A recombinant pseudorabies virus strain expressing a recombinant E2 protein of a porcine fever virus and application thereof

Abstract

The application discloses a recombinant pseudorabies virus strain expressing classical swine fever virus (CSFV) recombinant E2 protein and application, and relates to the technical field of biology.The TK / gE double gene deletion PRV attenuated strain is used as a virus carrier, an exogenous CSFV E2 gene with a transmembrane region removed is inserted after a PRV gC signal peptide sequence, and a recombinant virus rPRV-delTK / gE-gC-E2 for expressing the gC N terminal and fusing with the PRV gC is constructed; Western blotting analysis and indirect immunofluorescence detection prove that the gC-E2 protein is successfully expressed.The recombinant virus immunized mice can produce specific antibodies against E2; after immunizing piglets, the piglets can produce an antibody level equivalent to that of a classical CSFV attenuated vaccine strain C strain.The rPRV-delTK / gE-gC-E2 obtained in the research is a promising bivalent live vaccine candidate strain.

Description

TECHNICAL FIELD

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

[0002] The E2 protein is located on the surface of a porcine fever virus (CSFV) envelope, participates in virus infection, is responsible for binding to receptors on cells, is a main protective antigen of the 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 operation by using a 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 an exogenous gene can be inserted into the genes without affecting the in vitro and / or in vivo replication potential of the virus, so that the virus becomes a suitable carrier for expressing other exogenous 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.). After immunization, the host cells can be infected, the genome of the PRV can express the exogenous protein in the cells, the cells are lysed in the later stage of virus infection, the exogenous protein is released from the cells, and the humoral immunity is induced to produce antibodies against the exogenous protein.

[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 less effective against the prevalent CSFV 2.1d subgenotype. Therefore, it is extremely urgent to develop a vaccine against the prevalent strains of pseudorabies virus and swine fever virus. At present, there are many reports on PRV as a live carrier for expressing the E2 gene of swine fever virus, but there is no effective case that can be widely used in animal husbandry. SUMMARY

[0005] Based on the deficiencies in the prior art, the present research carries out a PRV gE / TK double gene deletion attenuated strain as a live carrier to express the E2 gene of the prevalent strain of CSFV, i.e. CSFV 2.1d E2 gene, and obtains a bivalent vaccine candidate strain rPRV-delTK / gE-gC-E2 which can induce the pig body to produce an antibody level equivalent to that of the classical CSFV vaccine strain C.

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

[0007] The present application provides a recombinant pseudorabies virus strain expressing a recombinant E2 protein of swine fever virus, wherein a genome of pseudorabies virus is used as a backbone of the recombinant pseudorabies virus strain, and an E2 gene of swine fever virus with a transmembrane region removed is used as an introduced gene.

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

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

[0010] Preferably, the pseudorabies virus is a PRV ZJ2013 strain, and the PRV ZJ2013 strain knocks out a TK gene and a gE gene; 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 E2 gene of swine fever virus with a transmembrane region removed is shown in SEQ ID NO. 1; the gC signal peptide sequence is shown in the sequence of 1-60 bp of SEQ ID NO. 2, and the gC gene sequence is shown in the sequence of 1108-2484 bp of SEQ ID NO. 2.

[0012] The present application further provides a preparation method of the recombinant pseudorabies virus strain, comprising the following steps:

[0013] (1) inserting a recombinant vector containing a sequence of porcine pestivirus E2 gene removing transmembrane region into the gC signal peptide sequence and the gC gene sequence of the TK gene and gE gene double gene deletion strain rPRV-delTK / gE, to obtain rPRV-delTK / gE-gC-E2;

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

[0015] Preferably, the TK gene nucleotide sequence is shown as SEQ ID NO. 4, the gE gene nucleotide sequence is shown as SEQ ID NO. 5; and the sequence of the porcine pestivirus E2 gene removing transmembrane region is shown as SEQ ID NO. 1.

[0016] Specifically, the recombinant vector further comprises homoA homologous arm, restriction endonuclease I-sceI, Kan gene sequence. The recombinant fragment P1 sequence comprises: EN-homoA-IsceI-Kan-homoA-Ec, wherein ENEC constitutes E2, and homA is used for removing the IsceI-Kan sequence in the second step of recombination.

[0017] The application further provides application of the recombinant pseudorabies virus strain in preparation of a vaccine for preventing or treating pseudorabies and swine fever.

[0018] The application further 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] The application has the following beneficial effects:

[0020] In the present study, a TK / gE double gene deletion PRV attenuated strain was used as a viral vector, and an exogenous CSFV E2 gene removing transmembrane region was inserted after the PRV gC signal peptide sequence to construct a recombinant virus rPRV-delTK / gE-gC-E2 located at the N terminal of gC and fused with PRV gC for expression; Western blotting analysis and indirect immunofluorescence detection proved that the gC-E2 protein was successfully expressed. The recombinant virus immunized mice could produce specific antibodies against E2; after immunizing piglets, the antibody level was equivalent to that of the classical CSFV attenuated vaccine strain C. The rPRV-delTK / gE-gC-E2 obtained in the present study is a promising bivalent live vaccine candidate strain. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 1 is a schematic diagram of rPRV-delTK / gE-gC-E2 fusion expression E2;

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

[0023] Figure 3 Figure for IFA detection of E2 expression in cells infected with recombinant strains;

[0024] Figure 4 Figure for Western blotting detection of E2 expression in cells infected with recombinant strains; wherein, 4. rPRV-delTK / gE infected cell supernatant concentrate; 5. rPRV-delTK / gE infected cell supernatant; 6. rPRV-delTK / gE infected cell; 7. rPRV-delTK / gE-gC-E2 infected cell supernatant concentrate; 8. rPRV-delTK / gE-gC-E2 infected cell; 9. rPRV-delTK / gE-gC-E2 infected cell supernatant;

[0025] Figure 5 Figure for PRV fusion E2 gene recombinant strain immunization test of mice;

[0026] Figure 6 Figure for CSFV E2 antibody level change of piglets immunized with rPRV-delTK / gE-gC-E2 strain;

[0027] Figure 7 Figure for PRV gB antibody level change of piglets immunized with A and B group strains. DETAILED DESCRIPTION

[0028] Example 1

[0029] 1. Experimental materials

[0030] PRV ZJ2013 strain (Porcine pesudorabies virus strain ZJ2013) isolated from a pig herd in Zhejiang, China in 2013 was preserved in China Center for Type Culture Collection on January 16, 2023, and the address of the preservation unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number is CCTCC NO: V202307. The TK gene of PRV ZJ2013 (the gene sequence is shown as SEQ ID NO. 4) was knocked out to obtain rPRV-ZJ2013, which was passaged in BHK-21 cell line. The TK gene of PRV ZJ2013 (the gene sequence is shown as SEQ ID NO. 4) and the gE gene (the gene sequence is shown as SEQ ID NO. 5) were knocked out to obtain rPRV-delTK / gE, which was passaged in BHK-21 cell line.

[0031] The monoclonal antibody 9011 against CSFV E2 protein was purchased from Beijing Jinuobaitai Biotechnology Co., Ltd. The CSFV C-Strain was preserved in the poultry disease room of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences.

[0032] 2. Bioinformatics analysis

[0033] The transmembrane region of E2 protein was analyzed by TMHMM software, and E2 (1-341aa) without the transmembrane region was selected for expression. The PRV gC signal peptide was predicted by online analysis software SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), and it was found that the gC signal peptide was located at 1-20 aa.

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

[0035] Referring to the CSFV E2 sequence (Genbank accession number KT953607), the E2 gene (1-1023nt) without the transmembrane region was codon-optimized (the gene sequence is shown as SEQ ID NO. 1) with pigs as the host. The sequence was designed by Genescript Company to synthesize a recombinant fragment P1, and the P1 sequence (the gene sequence is shown as SEQ ID NO. 3) consists of ENEC, homoA, IsceI-Kan, and homoA-Ec, wherein ENEC constitutes E2, and homoA is used for removing the IsceI-Kan sequence in the second step of recombination. Fragment P1 was synthesized by Genescript Company and cloned into T vector to obtain recombinant plasmid pT-E2-Kan (pT-EN-homoA-IsceI-Kan-homoA-Ec).

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

[0037] According to the prediction results of the gC protein signal peptide, the E2 gene was inserted between the gC signal peptide promoter-CDS, as shown in the schematic diagram Figure 1 , wherein the sequence of the target gene gC-sp-E2-linker-gC CDS is shown as SEQ ID NO. 2, wherein the sequence of the gC signal peptide gG-sp is the sequence of 1-60 bp shown in SEQ ID NO. 2, and the sequence of the gC CDS is the sequence of 1108-2484 bp shown in SEQ ID NO. 2.

[0038] The recombinant clone pPRV-delTK / gE-gC-E2 inserted with E2 was constructed by Red E / T two-step recombination method. In the first step of recombination, pT-E2-Kan was used as a template, and the primers pPRV-delTK / gE-gC-E2-F / pPRV-delTK / gE-gC-E2-R in Table 1 were used for PCR amplification, respectively.

[0039] Table 1

[0040]

[0041]

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

[0043] The PCR reaction conditions were as follows: denaturation at 95 ℃ for 45 s, annealing at 56 ℃ for 45 s, extension at 72 ℃ for 3 min, 30 cycles, and then extension at 72 ℃ for 10 min after the last cycle. After purification by gel recovery, 100 ng of the amplified PCR product was added to 50 μL of pPRV-dTK / gEGS1783 competent cells, mixed, and then transferred into a pre-cooled 1 mm electroporation cup for electroporation (1.8 kv, 25 uF, 200 Ω). Then, 1 mL of LB was immediately added, and the mixture was cultured at 32 ℃ for 1 h, and then spread on an LB plate containing 34 μg / mL Cam and 50 μg / mL Kan, and cultured at 32 ℃ for 30 h. The BAC plasmid was extracted, and identification was performed using the corresponding primers. The primer pair for gC identification was Kan-F / PRV-gC-R, and the PCR product size was 2539 bp. The pPRV-delTK / gE-gC-E2-kan mutant was obtained by screening.

[0044] The deletion of the Kan gene of the pPRV-delTK / gE-gC-E2-kan mutant was mediated by the homoA homologous arm introduced on the fragment P1. A single colony containing pPRV-delTK / gE-gC-E2-kan was picked, cultured overnight, and then 100 μL of the culture was inoculated into 2 mL of LB medium resistant to Cam, and cultured at 32 ℃ and 220 r / min until the OD450 value was 0.4-0.6. Then, 1% L-arabinose was added to a final concentration, and the culture was further cultured for 1 h to induce the expression of the restriction endonuclease I-SceI, and then transferred into a 42 ℃ water bath shaker for 30 min for Red recombination, and then cultured at 32 ℃ for 1 h.

[0045] The bacterial suspension was diluted 10 -3 ~10 -5Diluted and plated on Cam resistant plates containing 1% L-rabose, incubated at 32°C for 30h. When picking single colonies, inoculate on Kan resistant plates and Cam resistant plates, respectively. Pick colonies that grow on Cam resistant plates but not on Kan resistant plates. Identify with primer E2-F / PRV-gC-R in Table 1, a 1832bp fragment is expected, and pPRV-delTK / gE-gC-E2 is obtained.

[0046] As shown in Figure 2 PCR identification results are consistent with the expected results, indicating that pPRV-delTK / gE-gC-E2 is successfully constructed.

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

[0048] pPRV-delTK / gE-gC-E2 is transfected into BHK21 cells by calcium phosphate transfection method, and a recombinant virus containing GFP gene is obtained. The virus is harvested and named rPRV-delTK / gE-gC-E2-GFP.

[0049] Removal of vector sequences: Transfect pCAGGS-NLS / Cre plasmid into monolayer BHK21 cells, inoculate rPRV-delTK / gE-gC-E2-GFP after 24h, change the liquid after 90min, and spread 1% agarose gel. After 48h, pick up virus plaques without GFP, and obtain pure virus plaques without GFP after several rounds of screening. After PCR and sequencing identification, the recombinant virus with removed vector sequences is obtained and named rPRV-delTK / gE-gC-E2.

[0050] 6. Indirect immunofluorescence (IFA)

[0051] Infect BHK21 cells growing on slides with rPRV-dTK / gE and rPRV-delTK / gE-gC-E2. After 24h of growth, fix the monolayer cells with 4% paraformaldehyde (PFA) for 30min, discard the fixing solution, wash with PBS for three times, and then permeate the monolayer cells with 0.1% Triton for 10min. Discard the solution, wash with PBS for three times, dilute the anti-E2 monoclonal antibody 9011 1000 times, and incubate at 37°C for 1h, discard, wash with PBS for three times; then dilute the Cy3 anti-mouse secondary antibody (Beyotime, Shanghai, China) 1:1000, continue to incubate at 37°C for 1h, wash away the unbound dye with PBS, and observe and take pictures under a fluorescence microscope.

[0052] As shown in Figure 3As shown, the indirect immunofluorescence test results show that E2 is successfully expressed; the theoretical molecular weight of E2 is 38.2 kDa, and the theoretical molecular weight of gC is 51.97 kD; due to the presence of post-translational modification, it is reported in the literature that gC in PRV infected cells presents two forms, a precursor of 74 kD and a mature glycoprotein of 92 kD (Wei Jinfeng, 2019).

[0053] 7、Western blot analysis

[0054] BHK21 cells growing on the slide were infected with rPRV-dTK / gE and rPRV-delTK / gE-gC-E2. After 24 h, the monolayer cells were washed with pre-cooled PBS for 3 times. Then the cells were lysed with 1x loading buffer, denatured by heating at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was collected. After separation by SDS-PAGE, the proteins were transferred to NC membrane. The NC membrane was blocked with 10% skim milk (10% skim milk in PBST) at 4℃ overnight, washed once with PBST, incubated with 1:1000 diluted anti-E2 monoclonal antibody 9011 at 37℃ for 1 h, washed with PBST for 3 times, incubated with horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody (Joint Biotechnology) for 1 h, and washed with PBST for 3 times. Finally, ECL chemiluminescence developing solution was added for color development. Finally, the gel imaging system was used for photography.

[0055] The results of Western blotting detection showed that a specific band with a size of about 100 kD was detected in rPRV-delTK / gE-gC-E2 infected cells, indicating that E2 was expressed in fusion with PRV gC. Figure 4 )。

[0056] 8、Titer of recombinant virus

[0057] The recombinant virus was diluted by 10 times in DMEM medium to obtain recombinant virus with a dilution of 10 -3 ~10 -8 Then 0.1 mL of virus suspension was inoculated in each dilution into a 96-well culture plate covered with BHK-21 monolayer cells, 8 replicate wells were made for each dilution, and a non-virus inoculated cell control was set up. After incubation at 37℃ for 1 h, washing with PBS for 2 times. After incubation at 37℃ for 3-4 d, the plate was read under an inverted microscope, and the monolayer fused BHK-21 cells in the wells inoculated with virus dilution were observed for cytopathic effect. The virus titer was calculated by the Reed-Muench method.

[0058] The titer of rPRV-delTK / gE-gC-E2 was similar to that of rPRV-delTK / gE, both being 1.0 x 10 7.3TCID 50 / mL.

[0059] Performance determination of Example 2

[0060] Evaluation of immunogenicity in mice and rabbits.

[0061] Animal experiments were performed 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 Ethics Committee.

[0062] Twenty-four 6-week-old female Balb / c mice were randomly divided into 3 groups, 8 mice in each group. The control group, the 3rd group, the 5th group, the 8th group and the 9th group were respectively inoculated with 10 7 TCID 50 All mice were injected by muscle route. At 0, 1, 2, 3, 4, 5 weeks after immunization, 8 mice in each group were bled through the tail vein every week to detect antibodies.

[0063] Fifteen 20-day-old weaned piglets (all the pigs used have been tested to be negative for ASFV, PRRSV, PRV, CSFV, PCV2 antibodies) were randomly divided into 3 groups, 5 piglets in each group; divided into two immunizations, the second immunization was performed 7 weeks after the first immunization, and the immunization dose was the same as the first immunization. According to the ear tag number, the piglet number of each group is shown in Table 2.

[0064] Table 2 Grouping and immunization of piglets with recombinant strains

[0065]

[0066] Post-immunization monitoring: The principle of tracking by individual was adopted, and body temperature was detected within one week after immunization; nasal swabs were collected every two days after immunization to detect virus shedding. The serum of all pigs was collected at 0, 1, 2, 4, 7, 8, 9, 10, 11, 16, 20, 24 weeks after immunization to detect PRV gB antibody and CSFV E2 antibody levels.

[0067] PRV gB / gE antibody kit (IDEXX Laboratories, Inc, Westbrook, ME, USA) was used to identify PRV gB / gE specific antibodies in serum samples. E2 specific antibodies in serum samples were identified by blocking ELISA method (IDEXX Laboratories, Inc, Westbrook, ME, USA).

[0068] High-titer antibodies can be produced in mice immunized with rPRV-delTK / gE-gC-E2, while no E2 antibodies are produced after immunization with rPRV-delTK / gE Figure 5 ).

[0069] Regarding CSFV antibody levels, in piglets immunized with rPRV-delTK / gE-gC-E2, two weeks after the initial immunization, both groups of piglets began to show positive CSFV serum antibodies, and antibody levels gradually increased, but the antibody dispersion was large; one week after the second booster immunization, the CSFV antibody levels in piglets rose rapidly, and the antibodies were positive, with less dispersion. Figure 6 Five months after the second immunization, the average CSFV antibody blocking rate of rPRV-delTK / gE-gC-E2 piglets was approximately 80%. Figure 6 ).

[0070] Regarding PRV antibody levels, in piglets immunized in groups A (rPRV-delTK / gE-gC-E2) and B (rPRV-delTK / gE), serum PRV antibody levels rose rapidly one week after the initial immunization, with almost all groups turning positive. The average PRV antibody level in group A piglets was slightly lower than that in group B piglets. One week after the second booster immunization, PRV antibody levels in all groups reached their peak, and high antibody levels could be maintained for up to 6 months. Figure 7 ).

Claims

1. A recombinant pseudorabies virus strain expressing the recombinant E2 protein of classical swine fever virus, characterized in that, The pseudorabies virus genome was used as the backbone of the recombinant pseudorabies virus strain to remove the transmembrane region of classical swine fever virus. E2 Genes are used as import genes; The pseudorabies virus genome gC signal peptide sequence and gC Classical swine fever virus with insertion between gene sequences to remove transmembrane regions E2 The gene sequence was obtained, and the sequence of the target gene gC-sp-E2-linker-gC CDS is shown in SEQ ID NO.2, wherein the sequence of the gC signal peptide gC-sp is shown as the 1-60bp sequence in SEQ ID NO.

2. gC The gene sequence is as shown in SEQ ID NO.2, from 1108 to 2484 bp. The pseudorabies virus mentioned is the PRV ZJ2013 strain, with accession number CCTCC NO: V202307. The PRV ZJ2013 strain has been knocked out. TK Genes and gE Genes; the stated TK The gene nucleotide sequence is shown in SEQ ID NO.

4. gE The gene nucleotide sequence is shown in SEQ ID NO.5; The removal of transmembrane zone swine fever virus E2 The gene sequence is shown in SEQ ID NO.

1.

2. The method for preparing the recombinant pseudorabies virus strain as described in claim 1, characterized in that, Includes the following steps: (1) The classical swine fever virus containing the transmembrane region removed E2 Recombinant vector of gene sequence inserted into TK Genes and gE The gC signal peptide sequence of the double-gene deletion strain rPRV-delTK / gE is similar to... gC Between gene sequences, rPRV-delTK / gE-gC-E2 is obtained; (2) The rPRV-delTK / gE-gC-E2 obtained in step (1) was transfected into BHK-21 cells to obtain the recombinant pseudorabies virus strain.

3. The method for preparing the pseudorabies virus strain as described in claim 2, characterized in that, The recombinant vector also includes the homoA homologous arm and restriction endonuclease I- sce I, Kan Gene sequence.

4. The use of the recombinant pseudorabies virus strain according to claim 1 in the preparation of vaccines for the prevention or treatment of pseudorabies and classical swine fever.

5. A bivalent vaccine for the prevention or treatment of classical swine fever and pseudorabies, characterized in that, It includes the live virus or the inactivated virus of the recombinant pseudorabies virus strain described in claim 1.

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