Pseudorabies virus gD protein as well as preparation method and application thereof

Abstract

The invention discloses a pseudorabies virus variant gD protein as well as a preparation method and application thereof. The amino acid sequence of the pseudorabies virus variant gD protein is shown as a sequence 1 in a sequence table, and the nucleotide sequence of the pseudorabies virus variant gD protein is shown as a sequence 2 in the sequence table. The pseudorabies virus variant gD protein is used for indirect ELISA, can be used for detecting whether a pseudorabies virus variant gD protein antibody exists in pig blood or not, and is simple, convenient and rapid in detection, high in specificity, high in sensitivity, accurate and reliable. The pseudorabies virus variant gD protein can be prepared into a subunit vaccine, can be induced to generate a high-level anti-pseudorabies virus variant neutralizing antibody after immunizing a pig, has the advantages of strong immunogenicity, high safety and matching with a current epidemic strain, can distinguish vaccine immunization and wild virus infection animals through differential diagnosis, and has a wide application prospect. The method can be used for preventing and controlling the propagation and prevalence of pseudorabies. The gD protein of the pseudorabies virus variant can be used for detecting and diagnosing pseudorabies and preventing pseudorabies, and has good production and application prospects in development of novel diagnostic reagents and subunit vaccines of pseudorabies viruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary diagnostic reagents and veterinary vaccines. The present invention relates to a pseudorabies virus gD protein and its application, and specifically to a pseudorabies virus gD protein and the application of mammalian HEK293 cells expressing pseudorabies virus gD protein in the preparation of new diagnostic reagents and recombinant subunit vaccines. Background Art

[0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV) and mainly affects a variety of domestic animals and wild animals, with fever, itching, respiratory and nervous system disorders as the main symptoms. The World Health Organization (OIE) classifies it as a Class B animal disease. The prevention and control of the disease at home and abroad is mainly based on vaccine immunization. The matching PRV gB and gE antibody differential diagnosis method can eliminate and purify wild virus-infected pigs.

[0003] At present, the pseudorabies vaccines used in clinical practice in China are divided into three categories: the first is to inactivate the isolated wild virus or strong virus with formaldehyde, and then add adjuvant to emulsify it to make an inactivated vaccine, such as the inactivated pseudorabies vaccine Hubei A strain and Fujian A strain; the second is to develop a weak vaccine by repeatedly attenuating the isolated wild virus or strong virus through non-swine cells or chicken embryos, such as the live pseudorabies vaccine Bartha-K61 strain; the third is a gene-deficient vaccine constructed using genetic engineering technology, including the live pseudorabies vaccine HB-98 strain, HB2000 strain, SA215 strain and the live pseudorabies heat-resistant protectant vaccine C strain, as well as the inactivated pseudorabies vaccine HN1201-ΔgE strain, HNX-12 strain, and JS-2012-ΔgI / gE strain. These vaccines play a very important role in the prevention and control of pseudorabies in my country. In addition to gene-deleted inactivated vaccines and live vaccines targeting pseudorabies virus variants, subunit vaccines based on the gD gene of porcine pseudorabies virus have also become an important direction in the field of biological product research and development due to their high safety, good immune protection and ability to perform serological differential diagnosis.

[0004] Against the backdrop of the current market demand for widespread prevalence of pseudorabies virus variants, rapid and reliable disease diagnosis, and efficient and safe vaccines, the development of diagnostic reagents and subunit vaccines that match the currently prevalent variants has positive significance for the future control and purification of pseudorabies. Summary of the invention

[0005] One of the purposes of the present invention is to provide a pseudorabies virus gD protein. The antigenicity and immunogenicity of the gD protein obtained by HEK293 mammalian cell expression are similar to those of natural protein, and the gD protein has the characteristics of high glycosylation modification and strong immunogenicity.

[0006] A second object of the present invention is to provide nucleic acid molecules encoding the above-mentioned recombinant proteins, as well as recombinant eukaryotic vectors and recombinant cells into which these nucleic acid molecules are inserted for recombinant expression of the above-mentioned recombinant proteins.

[0007] The third object of the present invention is to provide a preparation method and use of the pseudorabies virus gD protein.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a pseudorabies virus gD protein, wherein the recombinant protein comprises a pseudorabies virus gG signal peptide sequence, a pseudorabies virus gD gene sequence with a transmembrane region truncated, and a purification tag 6×

[0010] His sequence.

[0011] Preferably, the pseudorabies virus gD gene has the amino acid sequence shown in Sequence 1; and the pseudorabies virus gG signal peptide has the amino acid sequence shown in Sequence 3.

[0012] The present invention also provides a nucleic acid molecule encoding the aforementioned recombinant protein, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a pseudorabies virus gG signal peptide and a nucleotide sequence encoding a pseudorabies virus gD protein.

[0013] Specifically, the nucleic acid molecule sequence encoding the pseudorabies virus gG signal peptide is shown in Sequence 4, and the nucleic acid molecule sequence encoding the pseudorabies virus gD protein is shown in Sequence 2.

[0014] The present invention also provides a recombinant vector, which comprises the nucleic acid molecule described above and further comprises an expression regulatory sequence operably linked to the nucleic acid molecule sequence.

[0015] In the present invention, "vector" refers to a nucleic acid carrier into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids, phages, cosmids, artificial chromosomes, phages and animal viruses. The types of animal viruses used as vectors include lentiviruses, adenoviruses, herpes viruses, poxviruses, baculoviruses, papillomaviruses, etc. A vector may contain a variety of control expression elements, including promoter sequences, reverse transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication start site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances.

[0016] In a specific embodiment of the present invention, the recombinant vector is constructed by inserting the nucleic acid molecule shown above into the pCDNA3.1 vector (Thermo Fisher Scientific).

[0017] The present invention also provides a method for constructing the aforementioned recombinant vector, which comprises inserting the aforementioned nucleic acid molecule into the pCDNA3.1 vector.

[0018] Specifically, the construction method of the recombinant vector of the present invention is as follows:

[0019] The signal peptide (coding sequence such as sequence 4), pseudorabies virus gD protein and 6×His sequence were combined respectively, and the termination codon TAA was added to the C-terminus of the gene sequence to artificially synthesize the complete gene sequence expressing the pseudorabies virus gD recombinant protein, which was cloned into the expression vector pCDNA3.1 by molecular cloning to construct a recombinant eukaryotic expression plasmid.

[0020] Specifically, the recombinant expression vector is constructed by inserting the nucleotide sequence shown in sequence 5 in the sequence table between the NheⅠ and NotⅠ enzyme recognition sites of the expression vector pCDNA3.1 to obtain a recombinant eukaryotic expression plasmid pCDNA3.1-gD expressing the pseudorabies virus gD recombinant protein.

[0021] The present invention also provides a recombinant cell, into which the aforementioned nucleic acid molecule is introduced or the aforementioned recombinant vector is transfected.

[0022] The term "recombinant cell" in the present invention refers to a cell into which a nucleic acid molecule or vector is introduced, including many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells Sf9, or fibroblasts, CHO cells, COS cells, BHK cells, HEK293 cells, etc.

[0023] In a specific embodiment of the present invention, the recombinant cell is a HEK293 cell.

[0024] The present invention also provides a method for producing pseudorabies virus gD recombinant protein using the above-mentioned recombinant cells, the method comprising culturing the above-mentioned recombinant cells under suitable conditions and recovering the recombinant protein, the method comprising the following steps: transfecting a host cell or the above-mentioned recombinant cell with a recombinant expression vector, harvesting the culture supernatant of the transfected cells multiple times, and / or culturing the recombinant cells in a target culture medium, harvesting the culture supernatant for purification, to obtain the above-mentioned pseudorabies virus gD recombinant protein.

[0025] The invention also provides a pseudorabies subunit vaccine and a preparation method and application thereof.

[0026] The pseudorabies subunit vaccine comprises pseudorabies virus gD recombinant protein and a pharmaceutically acceptable adjuvant, such as ISA 206VG adjuvant.

[0027] Furthermore, the subunit vaccine preparation method is as follows: the gD recombinant protein obtained by the above expression and purification is quantified by Nanodrop2000 A280, and mixed with ISA 206VG in a volume ratio of 1:1 to prepare a pseudorabies subunit vaccine.

[0028] The pseudorabies subunit vaccine provided by the present invention can induce the production of high-level neutralizing antibodies against pseudorabies virus variants after immunizing pigs, has the advantages of strong immunogenicity, good safety and matching with currently prevalent strains, and can be used to prevent and control the spread and prevalence of pseudorabies.

[0029] The present invention also provides a pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kit, which is characterized by comprising: pseudorabies virus gD recombinant protein, blocking solution (PBS buffer containing 10 mg / ml casein, pH 7.4), enzyme-labeled secondary antibody (HRP-labeled rabbit anti-pig IgG), PBST, color developing solution (TMB substrate solution), and stop solution (2 mol / L sulfuric acid).

[0030] Specifically, the pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kit comprises:

[0031] ELISA plate coated with Pseudorabies virus gD recombinant protein.

[0032] The kit may also include positive control serum and negative control serum. The positive control serum is the pig serum collected after immunization with pseudorabies inactivated vaccine, and the negative control serum is specific pathogen-free (SPF) pig serum.

[0033] The kit may also include: sample diluent, 20-fold concentrated washing solution, enzyme-labeled secondary antibody (HRP-labeled rabbit anti-pig IgG), substrate solution A, substrate solution B and stop solution.

[0034] The sample diluent contained 5 mg / ml casein in 0.01 M phosphate buffer (pH 7.4).

[0035] Substrate solution A is a citrate phosphate buffer containing 0.6 mg / ml urea hydrogen peroxide.

[0036] Substrate solution B is 0.2 mg / ml tetramethylbenzidine (TMB) solution.

[0037] The 20-fold concentrated washing solution is a 0.01 M phosphate buffer solution with a pH value of 7.4 and containing Tween-20 at a concentration of 0.8% to 1.2% (ml / ml).

[0038] The stop solution was 2M sulfuric acid solution.

[0039] If necessary, the kit may also include a sample dilution plate (2 plates, 96 wells / plate) for sample dilution.

[0040] The pseudorabies virus gD recombinant protein of the present invention can specifically and efficiently bind to specific antibodies in pig serum samples, has good antigenicity, and can distinguish vaccine-immunized and wild-type virus-infected animals through differential diagnosis of gD and gE antibodies in the serum of vaccine-immunized animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the result of genetic evolution tree analysis of PRV HNxy strain based on the amino acid sequence of gD gene.

[0042] Figure 2 This is a comparison of the nucleotide sequences of different PRV strains based on the gD gene.

[0043] Figure 3 This is the construction map of the recombinant eukaryotic expression plasmid of pseudorabies virus gD gene.

[0044] Figure 4 This is the SDS-PAGE detection result of the expression and purification of pseudorabies virus gD recombinant protein. Lane 1 is PageRuler pre-stained protein Marker (Thermo Fisher Scientific Company), lane 2 is the supernatant after HEK293 culture expression, lane 3 is the 30kD ultrafiltration concentrate, lane 4 is the target protein solution eluted with 100mM imidazole on a nickel column, lane 5 is the target protein solution eluted with 300mM imidazole on a nickel column, and lane 6 is the target protein finally harvested after gel filtration chromatography and concentration.

[0045] Figure 5 It is a schematic diagram of adding samples to the enzyme-linked immunosorbent assay plate of the kit of the present invention, N represents the addition of negative control serum, P represents the addition of positive control serum, and S1, S2, S3, S4, etc. represent the addition of serum to be tested. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are further described below in conjunction with specific examples. It is necessary for those skilled in the art to understand that the present invention may be modified and altered within the spirit and scope defined by the claims, but all fall within the protection scope of the present invention.

[0047] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources.

[0048] The plasmid pCDNA3.1 used in the following examples was purchased from Thermo Fisher Scientific; the transfection reagent PEI was purchased from Alfa Aesar; HEK293 cells were purchased from the American Type Culture Collection ATCC; HisTrap HP 5ml nickel column prepacked column and HiLoad Superdex 16 / 600 200pg gel filtration column were purchased from GE.

[0049] Example 1: Expression and purification of pseudorabies virus gD recombinant protein

[0050] Receptor diversity is the basis of PRV's pan-tropic infection. PRV glycoprotein gD can interact with at least one of the three receptors nectin1, nectin2 and CD55 and firmly bind to the cell. The gD protein then interacts with the receptor HVEM to invade the host (Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine [J]. Microbiol Mol Biol Rev. 2005; 69(3): 462-500). Current research reports show that mutations in the gD gene of the PRV epidemic variant significantly increase its affinity for human, porcine and mouse nectin-1 receptors, playing a key role in enhancing the ability of PRV variants to invade nerve cells (Wu HX, Qi HS, Wang B, et al. The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant[J]. J Biol Chem, 2023; 299(11): 105347. doi: 10.1016 / j.jbc.2023.105347).

[0051] The present invention is based on the whole genome sequence of pseudorabies virus PRV HNxy strain (China General Microbiological Culture Collection Center strain collection number CGMCC No.17696, recorded in patent document CN110527669A) to obtain the amino acid sequences of gG and gD genes. The results of genetic evolution tree analysis based on the gD amino acid sequence show that PRVHNxy strain belongs to genotype II ( Figure 1 ); The full length of the gD gene of the PRV HNxy strain is 1209 bp, encoding 402 amino acids. The nucleotide sequences of the gD gene of the PRV HNxy strain were compared with the current domestic epidemic variants, classical strains and attenuated vaccine strain Bartha-K61. The results showed that PRV HNxy had obvious differences from the classical strain Bartha-K61, Hubei A strain and Fujian A strain, and was highly consistent with the current epidemic variants ( Figure 2 In the present invention, the codon-optimized gD gene is expressed through a 293 mammalian cell expression system to obtain the target protein, which has two advantages: (1) the gD protein expressed by the mammalian eukaryotic system is highly glycosylated, similar to the characteristics of the natural gD protein, ensuring immunogenicity from the source; (2) the 293 cell expression system secretes and expresses the gD protein, and there are few impurity proteins in the supernatant. When the subunit vaccine is subsequently industrialized and scaled up, only one step of tangential flow ultrafiltration concentration is required to meet the purity requirement of more than 80%.

[0052] 1. Vector construction and synthesis

[0053] First, the pseudorabies virus gG signal peptide (sequence 3), the pseudorabies virus gD gene extracellular region (sequence 2) and the 6His sequence were combined, and the termination codon TAA was added to the C-terminus of the gene sequence to artificially synthesize the complete gene sequence expressing the pseudorabies virus gD recombinant protein (as shown in sequence 5); then, the fragment was cloned into the NheⅠ and NotⅠ enzyme recognition sites of the expression vector pCDNA3.1 by molecular cloning to construct the recombinant eukaryotic expression plasmid pCDNA3.1-gD ( Figure 3 ), this step was directly handed over to Suzhou GeneWeichi Gene Synthesis Company to complete.

[0054] 2. Cultivation of recombinant bacterial solution

[0055] Take the artificially synthesized positive bacterial liquid and puncture the bacteria, inoculate 5 ml of LB medium containing ampicillin antibiotics, and culture it at 37°C and 220 rpm overnight with shaking.

[0056] 3. Extraction of recombinant eukaryotic expression plasmid

[0057] Refer to the operating procedures of TIANGEN Plasmid Extraction Kit (Cat. No. DP103) and add anhydrous ethanol to the rinse solution PW according to the instructions before use.

[0058] (1) Column equilibration step: Add 500 μl of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0059] (2) Take 5 ml of overnight culture solution and add it to a centrifuge tube. Centrifuge at 12,000 rpm for 1 min and remove the supernatant as much as possible.

[0060] (3) Add 250 μl of solution P1 to the centrifuge tube containing the bacterial pellet and use a pipette or vortex oscillator to thoroughly resuspend the bacterial pellet.

[0061] (4) Add 250 μl of solution P2 to the centrifuge tube and gently invert it upside down 6 to 8 times to fully lyse the bacteria.

[0062] (5) Add 350 μl of solution P3 to the centrifuge tube and gently invert it 6 to 8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min.

[0063] (6) Use a pipette to transfer the supernatant collected in the previous step to the adsorption column CP3 (the adsorption column is placed in the collection tube). Be careful not to suck out the precipitate. Centrifuge at 12,000 rpm for 30 to 60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0064] (7) Add 600 μl of rinse solution PW to the adsorption column CP3, centrifuge at 12,000 rpm for 30 to 60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0065] (8) Repeat step (7).

[0066] (9) Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm for 2 min to remove the residual rinse solution in the adsorption column.

[0067] (10) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μl of elution buffer EB to the middle part of the adsorption membrane, leave it at room temperature for 2 min, and centrifuge it at 12,000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

[0068] (11) Take 2 μl of recombinant plasmid DNA and measure its concentration using Nanodrop2000 A260.

[0069] 4. Transfection of recombinant eukaryotic expression plasmid and expression of pseudorabies virus gD recombinant protein

[0070] (1) Transfection of vector DNA: HEK293 cells were inoculated in a cell culture dish with a diameter of 15 cm. When the cell density reached 1.2×10 7 ~1.5×10 7 The above recombinant expression plasmid pCDNA3.1-gD was transfected with PEI (Alfa Aesar) at a concentration of 1 mg / ml.

[0071] The specific operation is as follows: add 1ml OPTI-MEM culture medium to a 4ml Ep tube, then add 30μg of recombinant expression plasmid pCDNA3.1-gD in turn and mix gently; add 850μl OPTI-MEM culture medium to another 1.5ml Ep tube, then add 150μl PEI transfection reagent and mix; then add the OPTI-MEM culture medium containing the transfection reagent to the Ep tube containing the recombinant plasmid and mix gently, place at room temperature for 15min, then add to the culture dish with HEK293 cells, culture at 37℃, 5% CO2 for 5h, and replace the medium with serum-free DMEM culture medium.

[0072] (2) Expression of Pseudorabies Virus gD Recombinant Protein: After culturing the HEK293 cells transfected with the recombinant DNA plasmid for 72 h, the culture supernatant was harvested and stored at 4° C. for later use. Serum-free DMEM medium was added to the cell culture dish again, and the cells were cultured for another 72 h before the supernatant was harvested.

[0073] 5. Purification of Pseudorabies Virus gD Recombinant Protein

[0074] (1) 1000 ml of the culture supernatant was filtered through a 0.22 μm filter membrane and loaded onto a HisTrap HP 5 ml nickel pre-loaded column using a peristaltic pump at a flow rate of 1 ml / min. After loading, the impurities and unbound proteins were washed away using a buffer containing 20 mM imidazole (20 mM NaCl, 50 mM Tris-HCl, pH 8.0) on an AKTA purifier. The target protein was then washed with a buffer containing 100 mM imidazole and then with a buffer containing 300 mM imidazole. 20 μl of the protein sample was collected at each stage, 5 μl of 5× loading buffer was added, and the sample was placed on ice after boiling in a water bath for 10 minutes.

[0075] (2) Take 10 μl of protein sample for SDS-PAGE electrophoresis, and collect samples containing the target protein based on the SDS-PAGE electrophoresis results for the next gel filtration chromatography.

[0076] (3) The sample obtained from the previous step was purified by AKTA purification instrument through GE HiLoad Superdex16 / 600200pg column to obtain the target protein, and 10μl protein sample was taken for SDS-PAGE electrophoresis for identification ( Figure 4 The target protein was then concentrated using a 30 kD ultrafiltration tube.

[0077] (4) The protein concentration measured by Nanodrop2000 was 2 mg / ml, the volume was 40 ml, and the expression level of gD protein was calculated to be as high as 80 mg / L.

[0078] Example 2: Preparation of Pseudorabies Subunit Vaccine

[0079] Take 5 ml of ISA 206VG adjuvant and add it to a 20 ml beaker with a rotor. Turn on the magnetic stirrer and adjust it to gear 6. Take another 5 ml of the aqueous phase (containing the gD protein prepared in Example 1) and pour it into the beaker to make the final protein concentration of 150 μg / 2 ml. The pouring time is about 5 seconds. After the vortex is stable, emulsify for 5 minutes, turn off the magnetic stirrer, and fill the vaccine into a 20 ml bottle, cover and label it; store it at 4 degrees for later use.

[0080] Example 3: Immunization test of pseudorabies subunit vaccine in piglets

[0081] Thirteen weaned piglets with negative pseudorabies virus antibodies were screened for vaccine immune protection evaluation test and randomly divided into three groups. Five pigs were used as pseudorabies gD subunit vaccine immunization group, and 2 ml pseudorabies subunit vaccine was immunized through the neck muscle; 5 pigs were used as commercial inactivated vaccine (Kequanjing, porcine pseudorabies gE gene deleted inactivated vaccine, HNX-12 strain, batch number 20230101) immunization control group, and 2 ml pseudorabies inactivated vaccine was immunized through the neck muscle; 3 pigs were used as negative blank control, and 2 ml PBS was injected into the neck muscle. Serum was collected once a week after immunization to determine the titer of neutralizing antibodies against pseudorabies virus variants in the serum. 21 days after immunization, 1 ml of the virulent variant of pseudorabies PRV HNxy strain (China General Microbiological Culture Collection Center, culture collection number CGMCC No. 17696, recorded in patent document CN110527669A) was dripped into the nose of each animal, and the animal was observed for 14 days, and the clinical manifestations and the number of animal survival and death were recorded.

[0082] Table 1. Pseudorabies virus neutralizing antibody titers in sera of immunized piglets

[0083]

[0084] The results of neutralizing antibody test showed that the neutralizing antibody levels of the five piglets in the pseudorabies subunit vaccine immunization group were 1:3±0.36, 1:13.97±5.30 and 1:31.84±11.41 on days 7, 14 and 21 after immunization, respectively. Among them, the neutralizing antibody level on the day of challenge, that is, 21 days after immunization, reached 31.84±11.41. In comparison, the neutralizing antibody levels of the five piglets in the commercial inactivated vaccine immunization group were 1:3.06±0.57, 1:9.69±1.62 and 1:23.34±4.99 on days 7, 14 and 21 after immunization, respectively. However, the neutralizing antibody titers of the three animals in the blank control group remained negative (<1:2) 21 days after immunization.

[0085] 21 days after immunization, each animal was intranasally dripped with 1 ml of the virulent pseudorabies variant PRV HNxy strain (CGMCC No. 17696, the strain collection number of the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, recorded in the patent document CN110527669A). The body temperature test results 10 days after the challenge showed that the body temperature of the piglets in the pseudorabies gD subunit vaccine group and the commercial inactivated vaccine group was normal after the challenge, without a significant increase (Table 2), and no symptoms of pseudorabies appeared, with a protection rate of 100%. However, the body temperature of the three piglets in the blank control group increased significantly on the second day after the challenge (Table 2), of which two piglets died on the fourth and fifth days after the challenge, and another piglet was retained in high temperature from the second day after the challenge until the end of the experiment; all three animals had neurological symptoms of inability to stand on the hind legs and swinging limbs.

[0086] Table 2 Body temperature test results of immunized piglets 10 days after infection

[0087]

[0088]

[0089] This set of data shows that our pseudorabies gD subunit vaccine can be used as a candidate subunit vaccine for pseudorabies, and it has positive significance especially for infection with pseudorabies virus variant strains currently prevalent in the country.

[0090] Example 4: Preparation and effect verification of pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kit

[0091] 1. Preparation of pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kit

[0092] (1) Preparation of antigen-coated plate: The purified gD protein in Example 1 was diluted with a carbonate solution at pH 9.6 to a working solution of 1.0 μg / ml, and added to a 96-well polystyrene ELISA plate at 100 μl / well. The plate was placed at 2-8°C for 8-12 h to allow the coated antigen to fully bind to the ELISA plate. Then, 300 μl / well of a PBS buffer solution at pH 7.4 containing 10 mg / ml casein at pH 7.4 was added. The plate was blocked at 37°C for 2-3 h, and the liquid was dried. After the ELISA plate was dried, it was sealed with aluminum foil and stored at 2-8°C for later use.

[0093] (2) Positive control serum: The serum collected from pigs after immunization with the inactivated pseudorabies vaccine was used as the positive control serum of the kit (1 tube, 1.5 ml / tube).

[0094] (3) Negative control serum: Specific pathogen-free (SPF) porcine serum, used as the negative control serum for the kit (1 tube, 1.5 ml / tube).

[0095] (4) The sample diluent was prepared as 0.01 M phosphate buffer (pH 7.4) containing 5 mg / ml casein (1 bottle, 24 ml / bottle).

[0096] (5) Substrate solution A is prepared by mixing 0.6 mg / ml urea hydrogen peroxide in citrate phosphate buffer (1 bottle, 12 ml / bottle).

[0097] (6) Substrate solution B is prepared as a 0.2 mg / ml tetramethylbenzidine (TMB) solution (1 bottle, 12 ml / bottle).

[0098] (7) The 20-fold concentrated washing solution was prepared by using 0.01 M phosphate buffer with a pH of 7.4 and a concentration of 0.8% to 1.2% (ml / ml) of Tween-20 (2 bottles, 50 ml / bottle).

[0099] (8) Preparation of stop solution: 2M sulfuric acid solution (1 bottle, 12 ml / bottle).

[0100] (9) If necessary, the kit may also include a sample dilution plate (2 plates, 96 wells / plate) for sample dilution.

[0101] 2. How to use the pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kit

[0102] (1) Equilibration: Take the reagent kit out of the refrigerator and place it at room temperature for equilibration for 30 min before use; mix the liquid reagents before use.

[0103] (2) Preparation of washing solution: Before use, dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain washing buffer solution.

[0104] (3) Sample dilution: Dilute the serum to be tested in a serum dilution plate at a ratio of 1:20 (7 μl serum + 133 μl sample diluent). The negative and positive control sera have been diluted and can be used directly.

[0105] (4) Operation steps:

[0106] ① Sample addition: Take the antigen-coated plate and add the diluted serum to be tested, negative control serum and positive control serum to the antigen-coated plate, 100μl / well; set 1 well for each serum to be tested, 2 wells for negative control and positive control respectively, and the time span of the sample addition process should be as short as possible. Figure 5 The samples shown are as follows: N represents the addition of negative control serum, P represents the addition of positive control serum, and S1, S2, S3, S4, etc. represent the addition of serum to be tested.

[0107] ② Incubation: Vortex to mix, place in a 37℃ incubator, and react for 30 minutes.

[0108] ③ Wash the plate: discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 seconds, discard the washing solution, wash the plate 5 times in a row and pat dry.

[0109] ④ Add enzyme: Add 100 μl of rabbit anti-pig enzyme-labeled secondary antibody to each well.

[0110] ⑤ Incubation: Place in a 37°C incubator and react for 30 minutes.

[0111] ⑥ Wash the plate: discard the reaction solution, add 300μl of diluted washing buffer to each well, soak for 15s, discard the washing solution, wash the plate 5 times in a row and pat dry.

[0112] ⑦ Color development: Add 100 μl of substrate working solution to each well (substrate working solution is prepared by mixing equal amounts of substrate solution A and substrate solution B, prepared immediately before use), shake to mix, place in a 37°C incubator, and react for 15 minutes in the dark.

[0113] ⑧Termination: Add 50 μl of color stop solution to each well, shake and mix to terminate the reaction, and measure the results within 15 minutes.

[0114] (5) Judgment:

[0115] ①Test establishment conditions: Positive control well OD 450nm The value should be between 0.9 and 2.0. The OD of the negative control well 450nm The value should be ≤0.15;

[0116] ② Positive critical value (Cut-off value) = positive control well OD 450nm mean × 0.17;

[0117] ③ Sample OD 450nmValue ≥ positive control well OD 450nm Mean × 0.17, judged as positive; sample OD 450nm Value < OD of positive control well 450nm The mean value × 0.15 is considered negative; values ​​between the two are considered suspicious.

[0118] 3. Sensitivity test

[0119] Using 3 batches of pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kits (batches ZM2024001, ZM2024002, and ZM2024003) prepared according to the method described in step 1, 30 sera from wild-type pigs infected with pseudorabies virus and 50 sera from vaccine-immunized pigs were tested according to the method of step 2. The experimental results are shown in Table 3. A total of 78 positive samples were detected by the kit of the present invention, and 2 samples were not detected. The results show that the sensitivity of the kit to 30 sera from wild-type pigs infected with pseudorabies virus and 50 sera from vaccine-immunized pigs is 97.5%.

[0120] Table 3. Sensitivity test results

[0121]

[0122]

[0123] 4. Specificity test

[0124] According to the instructions for using the kit in step 2, the above three batches of pseudorabies virus gD protein antibody enzyme-linked immunosorbent assay kits (batches ZM2024001, ZM2024002, and ZM2024003) were used to test 60 healthy pig sera, 2 bovine viral diarrhea virus-positive sera (BVDV), 2 classical swine fever virus-positive sera (CSFV), and 2 porcine circovirus-positive sera (PCV2), respectively.

[0125] The specificity test results of the kit are shown in Table 4. The test results of 60 healthy pig sera were all negative, and the specificity of the three batches of kits was 100.00%; the test results of 2 bovine viral diarrhea virus positive sera (BVDV), 2 swine fever virus positive sera (CSFV), and 2 porcine circovirus positive sera (PCV2) were all negative, and the specificity of the three batches of kits was 100.00%.

[0126] Table 4. Specificity test results of pseudorabies virus gD protein antibody ELISA kit

[0127]

[0128] 5. Compliance test

[0129] The BioChek imported kit for detecting pseudorabies gB antibody in pigs and the kit of the present invention were used to simultaneously detect 60 healthy pig sera, 30 sera infected with wild pseudorabies virus and 50 sera immune to gE gene-deficient vaccine, so as to compare the coincidence rates of the detection results of the two kits.

[0130] How to use the BioChek imported test kit:

[0131] (1) Preparation of reagents: Before use, take 1 tablet of substrate and place it in a clean container, add 5.5 ml of substrate buffer solution, and mix until completely dissolved (about 10 minutes). Pour 1 bag of detergent powder completely into 1 L of deionized water and mix until completely dissolved. The other components in the kit can be used directly, but should be warmed to room temperature (22-27°C) before use.

[0132] (2) Sample preparation: dilute each sample at 1:50, i.e. add 245 μl of sample diluent to 5 μl of sample on the sample dilution plate, and record the positions of the sample and control on the pre-dilution plate. Mix well using a vortex mixer or oscillator, and use a new, unused pipette tip for each sample.

[0133] (3) Operation steps:

[0134] ① Take out the reaction plate coated with PRV gB from the sealed bag and record the position of the sample on the recording paper;

[0135] ② Add 100 μl of negative control to wells A1 and B1;

[0136] ③ Add 100 μl of positive control to wells C1 and D1;

[0137] ④ Add 100 μl of the diluted sample to the corresponding wells, cover the reaction plate with a lid, and incubate at room temperature (22-27°C) for 30 minutes;

[0138] ⑤ Pour out the contents in the air and wash 4 times with washing solution (350μl per well). Invert the reaction plate for the last time and tap it gently on absorbent paper;

[0139] ⑥Add 100 μl of enzyme-labeled secondary antibody to each well, cover the reaction plate with a lid, and incubate at room temperature (22-27°C) for 30 minutes;

[0140] ⑦Repeat step ⑤;

[0141] ⑧Add 100 μl of substrate reagent to each well, cover the reaction plate with a lid, and incubate at room temperature (22-27°C) for 15 min;

[0142] ⑨Add 100 μl of stop solution to each well to terminate the reaction and read the results within 30 minutes;

[0143] ⑩ Adjust the microplate reader to zero with air and measure the absorbance of each well at a wavelength of 405 nm.

[0144] (4) Results and determination: The relative content of antibodies in the sample can be calculated with reference to the positive control, and their relationship can be calculated by the S / P value (ratio of the sample to the positive control).

[0145] ① Calculation of S / P value: (average value of the sample to be tested - average value of the negative control) / (average value of the positive control - average value of the negative control) = S / P

[0146] ② Calculation of antibody titer:

[0147] Log 10 Titer = 1.1 × Log 10 (S / P)+3.361

[0148] Inverse log = titer

[0149] ③Judgment criteria:

[0150]

[0151] The test results of the kit of the present invention and the BioChek imported PRV gB antibody detection kit on 60 healthy pig sera, 30 pseudorabies virus wild-type infected sera and 50 gE gene-deficient vaccine immune sera are shown in Table 5. The number of positive sera detected by the kit of the present invention and the BioChek imported PRV gB antibody detection kit is 76, and the number of negative sera detected is 60. Among the 140 sera to be tested, the number of sera with consistent test results of the two kits is 135, and the compliance rate is 96.4%.

[0152] Table 5. Compliance test results

[0153]

Claims

1. A pseudorabies virus gD recombinant protein, the amino acid sequence of which is the amino acid sequence shown in Sequence 1 in the sequence table.

2. The gene encoding the pseudorabies virus gD recombinant protein according to claim 1, wherein the nucleotide sequence thereof is the nucleotide sequence shown in Sequence 2 in the sequence listing.

3. The eukaryotic expression vector of pseudorabies virus gD recombinant protein according to claim 1, characterized in that: The coding genes of the pseudorabies virus gD gene and the pseudorabies virus gG signal peptide described in claim 2 are cloned into the starting vector pCDNA3.1 to obtain a recombinant eukaryotic expression plasmid expressing the pseudorabies virus gD gene; wherein the amino acid sequence of the pseudorabies virus gG signal peptide is shown in Sequence 3, and the nucleotide sequence of the pseudorabies virus gG signal peptide is shown in Sequence 4.

4. The method for preparing the pseudorabies virus gD recombinant protein according to claim 1, characterized in that: The pseudorabies virus gD recombinant protein eukaryotic expression vector of claim 3 is transfected into HEK293 mammalian cells, and the pseudorabies virus gD protein is obtained through culture and purification.

5. A method for preparing a recombinant subunit vaccine of pseudorabies, characterized in that: The pseudorabies virus gD recombinant protein obtained in claim 4 is fully mixed with a pharmaceutically acceptable adjuvant to obtain a pseudorabies recombinant subunit vaccine.

6. Use of the pseudorabies virus gD recombinant protein according to claim 1 or prepared according to claim 3 or 4 in the preparation of a subunit vaccine for the prevention or treatment of porcine pseudorabies or in the preparation of a kit for detecting and diagnosing pseudorabies virus.

7. An enzyme-linked immunosorbent assay kit for pseudorabies virus gD protein antibody, comprising an enzyme-linked reaction plate and an enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with the pseudorabies virus gD protein according to claim 1.

8. The ELISA kit according to claim 7, characterized in that: The method for obtaining the enzyme-linked reaction plate is as follows: the pseudorabies virus gD protein is dissolved in 100 μl of a carbonate solution with a pH of 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate, with 0.1 μg of the pseudorabies virus gD protein in each well, and placed at 2 to 8° C. for 8 to 12 hours to allow the coated antigen to fully bind to the enzyme-linked reaction plate, and then 300 μl / well of a PBS buffer solution containing 10 mg / ml casein with a pH of 7.4 is added, and the plate is sealed at 37° C. for 2 to 3 hours, and after being dried, the enzyme-linked reaction plate is sealed and stored at 4° C. after drying.

9. The ELISA kit according to claim 7, characterized in that: , positive control serum and negative control serum; the positive control serum is the pig serum collected after immunization with pseudorabies inactivated vaccine; the negative control serum is specific pathogen-free (SPF) pig serum.

10. The enzyme-linked immunosorbent assay kit according to claim 7, characterized in that: The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody; The kit also includes substrate solution A and substrate solution B; the substrate solution A is a citric acid phosphate buffer solution containing 0.6 mg / ml hydrogen peroxide urea, and the substrate solution B is a 0.2 mg / ml tetramethylbenzidine solution, and the two are mixed in a ratio of 1:1 when used; the stop solution is a 2 mol / L sulfuric acid solution; The kit also includes a sample diluent and a 20-fold concentrated washing solution; the sample diluent is a phosphate buffer solution containing 5 mg / ml casein, 0.01 mol / L, and a pH value of 7.4; the concentrated washing solution is a phosphate buffer solution containing 0.8% to 1.2% Tween-20, 0.01 mol / L, and a pH value of 7.4.

Images