Recombinant pseudorabies virus strain for expressing hog cholera virus recombinant E2 protein and application of recombinant pseudorabies virus strain

Abstract

The invention discloses a recombinant pseudorabies virus strain for expressing hog cholera virus recombinant E2 protein and application, and relates to the technical field of biology. According to the research, a TK / gE double-gene deleted PRV attenuated strain is used as a virus vector, and after an exogenous CSFV E2 gene without a transmembrane region is inserted into a PRV gG signal peptide sequence, a recombinant virus rPRV-delTK / gE-gG-E2 which is positioned at a gG N end and is subjected to fusion expression with PRV gG is constructed; western blotting analysis and indirect immunofluorescence detection prove that the gG-E2 protein is successfully expressed. A recombinant virus immunized mouse can generate a specific antibody aiming at E2; after piglets are immunized, an antibody level equivalent to that of a classical CSFV attenuated vaccine strain C strain can be generated. The rPRV-delTK / gE-gG-E2 obtained by the research is a bigeminy live vaccine candidate strain with an extremely promising prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant pseudorabies virus strain expressing a recombinant E2 protein of a classical swine fever virus and an application thereof. Background Art

[0002] The E2 protein is located on the surface of the classical swine fever virus (CSFV) envelope, 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] Pseudorabies virus (PRV) has a 150kb genome and can be reverse genetically manipulated using bacterial artificial chromosome (BAC) technology to knock out virulence genes (TK, gE, and gI) to create a weaker strain with better immunogenicity. In addition, the PRV genome contains many non-essential genes, such as US4, US7, US8, and US9, into which exogenous genes can be inserted without affecting the virus's in vitro and / or in vivo replication potential, making it a suitable vector for expressing other porcine disease exogenous antigens ([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 arecombinant pseudorabies virus expressing the GP5 Of porcine reproductive and respiratory syndrome virus in piglets. Vet Immunol Immunopathol, 106: 309-319). After immunization, PRV can infect host cells. The genome of PRV can express exogenous proteins in cells. In the later stage of viral infection, cells lyse and exogenous proteins are released from cells, inducing humoral immunity and producing antibodies against exogenous proteins.

[0004] The existing PRV vaccine, Bartha-K61, provides only 50% protection against PRV variants. The C strain currently used to prevent classical swine fever is ineffective against the prevalent CSFV 2.1d subgenotype. Therefore, the development of vaccines against both pseudorabies virus and prevalent strains of classical swine fever virus is extremely urgent. While there have been numerous reports of using PRV as a live vector to express the classical swine fever virus E2 gene, no effective, widely adopted vaccine has yet been demonstrated for livestock farming. Summary of the Invention

[0005] Based on the deficiencies in the existing technology, this study used the PRV gE / TK double-gene deleted attenuated strain as a live vector to express the E2 gene of the CSFV epidemic strain CSFV 2.1d, and obtained a bivalent vaccine candidate strain rPRV-delTK / gE-gG-E2 that can induce pigs to produce antibodies at a level comparable to that of the classic CSFV vaccine strain C.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a recombinant pseudorabies virus strain expressing a recombinant E2 protein of a classical swine fever virus, wherein the pseudorabies virus genome is used as the skeleton of the recombinant pseudorabies virus strain, and the classical swine fever virus E2 gene with the transmembrane region removed is used as the introduced gene;

[0008] The swine fever virus E2 gene sequence with the transmembrane region removed is inserted between the gG signal peptide sequence and the gG gene sequence of the pseudorabies virus genome.

[0009] The classical swine fever virus E2 gene is the E2 gene from the new 2.1d subgenotype strain of classical swine fever virus.

[0010] Preferably, the pseudorabies virus is the pseudorabies virus strain PRV ZJ2013, and the TK gene and gE gene of the pseudorabies virus strain PRVZJ2013 are knocked out; the nucleotide sequence of the TK gene is shown in SEQ ID NO.4, and the nucleotide sequence of the gE gene is shown in SEQ ID NO.5.

[0011] More preferably, the sequence of the classical swine fever virus E2 gene with the transmembrane region removed is as shown in SEQ ID NO.1; the gG signal peptide sequence is as the 1-60bp sequence shown in SEQ ID NO.2, and the gG gene sequence is as the 1108-2556bp sequence shown in SEQ ID NO.2.

[0012] The present invention also provides a method for preparing the recombinant pseudorabies virus strain, comprising the following steps:

[0013] (1) Inserting a recombinant vector containing the swine fever virus E2 gene sequence with the transmembrane region removed between the gG signal peptide sequence and the gG gene sequence of the TK gene and gE gene double-gene deleted strain rPRV-delTK / gE to obtain rPRV-delTK / gE-gG-E2;

[0014] (2) Transfecting the rPRV-delTK / gE-gG-E2 obtained in step (1) into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

[0015] Preferably, the nucleotide sequence of the TK gene is shown as SEQ ID NO.4, the nucleotide sequence of the gE gene is shown as SEQ ID NO.5; and the sequence of the classical swine fever virus E2 gene with the transmembrane region removed is shown as SEQ ID NO.1.

[0016] Specifically, the recombinant vector also includes homology arms of homology to homology to A, restriction endonucleases I-sceI, and sequences of the Kan gene. The sequence of the recombination fragment P1 is: EN-homoA-IsceI-Kan-homoA-Ec, where ENEC forms E2, and homoA is used in the second recombination step to remove the IsceI-Kan sequence.

[0017] The present invention also provides the use of the recombinant pseudorabies virus strain in preparing vaccines for preventing or treating pseudorabies and swine fever.

[0018] The present invention also provides a bivalent vaccine for preventing or treating swine fever and pseudorabies, comprising the live virus or inactivated virus of the recombinant pseudorabies virus strain.

[0019] Beneficial effects of the present invention:

[0020] In this study, an attenuated PRV strain with a double TK / gE gene deletion was used as a viral vector. After inserting the exogenous CSFV E2 gene, which had its transmembrane domain removed, into the PRV gG signal peptide sequence, a recombinant virus, rPRV-delTK / gE-gG-E2, was constructed. The gG-E2 protein was expressed as a fusion protein at the N-terminus of gG. Western blotting analysis and indirect immunofluorescence assay confirmed successful expression of the gG-E2 protein. Immunization of mice with the recombinant virus produced antibodies specific to E2, and immunization of piglets produced antibodies at levels comparable to those of the classic attenuated CSFV vaccine strain, strain C. The rPRV-delTK / gE-gG-E2 obtained in this study is a highly promising candidate for a live, dual-dose vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of rPRV-delTK / gE-gG-E2 fusion expression of E2;

[0022] Figure 2 PCR identification diagram of pPRV-delTK / gE-gG-E2; wherein, a: first step Red recombination PCR identification; b: second step Red recombination PCR identification;

[0023] Figure 3 The expression of E2 in cells infected with the recombinant strain was detected by IFA;

[0024] Figure 4 The figure shows the expression of E2 in cells infected with the recombinant strain detected by Western blotting; 1. Concentrate of supernatant of cells infected with rPRV-delTK / gE-gG-E2; 2. Supernatant of cells infected with rPRV-delTK / gE-gG-E2; 3. Cells infected with rPRV-delTK / gE-gG-E2; 4. Concentrate of supernatant of cells infected with rPRV-delTK / gE; 5. Supernatant of cells infected with rPRV-delTK / gE; 6. Cells infected with rPRV-delTK / gE;

[0025] Figure 5 This is a mouse immunization experiment using a recombinant strain of PRV fused with the E2 gene;

[0026] Figure 6 Changes in CSFV E2 antibody levels after immunization of piglets with the rPRV-delTK / gE-gG-E2 strain;

[0027] Figure 7 This is a graph showing the changes in PRV gB antibody levels in piglets after immunization with group A and group B strains. DETAILED DESCRIPTION

[0028] Example 1

[0029] 1. Experimental Materials

[0030] The PRV ZJ2013 strain (Porcine pesudorabiesvirus strain ZJ2013) was isolated from a pig herd in Zhejiang, China in 2013 and deposited with the China Center for Type Culture Collection at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on January 16, 2023, under the accession number CCTCC V202307. The PRV ZJ2013 TK gene (sequence shown in SEQ ID NO. 4) was knocked out to generate rPRV-ZJ2013, which was then propagated in the BHK-21 cell line. The PRV ZJ2013 TK gene (sequence shown in SEQ ID NO. 4) and gE gene (sequence shown in SEQ ID NO. 5) were knocked out to generate rPRV-dTK / gE, which was then propagated in the BHK-21 cell line.

[0031] Monoclonal antibody 9011 against CSFV E2 protein was purchased from Beijing Jinnuo Baitai Biotechnology Co., Ltd. CSFV C-strain was maintained in the Poultry Disease Department of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences.

[0032] 2. Bioinformatics analysis

[0033] TMHMM software was used to analyze the transmembrane region of the E2 protein, and E2 (1-341 aa) without the transmembrane region was selected for expression. The online analysis software SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.O / ) was used to predict the PRV gG signal peptide. The gG signal peptide region was located at 1-20 aa.

[0034] 3. Design and synthesis of recombinant fragments containing the E2 gene

[0035] With reference to the CSFV E2 sequence (Genbank accession number KT953607), the E2 gene (nt 1-1023) was codon-optimized using the pig as the host (the gene sequence is shown in SEQ ID NO. 1). The designed sequence, fragment P1, was synthesized by Genescript for recombination. The P1 sequence (the gene sequence is shown in SEQ ID NO. 3) consists of: EN-homoA-IsceI-Kan-homoA-Ec, where ENEC constitutes E2, and homoA is used for the second recombination step to remove the IsceI-Kan sequence. Fragment P1 was synthesized by Genescript and cloned into a T-vector to generate the recombinant plasmid pT-E2-Kan (pT-EN-homoA-IsceI-Kan-homoA-Ec).

[0036] 4. Construction of PRV recombinant strain fused with E2 gene

[0037] According to the prediction results of gG protein signal peptide, E2 gene was inserted between gG signal peptide-CDS, as shown in the schematic diagram. Figure 1 As shown, the sequence of the target gene gG-sp-E2-linker-gG CDS is shown as SEQ ID NO.2, wherein the sequence of the gG signal peptide gG-sp is the 1-60bp sequence shown as SEQ ID NO.2, and the sequence of the gG CDS is the 1108-2556bp sequence shown as SEQ ID NO.2.

[0038] The E2-inserted recombinant clone, pPRV-delTK / gE-gG-E2, was constructed using the Red E / T two-step recombination method. In the first step, pT-E2-Kan was used as a template and PCR amplification was performed using the corresponding primers pPRV-delTK / gE-gG-E2-F and pPRV-delTK / gE-gG-E2-R, listed in Table 1.

[0039] Table 1

[0040]

[0041]

[0042] PCR reaction 25 μL system: 2× PCR buffer 12.5 μL, 2.5 mM dNTP 2 μL, 1 μL each of 10 μM forward and reverse primers, 0.1 μL template DNA, 0.25 μL KOD DNA polymerase, and 8.15 μL ddH2O.

[0043] PCR reaction conditions were: denaturation at 95°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 3 minutes, for 30 cycles, followed by an extension at 72°C for 10 minutes after the final cycle. After gel purification, 100 ng of the amplified PCR product was added to 50 μL of pPRV-dTK / gEGS1783 competent cells. The mixture was transferred to a pre-chilled 1 mm electroporation cuvette and electroporated (1.8 kV, 25 uF, 200 Ω). Immediately, 1 mL of LB was added, and the culture was incubated with shaking at 32°C for 1 hour. The culture was then plated on an LB plate containing 34 μg / mL Cam and 50 μg / mL Kan and incubated at 32°C for 30 hours. The BAC plasmid was extracted and identified using the corresponding primer pair: PRV-gG-F and PRV-gG-R. The PCR product was 4670 bp in size. The pPRV-delTK / gE-gG-E2-kan mutant was screened and obtained.

[0044] The Kan gene of the pPRV-delTK / gE-gG-E2-kan mutant was deleted by the homology arm introduced into fragment P1. A single colony containing pPRV-delTK / gE-gG-E2-kan was selected and cultured overnight. 100 μL of the colony was inoculated into 2 mL of Cam-resistant LB medium. The culture was incubated at 32°C and 220 rpm until the OD450 reached 0.4-0.6. L-arabinose was added to a final concentration of 1%. The culture was continued for 1 hour to induce expression of the restriction endonuclease I-SceI. The colony was then transferred to a 42°C shaking water bath for 30 minutes for Red recombination and incubated at 32°C for 1 hour.

[0045] The bacterial suspension was made into 10 -3 ~10-5 After dilution, plate the plate onto a Cam-resistant plate containing 1% L-arabinose and incubate at 32°C for 30 hours. To isolate individual colonies, plate them onto Kan-resistant and Cam-resistant plates, respectively. Select colonies that grow on the Cam-resistant plate but not on the Kan-resistant plate. Identify the clone using primers PRV-gG-F and PRV-gG-R (Table 1). A product with a fragment size of 3619 bp was obtained and sent for sequencing. The correctly sequenced clone was designated pPRV-delTK / gE-gG-E2.

[0046] like Figure 2 As shown, the PCR identification results were consistent with the expected results, indicating that pPRV-delTK / gE-gG-E2 was successfully constructed.

[0047] 5. Rescue of recombinant virus and removal of vector sequence

[0048] pPRV-delTK / gE-gG-E2 was transferred into BHK21 cells by calcium phosphate transfection to obtain recombinant virus containing GFP gene. The harvested virus was named rPRV-delTK / gE-gG-E2-GFP.

[0049] Vector sequence removal: BHK21 cell monolayers were transfected with the pCAGGS-NLS / Cre plasmid. 24 hours later, rPRV-delTK / gE-gG-E2-GFP was inoculated. 90 minutes later, the medium was changed and the cells were overlaid with 1% agarose gel. After 48 hours, viral plaques lacking GFP were selected. After several rounds of screening, pure GFP-free viral plaques were obtained. After PCR and sequencing, the recombinant virus, devoid of the vector sequence, was obtained and designated rPRV-delTK / gE-gG-E2.

[0050] 6. Indirect Immunofluorescence (IFA)

[0051] BHK21 cells grown on slides were infected with rPRV-dTK / gE and rPRV-delTK / gE-gG-E2. After 24 hours of growth, the monolayers were fixed with 4% paraformaldehyde (PFA) for 30 minutes. The fixative was discarded, the cells were washed three times with PBS, and then permeabilized with 0.1% Triton for 10 minutes. The fixative was discarded, the cells were washed three times with PBS, and the cells were incubated with anti-E2 monoclonal antibody 9011 at a 1000-fold dilution at 37°C for 1 hour. The cells were discarded and washed three times with PBS. Cy3 anti-mouse secondary antibody (Beyotime, Shanghai, China) was then diluted 1:1000 and incubated at 37°C for another 1 hour. Unbound dye was washed away with PBS, and the cells were observed and photographed using a fluorescence microscope.

[0052] like Figure 3As shown, the results of indirect immunofluorescence test showed that E2 was successfully expressed; the theoretical molecular weight of E2 is 38.2kDa and gG is 53.48kD; due to the existence of post-translational modification, the literature reported that in PRV-infected cells, gG is 70kD (Huang Wenxiang, 2020).

[0053] 7. Western blot analysis

[0054] BHK21 cells grown on slides were infected with rPRV-dTK / gE and rPRV-delTK / gE-gG-E2. After 24 hours, the cell monolayers were washed three times with ice-cold PBS. The cells were then lysed with 1× loading buffer, heat denatured at 100°C for 10 minutes, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected and separated by SDS-PAGE. The proteins were then transferred to a NC membrane. The NC membrane was blocked with 10% skim milk powder in PBST overnight at 4°C, washed once with PBST, incubated with a 1:1000 dilution of anti-E2 monoclonal antibody 9011 at 37°C for 1 hour, washed three times with PBST, and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Lianke Biotechnology) for 1 hour and washed three times with PBST. Finally, ECL chemiluminescence developer was added for color development. The gel was photographed using a gel imaging system.

[0055] Western blotting results showed that a specific band of approximately 135 kD was detected in the concentrated supernatant of rPRV-delTK / gE-gG-E2 infection, indicating that E2 and PRV gG were fused and expressed ( Figure 4 ).

[0056] 8. Titer of recombinant virus

[0057] The recombinant virus was diluted 10-fold in DMEM medium to obtain dilutions of 10-3 to 10-8. Then, 0.1 mL of the virus suspension was inoculated into a 96-well culture plate confluent with a monolayer of BHK-21 cells. Eight replicates were made for each dilution, and a control of uninfected cells was also established. After incubation at 37°C for 1 hour, the cells were washed twice with PBS. After culturing at 37°C for 3-4 days, the plates were read under an inverted microscope. Cytopathic effects were observed in the wells inoculated with the virus dilutions, and the virus titer was calculated using the Reed-Muench method.

[0058] The titer of rPRV-delTK / gE-gG-E2 was similar to that of rPRV-delTK / gE, both of which were 1.0×10 7.3 TCID 50 / mL or so.

[0059] Example 2 Performance Measurement

[0060] Immunogenicity evaluation in mice and rabbits.

[0061] Animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences (ZAAS). Animal experiments were approved by the Animal Experimentation Ethics Committee of ZAAS.

[0062] 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, group 5, group 8 and group 9 were inoculated with the recombinant virus for 10 7 TCID 50 All mice were injected intramuscularly. At 0, 1, 2, 3, 4, and 5 weeks after immunization, blood was collected from the tail vein of 8 mice in each group for antibody detection every week.

[0063] Fifteen 20-day-old weaned piglets (all tested negative for ASFV, PRRSV, PRV, CSFV, and PCV2 antibodies) were randomly divided into three groups of five piglets each. Each group received two immunizations, with a second immunization administered 7 weeks after the first, using the same dose as the first. The pigs in each group were numbered according to their ear tags as shown in Table 2.

[0064] Table 2 Recombinant strains for piglet group immunization

[0065]

[0066] Post-immunization monitoring: Follow-up records are kept on a per-pig basis, with body temperature monitored within one week after immunization. Nasal swabs are collected every two days after immunization to monitor virus excretion. Serum is collected from all pigs at 0, 1, 2, 4, 7, 8, 9, 10, 11, 16, 20, and 24 weeks after immunization to test for PRV gB and CSFV E2 antibody levels.

[0067] PRV gB / gE-specific antibodies were identified in serum samples using a PRV gB / gE antibody kit (IDEXX Laboratories, Inc, Westbrook, ME, USA) and E2-specific antibodies were identified in serum samples using a blocking ELISA method (IDEXX Laboratories, Inc, Westbrook, ME, USA).

[0068] RPRV-delTK / gE-gG-E2 immunized mice produced high titer antibodies, while rPRV-delTK / gE immunized mice did not produce E2 antibodies ( Figure 5 ).

[0069] Regarding the CSFV antibody level, in the piglets vaccinated with rPRV-delTK / gE-gG-E2, the CSFV serum antibodies of the two groups of piglets began to turn positive 2 weeks after the first vaccination, and the antibody levels gradually increased, but the antibody dispersion was large; 1 week after the second booster vaccination, the CSFV antibody levels of the piglets increased rapidly, the antibodies were positive, and the dispersion was small ( Figure 6 One month after the second vaccination, the CSFV antibody level in rPRV-delTK / gE-gG-E2 piglets decreased rapidly, and the individual antibody dispersion increased ( Figure 6 ).

[0070] Regarding the PRV antibody levels, the PRV serum antibody levels of piglets in group A (rPRV-delTK / gE-gG-E2) and group B (rPRV-delTK / gE) increased rapidly one week after the first vaccination, and the serum antibodies basically turned positive. The average PRV antibody level of piglets in group A was slightly lower than that of piglets in group B. One week after the second booster vaccination, the PRV antibody levels of piglets in each group reached the highest peak, and the high antibody levels could last for 6 months ( Figure 7 ).

Claims

1. A recombinant pseudorabies virus strain expressing a recombinant E2 protein of a classical swine fever virus, characterized in that: The pseudorabies virus genome is used as the backbone of the recombinant pseudorabies virus strain, and the classical swine fever virus E2 gene with the transmembrane region removed is used as the introduced gene; The swine fever virus E2 gene sequence with the transmembrane region removed is inserted between the gG signal peptide sequence and the gG gene sequence of the pseudorabies virus genome.

2. The recombinant pseudorabies virus strain according to claim 1, characterized in that The classical swine fever virus E2 gene is the E2 gene from the new 2.1d subgenotype strain of classical swine fever virus.

3. The recombinant pseudorabies virus strain according to claim 1, characterized in that The pseudorabies virus is the pseudorabies virus strain PRV ZJ2013, and the TK gene and gE gene of the pseudorabies virus strain PRV ZJ2013 are knocked out; the nucleotide sequence of the TK gene is shown in SEQ ID NO.4, and the nucleotide sequence of the gE gene is shown in SEQ ID NO.

5.

4. The recombinant pseudorabies virus strain according to claim 2, characterized in that The sequence of the classical swine fever virus E2 gene with the transmembrane region removed is shown in SEQ ID NO.1; The gG signal peptide sequence is the 1-60 bp sequence shown in SEQ ID NO.2, and the gG gene sequence is the 1108-2556 bp sequence shown in SEQ ID NO.

2.

5. The method for preparing the recombinant pseudorabies virus strain according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Inserting a recombinant vector containing the swine fever virus E2 gene sequence with the transmembrane region removed between the gG signal peptide sequence and the gG gene sequence of the TK gene and gE gene double-gene deleted strain rPRV-delTK / gE to obtain rPRV-delTK / gE-gG-E2; (2) Transfecting the rPRV-delTK / gE-gG-E2 obtained in step (1) into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

6. The method for preparing the pseudorabies virus strain according to claim 5, characterized in that: The nucleotide sequence of the TK gene is shown in SEQ ID NO.4, and the nucleotide sequence of the gE gene is shown in SEQ ID NO.5; the sequence of the classical swine fever virus E2 gene with the transmembrane region removed is shown in SEQ ID NO.

1.

7. The method for preparing the pseudorabies virus strain according to claim 6, characterized in that: The recombinant vector also includes homology arms of homology to homology, restriction endonuclease I-sceI, and Kan gene sequence.

8. Use of the recombinant pseudorabies virus strain according to any one of claims 1 to 4 in the preparation of a vaccine for preventing or treating pseudorabies and swine fever.

9. A bivalent vaccine for preventing or treating swine fever and pseudorabies, characterized in that: Comprising the live virus or inactivated virus of the recombinant pseudorabies virus strain according to any one of claims 1 to 4.

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