Porcine pseudorabies virus antigen, porcine pseudorabies subunit vaccine as well as preparation method and application of porcine pseudorabies virus antigen and porcine pseudorabies subunit vaccine

Abstract

The invention relates to the field of veterinary vaccines, in particular to a porcine pseudorabies virus antigen, a porcine pseudorabies subunit vaccine as well as a preparation method and application of the porcine pseudorabies virus antigen and the porcine pseudorabies subunit vaccine. The porcine pseudorabies virus antigen is porcine pseudorabies virus gD protein, and the amino acid sequence of the porcine pseudorabies virus gD protein is as shown in SEQ ID NO: 1. The porcine pseudorabies subunit vaccine prepared from the porcine pseudorabies virus antigen is simple in preparation method, high in neutralizing antibody value, strong in antigen immunity, capable of effectively activating immune response of a body and capable of playing a good immune protection role on porcine pseudorabies.

Description

Technical Field

[0001] The present invention relates to the field of veterinary vaccines, and in particular to a porcine pseudorabies virus antigen, a subunit vaccine, and a preparation method and application thereof. Background Art

[0002] Pseudorabies in pigs is an acute animal infectious disease caused by pseudorabies virus (PRV), which not only affects pigs, but also poses a potential threat to other animals and humans. In the prior art, although there are patents that have developed corresponding immune vaccines to deal with pseudorabies in pigs, such as patent CN116814561A, patent CN113425839A, etc. However, due to environmental factors, natural evolution of the virus, gene mutation, gene recombination and frequent use of vaccines, selective pressure may be exerted on the virus, prompting the virus to mutate continuously to escape the immune mechanism. The vaccines prepared from the existing classic PRV strains and variant strains can no longer effectively prevent and control the current popular pseudorabies in pigs.

[0003] Therefore, it is of great practical significance to find new porcine pseudorabies virus antigens and then develop new porcine pseudorabies subunit vaccines. Summary of the invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the prior art and firstly provide a porcine pseudorabies virus antigen.

[0005] The second technical problem to be solved by the present invention is to provide the coding gene of the above-mentioned porcine pseudorabies virus antigen.

[0006] The third technical problem to be solved by the present invention is to provide an expression vector.

[0007] The fourth technical problem to be solved by the present invention is to provide a recombinant cell,

[0008] The fifth technical problem to be solved by the present invention is to provide a method for preparing porcine pseudorabies virus antigen.

[0009] The sixth technical problem to be solved by the present invention is to provide the use of the porcine pseudorabies virus antigen in the preparation of a subunit vaccine for preventing and / or treating porcine pseudorabies.

[0010] The seventh technical problem to be solved by the present invention is to provide a porcine pseudorabies subunit vaccine.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0012] A porcine pseudorabies virus antigen, wherein the porcine pseudorabies virus antigen is the porcine pseudorabies virus gD protein;

[0013] The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO: 1.

[0014] Furthermore, the nucleotide sequence of the gene encoding the porcine pseudorabies virus gD protein is shown in SEQ ID NO:2.

[0015] The expression vector containing the gene encoding the porcine pseudorabies virus gD protein is also within the scope of protection of the present invention.

[0016] The recombinant cells containing the coding gene of the porcine pseudorabies virus gD protein or containing the expression vector are also within the scope of protection of the present invention.

[0017] A method for preparing a porcine pseudorabies virus antigen specifically comprises cloning the nucleotide sequence of the porcine pseudorabies virus gD protein into a eukaryotic expression vector to construct a recombinant plasmid, then transfecting the recombinant plasmid into expression cells, and obtaining the porcine pseudorabies virus antigen through culture, screening, protein expression, and purification.

[0018] Wherein, the eukaryotic expression vector is any one of pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, and the preferred eukaryotic expression vector is pcDNA3.1.

[0019] Wherein, the expression cell is a CHO cell among mammalian cells.

[0020] The culture is specifically carried out in a cell culture incubator at 36° C.-38° C. and containing 5% CO 2 for 48 hours, after which the liquid is discarded and replaced with a DMEM complete medium containing 0.8 mg / ml G418 for culture.

[0021] Wherein, the screening comprises detecting the expression yield of the monoclonal cell line protein by ELISA method, and screening monoclonal cells with the expression yield of the monoclonal cell line protein being greater than 1 g / L.

[0022] The protein expression was performed by inoculating monoclonal cells into commercial CHO serum-free medium at a cell density of 1.0×10 5 The cells were cultured at 36°C-38°C in a constant temperature shaker containing 5% CO2 at a rotation speed of 100 r / min; the cell density, activity and glucose content were detected every 24 hours, and when the glucose was lower than 2.5 g / L, glucose was supplemented to 3-4 g / L; feeding was performed on the 4th and 9th days of fermentation, and the added volume was 10% of the original culture medium; the temperature was lowered to 32°C on the 5th day of fermentation; and the cell fermentation liquid was collected on the 12th day.

[0023] The use of the porcine pseudorabies virus antigen in the preparation of a subunit vaccine for preventing and / or treating porcine pseudorabies is also within the scope of protection of the present invention.

[0024] A porcine pseudorabies subunit vaccine comprising the porcine pseudorabies virus antigen is also within the scope of protection of the present invention.

[0025] Wherein, the content of the porcine pseudorabies virus antigen is 30-125 μg / ml.

[0026] In some embodiments of the present invention, the content of the porcine pseudorabies virus gD protein is 40 μg / mL.

[0027] Wherein, the porcine pseudorabies subunit vaccine comprises an adjuvant, and the adjuvant is preferably ISA 201VG.

[0028] The preparation method of the above-mentioned pseudorabies subunit vaccine is specifically, preparing the pseudorabies virus antigen into an antigen solution, mixing it with an adjuvant, emulsifying it, and stirring it at 34-36° C. and 400-500 rpm for 15-20 minutes.

[0029] The concentration of the antigen solution is 30-125 μg / mL, and the adjuvant is ISA201.

[0030] In some embodiments of the present invention, the concentration of the antigen solution is 40 μg / mL, and the adjuvant is ISA201.

[0031] Specifically, the mass ratio of the antigen solution to the adjuvant is 1:1.

[0032] Beneficial effects:

[0033] The present invention provides a porcine pseudorabies virus antigen, whose amino acid sequence is shown in SEQ ID NO: 1. The present invention uses the porcine pseudorabies virus antigen to prepare a porcine pseudorabies virus subunit vaccine, which has a simple preparation method, a high vaccine neutralizing antibody value, strong antigen immunity, can effectively activate the body's immune response, and can play a good immune protection role against porcine pseudorabies. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0036] Example 1: Construction of cells expressing pseudorabies virus gD protein

[0037] 1. gD gene synthesis

[0038] The gD gene of the classical strain (JF797217.1) and the current popular strain (KP257591.1) was selected from GenBank as the research object. After sequence optimization and modification, a signal peptide sequence (METDTLLLWVLLLWVPGSTGD) was added to the N-terminal of the optimized gD sequence, and a His tag was added to finally obtain 5 sequences encoding gD genes, which were labeled as gD-1, gD-2, gD-3, and gD-4. The synthesis of all gD genes was completed by General Biotechnology Co., Ltd. Wherein, the amino acid sequences of the gD-1, gD-2, gD-3, and gD-4 are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, and the corresponding nucleotide sequences are shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0039] 2. Construction of recombinant plasmid

[0040] The coding gene of the recombinant protein of pseudorabies virus gD in step 1 was cloned into the eukaryotic expression vector pcDNA3.1. After verification, the recombinant plasmids pcDNA3.1-gD-1, pcDNA3.1-gD-2, pcDNA3.1-gD-3, and pcDNA3.1-gD-4 were obtained respectively.

[0041] 3. Cell transfection

[0042] (1) Subculture and expand the purchased CHO cells. At this time, the cells are in good condition and growing vigorously.

[0043] (2) One day before transfection (day –1), split the CHO cells from step 1 so that the final density of the CHO cells is 3.5×10 6 viable cells / mL and grow the cells overnight.

[0044] (3) The next day (day 0), the viable cell density and survival percentage were measured. When the cell density reached about 7 to 10 × 10 6 Live cells / mL. Transfection can be continued only when the survival rate is 95-99%.

[0045] (4) Dilute the cells from step 3 to a final density of 6 × 10 cells / mL using fresh ExpiCHO expression medium preheated to 37°C. 6 Gently shake the culture flask to mix the cells.

[0046] Note: Discard remaining cells; do not reuse high density cells for routine passaging.

[0047] (5) Use cold reagents (4°C) to prepare the ExpiFectamine CHO / plasmid DNA complex. There is no need to keep the reagents on ice during the complexation process. Simply remove the reagents from the refrigerator and start DNA complexation. The specific process is as follows:

[0048] A. Gently invert the ExpiFectamineCHO reagent bottle upside down 4 times to mix thoroughly.

[0049] B. Dilute 20 μL of recombinant plasmid DNA with 1 mL of cold OptiPRO medium. Mix by shaking or inverting the tube.

[0050] C. Dilute 80 μL of ExpiFectamineCHO reagent with 920 μL of OptiPRO medium. Mix by shaking or inverting the tube or gently pipetting 2 to 3 times.

[0051] D. Add the diluted ExpiFectamineCHO reagent to the diluted recombinant plasmid DNA. Shake or invert the test tube to mix well so that the ExpiFectamineCHO reagent and the recombinant plasmid DNA are fully in contact and form an ExpiFectamineCHO / plasmid DNA complex.

[0052] (6) Incubate the ExpiFectamineCHO / plasmid DNA complex obtained in step 5 at room temperature for 2 minutes, then slowly transfer the solution to the culture flask in step 4, gently shaking the culture flask during the addition process.

[0053] (7) After culturing for 48 hours in a cell culture incubator at 36-38°C and 5% CO2, the medium was discarded and replaced with DMEM complete medium containing 0.8 mg / ml G418 for further culturing.

[0054] 4. Screening of monoclonal cells

[0055] The 6-well plate cells were taken out from the incubator, the liquid was discarded, and pressure screening was performed with DMEM complete medium (containing 0.8 mg / ml G418). When the negative control cells were almost dead during the pressure screening, monoclonal cell screening was started.

[0056] Adjust the live cell density to 1.0 / well, inoculate 96-well plates, 200 μl per well, place at 36°C-38°C (preferably 37°C), and when the single cell line in the 96-well plate is amplified to more than 80-100%, detect the expression yield of the monoclonal cell line protein by ELISA. Digest the cells with higher expression values ​​and transfer them to 24-well plates. When the 24-well plates are full, take the supernatant for detection, detect the expression yield of the monoclonal cell line protein by ELISA, and screen the monoclonal cells with the expression yield of the monoclonal cell line protein greater than 1g / L.

[0057] 5. Protein Expression

[0058] Monoclonal cells were inoculated into commercial CHO serum-free medium at a cell density of 1.0×10 5 / ml, cultured in a constant temperature shaker at 36-38°C, 5% CO2, and a speed of 100r / min; the cell density, activity and glucose content were tested every 24 hours, and when the glucose was lower than 2.5g / L, glucose was supplemented to 3-4g / L; feeding was performed on the 4th and 9th days of fermentation, and the added volume was 10% of the original culture medium; the temperature was lowered to 32°C on the 5th day of fermentation; on the 12th day, the cell fermentation liquid was collected. After purification, 5 cell strains expressed proteins, which were named gD1, gD2, gD3, and gD4.

[0059] Example 2: Immunogenicity Assay

[0060] The five different protein fragments obtained in Example 1 were prepared into antigen solutions using PBS buffer as solvent. The 40 μg / mL antigen solution was mixed with an equal mass of ISA 201VG and emulsified, and then stirred at 34°C and 500 rpm for 15 min to prepare different groups of vaccine samples for KM mouse immunization experiments in the laboratory.

[0061] 60 healthy KM mice weighing about 20g (purchased from Xi'an Yifengda Biotechnology Co., Ltd.) were randomly divided into six groups. Groups 1 to 5 were immunized with vaccine samples of different antigen groups (subcutaneous injection, 0.2mL / mouse), and 10 non-immunized mice were set as the control group. 21 days after immunization, blood was collected from the eye sockets, and serum was separated under a sterile environment to determine the neutralizing antibody of pseudorabies virus in pigs. It was found that the protein group with the highest neutralizing antibody value was the gD1 protein in the first group. The specific test results are shown in Table 1.

[0062] Table 1 Grouping and results of KM mouse immunization experiment

[0063]

[0064] Example 3: Immune effect and safety evaluation

[0065] The gD1 protein obtained based on immunogenicity in Example 2 was subjected to immune effect and safety evaluation. The operation was specifically carried out according to the following steps:

[0066] The gD1 protein was purified by hollow fiber ultrafiltration to remove impurities such as cell debris. The purified gD1 protein was then quantitatively analyzed using a protein quantification kit. The quantified gD1 protein was prepared into an antigen solution using PBS buffer as a solvent, and then the 40μg / mL antigen solution was mixed with ISA201 adjuvant to prepare a vaccine. Specifically, the emulsification ratio of the aqueous phase (antigen solution) to the oil phase (ISA201 adjuvant) was 1:1 by mass. The oil phase was first introduced into a beaker, stirred slowly, and the aqueous phase was slowly added. After the aqueous phase was added, the shear emulsification equipment was started, and the mixture was emulsified for 15 minutes to form a stable oil-in-water emulsion, thereby obtaining a vaccine emulsion with good physical stability. After the emulsification is completed, the vaccine emulsion is aseptically quantitatively packaged (50mL / bottle), sealed with a cap, and the porcine pseudorabies virus subunit vaccine is obtained, which is stored at 2-8°C.

[0067] Healthy, well-developed, and uninfected 21-day-old piglets were selected as experimental animal models. After immunizing 21-day-old piglets with the pseudorabies virus subunit vaccine (injected into the neck muscle behind the ear, 2 mL / head), blood was collected and the neutralizing antibody level in the serum was quantitatively analyzed. The results showed that the neutralizing antibody level on day 21 was not less than 1:362.

[0068] In order to further verify the actual protective effect of the vaccine, the immunized pigs were challenged with the classic virulent strain (SC strain) and the current epidemic strain (JS strain). The challenge results showed that the vaccine can effectively prevent the attack of the classic virulent strain (SC strain) and the current epidemic strain (JS strain) at the same time, with a protection rate of 100%, and all immunized pigs had no fever and abnormal clinical symptoms (Table 2).

[0069] Table 2 Experimental groups and results

[0070]

[0071] The present invention provides a porcine pseudorabies virus antigen, a porcine pseudorabies subunit vaccine, and a method for preparing and applying the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A porcine pseudorabies virus antigen, characterized in that: The porcine pseudorabies virus antigen is the porcine pseudorabies virus gD protein; wherein the amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO:

1.

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

2.

3. An expression vector, characterized in that: Contains the coding gene according to claim 2.

4. A recombinant cell, characterized in that Contains the coding gene according to claim 2, or contains the expression vector according to claim 3.

5. A method for preparing porcine pseudorabies virus antigen, characterized in that: The nucleotide sequence of the porcine pseudorabies virus gD protein is cloned into a eukaryotic expression vector to construct a recombinant plasmid, which is then transfected into expression cells, and the porcine pseudorabies virus antigen is obtained through culture, screening, protein expression and purification.

6. The preparation method according to claim 5, characterized in that: The eukaryotic expression vector is any one of pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, and the expression cell is a CHO cell among mammalian cells.

7. Use of the porcine pseudorabies virus antigen according to claim 1 in the preparation of a subunit vaccine for preventing and / or treating porcine pseudorabies.

8. A porcine pseudorabies subunit vaccine, characterized in that: The porcine pseudorabies subunit vaccine comprises the porcine pseudorabies virus antigen according to claim 1.

9. The porcine pseudorabies subunit vaccine according to claim 8, characterized in that: The content of the porcine pseudorabies virus antigen is 30-125 μg / ml.

10. The porcine pseudorabies subunit vaccine according to claim 8, characterized in that: The porcine pseudorabies subunit vaccine comprises an adjuvant, and the adjuvant is preferably ISA 201VG.

Images