Pseudorabies virus gD protein as well as coding gene, preparation method and application thereof

Abstract

The invention discloses a pseudorabies virus gD protein as well as a coding gene, a preparation method and application thereof, and relates to the technical field of genetic engineering and immunology. The amino acid sequence of the pseudorabies virus gD protein is as shown in SEQ ID NO. 2. The nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention provides a PRV (Porcine Reproductive Virus)-gD protein derived from a PRV HeNLH / 2017 strain. The protein can be used as an immunogen to prepare a PRV subunit vaccine. According to the invention, the PRV-gD gene is further optimized to obtain an optimized sequence which is more suitable for efficiently expressing the target protein in CHO (Chinese Hamster Ovary) cells. By adopting the preparation method disclosed by the invention, the soluble expression efficiency of the target protein can be remarkably improved, and the product property is uniform and stable. The PRV subunit vaccine provided by the invention can improve the serum titer level and the neutralization titer level, thereby providing technical support for prevention and control of PRV.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and immunology, in particular to a pseudorabies virus gD protein, a coding gene thereof, a preparation method and application. BACKGROUND

[0002] Pseudorabies virus (PRV) is also known as Aujeszky virus, which belongs to the subfamily of alpha herpes virus and is the only natural host of PRV. After infection, it can cause acute death in piglets and sterility in breeding pigs.

[0003] The PRV virus particle is composed of four parts with different shapes, from inside to outside, it is the central linear double-stranded DNA genome, icosahedral nucleocapsid protein, protein membrane layer and lipid envelope containing viral glycoprotein. The genome of PRV is about 14 kb, and the content of G+C is up to 74%, which contains about 70 open reading frames, which can encode about 70-100 proteins associated with the virus, half of which are non-essential proteins and do not participate in the replication process of the virus, and the rest are mainly capsid proteins, envelope proteins, membrane proteins and various enzymes. At present, 11 kinds of proteins have been identified on the capsid membrane of the virus particle, including gB (UL27), gC (UL44), gD (US6), gE (US8), gG (US4), gH (UL22), gI (US7), gK (UL53), gL (UL1), gM (UL10), gN (UL49.5) 11 kinds of proteins, which all contain glycosylation modification sites (Mo Zhou, Muhammad Abid, et al. 2022). Among them, gD protein can combine with the receptor on the host cell membrane when PRV invades host cells, stimulate the membrane fusion mechanism, and help the virus invade host cells. When the gD gene is deleted, the virus can only transfer between cells, but cannot enter host cells, so it cannot infect the host, which can greatly reduce the infectivity of the virus. gD protein is the main immunogenic glycoprotein of PRV and the most effective virus neutralizing antibody target in PRV, which can stimulate the body to produce immune response and high titer neutralizing antibody, and is an effective antigen protein for preparing subunit vaccine.

[0004] At present, there is no PRV subunit vaccine approved by new veterinary drug, but in recent years, researches have shown that subunit vaccine has important significance for the prevention and control of PRV. Based on this, the present application intends to prepare pseudorabies virus gD protein through eukaryotic expression, and apply it to the preparation of PRV subunit vaccine, so as to provide technical support for the prevention and control of PRV. SUMMARY

[0005] The present application aims to provide a pseudorabies virus gD protein, a coding gene, a preparation method and an application thereof, so as to solve the problems of the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a pseudorabies virus gD protein, and the amino acid sequence of the pseudorabies virus gD protein is shown in SEQ ID NO. 2.

[0008] The present application also provides a coding gene of the above-mentioned pseudorabies virus gD protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

[0009] The present application also provides a biological material for preparing the above-mentioned pseudorabies virus gD protein, and the biological material is the substance in (1) or (2):

[0010] (1) a recombinant plasmid comprising the coding gene mentioned above;

[0011] (2) a recombinant CHO cell comprising the recombinant plasmid.

[0012] The present application also provides a preparation method of the above-mentioned pseudorabies virus gD protein, and the preparation method comprises the following steps: after cell culture of a recombinant CHO cell, a cell culture medium supernatant is separated and obtained, and the cell culture medium supernatant is purified to obtain the pseudorabies virus gD protein.

[0013] The recombinant CHO cell comprises a recombinant plasmid; the recombinant plasmid comprises the coding gene of the above-mentioned pseudorabies virus gD protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1; and / or

[0014] In the recombinant plasmid, an Ig Kappa secretion signal peptide sequence and a Kozak sequence are connected to the 5' end of the coding gene.

[0015] The present application also provides an application of the above-mentioned pseudorabies virus gD protein in preparing a PRV subunit vaccine.

[0016] The present application also provides an application of a combined immunization composition in preparing a PRV subunit vaccine, and the combined immunization composition comprises the above-mentioned pseudorabies virus gD protein and a gp96 N protein.

[0017] The amino acid sequence of the gp96 N protein is shown in SEQ ID NO. 6.

[0018] The present application also provides a PRV subunit vaccine, and the active ingredient comprises the above-mentioned pseudorabies virus gD protein.

[0019] Further, the PRV subunit vaccine further comprises a gp96 N protein.

[0020] The amino acid sequence of the gp96 N protein is shown in SEQ ID NO. 6.

[0021] Further, the mass ratio of the pseudorabies virus gD protein and the gp96 N protein is 3:(1-3).

[0022] Further, the PRV subunit vaccine further comprises a vaccine adjuvant. The vaccine adjuvant can be an ISA 201 adjuvant.

[0023] The present application discloses the following technical effects:

[0024] (1) The present application provides a PRV-gD protein derived from a PRV HeNLH / 2017 strain. The protein can be used as an immunogen to prepare a PRV subunit vaccine. The present application further optimizes the PRV-gD gene to obtain an optimized coding gene sequence that is more suitable for high-efficiency expression of the target protein in CHO cells. In addition, the application of an Ig Kappa secretion signal peptide sequence and a Kozak sequence further increases the soluble expression of the target protein, so that the expressed target protein has higher activity.

[0025] (2) The preparation method of the present application can significantly improve the efficiency of soluble expression of the target protein, and the product is uniform and stable. The expression amount of the target protein in the cell culture supernatant can reach 0.6 mg / mL, which is significantly higher than the yield of the target protein in other prokaryotic expression systems. In addition, the target protein expression system of the present application is a eukaryotic expression system, and the expressed target protein has high glycosylation modification, which is closer to the natural protein state and has higher biological activity.

[0026] (3) The present application provides a combined immunization composition comprising a gp96 N protein and a gD protein. The use of the composition for animal immunization can improve the serum titer level and the neutralization titer level, thereby providing higher protection rate for animals under attack and enhancing the TNF-α, INF-γ, and IL-2 cytokine levels. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0028] Figure 1Figure for identification result of bacterial liquid of recombinant plasmid pLVX-IRES-ZsGreen1-gD and pFASTBac1-gp96 N; wherein, A: PCR identification result of pLVX-IRES-ZsGreen1-gD bacterial liquid, M: Marker, lanes 1-2: pLVX-IRES-ZsGreen1-gD amplification products; B: PCR identification result of pFASTBac1-gp96 N bacterial liquid, M: Marker, lanes 1-3: pFASTBac1-gp96 N amplification products;

[0029] Figure 2 Figure for identification of PRV-gD recombinant protein soluble SDS-PAGE, wherein, M: Marker; lane 1: cell supernatant; lane 2: cell lysate;

[0030] Figure 3 Figure for Western Blot identification of PRV-gD recombinant protein; wherein, M: Marker; lane 1: cell supernatant; lane 2: cell lysate;

[0031] Figure 4 Figure for identification of PRV-gD recombinant protein purification result by SDS-PAGE, M: Marker; lane 1: before purification; lane 2: after purification;

[0032] Figure 5 Figure for identification result of gp96N protein preliminary expression; wherein, A: SDS-PAGE identification result, M: Marker, 1: cell supernatant, 2: cell lysate; B: Western blot identification result, M: Marker, 1: cell supernatant, 2: cell lysate;

[0033] Figure 6 Figure for identification of gp96N protein purification result by SDS-PAGE; wherein, M: Marker; lane 1: before purification; lane 2: after purification;

[0034] Figure 7 Figure for i-ELISA determination of mouse polyclonal serum antibody level; wherein, A: mouse polyclonal serum antibody level after first immunization for 28d; B: mouse polyclonal serum antibody growth and decline regularity chart;

[0035] Figure 8 Figure for significant analysis of neutralization titer of mouse polyclonal serum in different groups;

[0036] Figure 9The figure of the determination results of the spleen lymphocyte cytokines of the mice in different groups; wherein, A: the determination results of the IFN-γ content of the spleen lymphocyte of the mice; B: the determination results of the TNF-α content of the spleen lymphocyte of the mice; C: the determination results of the IL-2 content of the spleen lymphocyte of the mice. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not as limiting the application. It will be appreciated that certain features of the application, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All documents mentioned herein are hereby incorporated by reference in their entirety.

[0038] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for any numerical range recited herein, it is contemplated that every

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0040] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from a review of the description of the application and practice of the application. The description of the application and examples are illustrative only.

[0041] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0042] Construction of recombinant expression plasmid of Example 1

[0043] 1. Design and synthesis of PRV-gD protein coding region gene

[0044] The PRV-gD gene sequence of the present application is a DNA sequence optimized by CHO cell preferred codons, which is as follows:

[0045] The gD protein coding gene from PRV HeNLH / 2017 strain was modified, and the amino acids in the extracellular region were all replaced with the most frequently used nucleotide codon in CHO cells. At the same time, in order to avoid the high GC ratio of translated mRNA, the secondary structure of mRNA affects the translation efficiency, and to avoid some commonly used enzyme cutting sites, the optimal codon frequency is modified. Finally, on the basis of comprehensive consideration of multiple complex factors, a new PRV-gD coding gene is designed, and the nucleotide sequence is shown as SEQ ID NO. 1; the amino acid sequence of PRV-gD protein is shown as SEQ ID NO. 2.

[0046] SEQ ID NO. 1:

[0047] AAGCTTGCCACCATGGAGACAGATACACTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCTACAGGCGATGATGTGGATGCTGTGCCTGCCCCTACATTTCCTCCTCCCGCTTACCCTTATACAGAGTCTTGGCAGCTGACACTGACAACAGTGCCTTCTCCTTTTGTGGGCCCTGCTGATGTGTATCATACTAGACCTCTGGAAGATCCTTGTGGCGTGGTGGCTCTGATTTCTGATCCTCAGGTGGATAGACTGCTGAATGAGGCTGTTGCACATAGGAGACCTACATATAGAGCTCATGTTGCTTGGTATAGAATTGCTGATGGCTGTGCTCACCTGCTGTACTTTATTGAGTATGCTGATTGTGATCCTAGACAGATTTTTGGCAGATGTAGAAGAAGAACTA

[0048] Among them, the bold part represents the signal peptide, and the underlined part represents the 6×His tag.

[0049] SEQ ID NO. 2:

[0050] Among them, the bold part represents the signal peptide, and the underlined part represents the 6×His tag.

[0051] The obtained PRV-gD encoding gene was sent to Shanghai Shengong Bioengineering Co., Ltd. to synthesize into pUC57 vector to obtain pUC57-PRVgD plasmid.

[0052] 2. PCR amplification and double enzyme digestion recovery of PRV-gD gene

[0053] (1) Design and synthesis of primer sequence

[0054] The amplification primer was designed according to the PRV-gD encoding gene, and the forward primer contained an EcoR I restriction endonuclease site, an Ig Kappa secretion signal peptide and a Kozak sequence, and the reverse primer contained a BamH I restriction endonuclease site and a 6×His tag flanking the stop codon. The specific sequence is shown in Table 1.

[0055] Table 1. Primer list

[0056]

[0057]

[0058] Note: " indicates the enzyme digestion site; " indicates the 6×His tag; " indicates the Kozak sequence.

[0059] (2) PCR amplification

[0060] The pCGS3-PRV gD plasmid was used as the template, and the gD-F and gD-R primers were used for PCR amplification. The gD-F and gD-R primer amplification product contains EcoR I and BamH I double enzyme digestion sites; the amplification system is shown in Table 2, and the amplification program is shown in Table 3. The PCR amplification product was observed by 1% agarose gel electrophoresis, and the results showed that the PCR amplification product was about 1106 bp, which was consistent with the expectation, proving that the codon-optimized PRV-gD gene was obtained.

[0061] Table 2. PCR reaction system

[0062]

[0063] Table 3. Amplification program of target gene

[0064]

[0065] (3) Recovery and double enzyme digestion of target gene

[0066] According to the instructions of the universal DNA recovery kit, the gel was cut and the target gene was recovered, and the concentration of the recovered DNA was determined using a NanoDrop™ microspectrophotometer. The recovered target gene and the lentiviral vector pLVX-IRES-ZsGreenl (purchased from Shanghai Link-Med Biotech Co., Ltd.) were double-digested using EcoR I and BamH I restriction enzymes, and the enzyme digestion conditions were 37°C constant temperature enzyme digestion for 4h. The double enzyme digestion system is shown in Table 4.

[0067] Table 4 Double enzyme digestion system

[0068]

[0069] Subsequently, the empty vector plasmid pLVX-IRES-ZsGreenl and the double enzyme digestion product of the target gene were recovered, and T4 DNA ligase was used for 16°C ligation overnight to obtain the recombinant plasmid.

[0070] 3. Construction of recombinant PRV-gD protein expression bacteria

[0071] The recombinant plasmid was transformed into E. coli competent cells BL21, which were cultured on Amp + resistant solid medium, and single colonies were picked for bacterial liquid PCR identification, and positive colonies were selected (see Figure 1 ). The plasmid of the positive colonies was extracted for sequencing, and the sequencing results were correct, and the recombinant plasmid pLVX-IRES-ZsGreenl-gD was named.

[0072] Example 2 Construction of CHO cell strain stably expressing gD protein

[0073] 1. Lentivirus packaging and transduction of CHO cells

[0074] A three-plasmid co-transfection system was used to package the lentivirus according to the total mass of 3μg system. When about 50% of the cells died, the lentivirus suspension was collected and centrifuged, and then added dropwise to the CHO cells. The transduction efficiency and fluorescence intensity of the cells were observed under a microscope.

[0075] 2. Screening and identification of CHO-gD cell strain

[0076] The transduced CHO cells were cultured and subcloned to screen stable cell strains. Finally, a monoclonal cell strain with high fluorescence intensity and stable passage was obtained, and the constructed stable cell strain was named CHO-gD.

[0077] Example 3 Expression identification and purification of CHO-gD protein

[0078] 1. Primary expression and identification of CHO-gD protein

[0079] The CHO-gD cells were cultured at an initial density of 0.5 x 10 6 cells / mL, and 1 mL of cell suspension was collected every 24 h to count the cells and take samples, while 5% fresh SMM CHO-SI medium was added. When the cell viability decreased to about 80% of the previous day, the cell supernatant was collected, and the protein expression was verified by SDS-PAGE, and the results are shown in Figure 2 , and the Western blot verification results are shown in Figure 3 . The results show that the soluble gD protein with a size of about 55 kDa was successfully obtained.

[0080] 2. Purification of CHO-gD protein

[0081] The CHO-gD cells were inoculated at a density of 0.5 x 10 5 cells / mL, and 5% fresh SMM CHO-SI medium was added every 24 h. At 168 h, the cells were centrifuged at 2000 g for 30 min at 4°C, and then the cells at the bottom of the tube were discarded, and the supernatant was collected. The cell supernatant collected after centrifugation was added with a final concentration of 0.01 mol of protease inhibitor, and then the collected cell supernatant and various buffers needed during purification were pumped and filtered using a 0.22 μm filter under reduced pressure, and the filtered sample was collected and placed on ice for standby.

[0082] The supernatant was purified by Ni affinity chromatography column, and the treated sample was added to the column in batches, and the flow rate of the column was adjusted to 0.5 mL / min, and the sample was repeated 2-3 times. The elution method was: Banding Buffer: 800 mL DDW dissolved Na2HPO4·12H2O g, 17.55 g NaCl, 0.67 g NaH2PO4·2H2O, pH adjusted to 7, and DDW added to 2 L. Wash Buffer: Use Banding Buffer to prepare different concentrations of imidazole solution. The elution fractions were collected, and SDS-PAGE was used for identification, and the results are shown in Figure 4 , and the purified gD protein with a size of about 55 kDa and a purity of about 90% was obtained.

[0083] Example 4 Expression and purification of heat shock protein gp96N

[0084] The gene sequence of gp96N protein is derived from the gene sequence of pig-derived gp96 (GenBank Accession No. Y09136). The RNA of PK 15 cells is extracted, and the coding gene of the N-terminal fragment 22-370 amino acids of gp96 is amplified by RT-PCR and PCR. The specific nucleotide sequence is shown in SEQ ID NO. 5. The amino acid sequence of the heat shock protein gp96N encoded by the coding gene is shown in SEQ ID NO. 6.

[0085] SEQ ID NO. 5:

[0086]

[0087] SEQ ID NO.6:

[0088] EDEVDVDGTVEEDLGKSREGSRTDDEIVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALAGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEDDEYKAFYKSFSKESDDPM.

[0089] The coding gene shown in SEQ ID NO. 5 was connected to a pFASTBac1 vector (purchased from Invitrogen) to construct a pFASTBac1-gp96N vector, which was then transformed into DH5a competent cells, single colony was picked, and the bacterial liquid of the sequencing result correct was expanded to extract plasmid, which was transformed into DH10Bac competent cells to prepare a cosmid. The positive cosmid was transiently transfected into sf21 cells, and when the virus was amplified to P3 generation, the preliminary expression of gp96N protein was identified, and the results are shown in Figure 5 .

[0090] Further expansion was carried out, and the cells were inoculated in a triangular flask at a density of 2-3 x 10 6 cells / mL, and P3 generation infected virus was inoculated at a volume ratio of 2%, and cultured in a 28°C, 110 rpm shaking incubator in the dark for 72h, and the supernatant was collected for use. The protein was purified by Ni affinity chromatography combined with anion exchange chromatography, and the purification results are shown in Figure 6 .

[0091] Example 5 Evaluation of the immune effect of CHO-gD protein combined with heat shock protein gp96N immunization

[0092] 1. Mouse immunization strategy and challenge protection experiment scheme

[0093] The antigen mixture was obtained by mixing different groups of proteins according to Table 5, and an equal volume of ISA 201 adjuvant was heated to 31°C in a constant temperature water bath. A magnetic rotor was placed in the container with the adjuvant, and the container was placed on a magnetic stirrer for low-speed stirring for 5 min. During this period, the antigen mixture was slowly added. After stirring, the solution appeared as a milky white liquid. If no stratification occurred, the preparation was successful.

[0094] Table 5: Antigen dose for immunization by groups

[0095]

[0096] Fifty-six 6-8 week old female BALB / c mice were randomly selected and divided into 7 groups, with 8 mice in each group. The emulsion was transferred to a 1 mL sterile syringe, and the mice were immunized by subcutaneous injection at multiple points on the back. The immunization cycle was 14 days, and the mice were given a second booster immunization 14 days after the first immunization. The antigen dose was the same as that of the first immunization. Five LD 50 of PRV HeNLH / 2017 were used to challenge the immunized mice 28 days after the first immunization. The challenge was performed by intramuscular injection of the mice, and the mice were observed daily for 14 days after the challenge. The gD protein expressed by the baculovirus system was used as a control, and the preparation process was referred to the reference (Zhang, T., et al. (2020). "A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection." Vaccine 38(39): 6153-6161.).

[0097] 2. Determination of mouse serum antibody levels

[0098] (1) Determination of mouse serum specific antibody levels

[0099] Five mice were randomly selected from each group, and their tail tip blood was collected on days 0, 7, 14, 21, and 28 after the first immunization. The mouse tail tip blood was diluted in PBS at a volume ratio of 1:100, centrifuged at 5000 rpm for 10 min, and the supernatant was collected and stored at -20°C for later use. The purified gD protein was diluted to 2 μg / mL with CBS buffer, mixed well, and added to the enzyme-labeled strip at a volume of 100 μL / well. The strip was coated at 37°C for 2 h, and then blocked with 5% skim milk at 37°C for 2 h. The primary antibody was a mouse serum diluted by half, and the secondary antibody was a Goat Anti Mouse IgG / HRP diluted 1:5000. TMB color developing solution was added, and the color was developed in the dark for 10 min. 2% H2SO4 was used as a stop solution, and the reaction was terminated at a volume of 200 μL / well. The OD was read in an enzyme-labeled instrument.450 The results are shown in Table 1. Figure 7 The results show that the immune composition of the CHO-gD protein and the heat shock protein gp96N prepared in the present application can significantly improve the level of specific antibodies in the serum of mice and improve the level of humoral immune response of mice.

[0100] (2) Determination of the level of neutralizing antibodies in the serum of mice

[0101] The PK 15 cells were inoculated in the 96-well cell culture plate at a density of 1.7 x 10 4 cells / well one day in advance, and the next day, the serum collected from the mice 28 days after the first immunization was diluted with the maintenance medium, and then 50 μL of the PRV HeNLH / 2017 virus diluent diluted to 200 TCID 50 was added to the sample well, and negative control, positive control and cell control were set. The culture plate was placed in a 37°C incubator for 1 h. The PK15 cell culture plate inoculated the day before was placed in a biological safety cabinet, the culture medium was removed, the cell surface was washed with PBS, the sample and virus mixture at the end of the incubation were added, and the plate was placed in a 37°C 5% CO2 incubator for incubation. When the cells in the negative control appeared obvious lesions, the virus mixture in the culture plate was discarded, the cell surface was washed with sterile PBS, 50 μL / well of 4% tissue fixing solution was added, and the plate was fixed at room temperature for 20 min. After fixation, the fixing solution was discarded, 5% skim milk was added for blocking at 37°C for 2 h, Goat Anti Mouse IgG / HRP was added at 1:2000, and the plate was incubated at 37°C for 1 h. The plate was washed, AEC developing solution was added, and the plate was developed in the dark for 20 min. After the developing was completed, DDW was added to terminate the developing, and the plate was observed under a microscope. The results of the neutralization titer determination are shown in Table 2. Figure 8 The results show that the immune composition of the CHO-gD protein and the heat shock protein gp96N prepared in the present application can significantly improve the level of neutralizing antibodies in the serum of mice.

[0102] 3. Survival rate statistics of the challenged mice

[0103] After the mice were immunized twice with the same dose, a challenge protection experiment was performed on the mice, and the challenge dose was 5LD 50The mice were injected intramuscularly in the leg and observed for 14 days after challenge. The survival rate of the mice was observed during the observation period. The results showed that the mice immunized with PBS exhibited typical symptoms of itching at the challenge site 2 days after challenge, and 2 mice died 3 days later. Subsequent mice exhibited typical clinical symptoms such as biting the challenge site and disordered and dull hair, and all the remaining mice died 5 days later. The mice immunized with the CHO-gD protein died 1 day later, and the mice immunized with the baculovirus-expressed gD protein died 1 day later, indicating that immunization with 30 μg of the gD protein provided 80% protection for the mice. The mice immunized with the immunogen supplemented with the gp96 N protein exhibited a higher protection rate, and no mice died during the observation period. The mice immunized with the commercial inactivated vaccine served as a positive control, and no mice died during the observation period.

[0104] 4. Determination of the level of T cell immune response in mice

[0105] The spleen of the mouse was extracted, and mouse spleen lymphocytes were isolated. The cells were inoculated in a 96-well plate at a density of 2 x 10 5 TCID50 of the virus diluent was added to each well to stimulate the lymphocytes, and the plate was incubated in a 37°C 5% CO2 incubator for 48 hours. Then, the supernatant was collected. 50

[0106] The levels of IFN-γ, TNF-α and IL-2 in the mouse spleen lymphocytes were determined using a commercial kit. The results, as shown in Table 1, indicated that the immunological combination of the CHO-gD protein prepared in the present application and the heat shock protein gp96 N significantly increased the contents of the cytokines IFN-γ, TNF-α and IL-2, and enhanced the cellular immune response in mice. Figure 9

[0107] The above-described embodiments are merely preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.​​

Claims

1. A pseudorabies virus gD protein, characterized in that, The amino acid sequence of which is shown as SEQ ID NO.

2.

2. A gene encoding the gD protein of pseudorabies virus according to claim 1, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

1.

3. A biological material for preparing the pseudorabies virus gD protein according to claim 1, characterized by, The biological material is the substance described in (1) or (2): (1) a recombinant plasmid comprising the coding gene of the pseudorabies virus gD protein according to claim 2; (2) a recombinant CHO cell comprising the recombinant plasmid.

4. The method of preparing the pseudorabies virus gD protein according to claim 1, wherein the step of purifying the gD protein is performed by affinity chromatography using a monoclonal antibody against the gD protein. The pseudorabies virus gD protein is obtained by culturing the recombinant CHO cell, separating the supernatant of the cell culture medium, and purifying the supernatant of the cell culture medium; The recombinant CHO cell comprises the recombinant plasmid; the recombinant plasmid comprises the coding gene of the pseudorabies virus gD protein according to claim 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1; and / or In the recombinant plasmid, the 5' end of the coding gene is connected with an Ig Kappa secretion signal peptide sequence and a Kozak sequence.

5. The pseudorabies virus gD protein according to claim 1 in the preparation of a PRV subunit vaccine.

6. Use of a combination immunization composition in the manufacture of a PRV subunit vaccine, characterized in that, The combined immunization composition comprises the pseudorabies virus gD protein and the gp96 N protein according to claim 1. The amino acid sequence of the gp96 N protein is shown as SEQ ID NO.

6.

7. A PRV subunit vaccine, characterized in that, The active ingredient comprises the pseudorabies virus gD protein according to claim 1.

8. The PRV subunit vaccine according to claim 7, characterized in that, The PRV subunit vaccine further comprises a gp96 N protein. The amino acid sequence of the gp96 N protein is shown as SEQ ID NO.

6.

9. The PRV subunit vaccine according to claim 8, characterized in that, The mass ratio of the pseudorabies virus gD protein to the gp96 N protein is 3: (1-3).

10. The PRV subunit vaccine of claim 7, characterized in that, The PRV subunit vaccine further comprises a vaccine adjuvant.

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