A truncated protein of gD of pseudorabies virus of pig, a preparation method thereof, a subunit vaccine containing the truncated protein and application thereof

Abstract

The application discloses a pig pseudorabies virus gD truncated protein, a preparation method of the pig pseudorabies virus gD truncated protein, a subunit vaccine containing the truncated protein and application of the subunit vaccine, and the amino acid sequence of the gD truncated protein is shown as SEQ ID NO. 2 or SEQ ID NO. 4. The application finds that a C-terminal membrane proximal region (about 267-355 aa) of a PRV gD protein is a key functional region causing acute stress reaction of previously immunized piglets. Two truncated bodies T-266 and T-301 are determined through truncation screening, and a subunit vaccine is prepared in cooperation with a water adjuvant. Safety experiments show that no stress reaction occurs at a dose of 50-100 μg per head; and efficacy tests show that a neutralizing antibody level is equivalent to that of a full-length protein. Through the double strategies of truncating the gD protein and adopting the water adjuvant, the application overcomes the defects that the existing gD subunit vaccine causes stress reaction in previously immunized animals.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to a porcine pseudorabies virus gD truncated protein, its preparation method, and a subunit vaccine containing the truncated protein and its application. Background Technology

[0002] Porcine pseudorabies (PR) is an important porcine infectious disease caused by pseudorabies virus (PRV), and vaccination is its main means of prevention and control. PRV gD protein is a key glycoprotein on the viral envelope, playing a central role in viral adsorption and invasion, and is an important target antigen for inducing neutralizing antibodies. Marchioli et al. first reported the expression of gD protein in CHO cells and the evaluation of its vaccine efficacy in Marchioli CC, Yancey RJ, Petrovskis EA, et al. Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells[J]. Journal of Virology, 1987, 61(12): 3977-3982. In recent years, several institutions have been developing gD subunit vaccines, including the gD truncated protein (21-350aa) from Harbin Veterinary Research Institute (Chinese patent application CN202410634151.6, filed by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, entitled "A pseudorabies virus gD recombinant protein, subunit vaccine and its preparation method and application") and the gB-gD fusion protein from Keqian Biotechnology (Chinese patent application CN202610120715.3, filed by Wuhan Keqian Biotechnology Co., Ltd., entitled "A porcine pseudorabies virus gB-gD recombinant fusion protein subunit vaccine and its preparation method and application").

[0003] The PRV gD protein is approximately 402 amino acids in length and belongs to the type II transmembrane protein category. Li A et al., in Li A, Lu G, Qi J, et al. Structural basis of nectin-1 recognition by pseudorabies virus glycoprotein D[J]. PLoS Pathogens, 2017, 13(5): e1006314, resolved the crystal structure of the PRV gD and nectin-1 receptor complex, clarifying the structural division of the extracellular region: the N-terminal core receptor-binding domain (approximately 19-266 aa) forms a stable immunoglobulin-like fold, responsible for binding to the host receptor nectin-1, containing major B-cell antigen epitopes, and its folding is independent of the C-terminal sequence; the C-terminal proximal membrane region (approximately 267-355 aa, abbreviated as MPR) is mainly characterized by random coils, belonging to the intrinsic disordered region, and participates in the regulation of viral membrane fusion, see Schröter C, Klupp BG, Fuchs W, et al. The highly conserved proline at position 438 in pseudorabies virus gH is important for regulation of membrane fusion[J]. Journal of Virology, 2015, 89(1): 1-12, but this region cannot be resolved in crystal structure, and its other biological functions besides membrane fusion have not been reported in the prior art.

[0004] The applicant previously filed a related patent application (CN202310532797.9, invention title: Preparation method of porcine pseudorabies virus gD protein and subunit vaccine and application of porcine pseudorabies virus), involving a method for preparing the full-length extracellular region of porcine pseudorabies virus gD protein and a subunit vaccine. However, in subsequent experiments, the applicant unexpectedly discovered that when a subunit vaccine was prepared using a full-length gD extracellular protein containing the intact C-terminal membrane proximal region and an aqueous adjuvant, and then immunized, significant acute stress or allergic-like adverse reactions occurred in piglets that had previously been vaccinated with live pseudorabies vaccine or had been infected with wild-type virus (stable within the dose range of 30-200 μg / dose, showing a dose-independent effect). Based on the analysis, the applicant proposes the following mechanism hypothesis: the MPR region may induce an acute immune stress response in previously immunized or infected animals by cross-reacting with host membrane fusion-related proteins or activating specific signaling pathways. Existing literature reports gD protein truncation protocols (such as the 21-350aa truncated variant from the Harbin Veterinary Research Institute), but these still retain most of the MPR region (267-350aa). According to the applicant's data, the truncated variant retaining 267-330aa still induced stress responses in previously immunized animals (5 / 5), therefore existing truncation protocols cannot solve this problem. It is worth noting that existing studies on the function of the PRV gD protein MPR are limited to membrane fusion regulation, and no literature has ever reported an association between this region and immune side effects. Therefore, existing technologies neither recognize the existence of this problem nor provide any solutions.

[0005] Water adjuvants (such as MONTANIDE) TM GEL 01, among others, is a novel non-oil-based adjuvant with significant advantages: low viscosity and convenient injection, significantly reducing pain and stress responses in animals during injection; good biocompatibility, minimizing the formation of granulomas or local tissue damage; complete biodegradability in animals, eliminating the risk of drug residues; and the ability to simultaneously activate humoral and cellular immunity, inducing a more balanced immune response. Therefore, aqueous adjuvants are an ideal choice for preparing low-stress subunit vaccines.

[0006] Currently, oil adjuvants commonly used in veterinary vaccines (such as MONTANIDE) TM ISA 206, MONTANIDE TM While adjuvants such as ISA 201VG can effectively enhance the immune response, they have several drawbacks: they easily form granulomas or aseptic abscesses after injection, leading to local tissue damage; oil emulsions have high viscosity, making injection difficult and easily causing pain and stress in animals; oil adjuvants are metabolized slowly in animals, posing a risk of drug residues and affecting meat safety. Therefore, developing non-oil adjuvant vaccines with higher safety and lower stress response has significant clinical application value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a modified pseudorabies virus gD protein antigen and a matching low-stress adjuvant system. By truncating the C-terminal proximal membrane region of the gD protein that is associated with abnormal stress response, and using a water adjuvant, the risk of adverse reactions in previously immunized or previously infected animals is significantly reduced or eliminated while retaining effective immunogenicity.

[0008] This invention is based on a key discovery: the C-terminal proximal membrane region (approximately 267-355 aa) of the PRV gD protein is a critical functional region causing acute stress responses in previously immunized or infected pigs. Based on this discovery, this invention provides a truncated gD protein with the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4, and uses an aqueous adjuvant to prepare a subunit vaccine. This truncated protein completely retains its immunogenicity while eliminating the risk of stress responses; the aqueous adjuvant further reduces local and systemic stress responses caused by vaccine injection. Together, these factors achieve high safety and high immunogenicity of the vaccine.

[0009] According to a first aspect of the present invention, a truncated gD protein of porcine pseudorabies virus is provided, characterized in that the amino acid sequence of the truncated gD protein is as shown in SEQ ID NO.2 or SEQ ID NO.4. SEQ ID NO.2 corresponds to amino acids 1-266 of the PRV gD protein and has a 6×His tag fused to its C-terminus; SEQ ID NO.4 corresponds to amino acids 1-301 of the PRV gD protein and has a 6×His tag fused to its C-terminus.

[0010] According to a second aspect of the present invention, the present invention also provides a gene sequence encoding a truncated gD protein, said gene sequence being optimized with mammalian cell-preferred codons, wherein the codon-optimized gene sequence encoding the truncated protein shown in SEQ ID NO.2 is shown in SEQ ID NO.1; and the codon-optimized gene sequence encoding the truncated protein shown in SEQ ID NO.4 is shown in SEQ ID NO.3.

[0011] According to a third aspect of the present invention, the present invention provides a method for preparing the gD truncated protein, comprising the following steps: The codon-optimized gD truncated protein encoding gene (SEQ ID NO.1 or SEQ ID NO.3) was cloned into a eukaryotic expression vector to obtain a recombinant plasmid; 2) the recombinant plasmid obtained in step 1) was transfected into CHO cells, and CHO cell lines that stably express gD truncated protein were screened to obtain them; 3) the cell lines obtained in step 2) were domesticated to suspension culture to establish a high-expression cell line; and 4) the high-expression cell line obtained in step 3) was fermented and cultured, and the recombinant gD truncated protein was purified from the culture supernatant.

[0012] According to the technical solution of the present invention, preferably, the eukaryotic expression vector is pEE12.4. More preferably, the CHO cells are CHO-K1 cells.

[0013] According to a fourth aspect of the present invention, the present invention provides a low-stress response porcine pseudorabies virus subunit vaccine, comprising: 1) the gD truncated protein at a concentration of 50–100 μg / dose; and 2) an aqueous adjuvant.

[0014] According to the technical solution of the present invention, preferably, the volume ratio of the water adjuvant to the gD truncated protein is 1:9.

[0015] This invention uses a water-based adjuvant instead of a traditional oil-based adjuvant, wherein the water-based adjuvant is MONTANIDE. TM GEL 01. This aqueous adjuvant has low viscosity and is easy to inject, which can significantly reduce pain and stress response in animals during injection; the aqueous adjuvant has good biocompatibility at the injection site and is less likely to form granulomas or local tissue damage; the aqueous adjuvant is completely biodegradable in animals with no risk of drug residue; at the same time, it can effectively enhance humoral and cellular immune responses induced by gD truncated protein.

[0016] According to a fifth aspect of the invention, the invention also provides the use of the gD truncated protein or a subunit vaccine containing the truncated protein in the preparation of a vaccine for the prevention of porcine pseudorabies. Particularly, its use in the preparation of a low-stress vaccine suitable for pig herds previously vaccinated with a live pseudorabies vaccine or previously infected with wild-type virus.

[0017] The beneficial effects of this invention are as follows: 1) It has been discovered and confirmed that the proximal region of the C-terminal membrane of the PRV gD protein (approximately 267-355 aa) is a key functional region causing acute stress responses in previously immunized pigs. 2) Safety experiments show that no stress response was observed when gD truncated protein T-266 was administered at doses ranging from 30-200 μg / dose (0 / 5); no stress response was observed when gD truncated protein T-301 was administered at doses ranging from 30-200 μg / dose (0 / 5); while moderate to severe acute stress responses were observed when gD protein T-349 was administered at the same dose (5 / 5). 3) Efficacy tests show that the level of neutralizing antibodies induced by gD truncated protein was not significantly different from that induced by gD protein T-349. 4) The CHO-K1 cell lines 4C2 (T-266) and 7D2 (T-301) constructed in this invention express gD truncated protein at a yield of 2-3 g / L, with a purity of over 90% after one-step purification with Ni-NTA, demonstrating feasibility for large-scale production. 5) This invention uses an aqueous adjuvant (MONTANIDE GEL 01) instead of the traditional oil adjuvant. This aqueous adjuvant has low viscosity, is easy to inject, and significantly reduces pain and stress responses in animals during injection; it has good biocompatibility at the injection site, making it less likely to form granulomas or cause local tissue damage; the aqueous adjuvant is completely biodegradable in animals, with no risk of drug residues; simultaneously, it effectively enhances the humoral and cellular immune responses induced by gD truncated protein, synergistically achieving high safety and high immunogenicity of the vaccine. Attached Figure Description

[0018] Figure 1 This indicates the predicted structure of the gD protein; Figure 2 The diagram shows the PRV gD protein domain and the truncation strategy; A represents the division of the full-length gD protein domain; B represents the truncation positions of truncated body 1 (1-266aa) and truncated body 2 (1-301aa). Figure 3 The results of SDS-PAGE purification of gD truncated T-266 protein are shown; M is the protein marker, and 1 is the gD truncated T-266 protein. Figure 4 The results of SDS-PAGE for the purification of gD truncated T-301 protein are shown; M is the protein marker, and 1 is the gD truncated T-301 protein. Figure 5 The result of SDS-PAGE for the purification of gD protein T-349 is shown; M is the protein marker, and 1 is gD protein T-349. Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments and accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0020] Sources of reagents and materials CHO-K1 cells: sourced from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee / Cell Bank of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences; Cell culture medium and serum: both were purchased from Gibco, USA; eukaryotic expression vector pEE12.4: purchased from Shanghai Linyuan Biotechnology Co., Ltd.; Lipofectamine LTX: Purchased from Thermo Fisher Scientific, USA; MSX: purchased from Sigma-Aldrich; BCA reagent kit: purchased from Thermo Fisher Scientific. MONTANIDE TM ISA 201 VG: Purchased from ISABIK, France.

[0021] Sequence information description SEQ ID NO.1: Codon-optimized PRV gD truncated variant 1 (1-266aa) coding gene sequence, 819 bp; Source: Artificial sequence (codon-optimized); Description: Codon-optimized PRV gD truncated variant 1 protein coding gene, encoding amino acids 1-266, with 6×His at the C-terminus; SEQ ID NO.2: PRV gD truncated variant 1 (1-266 aa) amino acid sequence, 272 aa; Source: Artificial sequence; Description: PRV gD truncated variant 1 protein amino acid sequence, containing a C-terminal 6×His tag; SEQ ID NO.3: Codon-optimized PRV gD truncated variant 2 (1-301aa) coding gene sequence, 924 bp; Source: Artificial sequence (codon-optimized); Description: Codon-optimized PRV gD truncated variant 2 protein coding gene, encoding amino acids 1-301, with a 6×His tag at the C-terminus; SEQ ID NO.4: PRV gD truncated variant 2 (1-301aa) amino acid sequence, 307 aa; Source: Artificial sequence; Description: PRV gD truncated variant 2 protein amino acid sequence, containing a C-terminal 6×His tag.

[0022] Example 1: Design and sequence optimization of the gD truncated protein of porcine pseudorabies virus 1.1 Analysis of gD protein domains and determination of truncation strategy Three-dimensional structure prediction of PRV gD protein using AlphaFold2 ( Figure 1 The prediction results show that the extracellular region of the gD protein can be divided into two subregions with significantly different structural features: the N-terminal region (approximately 18-266 aa): forms a stable immunoglobulin-like fold structure and is the receptor-binding core domain; the C-terminal region (approximately 267-402 aa): is mainly characterized by random coils, with low AlphaFold prediction confidence (pLDDT < 70), and is an intrinsically disordered region, which is related to membrane fusion regulation function.

[0023] To determine the optimal truncation boundary, this invention designed the coding genes for three different truncation segments ( Figure 2 The amino acid sequences of T-266 (1-266aa) and T-301 (1-301aa) of gD protein are respectively compared with T-349 (1-349aa). T-266 and T-301 are preferred truncated variants, and their amino acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0024] 1.2 Codon Optimization and Gene Synthesis

[0025] Referring to the prior application CN202310532797.9, the nucleotide sequence of the extracellular region (T-349) was truncated. The gene sequence is shown in SEQ ID NO.1 (T-266) and SEQ ID NO.3 (T-301).

[0026] Example 2: Detection of receptor binding activity of gD truncated protein 2.1 Assay of nectin-1 receptor binding activity

[0027] The binding affinity of different truncated variants to the nectin-1 receptor of T-349 (gD extracellular protein (1-349aa)) was compared using ELISA.

[0028] 1) Coat ELISA plates with 100 ng / well of recombinant nectin-1 receptor protein and incubate overnight at 4°C; 2) Add serially diluted (0.1-100 nM) gD truncated protein or full-length gD protein and incubate at 37°C for 1 h; 3) Add anti-His tag-HRP antibody to detect binding signal and measure OD450 nm; 4) Calculate the half-maximal effect concentration (EC50) value.

[0029] Table 1 .

[0030] The results in Table 1 show that the receptor binding activity of the two truncated forms of nectin-1 was not significantly different from that of T-349 (P>0.05), indicating that truncating the proximal C-terminal membrane region does not affect the receptor recognition function of the N-terminal core receptor binding domain.

[0031] Example 3: Construction of recombinant expression plasmid and expression in CHO cells 3.1 Construction of Recombinant Plasmids The gD truncated protein-coding gene (SEQ ID NO.1 or SEQ ID NO.3) was constructed according to the patented method and cloned into the eukaryotic expression vector pEE12.4 via HindIII / EcoRI double restriction sites, constructing recombinant plasmids pEE12.4-gD-T1 and pEE12.4-gD-T2. The sequences were confirmed to be correct by restriction enzyme digestion and sequencing.

[0032] 3.2 CHO-K1 cell transfection and screening

[0033] 1) Recombinant plasmids were transfected into CHO-K1 cells using Lipofectamine LTX; 2) 24 h after transfection, use DMEM / F12 medium containing 25 μM MSX for pressure selection for 7-10 days; 3) Monoclonal screening was performed using the limiting dilution method, and gD protein expression levels were detected by ELISA; 4) Select the positive clone with the highest expression level for subsequent experiments.

[0034] 3.3 Suspension Acclimation and Fermentation The high-expression cell lines were gradually acclimatized to suspension culture: the medium was gradually transitioned from serum-containing medium to serum-free medium (CD-CHO:Ex-cell 302 = 6:4) over a period of approximately 7 weeks. The acclimatized cells were then fermented in shake flasks at an inoculation density of 2.5–3.5 × 10⁵ cells / mL, at a temperature of 37°C (reduced to 32°C from day 5 onwards), for a 12-day culture period, with feed added on days 4 and 9. Results showed that the 4C2 (T-266) and 7D2 (T-301) cell lines exhibited the highest expression levels of gD truncated protein, with yields reaching 2–3 g / L.

[0035] 3.4 Protein purification and identification The fermentation supernatant was purified by Ni-NTA affinity chromatography (washing with 20 mM imidazole, elution with 400 mM imidazole), and the purified protein was obtained after dialysis and medium exchange. SDS-PAGE analysis showed a purity of over 90%. Figure 3 , Figure 4 , Figure 5These are the purification results of gD truncated variant (T-266), gD truncated variant (T-301), and the gD protein T-349 described in the prior application.

[0036] Example 4: Vaccine preparation and safety evaluation 4.1 Vaccine Preparation

[0037] MONTANIDE TM Preparation of GEL 01 adjuvant (aqueous adjuvant) vaccine: Take 50 ml of protein solution, add an appropriate amount of sterile PBS and mix well; at room temperature, add 10 ml of MONTANIDE at an adjuvant:antigen (v:v) ratio of 1:9. TM GEL 01 adjuvant is slowly added to the antigen solution while stirring. After the addition is complete, stirring continues for 10 minutes to form a homogeneous emulsion. Once emulsification is successful, the emulsion is dispensed, sealed, and labeled, and stored at 2–8°C. Specific vaccine information is shown in Table 2 below, where vaccine 13 is a control without added protein.

[0038] Table 2 .

[0039] 4.2 Safety Experiment in Pigs Sixty-five 4-5 week old piglets (piglets immunized with live pseudorabies vaccine via intranasal drip at birth) were randomly divided into 13 groups of 5 piglets each. The control group (vaccine 13) received an intramuscular injection of 1 ml of adjuvant without protein. The remaining 12 groups served as subunit vaccine immunization groups, with each group receiving an intramuscular injection of vaccine 1-vaccine 12 prepared in section 4.1. Stress responses were observed post-immunization. The results are shown in Table 3: Inoculation with gD truncated protein T-266 at doses between 30 and 200 μg / piglet resulted in no stress. Inoculation with gD truncated protein T-301 at doses between 30 and 200 μg / piglet resulted in stress in only one piglet in the 200 μg / piglet group. Inoculation with gD protein T-349 at doses between 30 and 200 μg / piglet resulted in acute stress in all piglets. Stressful piglets exhibit the following symptoms: depression, restlessness, rapid breathing, panting, flushed or cyanotic skin, muscle tremors, stiff limbs or convulsions, as shown in Table 3 below.

[0040] Table 3 .

[0041] The above results indicate that the one case of stress response observed with T-301 at a dose of 200 μg / dose was within the overdose range (2-4 times the recommended immunization dose of 50-100 μg / dose), and only manifested as mild stress symptoms. In contrast, gD extracellular protein T-349 resulted in a 100% moderate to severe acute stress response (5 / 5) at a dose of 30 μg / dose, demonstrating that T-301 has a significantly better safety profile than gD extracellular protein T-349. Furthermore, T-266 did not exhibit any stress response in any of the dose groups (including 200 μg / dose), completely eliminating immune side effects and demonstrating the significant effectiveness of the truncation strategy of this invention.

[0042] 4.3 Efficacy test in pigs

[0043] Vaccines 2, 6, and 10 were selected and administered their second dose 21 days after the first immunization. Vaccine 13 was used as an adjuvant for continued vaccination. Blood samples were collected 7 days after the second immunization, serum was separated, and neutralizing antibodies were detected. The neutralizing antibody preparation steps are as follows: Dilute the serum sample 1:4 with DMEM and heat-inactivate it at 56℃ for 30 minutes; serially dilute the inactivated serum with DMEM, and add an equal volume of virus solution containing 200 TCID50 / 0.1 ml (the pseudorabies virus LY strain was isolated by the company's researchers from the brain tissue of a pig suspected of having pseudorabies in a pig farm in Longyou, Zhejiang Province) to the diluted serum sample, mix thoroughly, and incubate at 37℃ for 1 hour; take 100 μl of serum-virus mixture and add it to 96-well cell culture plates (with a monolayer of Vero cells), adding 4 wells for each dilution, and incubate at 37℃ for 1 hour; discard the supernatant, add 200 μl of DMEM containing 2% FBS, and incubate at 37℃ and 5% CO2 for 96 hours. Observe the CPE daily, and calculate according to the Reed-Muench method. The highest dilution with a CPE of 50% is taken as the neutralizing antibody titer of the serum. The results are shown in Table 4 below: There was no significant difference in the neutralizing antibodies induced by vaccines 2, 6, and 10. The control adjuvant group (vaccine 13) did not induce any neutralizing antibodies against pseudorabies virus. This indicates that the modified gD truncated proteins T-266 and T-301, while ensuring improved safety, did not alter the immunogenicity of the gD protein, as shown in Table 4 below.

[0044] Table 4 .

[0045] The present invention has been illustrated by the above embodiments; however, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments is to assist those skilled in the art in practicing the present invention. Any person skilled in the art can readily make further improvements and modifications without departing from the spirit and scope of the present invention; therefore, the present invention is limited only by the content and scope of the claims, and is intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A truncated gD protein of porcine pseudorabies virus, characterized in that, The amino acid sequence of the gD truncated protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. A gene sequence encoding the gD truncated protein of claim 1, characterized in that, The gene sequences were optimized using mammalian cell-preferred codons. The codon-optimized gene sequence encoding the gD truncated protein as shown in SEQ ID NO.2 is shown in SEQ ID NO.1; and the codon-optimized gene sequence encoding the gD truncated protein as shown in SEQ ID NO.4 is shown in SEQ ID NO.

3.

3. A method for preparing the gD truncated protein of claim 1, characterized in that, Includes the following steps: 1) The codon-optimized gD truncated protein encoding gene was cloned into a eukaryotic expression vector to obtain a recombinant plasmid; wherein the codon-optimized gD truncated protein encoding gene is shown in SEQ ID NO.1 and SEQ ID NO.3; 2) Transfect the recombinant plasmid described in step 1) into CHO cells and screen to obtain CHO cell lines that stably express gD truncated protein; 3) The CHO cell line obtained in step 2) is adapted to suspension culture to establish a high-expression cell line; 4) The high-expression cell line described in step 3) was purified from the culture supernatant to obtain recombinant gD truncated protein.

4. The method according to claim 3, characterized in that, The eukaryotic expression vector is pEE12.

4.

5. The method according to claim 3, characterized in that, The CHO cells are CHO-K1 cells.

6. A low-stress-response porcine pseudorabies virus subunit vaccine, characterized in that, include: 1) The gD truncated protein as described in claim 1, at a concentration of 50–100 μg / dose; 2) Pharmaceutically acceptable aqueous adjuvants.

7. The subunit vaccine according to claim 6, characterized in that, The volume ratio of the aqueous adjuvant to the gD truncated protein is 1:

9.

8. The subunit vaccine according to claim 6 or 7, characterized in that, The aqueous adjuvant is MONTANIDE. TM GEL01.

9. The use of a gD truncated protein as described in claim 1 or a subunit vaccine comprising the truncated protein as described in claim 6 or 7 in the preparation of a vaccine for the prevention of porcine pseudorabies.

Images