Heat-resistant protective agent live vaccine for highly pathogenic porcine reproductive and respiratory syndrome

Abstract

The invention discloses a heat-resistant protective agent live vaccine for highly pathogenic porcine reproductive and respiratory syndrome, and belongs to the technical field of protective agent vaccines.The heat-resistant protective agent live vaccine is prepared by the following steps: S1, cell culture: carrying out bioreactor cell microcarrier suspension culture according to a monkey embryo kidney epithelial cell Marc-145 microcarrier cell suspension culture process; according to the preparation method, the first antigen liquid and the second antigen liquid are extracted and mixed, the pig alpha interferon is added to be matched with the aluminum hydroxide adjuvant to obtain the composite immunologic adjuvant, and the antigen vaccine is prepared by adopting a suspension culture process; the vaccine can effectively improve the virus titer, achieves the effect of simultaneously preventing the high-pathogenicity porcine reproductive and respiratory syndrome virus and the haemophilus parasuis disease, avoids the condition of mixed infection, and greatly improves the immune regulation effect of the vaccine and enhances the vaccine effect by virtue of the composite immunologic adjuvant.

Description

Technical Field

[0001] The present invention relates to the technical field of protectant vaccines, and more particularly to a heat-resistant protectant live vaccine for highly pathogenic porcine reproductive and respiratory syndrome. Background Art

[0002] At present, highly pathogenic porcine reproductive and respiratory syndrome is one of the main epidemic diseases that threatens my country's pig industry. It mainly causes reproductive disorders such as miscarriage in late pregnancy, premature birth, stillbirth, etc. in sows. At the same time, it is easy to mix or secondary infection with Haemophilus parasuis. The treatment of this disease requires the development of vaccines to immunize the pig herd.

[0003] However, most of the existing vaccines are only inactivated vaccines that can prevent highly pathogenic porcine reproductive and respiratory syndrome alone, and do not have the function of simultaneously preventing Haemophilus parasuis disease, which is easily co-infected with porcine reproductive and respiratory syndrome. Therefore, the vaccine has poor comprehensiveness in prevention, and the adjuvants used in the preparation of the vaccine do not have comprehensive immunomodulatory effects, which greatly reduces the effectiveness of the vaccine. At the same time, most of them have the disadvantages of low antigen efficiency and poor protective effect in cell proliferation and freeze-drying processes. Therefore, it is necessary to develop a heat-resistant protectant live vaccine for highly pathogenic porcine reproductive and respiratory syndrome with good preventive effect. Summary of the invention

[0004] (1) Technical issues to be solved

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a heat-resistant protectant live vaccine for highly pathogenic porcine reproductive and respiratory syndrome, which has the characteristics of preventing both highly pathogenic porcine reproductive and respiratory syndrome and Haemophilus parasuis disease, having good vaccine immunomodulatory effects and good protective and therapeutic effects.

[0006] (2) Technical solution

[0007] To achieve the above object, the present invention provides a highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine, comprising the following preparation steps:

[0008] S1. Cell culture

[0009] The bioreactor cell microcarrier suspension culture was carried out according to the monkey embryonic kidney epithelial cell Marc-145 microcarrier cell suspension culture process;

[0010] After the number of cells has proliferated to an appropriate scale, the stirring paddle is turned off, and the culture supernatant is discharged after complete sedimentation, and then the microcarriers are rinsed aseptically, and then a pancreatic enzyme solution is added for digestion, and then the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for secondary suspension culture, and the above steps are repeated, and the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for tertiary culture;

[0011] S2. Viral antigen extraction

[0012] The microcarrier suspension culture of HP-PRRS virus is carried out in a bioreactor according to the suspension culture process of HP-PRRS virus, and the cell culture of HP-PRRS virus harvested from the suspension culture is frozen and thawed, and the antigen liquid is filtered with a filter rod to remove the microcarrier to obtain the first antigen liquid;

[0013] Taking Haemophilus parasuis for proliferation and cultivation, harvesting a bacterial solution containing Haemophilus parasuis, concentrating it, inactivating and washing it, separating it to obtain an inactivated Haemophilus parasuis concentrate, adding Tween-80 to the concentrate to obtain a second antigen solution;

[0014] The first antigen liquid and the second antigen liquid are mixed in proportion to obtain a mixed antigen liquid;

[0015] S3. Selection and preparation of adjuvants

[0016] The porcine interferon alpha protein with antiviral activity produced by expressing the porcine interferon alpha gene in an expression system by recombinant DNA technology is used as porcine interferon alpha and combined with aluminum hydroxide adjuvant to obtain a composite immune adjuvant;

[0017] S4. Preparation of heat-resistant protective agent

[0018] Sodium L-glutamate, tryptone, hydrolyzed milk protein, enzymatic casein, sucrose, thiourea, gelatin, trehalose, D-sorbitol, glycine and arginine were prepared into 5 kinds of protective agent formulas in different proportions to obtain 5 kinds of heat-resistant protective agents, and the best lyophilized protective agent was obtained by selecting different lyophilization curves for lyophilization;

[0019] S5. Seedling matching

[0020] The first antigen solution and the second antigen solution were diluted with PBS solution, and the composite adjuvant was added and stirred, and then mixed with the heat-resistant protective agent in a ratio of 3:1, and then quantitatively packaged and freeze-dried to obtain a highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protective agent live vaccine.

[0021] When using the highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine of the present technical solution, the virus titer can be effectively increased by preparing the antigen vaccine through the suspension culture process. The vaccine safety is ensured and the vaccine antigen efficiency is improved by antigen dilution and proportional matching of the heat-resistant protectant. Through the virus attack experiment, it is proved that the highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine has a good protective effect on the strain in pigs immunized, and can be used for the prevention and protection of pig herds from HP-PRRS.

[0022] Furthermore, before inoculating the cells in the bioreactor used in S1, the warm culture medium is sterile filtered and then injected into the bioreactor tank, which is rinsed multiple times, and the supernatant is discharged after the microcarriers are completely settled.

[0023] Furthermore, the preheating temperature of the pancreatic enzyme solution in S1 is 37 degrees.

[0024] Furthermore, the freeze-thaw temperature in S2 is -40 degrees.

[0025] Furthermore, the freeze-drying curve in S4 includes four freeze-drying curves, and each freeze-drying curve includes a pre-freezing stage, a sublimation drying stage and an analytical drying stage.

[0026] Furthermore, the filter rod in S3 adopts a deep filter element made of PP2 material.

[0027] Furthermore, the best time for inoculation in S2 is the fourth day of cell growth, and the best time for collecting the virus is 70-74 hours after infection.

[0028] Furthermore, the pH value of the PBS solution is 7.2.

[0029] (3) Beneficial effects

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1. The present invention extracts the first antigen liquid and the second antigen liquid, mixes them, and adds porcine interferon alpha and aluminum hydroxide adjuvant to obtain a composite immune adjuvant. The antigen vaccine prepared by the suspension culture process can effectively increase the virus titer, achieve the effect of preventing highly pathogenic porcine reproductive and respiratory syndrome virus and Haemophilus parasuis at the same time, and avoid the occurrence of mixed infection. At the same time, with the help of the composite immune adjuvant, the immune regulation effect of the vaccine is greatly improved, and the vaccine effect is enhanced. The vaccine safety is guaranteed and the vaccine antigen efficiency is improved by antigen dilution and proportional preparation of heat-resistant protective agents. Through the virus attack experiment, it is proved that the live vaccine of highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protective agent immunized pigs has a good protective effect on the strain, and is used for the prevention and protection of HP-PRRS in pigs;

[0032] 2. The present invention improves production efficiency and stability through microcarrier suspension culture technology, while reducing production costs and improving vaccine quality. By selecting heat-resistant protective agents and optimizing freeze-drying curves, long-term storage of vaccines at 2-8°C is achieved, solving the problems of inconvenient vaccine storage and high storage costs. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The present invention provides a technical solution: a highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine, comprising the following production steps:

[0035] S1. Cell culture

[0036] The bioreactor cell microcarrier suspension culture was carried out according to the monkey embryonic kidney epithelial cell Marc-145 microcarrier cell suspension culture process;

[0037] After the number of cells has proliferated to an appropriate scale, the stirring paddle is turned off, and the culture supernatant is discharged after complete sedimentation, and then the microcarriers are rinsed aseptically, and then a pancreatic enzyme solution is added for digestion, and then the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for secondary suspension culture, and the above steps are repeated, and the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for tertiary culture;

[0038] S2. Viral antigen extraction

[0039] The microcarrier suspension culture of HP-PRRS virus is carried out in a bioreactor according to the suspension culture process of HP-PRRS virus, and the cell culture of HP-PRRS virus harvested from the suspension culture is frozen and thawed, and the antigen liquid is filtered with a filter rod to remove the microcarrier to obtain the first antigen liquid;

[0040] Taking Haemophilus parasuis for proliferation and cultivation, harvesting a bacterial solution containing Haemophilus parasuis, concentrating it, inactivating and washing it, separating it to obtain an inactivated Haemophilus parasuis concentrate, adding Tween-80 to the concentrate to obtain a second antigen solution;

[0041] The first antigen liquid and the second antigen liquid are mixed in proportion to obtain a mixed antigen liquid;

[0042] S3. Selection and preparation of adjuvants

[0043] The porcine interferon alpha protein with antiviral activity produced by expressing the porcine interferon alpha gene in an expression system by recombinant DNA technology is used as porcine interferon alpha and combined with aluminum hydroxide adjuvant to obtain a composite immune adjuvant;

[0044] S4. Preparation of heat-resistant protective agent

[0045] Sodium L-glutamate, tryptone, hydrolyzed milk protein, enzymatic casein, sucrose, thiourea, gelatin, trehalose, D-sorbitol, glycine and arginine were prepared into 5 kinds of protective agent formulas in different proportions to obtain 5 kinds of heat-resistant protective agents, and the best lyophilized protective agent was obtained by selecting different lyophilization curves for lyophilization;

[0046] S5. Seedling matching

[0047] The first antigen solution and the second antigen solution were diluted with PBS solution, and the composite adjuvant was added and stirred, and then mixed with the heat-resistant protective agent in a ratio of 3:1, and then quantitatively packaged and freeze-dried to obtain a highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protective agent live vaccine.

[0048] Specifically, before the bioreactor cells are inoculated, the warm culture medium used in S1 is sterile filtered and then injected into the bioreactor tank, which is then rinsed multiple times, and the supernatant is discharged after the microcarriers are completely settled.

[0049] Specifically, the preheating temperature of the pancreatic enzyme solution in S1 is 37 degrees, and the freeze-thaw temperature in S3 is -40 degrees.

[0050] Specifically, the freeze-drying curve in S4 includes four freeze-drying curves, and each freeze-drying curve includes a pre-freezing stage, a sublimation drying stage, and an analytical drying stage.

[0051] Specifically, the filter rod in S2 uses a PP2 material deep filter element, the best time to inoculate the cells in S2 is the fourth day of cell growth, the best time to collect the cells is 70-74 hours after inoculation, and the pH value of the PBS solution is 7.2.

[0052] Example

[0053] 1. Four freeze-drying curves were used to freeze-dry the heat-resistant protective agent 1, as shown in Table 1;

[0054] Table 1: Freeze-drying curve (all incubated at room temperature)

[0055]

[0056]

[0057] Protective agent formula 1

[0058] Take 30g of sodium L-glutamate, 15g of tryptone, 80g of hydrolyzed milk protein, 30g of enzymatic casein, 100g of sucrose, 120g of gelatin and 20g of trehalose according to the proportion to prepare a heat-resistant protective agent 1;

[0059] Protective agent formula 2

[0060] Take 30g of sodium L-glutamate, 15g of tryptone, 80g of hydrolyzed milk protein, 30g of enzymatic casein, 120g of sucrose, 2.5g of thiourea, 120g of gelatin and 18g of trehalose according to the proportion to prepare a heat-resistant protective agent 2;

[0061] Protective agent formula 3

[0062] Take 30g of sodium L-glutamate, 80g of hydrolyzed milk protein, 100g of sucrose, 100g of gelatin, 25g of trehalose, 30g of D-sorbitol, 2.5g of glycine and 10g of arginine according to the proportion to prepare heat-resistant protective agent 3;

[0063] Protective agent formula 4

[0064] Take 30g of sodium L-glutamate, 80g of hydrolyzed milk protein, 100g of sucrose, 2.5g of thiourea, 100g of gelatin, 20g of trehalose, 30g of D-sorbitol, 2.5g of glycine and 10g of arginine according to the proportion to prepare a heat-resistant protective agent 4;

[0065] Protective agent formula 5

[0066] Take 30g of sodium L-glutamate, 80g of hydrolyzed milk protein, 100g of sucrose, 2.5g of thiourea, 120g of gelatin, 25g of trehalose, 30g of D-sorbitol, 2.5g of glycine and 10g of arginine according to the proportion to prepare a heat-resistant protective agent 5;

[0067] In this experiment, five kinds of heat-resistant protective agents were subjected to freeze-drying experiments through four freeze-drying curves, and the best freeze-drying protective agent was heat-resistant protective agent 2, and the best freeze-drying curve was freeze-drying curve 4. After freeze-drying, three batches of vaccine products were prepared according to the optimized process, and all tests met the quality standard requirements.

[0068] 2. Immune challenge experiment

[0069] 40 28-day-old PRRSV antigen and antibody negative piglets were selected and randomly divided into 4 groups, with 10 piglets in each group for isolation and rearing. Three groups of piglets were injected with vaccine in the muscle behind the ear root, and one group was the control group, which was not vaccinated. Each group of piglets was observed after injection, and blood was collected from each group of piglets for PRRSV antibody detection 28 days after vaccination, and then each group of piglets was injected with strong virus. After the virus attack, the piglets and their feeding and coughing symptoms were recorded, as shown in Table 2;

[0070] Table 2: Results of the 28-day piglet immune challenge experiment

[0071]

[0072] As shown in Table 2, the mortality rate of piglets challenged with virus after vaccination in the experimental group and the control group was higher in the control group that was not vaccinated. The vaccination provided 100% protection to the piglets in the experimental group, which greatly reduced the morbidity and mortality of piglets after PRRSV infection and reduced breeding losses.

[0073] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine, characterized in that: The production steps include: S1. Cell culture The bioreactor cell microcarrier suspension culture was carried out according to the monkey embryonic kidney epithelial cell Marc-145 microcarrier cell suspension culture process; After the number of cells has proliferated to an appropriate scale, the stirring paddle is turned off, and the culture supernatant is discharged after complete sedimentation, and then the microcarriers are rinsed aseptically, and then a pancreatic enzyme solution is added for digestion, and then the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for secondary suspension culture, and the above steps are repeated, and the cell suspension and microcarriers are added to a larger capacity bioreactor, and the growth medium is added for tertiary culture; S2. Viral antigen extraction The microcarrier suspension culture of HP-PRRS virus is carried out in a bioreactor according to the suspension culture process of HP-PRRS virus, and the cell culture of HP-PRRS virus harvested from the suspension culture is frozen and thawed, and the antigen liquid is filtered with a filter rod to remove the microcarrier to obtain the first antigen liquid; Taking Haemophilus parasuis for proliferation and cultivation, harvesting a bacterial solution containing Haemophilus parasuis, concentrating it, inactivating and washing it, separating it to obtain an inactivated Haemophilus parasuis concentrate, adding Tween-80 to the concentrate to obtain a second antigen solution; The first antigen liquid and the second antigen liquid are mixed in proportion to obtain a mixed antigen liquid; S3. Selection and preparation of adjuvants The porcine interferon alpha protein with antiviral activity produced by expressing the porcine interferon alpha gene in an expression system by recombinant DNA technology is used as porcine interferon alpha and combined with aluminum hydroxide adjuvant to obtain a composite immune adjuvant; S4. Preparation of heat-resistant protective agent Sodium L-glutamate, tryptone, hydrolyzed milk protein, enzymatic casein, sucrose, thiourea, gelatin, trehalose, D-sorbitol, glycine and arginine were prepared into 5 kinds of protective agent formulas in different proportions to obtain 5 kinds of heat-resistant protective agents, and the best lyophilized protective agent was obtained by selecting different lyophilization curves for lyophilization; S5. Seedling matching The first antigen solution and the second antigen solution were diluted with PBS solution, and the composite adjuvant was added and stirred, and then mixed with the heat-resistant protective agent in a ratio of 3:1, and then quantitatively packaged and freeze-dried to obtain a highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protective agent live vaccine.

2. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: Before inoculating the cells in the bioreactor used in S1, the warm culture medium is sterile filtered and then injected into the bioreactor tank, which is then rinsed multiple times, and the supernatant is discharged after the microcarriers are completely settled.

3. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The preheating temperature of the pancreatic enzyme solution in S1 is 37 degrees.

4. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The freeze-thaw temperature in S2 is -40 degrees.

5. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The freeze-drying curve in S4 includes four freeze-drying curves, each of which includes a pre-freezing stage, a sublimation drying stage and an analytical drying stage.

6. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The filter rod in S2 is made of PP2 material deep filter element.

7. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The best time for inoculation in S2 is the fourth day of cell growth, and the best time for harvesting the virus is 70-74 hours after infection.

8. The highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine according to claim 1, characterized in that: The pH value of the PBS solution is 7.2.

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