Stable-expression porcine pseudorabies virus gD recombinant protein, vaccine composition as well as preparation method and application of vaccine composition

Abstract

The invention relates to the technical field of biological medicine, in particular to stably expressed porcine pseudorabies virus gD recombinant protein, a vaccine composition and a preparation method and application of the vaccine composition. The invention provides a porcine pseudorabies virus gD recombinant protein capable of being stably expressed. The amino acid sequence of the porcine pseudorabies virus gD recombinant protein is shown as SEQ ID NO: 7. The porcine pseudorabies virus gD recombinant protein is stably expressed in monoclonal cells, the passage frequency is stable, and when 48 generations are continuously passed, the porcine pseudorabies virus gD recombinant protein still has good protein expression quantity. And a good foundation is laid for subsequent application in preparation of a stable porcine pseudorabies subunit vaccine composition or a porcine pseudorabies virus vaccine potency test preparation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a stably expressed porcine pseudorabies virus gD recombinant protein, a vaccine composition, and a preparation method and application thereof. Background Art

[0002] Porcine Pseudorabies, also known as swine fever and scrapie or porcine infectious encephalomyelitis, is an acute infectious disease caused by the Porcine Pseudorabies Virus (PRV) that infects pigs, cattle, sheep, dogs, cats, rabbits, and even humans.

[0003] Although there are vaccines for pseudorabies in pigs on the market, such as JS-A1 strain, Bartha-K61 strain, HB-98 strain, HB2000 strain, etc., the virus strain has been in a process of continuous evolution. In order to adapt to the mutation of the virus, further improve the immune effect of the vaccine, achieve accurate prevention and control and purification of pseudorabies in pigs, and promote the development of vaccine and diagnostic technology, it is necessary to continuously explore and prepare new recombinant proteins of pseudorabies virus that can be stably expressed in pigs to deal with new virus strains. 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 stably expressed porcine pseudorabies virus gD recombinant protein.

[0005] The second technical problem to be solved by the present invention is to provide the above-mentioned coding gene for the stably expressed porcine pseudorabies virus gD recombinant protein.

[0006] The third technical problem to be solved by the present invention is to provide a recombinant expression vector containing the above encoding gene.

[0007] The fourth technical problem to be solved by the present invention is to provide a recombinant cell containing the above encoding gene or the above recombinant expression vector.

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

[0009] The sixth technical problem to be solved by the present invention is to provide a use of a stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of a subunit vaccine composition 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 virus subunit vaccine composition.

[0011] The eighth technical problem to be solved by the present invention is to provide an application of a stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of a porcine pseudorabies virus vaccine efficacy test preparation.

[0012] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0013] A stably expressed porcine pseudorabies virus gD recombinant protein, the amino acid sequence of the porcine pseudorabies virus gD recombinant protein is shown in SEQ ID NO:7.

[0014] Among them, when the porcine pseudorabies virus gD recombinant protein is continuously passaged for 48 generations in monoclonal cells, the protein expression level does not decrease and still has a good protein expression level.

[0015] The nucleotide sequence of the gene encoding the porcine pseudorabies virus gD recombinant protein is shown in SEQ ID NO:8.

[0016] Specifically, the coding gene was obtained by selecting the gD genes of the classic strain (JF797217.1) and the current epidemic strain (KP257591.1) from GenBank as research objects. After sequence optimization and modification, a signal peptide sequence (METDTLLLWVLLLWVPGSTGD) was added to the N-terminus of the optimized gD sequence, and a His tag was added.

[0017] A recombinant expression vector containing the gene encoding the porcine pseudorabies virus gD recombinant protein is also within the scope of protection of the present invention.

[0018] A recombinant cell containing the coding gene of the porcine pseudorabies virus gD recombinant protein or a recombinant expression vector is also within the scope of protection of the present invention.

[0019] A method for preparing a stably expressed porcine pseudorabies virus gD recombinant protein comprises the following steps: transfecting a recombinant plasmid expressing the porcine pseudorabies virus gD recombinant protein into mammalian cells, and performing culture, pressure screening, monoclonal cell screening, and protein expression purification to obtain the protein.

[0020] The recombinant plasmid expressing the recombinant protein of pseudorabies virus gD is obtained by cloning the coding gene of the recombinant protein of pseudorabies virus gD into a eukaryotic expression vector.

[0021] Specifically, the eukaryotic expression vector includes but is not limited to pcDNA3.1. All eukaryotic expression vectors in the prior art that can be used for cloning the recombinant protein of pseudorabies virus gD are within the scope of protection of the present invention, such as pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.

[0022] Wherein, the mammalian cells are preferably CHO cells, and the cell confluence during transfection is 80% to 90%.

[0023] The culture conditions are as follows: 36° C. to 38° C. in a cell culture incubator containing 5% CO 2 for 48 hours.

[0024] Wherein, the pressure screening is to screen positive cell lines through G418.

[0025] Specifically, pressure screening was performed in DMEM complete medium containing 0.8 mg / mL G418.

[0026] Among them, the monoclonal cell screening starts when the negative control cells are basically dead after pressure screening. Adjust the live cell density to 1.0 / well, inoculate a 96-well plate with 200 μL per well, and place it at 36°C to 38°C. When the single cell line in the 96-well plate is amplified to more than 80% to 100%, the expression yield of the monoclonal cell line protein is detected by ELISA. Digest the cells with higher expression values ​​and transfer them to a 24-well plate. When the 24-well plate is full, take the supernatant for detection, and detect the protein expression yield of the monoclonal cells by ELISA.

[0027] 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 36-38°C in a constant temperature shaker containing 5% CO2 at a speed of 100 r / min; the cell density, activity and glucose content were detected every 24 hours, and when the glucose content 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.

[0028] The use of the stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of a subunit vaccine composition for preventing and / or treating porcine pseudorabies is also within the scope of protection of the present invention.

[0029] A porcine pseudorabies virus subunit vaccine composition comprises an immunizing amount of porcine pseudorabies virus gD recombinant protein.

[0030] The use of the porcine pseudorabies virus gD recombinant protein in the preparation of a porcine pseudorabies virus vaccine efficacy test preparation is also within the scope of protection of the present invention.

[0031] Beneficial effects: The present invention provides a porcine pseudorabies virus gD recombinant protein (i.e., gD4) that can be stably expressed, and the amino acid sequence of the porcine pseudorabies virus gD recombinant protein is shown in SEQ ID NO: 7. It is obtained by transfecting a recombinant plasmid expressing the porcine pseudorabies virus gD recombinant protein into mammalian cells, and then culturing, pressurized screening, monoclonal cell screening, and protein expression. The monoclonal cell of the porcine pseudorabies virus gD recombinant protein has a stable number of passages when stably expressed, and when continuously passaged to 48 generations, after protein expression, gD4 still has a good protein expression amount. It lays a good foundation for the subsequent preparation of a stable porcine pseudorabies subunit vaccine composition or a porcine pseudorabies virus vaccine efficacy test preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0033] Figure 1 The results of identification of gD4 protein expressed in cells of different generations. Among them, M is Marker; N is negative control; P is positive control; 1 is F10 generation; 2 is F16 generation; 3 is F22 generation; 4 is F38 generation; 5 is F48 generation. 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 porcine 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 36-38°C (preferably 37°C), and when the single cell line in the 96-well plate is amplified to more than 80-100%, detect the protein expression yield of the monoclonal cell line 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 protein expression yield of the monoclonal cells by ELISA, and screen the monoclonal cells with protein expression yield 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, the expression proteins of 5 monoclonal cells were obtained, which were named gD1, gD2, gD3, and gD4 in sequence.

[0059] Example 2: Stability evaluation of gD protein cells

[0060] The four gD protein expressing cells frozen in Example 1 were inoculated into 60 mL of basal culture medium to a cell density of 1.0×10 5 / mL, placed at 36-38℃, 5% CO2 shaker for suspension culture. When the cell density reaches 3×10 5 / mL, and when the cell viability reached more than 90%, the cells were subcultured every 2 to 3 days. For continuous subculture, F10, F16, F22, F38 and F48 cells were taken for protein expression. On the 12th day, the cell culture supernatant was harvested for protein expression detection. The results are detailed in Table 1. Figure 1 The results show the identification of gD4 protein expressed in cells of different generations.

[0061] Table 1 Expression of gD protein in different generations

[0062]

[0063] From Table 1 and Figure 1 It can be seen that the gD4 protein has a good protein expression level after 48 consecutive passages, indicating that its passage number is stable, which lays a good foundation for the subsequent preparation of stable porcine pseudorabies subunit vaccine or porcine pseudorabies virus vaccine efficacy test preparation.

[0064] Example 3: Immune effect and safety evaluation

[0065] The immune effect and safety evaluation of gD4 protein expressed by gD-4 cell line were determined. The specific steps are as follows:

[0066] The gD4 protein was purified by hollow fiber ultrafiltration to remove impurities such as cell debris. The purified gD4 protein was then quantitatively analyzed using a protein quantification kit. The quantified gD4 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 (muscular injection behind the ear and neck, 2 mL / head), blood was collected and the neutralizing antibody level in the serum was quantitatively analyzed. The results showed that the neutralizing antibody on the 21st day was not less than 1:102. This result fully proves that the pseudorabies virus subunit vaccine can effectively stimulate the piglet body to produce high levels of neutralizing antibodies, providing a strong immune defense against the invasion of pseudorabies virus.

[0068] In order to verify the actual protective effect of the vaccine, the experiment set up a virus challenge phase. The immunized pigs were challenged with the classic strong virus (SC strain) and the current epidemic strain (JS strain). The challenge results showed that the vaccine can effectively prevent the attack of the classic strong virus (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.

[0069] In summary, the porcine pseudorabies virus subunit vaccine of the present invention can not only induce the body to produce high levels of neutralizing antibodies after immunizing 21-day-old piglets, but also shows good protection effect and safety in the challenge experiment (Table 2).

[0070] Table 2 Experimental groups and results

[0071]

[0072] The present invention provides a stably expressed porcine pseudorabies virus gD recombinant protein, a vaccine composition, and a preparation method and application thereof. 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 protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

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

7.

2. The porcine pseudorabies virus gD recombinant protein according to claim 1, characterized in that: When the porcine pseudorabies virus gD recombinant protein is continuously propagated for 48 generations in monoclonal cells, the protein expression level does not decrease.

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

8.

4. A recombinant expression vector, characterized in that: Contains the coding gene according to claim 3.

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

6. A method for preparing a stably expressed porcine pseudorabies virus gD recombinant protein, characterized in that: The recombinant plasmid containing the recombinant protein of porcine pseudorabies virus gD is transfected into mammalian cells, and the cells are cultured, screened under pressure, screened for monoclonal cells, and purified for protein expression to obtain the recombinant protein.

7. The preparation method according to claim 6, characterized in that: The recombinant plasmid expressing the recombinant protein of pseudorabies virus gD is obtained by cloning the coding gene of the recombinant protein of pseudorabies virus gD into a eukaryotic expression vector.

8. Use of the stably expressed porcine pseudorabies virus gD recombinant protein according to claim 1 in the preparation of a subunit vaccine composition for preventing and / or treating porcine pseudorabies.

9. A porcine pseudorabies virus subunit vaccine composition, characterized in that: The porcine pseudorabies virus subunit vaccine composition comprises the porcine pseudorabies virus gD recombinant protein described in claim 1.

10. Use of the recombinant porcine pseudorabies virus gD protein of claim 1 in the preparation of a porcine pseudorabies virus vaccine efficacy test preparation.

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