Pseudorabies virus gD recombinant protein, subunit vaccine as well as preparation method and application of pseudorabies virus gD recombinant protein and subunit vaccine

Abstract

The invention relates to the technical field of biology, in particular to a pseudorabies virus gD recombinant protein, a subunit vaccine and a preparation method and application thereof.The gD recombinant protein sequentially comprises a signal peptide, a connecting peptide, the 21st-350th site of the gD protein, an enzyme cutting site and a His tag from the N end to the C end, the amino acid sequence of the 21st-350th site of the gD protein is shown as SEQ ID No.4, and the amino acid sequence of the 21st-350th site of the gD protein is shown as SEQ ID No.6. The pseudorabies virus gD recombinant protein has high immunogenicity, the prepared subunit vaccine can generate a specific antibody with neutralizing activity, mice do not die after challenge, the protection rate is 100%, the protection rate after challenge of pigs is 100%, pathological sections only show slight pathological changes, and the pseudorabies virus gD recombinant protein has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a pseudorabies virus gD recombinant protein, a subunit vaccine, and a preparation method and application thereof. Background Art

[0002] Pseudorabies virus (PRV) is the causative agent of pseudorabies, which was first confirmed by Hungarian veterinarian Aladár Aujeszky in 1902, so pseudorabies is also called Aujeszky's disease. The natural host of PRV is pigs, but it can also infect various mammals, such as cattle, sheep, dogs, cats and mice. Pseudorabies is a highly contagious disease that can be transmitted through saliva, nasal secretions and aerosols. The clinical symptoms of piglets infected with PRV are coughing, sneezing, fever, constipation, ataxia and salivation, resulting in a high mortality rate, especially in piglets less than 1 month old infected with PRV, with a mortality rate close to 100%. The clinical symptoms of sows are abortion, stillbirth or mummified fetuses. Some non-natural hosts, such as mice, also have a mortality rate close to 100% when infected with PRV.

[0003] The diameter of the virus particle of PRV is about 100-150nm, and it includes the core, capsid, shell and envelope from the inside to the outside. The core and capsid form the nucleocapsid, and the linear double-stranded DNA (dsDNA) genome is wrapped by the icosahedral capsid. The average GC content of the pseudorabies virus genome is about 74%, the length is about 150kb, and it has at least 70 open reading frames (ORFs), encoding more than 100 viral proteins, and mature virus particles contain about 50 viral structural proteins. PRV infection completes the adsorption process by binding glycoproteins (such as gB and gC) on the envelope of the virus particles to heparanase proteoglycans on the cell surface. gD binds to receptors on the cell membrane to assist the virus in invading the cell. Known PRV receptors on the cell membrane include Nectin-1, HVEM and 3-O-heparan sulfate. The fusion process of the viral envelope and the cell membrane is mediated by gB, gH and gL. Studies have shown that several PRV subunit vaccines, such as gD and gB, expressed as protective antigens of PRV in prokaryotic or eukaryotic expression systems, are highly safe and can be used in special situations such as pregnant sows that are not suitable for live vaccines. Subunit vaccines do not have safety risks such as reversion to virulence and viral recombination mutations.

[0004] Among the 11 PRV glycoproteins, gB, gC, and gD can stimulate the body to produce neutralizing antibodies. Therefore, the expressed recombinant gB, gC, and gD proteins can induce protective immunity against PRV attack. It is reported that gD recognizes receptors of the poliovirus receptor family on the cell surface, mediates the stable binding of the virus to the target cell, and participates in the adsorption and invasion process of viral infection, so anti-gD antibodies can induce better protective responses by inhibiting the binding between gD and viral receptors. Pigs and mice injected with anti-gB, gC, and gD monoclonal antibodies can resist the virulent attack of PRV. In addition, animals infected with recombinant vaccinia virus or adenovirus vaccines expressing PRV gD protein also acquired immunity and could resist the attack of virulent strains. It is worth noting that the gD protein of human herpes simplex virus type 2 (HSV-2) induces high-titer antibodies in humans, which may also be an important component of HSV-2 subunit vaccines.

[0005] In this application, PRV proteins were screened to detect immunogenic proteins that react with PRV-positive sera, and several envelope glycoproteins were found to have relatively strong immunogenicity. Therefore, we first used the Bac-to-Bac expression system to secrete and express PRV gD, and conducted protection tests in mice and pigs, respectively. Animal experiments showed that gD recombinant protein can provide 100% protection for experimental animals, while reducing histopathological damage in mice and pigs, indicating that the PRVgD recombinant protein expressed in the insect expression system has the potential to be a candidate vaccine for PRV subunits. Summary of the invention

[0006] The primary purpose of the present invention is to provide a pseudorabies virus gD recombinant protein, wherein the gD recombinant protein comprises a signal peptide, a connecting peptide, gD protein positions 21-350, a restriction site, and a His tag in sequence from the N-terminus to the C-terminus, and the amino acid sequence of the gD protein positions 21-350 is shown in SEQ ID No.4.

[0007] Preferably, the signal peptide is one or more of BV GP64, Melittin, Chitinase, and HIV-ENV.

[0008] Preferably, there are at least 6 His tags.

[0009] Preferably, there are 6 His tags.

[0010] The second object of the present invention is to provide the biomaterial related to the gD recombinant protein as shown in any one of the following B1)-B5):

[0011] B1) a nucleic acid molecule encoding the gD recombinant protein;

[0012] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0013] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0014] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0015] B5) A transgenic animal cell line containing the nucleic acid molecule described in B1) or a transgenic animal cell line containing the expression cassette described in B2).

[0016] The third object of the present invention is to provide the use of the gD recombinant protein as shown in any one of the following P1-P3:

[0017] P1. Application of the gD recombinant protein in the preparation of anti-pseudorabies virus serum;

[0018] P2. Use of the gD recombinant protein in the preparation of pseudorabies virus antibodies;

[0019] P3. Application of the gD recombinant protein in the preparation of an anti-pseudorabies virus vaccine;

[0020] The gD recombinant protein includes a signal peptide, a connecting peptide, gD protein positions 21-350, a restriction site, and a His tag in sequence from the N-terminus to the C-terminus. The amino acid sequence of the gD protein positions 21-350 is shown in SEQ ID No.4.

[0021] The fourth object of the present invention is to provide a pseudorabies subunit vaccine, wherein the pseudorabies subunit vaccine comprises the gD recombinant protein.

[0022] Preferably, the pseudorabies subunit vaccine further comprises an adjuvant.

[0023] Preferably, the adjuvant is a chemical immune adjuvant.

[0024] The fifth object of the present invention is to provide the use of the pseudorabies subunit vaccine in the preparation of a product for preventing or treating pseudorabies.

[0025] The sixth object of the present invention is to provide a method for preparing the pseudorabies subunit vaccine, wherein the method comprises emulsifying the envelope protein gD of the pseudorabies virus JM strain.

[0026] The beneficial effects of the present invention are as follows: (1) The present invention provides a pseudorabies virus gD recombinant protein, which has strong immunogenicity and has the potential to prepare subunit vaccines. (2) The subunit vaccine prepared by the pseudorabies virus gD recombinant protein can produce specific antibodies with neutralizing activity. When mice and pigs are challenged with the virus, no death occurs, indicating that the protection rate of gD is 100%, and pathological sections only show slight pathological changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Identification of the immunogenicity of PRV protein

[0028] Figure 2 Analysis of PRV gD protein

[0029] Note: A. Signal peptide prediction B. Transmembrane domain prediction C. Recombinant gD protein design

[0030] Figure 3 Expression, purification and identification of PRV gD protein Note: A. SDS-PAGE identification of recombinant gD protein expression and purification 1. Cell culture supernatant; 2. Purified protein; B. Western blotting identification of purified protein

[0031] Figure 4 Mice immunized with gD were protected from PRV-JM strain challenge Note: A. Indirect ELISA test results of gD antibody in experimental mice; B. Survival rate of mice after challenge; C. Pathological changes in organs of experimental mice

[0032] Figure 5 Detection of PRV antibodies in immunized pigs Note: A. Indirect ELISA test results of gD antibodies in experimental pigs; B. Neutralizing antibody titer test results in experimental pigs

[0033] Figure 6 gD-immunized pigs can resist PRV-JM strain attack Note: A. Changes in body temperature of experimental pigs after virus attack; B. Survival rate of experimental pigs; C. Pathological changes in organs of experimental pigs DETAILED DESCRIPTION

[0034] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0035] Example 1: Pseudorabies subunit vaccine

[0036] 1. Cells, viruses and plasmids

[0037] HEK293T cells, PK-15 cells and Vero cells (laboratory storage) were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum in an atmosphere containing 5% CO. 2 Cultured in an incubator at 37°C. Sf9 cells were cultured in a 28°C shaking incubator in Sf9 medium (Yishengke IB905, China). The PRV-JM strain was isolated and preserved by our laboratory (GeneBank: OK338077.1) and amplified in PK-15 cells. The full-length sequences of each PRV gene were amplified and cloned into the pCAGGS-Flag vector, respectively, and named Flag-X (X is the name of each gene). The PRV gD gene was truncated and optimized, the corresponding cDNA was synthesized, and cloned into the pFASTBac-EGFP vector, named pFastBac-EGFP-gD.

[0038] 2. Experimental Methods

[0039] 2.1 Indirect immunofluorescence

[0040] HEK293T cells were transfected with plasmids encoding PRV proteins, and the cells were fixed with 4% paraformaldehyde 24 h later and permeabilized with 0.3% TritonX-100. After blocking, PRV-positive serum and anti-Flag antibody were used as primary antibodies, and FITC-labeled goat anti-pig IgG (Biolong; China) or Alexa Fluor488 anti-mouse IgG (Invitrogen; USA) were used for immunofluorescence staining. All samples were observed and photographed using a Zeiss fluorescence microscope.

[0041] 2.2 Expression, purification and western blotting of PRV gD protein

[0042] The signal peptide and transmembrane domain of gD were analyzed respectively. The signal peptide of gD was analyzed using the website (https: / / www.novopro.cn / tools / signalp.html). The transmembrane domain of gD was analyzed using the website (https: / / www.novopro.cn / tools / tmhm.html). The sequence of gD was codon-optimized using the website (https: / / sg.idtdna.com / CodonOpt) and fully synthesized by Jilin Kumei Biotechnology Co., Ltd. The gD protein was expressed using the Bac-to-Bac system: the transfer plasmid pFastBac-EGFP-gD was transformed into DH10Bac competent cells (Huayueyang Biotechnology Co., Ltd.) to produce a recombinant baculovirus named Bac-gD. Sf9 cells were infected with Bac-gD (P3) to express the protein. The supernatant of Sf9 cells cultured for 72 hours was collected for protein purification. The Sf9 culture supernatant and purified proteins were subjected to SDS-PAGE and transferred to a PVDF membrane. Mouse anti-His antibody (Sigma-Aldrich, USA) was used as the primary antibody. Western blotting was performed using 800CW goat anti-mouse IgG (H+L) (926-32210) as the secondary antibody (LI-COR; USA). The membrane was observed using the Odyssey CLX infrared fluorescence imaging system (LI-COR, USA).

[0043] 2.3 Indirect ELISA detection method

[0044] The indirect ELISA method established in our laboratory was used to detect the experimental serum. The gD protein coating concentration was 0.5ug / mL, the coating condition was 4℃ overnight, the experimental serum dilution was 1:200, and the incubation condition was 25℃ for 20min; HRP-goat anti-swine IgG (Biolong; China) diluted 1:30000 or HRP-goat anti-mouse IgG (Sigma-Aldrich; USA) diluted 1:10000 was used as the secondary antibody, and the incubation condition was 37℃ for 20min; TMB substrate was used for color development at 37℃ for 15min, 2M H 2 SO 4 OD was read using an ELISA reader. 450nm Calculate (sample OD 450nm Value - negative control OD 450nm value) / (positive control OD 450nm Value - negative control OD 450nm value).

[0045] 2.4 Vaccination and challenge of mice

[0046] Four-week-old female BALB / c mice (n=10) were randomly divided into two groups: PBS group and gD group. The gD group was immunized by intramuscular injection with 0.1 ml PBS mixed with 50 μg protein and an equal volume of ISA201 adjuvant in a 1:1 ratio. The PBS group was immunized with 0.1 ml PBS mixed with an equal volume of ISA201 adjuvant in a 1:1 ratio. All mice were immunized twice on days 1 and 14, and challenged with 3×10 4 TCID 50 / mouse). Blood was collected once a week before and after immunization. After challenge, the clinical symptoms and mortality of mice were recorded every day. After the death of mice, the heart, liver, spleen, lung, kidney, brain, tonsil and lymph node were collected. The lesions were observed and histopathological analysis was performed. 14 days after challenge, the mice were euthanized, and the heart, liver, spleen, lung, kidney, brain tissue, tonsil and lymph node were collected for further experiments.

[0047] 2.5 Vaccination and challenge of piglets

[0048] Eight healthy piglets without PRRSV, CSFV, PCV2 and PRV infection were randomly divided into two groups: PBS group (n = 3) and gD group (n = 5). The gD group was immunized by intramuscular injection with 500ug protein mixed with ISA201 adjuvant in a ratio of 1:1. The PBS group was immunized with 0.5ml PBS mixed with ISA201 adjuvant in a ratio of 1:1. All pigs were immunized twice on the 1st and 14th days, and PRV-JM strain (10 5 TCID 50 During the immunization period, blood was collected once a week and once every 3 days after the challenge to detect the neutralizing antibody and specific antibody titer of the immunized serum. After the challenge, the rectal temperature and death of the pigs were recorded every day. The surviving pigs were euthanized 28 days after the death of the pigs or the challenge, and the heart, liver, spleen, lungs, kidneys, brain, tonsils and lymph nodes were collected to observe the lesions and conduct the next experiment.

[0049] 2.6 Neutralization test

[0050] The collected serum samples were inactivated at 56°C for 30 minutes. The serum was diluted 2-fold using DMEM containing 2% serum. The diluted serum was mixed with an equal volume of 100 TCID 50 The mixture was added to Vero cells cultured in 96-well plates and incubated at 37°C for 2-5 days. The cells were examined microscopically to determine the cytopathic effect (CPE). The serum neutralization titer was calculated using the Reed-Muench method.

[0051] 2.7 Quantification of viral load in tissues and organs

[0052] 100 mg of each lung, brain, tonsil and lymph node tissues were ground in PBS (weight / volume = 1:3) under sterile conditions. After complete grinding, the samples were centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected, and genomic DNA was extracted using a genomic DNA extraction kit (Tiangen, China). qPCR was performed on the QuantStudio 5 real-time PCR system using gB-specific primers and probes to detect tissue viral load. Primers (5'-ACGCACGGGCGTGATC-3' and 5'-ACCCGGTCCCGAGCA-3') and probe gB-TaqMan-probe (FAM-CTCGCGCGCGCGACCTCACGAGCCTCGCAC-MGB).

[0053] 2.8 Statistical analysis

[0054] All data were analyzed using GraphPad Prism software. Statistical analysis among multiple groups was performed using one-way analysis of variance (SPSS). p values ​​less than 0.05 were considered statistically significant.

[0055] 3. Results

[0056] 3.1 Screening and identification of immunogenic PRV proteins

[0057] Each PRV protein expression plasmid was transfected into HEK293T cells, and PRV immunogenic proteins were screened by IFA using anti-PRV positive serum and anti-Flag serum. Figure 1 Eukaryotically expressed PRV proteins that can react with PRV-positive serum are shown, and cells transfected with these proteins show varying degrees of green fluorescence. Among them, the main envelope proteins gB (UL27), gC (UL44) and gD (US6) have strong immunogenicity. At the same time, according to the results of PRV vaccine immunization and virus challenge tests in this laboratory: gD antibodies detected higher antibody levels 2 weeks after immunization, and responded quickly after challenge and lasted for a long time, and the overall effect was better than gB and gC proteins. Therefore, this project selected gD protein as the subunit vaccine antigen.

[0058] 3.2 Expression, purification and detection of gD

[0059] The insect expression system was selected for secretory expression of gD protein. First, the hydrophobicity, signal peptide and transmembrane region of gD were analyzed through online websites, and it was found that aa 1-17 of gD was the signal peptide, aa 18-353 was the extracellular domain, aa 354-376 was the transmembrane region, and aa 377-402 was the intracellular domain ( Figure 2 AB). In order to increase hydrophilicity, according to the hydrophobicity analysis results ( Figure 2 C), the cDNA corresponding to aa 21-350 of gD was cloned into a vector to construct a recombinant expression plasmid, the nucleotide sequence of aa 21-350 of gD was shown in SEQ ID No.3, the amino acid sequence was shown in SEQ ID No.4, and a signal peptide was fused to the N-terminus of aa 21-350 of the gD protein, and a 6×His tag was added to the C-terminus of gD to facilitate purification, to obtain a gD recombinant protein, the nucleotide sequence of which was shown in SEQ ID No.1, and the amino acid sequence was shown in SEQ ID No.2 ( Figure 2 E). The purified protein was detected by SDS-PAGE and western blotting, and the target protein band was detected ( Figure 3 ).

[0060] 3.3 Mouse immune protection experiment

[0061] The experimental results are as follows Figure 4 As shown, the gD group produced higher levels of specific antibodies after immunization ( Figure 4 A), when mice did not die after the challenge, it showed that the protection rate of gD was 100% ( Figure 4 B). Mice that survived 14 days after the challenge were autopsied, and pathological sections showed only slight pathological changes ( Figure 4 C). Mice in the PBS group developed obvious pruritus and even hind limb fractures after being challenged with PRV-JM strain, and all died on the 4th day after challenge. However, due to the early death time, the pathological changes were not significant ( Figure 4 C).

[0062] 3.4 Piglet immune protection experiment

[0063] The experimental results are as follows Figure 5 and 6 As shown in Figure 2, pigs in the gD group produced higher levels of specific antibodies against gD on day 14 after immunization ( Figure 5 A), and can produce high levels of neutralizing antibodies ( Figure 5 B). After the challenge, the experimental pigs in the PBS group showed clinical symptoms such as skin itching and loss of appetite, and died on the 6th day. The rectal temperature of the experimental pigs in the gD group only showed a brief transient increase, no other clinical symptoms, and all survived ( Figure 6 AB).

[0064] Autopsy revealed no significant pathological changes in the lungs, mandibular lymph nodes, and tonsils of the experimental pigs in the gD group ( Figure 6C). The lungs of the experimental pigs in the PBS group showed extensive inflammatory cell infiltration and congestion, accompanied by widening of the alveolar septum; the mandibular lymph nodes showed a significant decrease in lymphocytes, and scattered hemorrhages in the paracortical area; the lungs showed extensive inflammatory cell infiltration and congestion, accompanied by widening of the alveolar septum ( Figure 6 C).

[0065] In summary, the present invention provides a pseudorabies virus gD recombinant protein, which has strong immunogenicity and has the potential to prepare subunit vaccines. The subunit vaccine prepared by the pseudorabies virus gD recombinant protein can produce specific antibodies with neutralizing activity, and the protection rate is 100% after the mice are challenged with the virus. The virus load of the immunized pigs after the virus is low, and the pathological sections only show slight pathological changes, and the protection rate is 100%, which has broad application prospects.

Claims

1. A pseudorabies virus gD recombinant protein, characterized in that: The gD recombinant protein includes a signal peptide, a connecting peptide, gD protein positions 21-350, a restriction site, and a His tag in sequence from the N-terminus to the C-terminus. The amino acid sequence of the gD protein positions 21-350 is shown in SEQ ID No.

4.

2. The pseudorabies virus gD recombinant protein according to claim 1, characterized in that The signal peptide is one or more of BV GP64, Melittin, Chitinase and HIV-ENV.

3. The pseudorabies virus gD recombinant protein according to claim 1, characterized in that The His tags are provided with at least 6.

4. The biological material related to the gD recombinant protein according to any one of claims 1 to 3 is any one of the following B1) to B5): B1) a nucleic acid molecule encoding the gD recombinant protein according to any one of claims 1 to 3; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic animal cell line containing the nucleic acid molecule described in B1) or a transgenic animal cell line containing the expression cassette described in B2).

5. Use of the gD recombinant protein as claimed in claim 1 in any one of the following P1-P3: P1. Application of the gD recombinant protein in the preparation of anti-pseudorabies virus serum; P2. Use of the gD recombinant protein in the preparation of pseudorabies virus antibodies; P3. Application of the gD recombinant protein in the preparation of an anti-pseudorabies virus vaccine; The gD recombinant protein includes a signal peptide, a connecting peptide, gD protein positions 21-350, a restriction site, and a His tag in sequence from the N-terminus to the C-terminus. The amino acid sequence of the gD protein positions 21-350 is shown in SEQ ID No.

4.

6. A pseudorabies subunit vaccine, characterized in that: The pseudorabies subunit vaccine comprises the gD recombinant protein according to any one of claims 1 to 3.

7. The pseudorabies subunit vaccine according to claim 6, characterized in that: The pseudorabies subunit vaccine also includes an adjuvant.

8. The pseudorabies subunit vaccine according to claim 7, characterized in that The adjuvant is a chemical immune adjuvant.

9. Use of the pseudorabies subunit vaccine according to any one of claims 6 to 8 in the preparation of a product for preventing or treating pseudorabies.

10. A method for preparing the pseudorabies subunit vaccine according to any one of claims 6 to 8, characterized in that: The method is to emulsify the envelope protein gD of the pseudorabies virus JM strain.

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