Process and application of porcine pseudorabies virus gene deletion inactivated vaccine

Abstract

The invention relates to the technical field of veterinary biological products, and discloses a porcine pseudorabies virus gene deletion inactivated vaccine preparation process, which comprises: S1, carrying out serum-free suspension culture on a porcine pseudorabies virus; s2, virus content detection; s3, an antigen treatment and purification process; and S4, proportioning and emulsifying the inactivated vaccine. The prepared suspension virus is high in content, short in culture period, simple and stable in purification process and easy to operate, the production cost can be greatly reduced, meanwhile, vaccine preparation proportion of antigens is optimized, the purification loss rate is low, antigen purity is high, immunogenicity is enhanced, side reactions of pigs can be reduced, vaccine batch difference is reduced, the quality is easy to control, and the method is suitable for large-scale popularization and application. The vaccine yield and quality can be remarkably improved, the vaccine protection rate is increased, the vaccine stability duration is long, the immune effect is enhanced, the inter-batch difference is reduced, and the immune duration is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a process and application of a porcine pseudorabies virus gene-deleted inactivated vaccine. Background Art

[0002] Pseudorabies (PR) is an important infectious disease of pigs caused by pseudorabies virus (PRV). The main clinical symptoms include central nervous system disorders in newborn piglets, respiratory symptoms in weaned piglets and fattening pigs, and reproductive disorders in pregnant sows, causing huge economic losses to the world's pig farming industry.

[0003] At present, the prevention and control of pseudorabies mainly relies on vaccine immunity. Studies have shown that PRV gE protein is the main glycoprotein and the main virulence protein encoded by the gE gene. The gI glycoprotein and the gE glycoprotein together form a non-covalent complex. Since gE / gI proteins are non-essential proteins for viral replication, the deletion of the gE / gI gene can reduce the toxicity of the virus without affecting or even improving the immunogenicity of the virus. Therefore, gE / gI gene-deficient vaccines have been widely developed and used. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the shortcomings of the prior art, the present invention provides a process and application of a porcine pseudorabies virus gene-deleted inactivated vaccine, which has the advantages of high suspended virus content, short culture cycle, simple, stable and easy-to-operate purification process, which can greatly reduce production costs, and at the same time optimize the antigen-vaccine ratio, low purification loss rate, high antigen purity and enhanced immunogenicity, which can reduce adverse reactions in pigs, reduce batch-to-batch differences in vaccines, and easily control quality. It can significantly improve vaccine production and quality, improve vaccine protection rate, and has the advantages of long vaccine stability period, enhanced immune effect, reduced batch-to-batch differences, and enhanced immune duration.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine, comprising the following steps:

[0008] S1: Serum-free suspension culture of pseudorabies virus: BHK full suspension cells were quickly taken out from liquid nitrogen and quickly thawed in a 37°C water bath. After the BHK full suspension cells were completely thawed, about 20 times the volume of Tac-S101 BHK cell serum-free culture medium was added to the recovered cells, and the cells were gently dispersed and centrifuged at 800 rpm / min for 5 min. The supernatant was poured out, and Tac-S101 BHK cell serum-free culture medium was added to resuspend the cells evenly by blowing, inoculated into a 125 ml shake bottle, and placed on a 5% CO2, 37°C incubator equipped with an orbital shaker (shaking speed of 125 rpm) for suspension culture. After 48 hours of culture, the cells were diluted and passaged according to a certain ratio. After the third generation of cells was stable, they could be inoculated with virus culture. When the cells were passaged to the fourth generation, they were cultured for 48-60 hours and inoculated with serum-free suspension culture PRV according to MOI = 0.1-1.0. ZJ1G strain virus was added to the cell maintenance medium according to the volume ratio of BHK cell culture medium to adjust the cell density to 2.0-3.0×10 6 / ml, and culture until the cell density is 1.0-1.5×10 6 The virus solution was harvested when the cell viability was 50-60%, and the culture time after virus inoculation was 48-72 hours. 50 Detection no less than 10 8.5 / 0.1ml;

[0009] S2: Virus content detection: The cultured virus was diluted 10 times in serum-free cell culture medium, and 10 -7 , 10 -8 , 10 -9 Three dilutions were prepared. Each dilution was inoculated into 6 wells of a 96-well culture plate with PK15 adherent cell monolayers, 0.1 ml per well. A negative control was set up. The cells were cultured in an incubator at 37°C with 5% CO2 for 72 hours. The number of wells showing cytopathic effect (CPE) at each dilution was observed under a microscope. The virus TCID was calculated according to the Reed-Muench method. 50 ;

[0010] S3: Antigen treatment and purification process: The harvested virus solution was frozen and thawed three times, centrifuged at 4°C, and the supernatant was first filtered through a filter, then filtered through a 500KD hollow fiber ultrafiltration, concentrated 5-10 times, and then reduced with PBS. This was repeated three times (hollow fiber ultrafiltration part). After purification, the virus content loss of the virus solution did not exceed 0.2 titers, and the removal rate of foreign proteins reached 85%-90%;

[0011] S4: Inactivated vaccine proportioning and emulsification: dilute the purified antigen to a virus content of 10 7.0 TCID 50 / ml, add formaldehyde solution to the diluted virus solution to make the final formaldehyde concentration in the virus solution 0.2%, place it in a constant temperature shaker, shake and inactivate for 18 hours, add 0.2% sodium metabisulfite to terminate the inactivation, mix the completely inactivated antigen with the sterile immunopotentiator QS201 to obtain an aqueous phase; then put the 15A adjuvant and the mixed aqueous phase into an emulsification tank according to the proportion, stir slowly at 300r / min for 10min, mix and emulsify to obtain an inactivated vaccine.

[0012] Preferably, after adjusting the cell density in S1, 0.5% NaHCO3 solution is added.

[0013] Preferably, the pore size of the filter in S3 is 0.6 μm.

[0014] Preferably, the centrifugal speed in S3 is 4000 rpm and the centrifugal time is 15-20 min.

[0015] Preferably, the temperature of the constant temperature shaker in S4 is 37°C.

[0016] Preferably, the ratio of the completely inactivated antigen to the sterile immunopotentiator QS201 in S4 is 79:1.

[0017] Preferably, the ratio of the 15A adjuvant to the mixed aqueous phase in S4 is 3.8:16.2.

[0018] The present invention also provides a porcine pseudorabies virus gene-deleted inactivated vaccine using the above-mentioned vaccine.

[0019] Preferably, the vaccine is a porcine pseudorabies virus gene-deleted inactivated vaccine.

[0020] Compared with the prior art, the present invention provides a gene-deleted inactivated vaccine process and application of porcine pseudorabies virus, which has the following beneficial effects:

[0021] The suspended virus prepared by the present invention has a high content, a short culture cycle, and a simple, stable and easy-to-operate purification process, which can greatly reduce production costs. At the same time, the antigen-vaccine ratio is optimized, the purification loss rate is low, the antigen purity is high, and the immunogenicity is enhanced, which can reduce adverse reactions in pigs, reduce batch-to-batch differences in vaccines, and is easy to control quality. It can significantly improve vaccine production and quality, improve vaccine protection rate, and ensure a long stable vaccine period, enhanced immune effects, reduced batch differences, and enhanced immune duration. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] A preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine comprises the following steps:

[0024] S1: Serum-free suspension culture of pseudorabies virus: BHK full suspension cells were quickly taken out from liquid nitrogen and quickly thawed in a 37°C water bath. After the BHK full suspension cells were completely thawed, about 20 times the volume of Tac-S101 BHK cell serum-free culture medium was added to the recovered cells, and the cells were gently dispersed and centrifuged at 800 rpm / min for 5 min. The supernatant was poured out, and Tac-S101 BHK cell serum-free culture medium was added to resuspend the cells evenly by blowing, inoculated into a 125 ml shake bottle, and placed on a 5% CO2, 37°C incubator equipped with an orbital shaker (shaking speed of 125 rpm) for suspension culture. After 48 hours of culture, the cells were diluted and passaged according to a certain ratio. After the third generation of cells was stable, they could be inoculated with virus culture. When the cells were passaged to the fourth generation, they were cultured for 48-60 hours and inoculated with serum-free suspension culture PRV according to MOI = 0.1-1.0. ZJ1G strain virus was added to the cell maintenance medium according to the volume ratio of BHK cell culture medium to adjust the cell density to 2.0-3.0×10 6 / ml, and add 0.5% NaHCO3 solution, and culture until the cell density is 1.0-1.5×10 6 The virus solution was harvested when the cell viability was 50-60% and the inoculation time was 48-72 hours. 50 Detection no less than 10 8.5 / 0.1ml;

[0025] S2: Virus content detection: The cultured virus was diluted 10 times in serum-free cell culture medium, and 10 -7 , 10 -8 , 10 -9 Three dilutions were prepared. Each dilution was inoculated into 6 wells of a 96-well culture plate with PK15 adherent cell monolayers, 0.1 ml per well. A negative control was set up. The cells were cultured in an incubator at 37°C with 5% CO2 for 72 hours. The number of wells showing cytopathic effect (CPE) at each dilution was observed under a microscope. The virus TCID was calculated according to the Reed-Muench method. 50 ;

[0026] S3: Antigen treatment and purification process: After the harvested virus liquid is repeatedly frozen and thawed 3 times, it is centrifuged at 4000rpm for 15-20min at 4℃. The supernatant is first filtered through a 0.6μm filter, then filtered through a 500KD hollow fiber ultrafiltration, concentrated 5-10 times, and then reduced with PBS. This is repeated 3 times (hollow fiber ultrafiltration part). After purification, the virus content loss of the virus liquid does not exceed 0.2 titers, and the removal rate of foreign proteins reaches 85%-90%;

[0027] <![CDATA[Virus content (TCID 50 / 0.1 ml)]]> Protein content (mg / ml) Before purification 8.85 2.18 After purification 8.69 0.28

[0028] S4: Inactivated vaccine proportioning and emulsification: dilute the purified antigen to a virus content of 10 7.0 TCID 50 / ml, add formaldehyde solution to the diluted virus solution to make the final formaldehyde concentration in the virus solution 0.2%, place it in a constant temperature shaker at 37℃, shake and inactivate for 18 hours, add 0.2% sodium metabisulfite to terminate the inactivation, mix the completely inactivated antigen and sterile immunopotentiator QS201 in a ratio of 79:1 to obtain an aqueous phase; then put the 15A adjuvant and the mixed aqueous phase into an emulsification tank in a ratio of 3.8:16.2, stir slowly at 300r / min for 10 minutes, mix and emulsify to obtain an inactivated vaccine.

[0029] Comparison of immunogenicity effects

[0030]

[0031] Effectiveness test

[0032]

[0033] Body temperature changes 14 days after infection (body temperature > 40°C is considered elevated)

[0034]

[0035]

[0036] Clinical symptoms 14 days after infection

[0037] serial number Group Clinical symptoms 1 Patent Group No clinical symptoms 2 Purification control group No clinical symptoms 3 Vaccine control group No clinical symptoms 4 Original vaccine group One pig showed signs of anorexia 5 Blank control group Four showed anorexia

[0038] Neutralizing antibody detection before virus attack

[0039]

[0040] Post-challenge efficacy test

[0041]

[0042]

[0043] It can be seen from the above results that the suspended virus prepared by the present invention has a high content, a short culture cycle, a simple, stable and easy-to-operate purification process, which can greatly reduce production costs. At the same time, the antigen-vaccine ratio is optimized, the purification loss rate is low, the antigen purity is high and the immunogenicity is enhanced, which can reduce the adverse reactions of pigs, reduce the batch differences of vaccines, and make the quality easy to control. It can significantly improve the vaccine yield and quality, improve the vaccine protection rate, and the vaccine has a long stable duration, enhanced immune effect, reduced batch differences, and enhanced immune duration.

[0044] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation process for a porcine pseudorabies virus gene-deleted inactivated vaccine, It is characterized in that The steps include: S1: Serum-free suspension culture of pseudorabies virus: BHK whole suspension cells were quickly taken out from liquid nitrogen and thawed rapidly in a 37°C water bath. After the BHK whole suspension cells were completely thawed, about 20 times the volume of Tac-S101 BHK cell serum-free medium was added to the resuscitated cells, and the cells were gently dispersed and centrifuged at 800 rpm / min for 5 min. The supernatant was discarded, and Tac-S101 BHK cell serum-free medium was added to resuspend the cells evenly by blowing, and inoculated into a 125 ml shaking bottle, and placed on a 5% CO2-loaded orbital shaker (shaking speed was 125 rpm). 2 , suspension culture was performed in a 37°C incubator. After 48 hours of culture, the cells were diluted and subcultured according to a certain ratio. After the third generation of cells was stable, they could be inoculated with virus. When the cells were subcultured to the fourth generation, after 48-60 hours of culture, they were inoculated with serum-free suspension culture PRV ZJ1G strain virus at MOI = 0.1-1.

0. Cell maintenance medium was added according to the volume ratio of BHK cell culture medium to adjust the cell density to 2.0-3.0×10 6 / ml, and culture until the cell density is 1.0-1.5×10 6 The virus solution was harvested when the cell viability was 50-60%, and the culture time after virus inoculation was 48-72 hours. 50 Detection no less than 10 8.5 / 0.1ml; S2: Virus content detection: The cultured virus was diluted 10 times in serum-free cell culture medium, and 10 -7 , 10 -8 , 10 -9 Three dilutions were added, and each dilution was inoculated into 6 wells of 96-well culture plates with PK15 adherent cell monolayers, 0.1 ml per well. A negative control was also set up at 37°C with 5% CO. 2 The cells were cultured in an incubator for 72 hours, and the number of wells showing cytopathic effect (CPE) at each dilution was observed under a microscope. The virus TCID was calculated according to the Reed-Muench method. 50 ; S3: Antigen treatment and purification process: The harvested virus solution was frozen and thawed three times, centrifuged at 4°C, and the supernatant was first filtered through a filter, then filtered through a 500KD hollow fiber ultrafiltration, concentrated 5-10 times, and then reduced with PBS. This was repeated three times (hollow fiber ultrafiltration part). After purification, the virus content loss of the virus solution did not exceed 0.2 titers, and the removal rate of foreign proteins reached 85%-90%; S4: Inactivated vaccine proportioning and emulsification: dilute the purified antigen to a virus content of 10 7.0 TCID 50 / ml, add formaldehyde solution to the diluted virus solution to make the final formaldehyde concentration in the virus solution 0.2%, place it in a constant temperature shaker, shake and inactivate for 18 hours, add 0.2% sodium metabisulfite to terminate the inactivation, mix the completely inactivated antigen with the sterile immunopotentiator QS201 to obtain an aqueous phase; then put the 15A adjuvant and the mixed aqueous phase into an emulsification tank according to the proportion, stir slowly at 300r / min for 10min, mix and emulsify to obtain an inactivated vaccine.

2. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: After adjusting the cell density in S1, add 0.5% NaHCO 3 Solution.

3. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: The pore size of the filter in S3 is 0.6 μm.

4. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: The centrifugal speed in S3 is 4000 rpm and the centrifugal time is 15-20 min.

5. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: The temperature of the constant temperature shaker in S4 is 37°C.

6. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: The ratio of the completely inactivated antigen to the sterile immune enhancer QS201 in the S4 is 79:

1.

7. The preparation process of a porcine pseudorabies virus gene-deleted inactivated vaccine according to claim 1, Features: The ratio of the 15A adjuvant to the mixed aqueous phase in the S4 is 3.8:16.

2.

8. Use of the preparation process of a porcine pseudorabies virus gene-deficient inactivated vaccine according to any one of claims 1 to 7 in the preparation of a porcine pseudorabies virus gene-deficient inactivated vaccine.

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