A recombinant pseudorabies virus strain expressing a full-length e2 protein of a classical swine fever virus and application thereof

Abstract

The application discloses a recombinant pseudorabies virus strain expressing a full-length E2 protein of a mutant of a classical swine fever virus and application, and relates to the technical field of biology.The recombinant full-length E2 protein provided by the application has a hydrophobicity score lower than 3.25, and has a high protein expression level; meanwhile, the transmembrane region of the E2 protein has a hydrophobicity threshold value of 3.25-3.51.The finding has guiding significance for expression of the E2 protein or other transmembrane proteins in eukaryotic cells.The application constructs a recombinant PRV expressing a classical swine fever virus (CSFV) E2 protein, and high-level E2 antibodies and cellular immunity can be induced after immunization of mice; after immunization of rabbits, the rabbits can resist lethal attack of PRV ZJ2013 and fever reaction caused by the classical swine fever.The recombinant pseudorabies virus strain provided by the application can be used as a double vaccine for preventing CSFV and PRV infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a recombinant pseudorabies virus strain expressing a full-length E2 protein of a mutant of classical swine fever virus and application. BACKGROUND

[0002] The E2 protein is located on the surface of the envelope of the classical swine fever virus (CSFV), participates in viral 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] The pseudorabies virus (PRV) has a genome of 150 kb, and can be subjected to reverse genetic manipulation by using the 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 and 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 foreign antigens of other swine 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.). After immunization, the PRV genome can express foreign proteins in host cells, and then the foreign proteins are secreted out of the cells to induce humoral immunity and produce antibodies against the foreign proteins.

[0004] The existing PRV vaccine, i.e. Bartha-K61 vaccine, only provides 50% protection against PRV variants. The C-Strain currently used for the prevention of 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 swine fever virus (CSFV). There have been many reports on PRV vaccines expressing the E2 gene of swine fever, but there is no commercialized PRV and swine fever bivalent vaccine widely used in animal husbandry. The vaccine with gE / gI / TK gene deletion is safe and can completely prevent PRV. The E2 glycoprotein of swine fever virus is mainly used for the development of swine fever vaccines. Multivalent combined vaccines, especially viral vector vaccines expressing foreign proteins, are an effective strategy against various pig diseases and combined infections. SUMMARY

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

[0006] Analysis of the E2 transmembrane region found that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating one amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant E2 (E2FL-muta3 or E2FL-muta4) could achieve high expression, and the expressed full-length mutant E2 could be located on the cell membrane. After immunizing mice with the PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4, specific cellular immunity and humoral immunity against E2 protein were produced, and the level of E2 antibody produced was significantly higher than that of the PRV vector vaccine expressing truncated E2. After immunizing rabbits with the constructed vector vaccine, the lethal challenge of virulent PRV ZJ2013 and the fever reaction caused by swine fever virus were prevented at the same time.

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

[0008] The present application provides a swine fever virus mutant full-length E2 protein, and the coding gene sequence of the swine fever virus mutant full-length E2 protein is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0009] The swine fever virus mutant full-length E2 protein comprises a signal peptide, an extracellular region of E2 protein, a transmembrane region of E2 protein, and a cytoplasmic tail of E2 protein. More specifically, the mutant part is the transmembrane region of E2 protein.

[0010] The present application also provides a nucleic acid molecule encoding the swine fever virus mutant full-length E2 protein.

[0011] The present application also provides a biological material related to the nucleic acid molecule, comprising any one of the following:

[0012] (a) an expression cassette comprising the nucleic acid molecule;

[0013] (b) a recombinant vector comprising the nucleic acid molecule or the expression cassette of (a).

[0014] The application also provides a bacterial artificial chromosome comprising the nucleic acid molecule.

[0015] The application also provides a recombinant pseudorabies virus strain expressing a mutated full-length E2 protein of classical swine fever virus, wherein the nucleic acid molecule is inserted into the genome of the pseudorabies virus; and the pseudorabies virus is a PRV ZJ2013 strain.

[0016] 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. 3, the nucleotide sequence of the gE gene is shown in SEQ ID NO. 4, and the nucleotide sequence of the 11K gene is shown in SEQ ID NO. 5.

[0017] The application also provides a preparation method of the recombinant pseudorabies virus strain, comprising the following steps:

[0018] (1) replacing the gE gene and the 11K gene of pPRV-HA2-dTK with an expression cassette comprising the nucleic acid molecule sequence to obtain pPRV-dTK / gE-E2;

[0019] (2) transfecting the pPRV-dTK / gE-E2 obtained in step (1) into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

[0020] Specifically, the expression cassette further comprises a promoter CMV, a BGH-pA human beta globin gene and a polyadenylation tail.

[0021] The application also provides application of the recombinant pseudorabies virus strain in preparation of a vaccine for preventing or treating pseudorabies and classical swine fever.

[0022] The application also provides a bivalent vaccine for preventing classical swine fever and pseudorabies, comprising live viruses or inactivated viruses of the recombinant pseudorabies virus strain.

[0023] The application has the following beneficial effects:

[0024] (1) The recombinant full-length E2 protein provided by the application has a hydrophobicity score lower than 3.25 and a high protein expression level, and the transmembrane region of the E2 protein has a hydrophobicity threshold of 3.25-3.51. This finding not only has guiding significance for expression of the E2 protein in eukaryotic cells, but also has important significance for efficient expression of other transmembrane proteins in eukaryotic cells.

[0025] (2)The application constructs a recombinant PRV expressing the E2 protein of classical swine fever virus (CSFV), which can induce E2 antibodies and specific cellular immunity against the E2 protein after immunization of mice. The immunized rabbits can prevent lethal challenge of PRV ZJ2013 and fever caused by swine fever.

[0026] (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

[0027] Figure 1 Figure 1 is a diagram of the spatial structure of E2 protein; wherein A is the spatial structure of a homodimer formed by full-length E2 protein, annotated with the structure of the membrane. Green and red represent the two strands that make up the E2 dimer. B is the hydrophobic and hydrophilic region of the E2 protein. The hydrophobic region in the spatial structure of the E2 protein is analyzed and annotated using Discovery Studio software, the area of the hydrophobic region is calculated, and the amino acids involved in the formation of the hydrophobic region are labeled. The box represents the amino acids of the transmembrane region of the E2 protein.

[0028] Figure 2 Figure 2 is a diagram of the mutated E2 transmembrane region; wherein A is a summary of various characteristics of viral envelope proteins, taking CSFV E2 protein, SARS-CoV-2 (new coronavirus) S protein, AIV (avian influenza virus) HA protein, PRV transmembrane region gG protein, and MDV (Marek's disease virus) gB protein as examples. The oval circle represents a special value. B is a reference for mutating the E2 transmembrane region with 339-366 aa amino acids of 2.1d subtype KT953607 and QEP54409. Taking KT953607 as a reference, E2FL-muta1 and E2FL-muta2 are obtained. Taking QEP54409 as a reference, E2FL-muta3, E2FL-muta4, E2FL-muta5, E2FL-muta6, and E2FL-muta7 are obtained by mutagenesis. The mutated glycine is shaded green, and the mutated alanine is shaded gray. The two blue boxes indicate that KT953607 and QEP54409 differ at 348 AA. Above each sequence is the predicted secondary structure (red column represents a-helix, blue arrow represents β-turn, and yellow arrow represents β-sheet). The hydrophobicity of each amino acid can be represented by the height of the red column. The higher the column, the brighter the color, and the more hydrophobic the amino acid. The lower the column, the darker the color, and the less hydrophobic the amino acid. The column is gray or has no column, indicating that the amino acid is hydrophilic. The number of mutated amino acids, the hydrophobicity score (HYB score), and the similarity of the secondary structure (SS) to the original sequence of each sequence are shown below. The red box represents the mutated sequence with the same secondary structure as the original sequence.

[0029] Figure 3 Figure 8. Construction map of recombinant PRV expressing CSFV E2. A, Construction map of pPRV-HA2-dTK, pPRV-dTK / gE-E2. TK gene was deleted from PRV genome and inserted into pHA2 plasmid to obtain pPRV-HA2-dTK. gE and 1 IK genes were deleted from pPRV-HA2-dTK and E2 expression cassette was inserted to obtain pPRV-dTK / gE-E2. B, Different forms of E2 expression. Extracellular means extracellular domain 1-338 AA of E2 protein, SP means signal peptide sequence of 18 aa of E2 protein itself, TM means transmembrane domain (339-366 aa) of E2 protein, * means mutated amino acid. Dash line means the same amino acid as its reference sequence. Tail means cytoplasmic domain (367-373 aa) of E2 protein. E2 full length (E2 FL) contains signal peptide, extracellular domain, transmembrane domain and tail of E2 protein.

[0030] Figure 4 Figure 9. RFLP identification of recombinant PRV expressing CSFV E2. Genomes of different recombinant strains were digested with BamHI. Arrows indicate that E2 gene was successfully inserted in Group A to Group D. Control group is pPRV-dTK / gE.

[0031] Figure 5 Figure 10. Determination of E2 protein expression by WB method as described in Materials and Methods. GFP was used as internal control of E2 protein (green fluorescent protein on pHA2 plasmid and pHA2 inserted into PRV genome). Marker in the figure is 60 kD.

[0032] Figure 6 Figure 11. Determination of E2 protein expression by IFA method.

[0033] Figure 7 Figure 12. IFA detection of E2 protein surface localization on ST cell membrane (IFA control group: recombinant PRV strain expressing E2 without transmembrane domain).

[0034] Figure 8 Figure 13. Detection of CSFV E2 protein specific antibodies in mice in vivo.

[0035] Figure 9 Figure 14. Detection of PRV gB, gE antibodies in immunized mice.

[0036] Figure 10 Figure 15. Statistical chart of CSFV E2 protein specific cellular immunity ELISPOT in mice.

[0037] Figure 11ELISPOT spot map for mouse CSFV E2 protein specific cellular immunity (PMA, E2, DMEM).

[0038] Figure 12 Fig. for rabbit challenge test; wherein, A is the timeline of immunization time and challenge time of rabbit test; B, C groups are immunized with DMEM, control group (rPRV-dTK / gE) and Group A to Group D, respectively, on the 0th day of priming and the 7th day of boosting. Each group is challenged with porcine fever virus C-Strain 10 6 TCID 50 At 35 dPV, the rabbits were challenged, and rectal temperature was measured every 6 h from 0 to 96 h after challenge (B). On day 42, the rabbits were challenged with 10 6 TCID 50 The rabbits were challenged, and the survival curve was drawn by observing for 14 days (C). DETAILED DESCRIPTION

[0039] Construction of recombinant full-length E2 protein

[0040] PRV ZJ2013 strain was isolated from a pig population in Zhejiang, China in 2013 and preserved in the China Center for Type Culture Collection on January 16, 2023, with the preservation number CCTCC NO: V202307. The TK gene (gene sequence as shown in SEQ ID NO. 3) and gE gene (gene sequence as shown in SEQ ID NO. 4) of PRV ZJ2013 were knocked out to obtain rPRV-dTK / gE, which was passaged in a 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, competent cells containing pPRV-HA2-dTK and pEP-kan-S plasmids were preserved in the laboratory, and the specific construction process is described in the patent with the 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]. Viruslogy, 2010, 26(4): 330-335.”

[0041] 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, and the CSFV C-Strain rabbit polyclonal antibody (pAb) was obtained after the rabbit was immunized with C-Strain multiple times. The CSFV C-Strain strain was preserved in the poultry disease room of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences.

[0042] The E2 protein of KT953607 of subtype 2.1d was selected for research. The spatial structure of the E2 protein was modeled.

[0043] 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 using Swiss model with 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 RoseTTAFold tool, and the cell membrane of the transmembrane region was labeled using Discovery Studio software. The hydrophobic region and the hydrophobic score of the spatial structure of the E2 protein were analyzed and annotated using Discovery Studio software, the area of the hydrophobic region 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.

[0044] The E2 protein can form a homodimer and has a transmembrane domain. Since the amino acid sequence similarity between 4JNT and E2 protein reaches 80%, 4JNT is used as a template for homology modeling using Swiss model, and the spatial structure of the transmembrane region of E2 protein is predicted from scratch using Rosettafold tool. The membrane structure is 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 inside the plasma membrane Figure 1 A). The corresponding amino acids of the transmembrane region are 339-366 aa. The hydrophobic region of the surface of the E2 protein was analyzed using Discovery Studio software, and it was found that the E2 protein has two main hydrophobic regions. One of the hydrophobic regions (hydrophobic region 1) overlaps with the transmembrane region, and the surface area is Another hydrophobic region (hydrophobic region 2) is between 294-329 aa, and the surface area is ( Figure 1 B).

[0045] It is known that E2 with a transmembrane region cannot be expressed in vitro, while E2 without a transmembrane region can be expressed in vitro. E2 belongs to envelope proteins. Comparison is made among viral envelope proteins expressed in vitro, such as SARS-CoV-2 S protein, AIV HA protein, PRV gG protein, MDV gB protein, and analysis is made on the length of the transmembrane domain, the number of continuous hydrophobic amino acids, the hydrophobic score, the charged amino acids in the transmembrane region, and the secondary structure of the transmembrane domain of the envelope proteins. Figure 2 As shown in FIG. 1A, the hydrophobic score of the E2 protein is significantly higher than that of other proteins. Therefore, the transmembrane region is a key factor limiting the expression of E2. According to the sequence of the transmembrane region of the E2 protein, amino acid mutations are made. As can be seen from the alignment of 137 E2 sequences, the transmembrane region of E2 is relatively conservative except for two amino acids V and A at 348 aa. Figure 2 B). E2FL-muta1 and E2FL-muta2 are obtained from KT953607 as a template, and E2FL-muta3, E2FL-muta4, E2FL-muta5, E2FL-muta6 and E2FL-muta7 are obtained from QEP54409 as a template. Mutated sequences Figure 2 B) shows that the hydrophobicity of the mutated amino acids is reduced, and the hydrophobic score of the mutated transmembrane sequence is also reduced to different degrees. The secondary structure of E2FL-muta3 and E2FL-muta4 is consistent with the original sequence, and the secondary structure of the remaining sequences is similar to the original sequence Figure 2 B). E2FL-muta1 and E2FL-muta2, E2FL-muta3 and E2FL-muta4 are selected for subsequent testing.

[0046] Example 2 Construction of recombinant PRV inserted with full-length E2 gene

[0047] 1. Construction of recombinant full-length E2 expression cassette

[0048] The E2 nucleotide sequence (GenBank accession number: KT953607) is codon-optimized by Genescript. PstI and NotI enzyme digestion sites are added at both ends of the optimized sequence. The pEP-kan-S plasmid and the optimized full-length E2 fragment (the gene sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2) are digested by PstI and NotI, and then ligated to obtain the plasmid pEP-E2-kan (the E2 gene is located below the human cytomegalovirus promoter, the BGH polyA terminator is in front, the I-SecI homing endonuclease site and the kan resistance gene are located behind the BGH polyA gene), and the plasmid is confirmed by sequencing.

[0049] 2. Construction of recombinant PRV inserted with full-length E2 gene

[0050] An E2 expression cassette was inserted at the gE and 11K gene sites of the PRV. For detailed instructions on E2 insertion, see [link to documentation]. Figure 3 AB. In short, the amplified E2 expression cassette contains approximately 50 bp homologous arms at both ends, and is inserted into the gE gene (gene sequence shown in SEQ ID NO.4) and 11K gene (gene sequence shown in SEQ ID NO.5) of pPRV-HA2-dTK through two-step RED-mediated recombination. For details, refer 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.).

[0051] Using pEP-E2-kan as a template, a fragment of approximately 3305 bp was amplified using primers PRV-dgE-E2-in-F and PRV-dgE-E2-in-R (Table 1). This fragment was then electroporated into competent cells containing pPRV-HA2-dTK to obtain the pPRV-dTK / gE-E2 mutants of Group A-Group D. Using pEP-E2-kan as a template, a fragment of approximately 1144 bp was amplified using primers PRV-dgE-F and PRV-dgE-R (Table 1). This fragment was then electroporated into competent cells containing pPRV-HA2-dTK to obtain the pPRV-dTK / gE mutants. Figure 3 A).

[0052] Table 1 PCR Primers

[0053]

[0054]

[0055] The BAC plasmids of pPRV-dTK / gE and pPRV-dTK / gE-E2 were extracted by alkaline lysis method, and BHK-21 cells were transfected by Morgan's calcium phosphate transfection method. The cells were further cultured at 37°C and 5% CO2. Then the viruses were rescued and named as Control: rPRV-dTK / gE, Group A: rPRV-dTK / gE-18sig / E2FL / muta1, Group B: rPRV-dTK / gE-18sig / E2FL / muta2, Group C: rPRV-dTK / gE-18sig / E2FL / muta3, Group D: rPRV-dTK / gE-18sig / E2FL / muta4 Figure 3 B).

[0056] The genome of recombinant virus rPRV-dTK / gE-E2 was digested by BamHI enzyme, and the insertion of E2 gene was identified by restriction fragment length polymorphism (RFLP) and PCR identification and sequencing with primers PRV-E2-identify-2F and PRV-E2-identify-2R (Table 1), and the results showed that four recombinant viruses were successfully constructed. Figure 4 ) The four recombinant viruses and the control rPRV-dTK / gE were used to infect BHK-21 cells at an MOI of 0.02, and the viruses were collected from the infected cells and cell culture supernatant after 72 hours, and the total viruses were obtained and used to infect BHK-21 cells by standard TCID 50 The viral titers were determined by the test. The results showed that all the recombinant strains had similar titers.

[0057] ST cells were infected with rPRV-dTK / gE or rPRV-dTK / gE-E2 (MOI = 1). After 24 h, the monolayer cells were washed 3 times with pre-cooled PBS. Then they 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 12000 rpm for 10 min, and electrophoresed on a 12% SDS-polyacrylamide gel (PAGE), and then electrotransferred to a nitrocellulose (NC) membrane. After blocking the membrane with 10% non-fat milk, the anti-E2 monoclonal antibody 9011 was used as the primary antibody, and the horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (Joint Biotech, Hangzhou, China) was used as the secondary antibody. Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Bao Biological, Dalian, China) was used for color development. GFP was used as an internal reference. The results showed that the E2 monoclonal antibody 9011 specific band appeared in the ST cells infected with Group C and Group D, but not in the control (rPRV-dTK / gE) and Group A and Group B (seeFigure 5 ).

[0058] Indirect immunofluorescence assay (IFA): ST cells grown on spread sheets were infected with rPRV-dTK / gE-E2 (MOI=1). After 24 h, monolayers of cells were fixed with 4% paraformaldehyde (PFA) and infiltrated with 0.1% Triton. After incubation with anti-E2 monoclonal antibody 9011 and Cy3 anti-mouse secondary antibody (Beyotime, Shanghai, China), cells were directly observed under a confocal microscope. The results showed that ST cells infected with Group C and Group D were positive for E2 protein immunofluorescence, while those infected with Group A, Group B, and the control group were negative (see [link to study]). Figure 6 ).

[0059] In addition, IFA observation of E2 protein on the surface of ST cells revealed that the mutant full-length E2 expressed by Group C and Group D was localized on the ST cell surface, while the E2 protein expressed by the IFA control group (recombinant PRV strain expressing transmembrane-depleted E2) (encoding gene sequence shown in SEQ ID NO. 6) could not be localized on the cell membrane. Figure 7 ).

[0060] Example 3 Performance Measurement

[0061] 1. Detection of CSFV E2-specific antibodies in immunized mice

[0062] Animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" issued by the Zhejiang Academy of Agricultural Sciences (ZAAS). The animal experiments were approved by the ZAAS Animal Experimentation Ethics Committee.

[0063] Twenty-four 6-week-old female Balb / c mice were randomly divided into three groups of eight each. The control group, Group C, and Group D were each inoculated with 10 [units of vaccine / vaccination]. 7 TCID 50 All mice were administered an intramuscular injection, followed by a booster immunization at the same dose and route one week later. Antibody levels were measured in blood samples from five mice in each group via the tail vein at weeks 0, 1, 2, 3, 4, and 5 post-immunization. Spleens from three mice in each group were collected two weeks after the initial immunization for cellular immunoassay.

[0064] Specific E2 antibodies in the serum of immunized mice were detected using indirect ELISA. Results showed that no E2 antibodies were detected in the control group mice after immunization. In Group C and Group D immunized mice, E2-specific antibodies were detectable in the serum 7 days after the initial immunization, and antibody levels increased further with prolonged immunization time. Figure 8 The antibody level reached above 0.5 by day 28. There was no significant difference in antibody levels between Group C and Group D at different time points after immunization.

[0065] 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 did not produce antibodies against gE( Figure 9 ).

[0066] 2. Cellular immunity detection of mice immunized with recombinant PRV strain expressing E2 protein

[0067] The sequences of 137 E2 proteins were aligned. The conserved regions of E2 protein were analyzed using Gblock software, and polypeptides were synthesized from the conserved regions. The synthesis and purification of polypeptides were completed by GenScript Biotech Co., Ltd. (Shanghai, China). The purity of polypeptides was more than 95%, and they were stored at -80°C after lyophilization. Table 2 shows the sequences of polypeptides.

[0068] Table 2 E2 overlapping peptide library

[0069] 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

[0070] 14 days after the first immunization, the spleens of 3 mice in each group were taken for cellular immunity detection. The spleens were separated using mouse lymphocyte separation medium (Dakewe, Guangzhou, China), and the spleen cells were cultured in RPMI-1640 medium supplemented with 10% FBS.

[0071] ELISpot method: The mouse IFN-γ pre-coated ELISpot kit (Dakewe, Guangzhou, China) was used to detect the secretion frequency of spleen cells in each group. The spleen cells of each mouse were added to each well in portions, about 10 5 cells per well, in six replicates. The spleen cells were incubated at 37°C, 5% CO2 for 20h with RPMI 1640 medium, PMA, E2 overlapping peptide library (10 μg / ml). ELISpot test was performed. Finally, the plates were quantified using an automatic ELISpot reader (Mabtech, USA). The frequency of peptide-specific T cells was expressed as the number of spot forming units (SFU) per 10 5 spleen cells.

[0072] The results showed that Group C and Group D could induce IFN-γ response, and no E2-specific T cells were detected in the control group (rPRV-dTK / gE) Figure 10 ). In addition, the results of spleen cells in each group stimulated by DMEM were negative, while the results of spleen cells stimulated by PMA were positive Figure 11 ). In summary, the mice immunized with Group C and Group D could induce E2-specific cellular immunity.

[0073] 3. Challenge protection experiment of GroupD recombinant virus on rabbits

[0074] Rabbits were immunized with recombinant PRV strains expressing full-length E2. Fifteen 2-month-old rabbits free of CSFV, PRV were confirmed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) and were used for vaccine evaluation experiment. The rabbits were randomly divided into three groups, with 5 rabbits in each group. GroupD was intramuscularly injected with 1 ml of rPRV-dTK / gE-E2 at different doses (10 7 TCID 50 ) of DMEM and rPRV-dTK / gE as controls. Seven days after the first vaccination, a booster was given. Thirty-five days after the first vaccination, each group was challenged with 10 6 TCID 50 CSFV C-Strain. Forty-two days after the first vaccination, each group was challenged with 10 6 TCID 50 PRV ZJ2013, and observed for 14 days.

[0075] The results showed that after CSFV challenge, the rabbits in GroupD did not have elevated body temperature after virus inoculation, indicating that GroupD could protect them from the fever reaction caused by C-Strain challenge ( Figure 12 A). The rectal temperatures of rabbits in the DMEM and rPRV-dTK / gE groups began to rise at 48 h, sharply rose to about 41°C, and maintained high temperature for at least 16 h, indicating that C-Strain of swine fever caused a typical fever reaction in rabbits ( Figure 12 B). After 96 h, all rabbits returned to normal temperature and could eat normally.

[0076] On the 42nd day, rabbits were challenged with 10 6 TCID 50 PRV ZJ2013 strain and observed for 14 days ( Figure 12 A). Three days after the challenge, all rabbits in the DMEM immunization group had pruritus symptoms and one died. Within 5 days after the challenge, all rabbits in the DMEM immunization group died. Rabbits in the rPRV-dTK / gE group and GroupD group had no clinical symptoms and were healthy and survived ( Figure 12 C). Therefore, the GroupD candidate vaccine strain can protect rabbits from PRV ZJ2013 strain challenge. It is proved that GroupD can protect rabbits from both lethal challenge of PRV and fever reaction caused by C-Strain of swine fever.

[0077] This study successfully constructed the recombinant PRV expressing the mutant full-length E2 protein of CSFV 2.1d subtype for the first time. The PRV expressing full-length E2(Group C: rPRV-dTK / gE-18sig / E2FL / muta3, Group D: rPRV-dTK / gE-18sig / E2FL / muta4) could induce high levels of E2 antibody and cellular immunity in immunized mice. The advantage is that the recombinant PRV expressing E2 is a live vector vaccine that lacks the gE gene, which can distinguish between wild-type infection and vaccine immunization by detecting gE antibodies. Compared with the attenuated CSFV vaccine, the recombinant PRV-CSFV live vector vaccine only detects E2 antibodies of CSFV after immunization, and by detecting antibodies to other structural proteins of CSFV, it can also distinguish between antibodies to the vaccine and the wild virus.

Claims

1. A mutated full-length E2 protein of a porcine pestivirus, characterized in that, The coding gene sequence of the mutated full-length E2 protein of the porcine fever virus is shown as SEQ ID NO. 1 or SEQ ID NO.

2.

2. A nucleic acid molecule encoding the mutated full-length E2 protein of the porcine fever virus according to claim 1.

3. Biological material associated with the nucleic acid molecule of claim 2, characterized in that, For any one of the following: (a) an expression cassette containing the nucleic acid molecule according to claim 2; (b) a recombinant vector containing the nucleic acid molecule according to claim 2 or the expression cassette in (a).

4. A bacterial artificial chromosome, characterized in that, The nucleic acid molecule according to claim 2.

5. A recombinant pseudorabies virus strain expressing a mutated full-length E2 protein of a porcine pestivirus, characterized in that, Inserting the expression cassette containing the nucleic acid molecule according to claim 2 into the genome of the pseudorabies virus; the pseudorabies virus is the PRV ZJ2013 strain, and the preservation number is CCTCC NO: V202307; 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. 3, gE gene nucleotide sequence is shown as SEQ ID NO. 4, 11K gene nucleotide sequence is shown as SEQ ID NO.

5.

6. The recombinant pseudorabies virus strain of claim 5, wherein, The expression cassette further comprises the promoter CMV, the BGH-pA human beta globulin gene-poly A tail.

7. The recombinant pseudorabies virus strain according to claim 5 for use in the preparation of a vaccine for preventing or treating pseudorabies and porcine fever.

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

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