in virus infection, is responsible for binding to receptors on cells, is the main protective antigen of CSFV, and can induce the production of neutralizing antibodies.

[0003] Pseudorabies virus (PRV) has a genome of 150 kb, and can be used for reverse genetic manipulation by bacterial artificial chromosome (BAC) technology. The virulence genes (TK, gE and gI) are knocked out to become a weak strain with good immunogenicity. In addition, the PRV genome contains many non-essential genes, such as US4, US7, US8, US9, and foreign genes can be inserted into these genes without affecting the in vitro and / or in vivo replication potential of the virus, making it a suitable carrier for expressing other foreign antigens of pig diseases ([1] Cong X, Lei JL, Xia SL, et al. 2016. Pathogenicity and immunogenicity of a gE / gI / TK gene-deleted pseudorabies virus variant in susceptible animals. Vet Microbiol, 182: 170-177. [2] Qiu HJ, Tian ZJ, Tong GZ, et al. 2005. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets. Vet Immunol Immunopathol, 106: 309-319.). The PRV vector expressing foreign proteins can infect host cells after immunization, and the genome of PRV can express foreign proteins in cells, and then the foreign proteins are secreted out of cells to induce humoral immunity and produce antibodies against foreign proteins.

[0004] The existing PRV vaccine, Bartha-K61 vaccine, only provides 50% protection against PRV variants. The C-Strain currently used to prevent swine fever is not effective against the prevalent 2.1d subgenotype. Therefore, it is necessary to develop a vaccine against the prevalent strains of pseudorabies virus (PRV) and classical swine fever virus (CSFV). There have been many reports of PRV vaccines expressing the E2 gene of swine fever, but there is currently no commercial PRV and swine fever dual genetic engineering vector vaccine. The gE / gI / TK gene-deleted vaccine is safe and can completely prevent PRV. The E2 glycoprotein of swine fever virus is mainly used to develop swine fever vaccines. Multi-vaccine, especially viral vector vaccine expressing foreign proteins, is an effective strategy against various pig diseases combined with infection. SUMMARY

[0005] Based on the deficiencies in the prior art, the present application provides a recombinant pseudorabies virus strain expressing a classical swine fever virus E2 protein and an application thereof.

[0006] The applicant found that only when the E2 transmembrane region is removed and a signal peptide is added, the E2 protein can be expressed in vitro. The signal peptide can affect the expression of the protein, and the E2 protein of CSFV carries a 33 aa leader sequence. The applicant found that there is an 18 aa E2 signal peptide through signal peptide characteristic analysis and verification. It has been reported that the porcine albumin signal peptide (Halb) is a well-characterized secretory signal peptide and can be used to express E2 protein. Accordingly, the applicant used the porcine albumin signal peptide to replace the E2 signal peptide, and the expression amount of the E2 protein was obviously improved.

[0007] The specific technical solutions of the present application are as follows:

[0008] The present application provides a classical swine fever virus recombinant E2 protein, wherein the E2 protein coding gene sequence of the classical swine fever virus removing the transmembrane region is shown as SEQ ID NO. 1 or SEQ ID NO. 6.

[0009] The present application also provides a nucleic acid molecule encoding the classical swine fever virus recombinant E2 protein.

[0010] The present application also provides biological materials related to the nucleic acid molecule, including any of the following:

[0011] (a) an expression cassette containing the nucleic acid molecule;

[0012] (b) a recombinant vector containing the nucleic acid molecule or the expression cassette in (a).

[0013] The present application also provides a bacterial artificial chromosome comprising the nucleic acid molecule.

[0014] The application further provides a recombinant pseudorabies virus strain expressing a classical swine fever virus recombinant E2 protein, wherein the nucleic acid molecule is inserted into a genome of the pseudorabies virus; and the pseudorabies virus is a PRV ZJ2013 strain.

[0015] Preferably, the TK gene, the gE gene and the 11K gene of the PRV ZJ2013 strain are knocked out; the nucleotide sequence of the TK gene is shown in SEQ ID NO. 2, the nucleotide sequence of the gE gene is shown in SEQ ID NO. 3, and the nucleotide sequence of the 11K gene is shown in SEQ ID NO. 4.

[0016] The application further provides a preparation method of the recombinant pseudorabies virus strain, comprising the following steps:

[0017] (1) replacing the gE gene and the 11K gene of pPRV-HA2-dTK with an expression cassette containing the sequence of the nucleic acid molecule to obtain pPRV-dTK / gE-E2;

[0018] (2) transfecting the pPRV-dTK / gE-E2 obtained in step (1) into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

[0019] Specifically, the expression cassette further comprises a promoter CMV, a BGH-pA human beta globulin gene and a polyadenylic acid tail.

[0020] The application further provides application of the recombinant pseudorabies virus strain in preparation of a vaccine for preventing or treating pseudorabies and classical swine fever.

[0021] The application further provides a bivalent vaccine for preventing classical swine fever and pseudorabies, comprising live viruses or inactivated viruses of the recombinant pseudorabies virus strain.

[0022] The application has the following beneficial effects:

[0023] (1) The recombinant E2 protein provided by the application successfully expresses the E2 protein by using the 18 aa signal peptide of the E2 protein itself and the signal peptide of porcine albumin.

[0024] (2) The recombinant PRV expressing the E2 protein of the classical swine fever virus (CSFV) constructed by the application can produce antibodies and specific cellular immunity against the E2 protein after immunization of mice.

[0025] (3) The recombinant pseudorabies virus strain provided by the application can be used as a bivalent vaccine for preventing CSFV and PRV infection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1A spatial structure model of the E2 protein is provided; where A represents the spatial structure of the homodimer formed by the full-length E2 protein, annotated with membrane structures. Green and red represent the two strands that make up the E2 dimer. B represents the hydrophobic and hydrophilic regions of the E2 protein. The hydrophobic regions in the E2 protein's spatial structure were analyzed and annotated using Discovery Studio software, the area of ​​the hydrophobic regions was calculated, and the amino acids involved in the formation of the hydrophobic regions were labeled. Boxes represent amino acids in the transmembrane region of the E2 protein.

[0027] Figure 2 The diagram shows the construction of recombinant PRV expressing classical swine fever virus E2. A represents the construction of pPRV-HA2-dTK and pPRV-dTK / gE-E2. The TK gene is deleted from the PRV genome and inserted into the pHA2 plasmid to obtain pPRV-HA2-dTK. The gE and 11K genes are deleted from pPRV-HA2-dTK, and an E2 expression cassette is inserted to obtain pPRV-dTK / gE-E2. B shows different expression forms of the constructed E2. The extracellular domain represents the extracellular domain 1-338 aa of the E2 protein; SP represents the 18 aa signal peptide sequence of the E2 protein itself; TM represents the transmembrane domain (339-366 aa) of the E2 protein; and the tail represents the cytoplasmic domain (367-373 aa) of the E2 protein. The extracellular region of the signal peptide, the transmembrane region of E2, and the tail represent the full-length E2 (E2FL).

[0028] Figure 3 RFLP diagram for identifying recombinant PRV expressing classical swine fever virus E2. The genomes of different recombinant strains were digested with BamHI. Arrows indicate that Groups 1-5 all successfully inserted the E2 gene. The control group was pPRV-dTK / gE.

[0029] Figure 4 For use of TCID 50 Viral titer plots were determined for the control group and Groups 1-5.

[0030] Figure 5 To determine the E2 protein expression map using the Western blot method described in Materials and Methods, GFP was used as an internal control for E2 protein. (Green fluorescent protein on the pHA2 plasmid and pHA2 insertion into the PRV genome were also included). The marker in the figure is 60 kDa.

[0031] Figure 6 The expression profile of E2 protein was determined by IFA for the control group and Groups 1-5.

[0032] Figure 7 This image shows the detection of CSFV E2 protein-specific antibodies in mice.

[0033] Figure 8 Detection of PRV gB, gE antibody in immunized mice.

[0034] Figure 9 Statistical diagram of ELISPOT of mouse CSFV E2 protein specific cellular immunity.

[0035] Figure 10 Spot diagram of ELISPOT of mouse CSFV E2 protein specific cellular immunity. DETAILED DESCRIPTION

[0036] Example 1 Construction of recombinant E2 protein

[0037] PRV ZJ2013 strain (Porcine pesudorabies virus strain ZJ2013) isolated from a pig herd in Zhejiang, China in 2013 was deposited at China Center for Type Culture Collection on January 16, 2023, and the address of the depositing unit is No. 299, Bajiyi Road, Wuchang District, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO: V202307. The TK gene (the gene sequence is shown as SEQ ID NO. 2) and gE gene (the gene sequence is shown as SEQ ID NO. 3) of PRV ZJ2013 were knocked out to obtain rPRV-dTK / gE, which was passaged in BHK-21 cell line. CSFV C-Strain was propagated in ST cells (ATCC CRL-1746). BHK-21 cells and ST cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (ZETA) and cultured at 37°C with 5% CO2. In addition, the competent cells containing pPRV-HA2-dTK and pEP-kan-S plasmids were stored in the laboratory, and the specific construction process is described in the patent with application number ZL201210100785.0 and the literature “Yin W L, Yin L B, Ye W C, et al. Construction of infectious bacterial artificial chromosome clone of porcine pseudorabies virus Zhejiang strain [J]. Virus, 2010, 26(4): 330-335.”

[0038] The monoclonal antibody 9011 against CSFV E2 protein was purchased from Beijing Jinuobaitai Biotechnology Co., Ltd. The 2B6 monoclonal antibody was a gift from Professor Fang Weihuan. CSFV C-Strain was stored in the Avian Disease Room of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences.

[0039] Modeling of E2 protein spatial structure.

[0040] The mature E2 protein of CSFV contains an N-terminal extracellular region, a transmembrane region and a C-terminal cytoplasmic region. The extracellular region of the E2 protein of CSFV was constructed by homology modeling using the Swiss model, with the bovine viral diarrhea virus type 1 E2 envelope protein (PDB ID: 4JNT) as a template. The transmembrane region of the E2 protein was simulated using the RoseTTAFold tool. The cell membrane of the transmembrane region was labeled using Discovery Studio software. The hydrophobic regions and hydrophobic scores of the spatial structure of the E2 protein were analyzed and annotated using Discovery Studio software. The hydrophobic region area was calculated, and the amino acids involved in the formation of the hydrophobic region were labeled. The secondary structure (SS) of the mutated protein sequence was predicted using Discovery Studio software.

[0041] The signal peptide affects the expression of the protein. Two signal peptides were selected for study in the present patent: one is the 18 aa E2 signal peptide, and the other is the pig albumin signal peptide (Halb).

[0042] The E2 protein can form a homodimer and has a transmembrane domain. Since the amino acid sequence similarity between 4JNT and the E2 protein reaches 80%, the 4JNT was used as a template for homology modeling using the Swiss model, and the spatial structure of the transmembrane region of the E2 protein was predicted from scratch using the Rosettafold tool. The membrane structure was labeled in Discovery Studio software, and the final appearance is shown in Figure 1 A. The transmembrane region of E2 is composed of two α-helices connected by a β-turn embedded in the membrane structure and a β-turn located on the inside of the plasma membrane (A). Figure 1 A). The amino acids corresponding to the transmembrane region are 339-366 aa. Analysis of the hydrophobic region on the surface of the E2 protein using Discovery Studio software found that the main hydrophobic region of the E2 protein, hydrophobic region 1, overlaps with the transmembrane region, with a surface area of 2 Figure 1 B).

[0043] Five experiments were designed in the present experiment to characterize the expression of the E2 protein by grouping the signal peptides and transmembrane regions of the E2 protein expressed in different forms.

[0044] Example 2 Construction of recombinant PRV inserted with E2 gene

[0045] 1. Construction of recombinant E2 expression cassette

[0046] E2 nucleotide sequence (GenBank Accession No. KT953607) was codon-optimized by Genescript. PstI and NotI enzyme cutting sites were added at both ends of the optimized sequence. After pEP-kan-S plasmid and the optimized E2 fragment (gene sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 6) were cut by PstI and NotI, the plasmid pEP-E2-kan (E2 gene is located under the human cytomegalovirus promoter, BGH polyA terminator, I-SecI homing endonuclease site and kan resistance gene are located behind the BGH polyA gene) was obtained by ligation, and the plasmid was confirmed by sequencing.

[0047] 2. Construction of recombinant PRV inserted with E2 gene

[0048] E2 expression cassette was inserted at the positions of gE and 11K genes of PRV. The detailed process of inserting E2 is shown in Figure 2 A-B. Briefly, the amplified E2 expression cassette contains about 50 bp homologous arms at both ends, and is inserted into the gE gene (gene sequence as shown in SEQ ID NO. 3) and 11K gene (gene sequence as shown in SEQ ID NO. 4) of pPRV-HA2-dTK by two-step RED-mediated recombination. The specific process is referred to the literature (Yin W, Yin L, Ye W, et al. 2010. Construction of an Infectious Clone of Pseudorabies Virus Strain ZJ Genome Maintained as a Bacterial Artificial Chromosome. Chinese Journal of Virology, 26: 330-335.).

[0049] A fragment of about 3305 bp was amplified from pEP-E2-kan as a template using primers PRV-dgE-E2-in-F and PRV-dgE-E2-in-R (Table 1), and was electroporated into pPRV-HA2-dTK-containing competent cells to obtain pPRV-dTK / gE-E2 mutants of Group 1-Group 5. A fragment of about 1144 bp was amplified from pEP-E2-kan as a template using primers PRV-dgE-F and PRV-dgE-R (Table 1), and was electroporated into pPRV-HA2-dTK-containing competent cells to obtain pPRV-dTK / gE mutants of Group 6-Group 8 (A). Figure 2 A).

[0050] Table 1 PCR primers

[0051]

[0052] BAC plasmids of pPRV-dTK / gE and pPRV-dTK / gE-E2 were extracted using alkaline lysis and transfected into BHK-21 cells using Morgan's calcium phosphate transfection method. Cells were further cultured at 37°C and 5% CO2. The virus was then rescued and named as follows: Control: rPRV-dTK / gE; Group 1: rPRV-dTK / gE-18sig / E2FL; Group 2: rPRV-dTK / gE-E2dT; Group 3: rPRV-dTK / gE-18sig / E2dT; Group 4: rPRV-dTK / gE-E2FL; Group 5: rPRV-dTK / gE-Halb / E2dT. Figure 2 B).

[0053] The genome of the recombinant virus rPRV-dTK / gE-E2 was digested with BamHI, and the insertion of the E2 gene was identified by restriction fragment length polymorphism (RFLP). PCR identification and sequencing were performed using primers PRV-E2-identify-2F and PRV-E2-identify-2R (Table 1). The results showed that five recombinant viruses were successfully constructed. Figure 3 Five recombinant viruses and the control rPRV-dTK / gE were infected into BHK-21 cells at an MOI of 0.02. Total virus was obtained from infected cells and cell culture supernatant after 72 hours and then inoculated into BHK-21 cells. Figure 3 Standard TCID is used on ) 50 The viral titer was determined experimentally. The results showed that all recombinant strains had similar titers. Figure 4 ).

[0054] ST cells were infected with rPRV-dTK / gE or rPRV-dTK / gE-E2 (MOI = 1). After 24 h, the monolayer cells were washed with pre-cooled PBS for 3 times. Then the cells were lysed in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 1% SDS, 1% β-mercaptoethanol, 5% glycerol, bromophenol blue) for 5 min. The cell lysate was centrifuged at 12,000 rpm for 10 min, and then electrophoresed on 12% SDS-polyacrylamide gel (PAGE) and electro-transferred to nitrocellulose (NC) membrane. After blocking the membrane with 10% non-fat milk, the primary antibody was anti-E2 monoclonal antibody 9011, and the secondary antibody was horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (Join Biotech, Hangzhou, China). Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Bao Biotech, Dalian, China) was used for color development. GFP was used as an internal reference. The results showed that specific bands recognized by E2 monoclonal antibody 9011 appeared in ST cells of Group 3 and Group 5, but not in the control group (rPRV-dTK / gE) and Group 1, Group 2, and Group 4 (see Fig. 2). Figure 5 It can be seen from the size of the specific bands that Group 5 and Group 3 successfully expressed E2 protein.

[0055] Indirect immunofluorescence (IFA): ST cells growing on slides were infected with rPRV-dTK / gE-E2 (MOI = 1). After 24 h, the monolayer cells were fixed with 4% PFA and permeabilized with 0.1% Triton. After incubation with anti-E2 monoclonal antibody 9011 and Cy3 anti-mouse secondary antibody (Biogen, Shanghai, China), direct observation was performed by confocal microscopy. The results showed that ST cells were positive for E2 protein immunofluorescence after infection with Group 3 and Group 5, but negative after infection with Group 1, Group 2, Group 4, and the control group (see Fig. 3). Figure 6

[0056] Example 3 Performance determination

[0057] 1. Antibody detection of mice immunized with recombinant PRV strains expressing E2 protein

[0058] Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang Academy of Agricultural Sciences (ZAAS). The animal experiments were approved by the ZAAS Animal Experiment Ethics Committee.

[0059] Twenty-four 6-week-old female Balb / c mice were randomly divided into 3 groups, with 8 mice in each group. The control group, Group 3, and Group 5 were inoculated with 10 7 TCID​50 All mice were immunized intramuscularly, and boosted with the same dose and route one week later. Blood samples were collected from the tail vein of 5 mice per group at weeks 0, 1, 2, 3, 4, 5 post-immunization for antibody detection. Spleens were collected from 3 mice per group at 2 weeks post-primary immunization for cellular immune detection.

[0060] Specific E2 antibodies in sera of immunized mice were detected by indirect ELISA. The results showed that no E2 antibodies were detected in control mice at any time post-immunization. E2 antibodies were detectable in Group 3 and Group 5 mice at 14 days post-primary immunization, and the antibody levels increased further Figure 7 ) and reached above 0.3 at day 28. There was no significant difference in antibody levels between Group 3 and Group 5 at different time points post-immunization.

[0061] PRV gB / gE antibody detection kit (IDEXX Laboratories, Inc, Westbrook, ME, USA) was used for detection, and the results showed that all groups produced antibodies against PRV gB, but not against gE Figure 8 A-B).

[0062] 2. Cellular immune detection of mice immunized with recombinant PRV strains expressing E2 protein

[0063] The sequences of 137 E2 proteins were aligned. The conserved region of E2 protein was analyzed using Gblock software, and the polypeptide was synthesized from the conserved region. The synthesis and purification of the polypeptide were completed by GenScript Biotech Co., Ltd. (Shanghai, China). The purity of the polypeptide was more than 95%, and it was stored at -80°C after lyophilization. Table 2 shows the sequence of the polypeptide.

[0064] Table 2. E2 overlapping peptide library

[0065] Polypeptide sequence (N'to C') Number of amino acids CTAVSPTTLRTEVVK 15 TFKREKPFPHRVDCATTIVEKED 23 SCKEDYRYAISSTNEIGPLGAEG 23 LTTTWREYSHSLQLDDGTVRAICTAG 26 VSRRYLASL 9 YAISSTNEI 9 SAFYLVCPI 9 CTFNYTKTL 9 TSVTFELLF 9 TWREYSHSL 9 DSYFQQYML 9 KTFKREKPF 9 LPTSVTFEL 9 GEYQYWFDL 9 RYAISSTNE 9 KPFPHRVDC 9 SPTTLRTEV 9 CTAVSPTTL 9 AGPVRKTSC 9

[0066] At 14 days post-primary immunization, 3 mice per group were sacrificed for cellular immune detection. The spleens were isolated using mouse lymphocyte isolation medium (Dako, Guangzhou, China), and the splenocytes were cultured in RPMI-1640 medium supplemented with 10% FBS.

[0067] ELISpot method: Mouse IFN-γ pre-coated ELISpot kit (Dako, Guangzhou, China) was used to detect the secretion frequency of splenocytes in each group. The splenocytes of each mouse were split into six replicates, with approximately 10 5Cell. Splenocytes were incubated at 37°C, 5% CO2 for 20h in RPMI 1640 medium, PMA, E2 overlapping peptide library (10 μg / ml) stimulation, respectively. ELISpot assay was performed. Finally, the plates were quantified using an automated ELISpot reader (Mabtech, USA). The frequency of peptide-specific T cells was expressed as the number of spots forming units (SFU) per 10 5 splenocytes.

[0068] The results showed that Group 3 and Group 5 were able to induce IFN-γ response, and no E2-specific T cells were detected in the control group (rPRV-dTK / gE) ( Figure 9 ). In addition, the results of splenocytes of each group stimulated by RPMI 1640 were negative, while the results of PMA stimulation were positive ( Figure 10 ). In summary, Group 3 and Group 5 immunization can induce E2-specific cellular immunity in mice.

[0069] The recombinant PRV strain expressing E2 protein constructed in this experiment can efficiently express E2 protein in vitro and in vivo, and can induce humoral and cellular immunity in animals, which is a promising vaccine candidate strain. Its advantages are: the deleted gene of the recombinant PRV strain expressing E2 protein contains the gE gene, and the antibody detection method can be used to distinguish wild virus infection and vaccine immunization. Compared with the attenuated vaccine of swine fever, only E2 antibody of CSFV is detected after immunization of recombinant PRV-CSFV live vector vaccine, and by detecting the antibody against other structural proteins of swine fever virus, the antibody against vaccine and wild virus can also be distinguished.

Claims

1. A recombinant pseudorabies virus strain expressing a recombinant E2 protein of a porcine pestivirus, characterized in that, In the pseudorabies virus genome, an expression cassette containing a nucleic acid molecule encoding a recombinant E2 protein of porcine pestivirus is inserted, and the sequence of the nucleic acid molecule is shown as SEQ ID NO. 1; the pseudorabies virus is a pseudorabies virus strain PRV ZJ2013 strain, the preservation number is: CCTCC NO: V202307, and the pseudorabies virus strain PRV ZJ2013 strain further knocks out TK genes, gE genes and 11K genes; the TK gene nucleotide sequence is shown as SEQ ID NO. 2, gE gene nucleotide sequence is shown as SEQ ID NO. 3, 11K gene nucleotide sequence is shown as SEQ ID NO.

4.

2. The method of producing the recombinant pseudorabies virus strain of claim 1, characterized in that, comprising the following steps: (1) Replace pPRV-HA2-dTK with an expression cassette containing the nucleic acid molecule sequence shown in SEQ ID NO.

1. gE Genes and 11K Gene, obtain pPRV-dTK / gE-E2; Wherein, TK The gene is deleted from the pseudorabies virus genome, inserted into the pHA2 plasmid to obtain pPRV-HA2-dTK; the gene is deleted from pPRV-HA2-dTK gE And 11K The gene is inserted into an expression cassette containing a nucleic acid molecule encoding a recombinant E2 protein of porcine fever virus to obtain pPRV-dTK / gE-E2; (2) Transfect the pPRV-dTK / gE-E2 obtained in step (1) into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

3. The method for preparing the recombinant pseudorabies virus strain as described in claim 2, characterized in that, The expression cassette further comprises a promoter CMV, a terminator BGH-pA human beta globin gene-poly A tail.

4. The recombinant pseudorabies virus strain of claim 1 for use in the preparation of a vaccine for preventing pseudorabies and swine fever.

5. A bivalent vaccine for the prevention of swine fever and pseudorabies, characterized in that, The live virus or inactivated virus comprising the recombinant pseudorabies virus strain of claim 1.

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