A vaccine freeze-dried heat-resistant protective agent and its preparation method and application

Abstract

The invention discloses a vaccine freeze-dried heat-resistant protective agent and a preparation method and application thereof. The freeze-dried heat-resistant protective agent consists of the following components in weight percentage: 0.6-1.0% of gelatin, 4.5-6.5% of brain heart infusion, 3.5-5.5% of tryptone, 6-10% of trehalose, 2.0-3.0% of sucrose, 1.5-2.5% of sodium thiosulfate, 0.6-1.2% of polyvinyl pyrrolidone (PVPK30), 0.4-0.6% of dextran, 0.3-0.5% of mannitol, 0.2-0.4% of glycine, 0.04-0.06% of sodium glutamate, 0.2-0.3% of L-arginine, and the balance is water for injection. The freeze-dried heat-resistant protective agent of the vaccine disclosed by the invention has components that interact with each other, thereby improving the temperature tolerance of porcine infectious gastroenteritis, porcine epidemic diarrhea and porcine rotavirus triple live vaccines during freeze-drying, reducing the loss of efficacy, and the virus loss rate before and after freeze-drying does not exceed 0.2 titers.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine protective agents, in particular to a vaccine freeze-dried heat-resistant protective agent and a preparation method and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE) and porcine rotavirus disease are three serious viral diarrhea diseases in pigs. The three pathogens can infect pigs of all ages and can cause symptoms such as vomiting, watery diarrhea and dehydration. The harm to piglets under 10 days old is particularly serious, with high morbidity and mortality rates, which can reach 100%. Live vaccines for porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE) and porcine rotavirus disease and most veterinary live vaccine products use conventional lyophilized protective agents. The main ingredients of conventional protective agents are skim milk, sucrose, gelatin, etc., with simple formulations, easy preparation, good solubility, but poor thermal stability and protective function. The storage conditions of the products are relatively harsh, and most of them need to be stored below -15°C. During the production, transportation and use of vaccines, if the cold chain system is not properly controlled, the temperature of the vaccine storage environment will rise, which often leads to a decrease in vaccine efficacy or failure, resulting in immune failure. Heat-resistant lyophilization protective agents consider more carefully the effects of physical and chemical changes that may occur in lyophilized materials on the survival of live vaccines when biologically active products are stored at higher temperatures and longer storage times than conventional lyophilization protective agents. By adding fillers, antifreeze agents, antioxidants, acid-base adjusters, buffers, hydroxyl neutralizers, low molecular weight compounds, and high molecular weight compounds, the virus can be stored for more than 24 months at 2-8°C without a significant decrease in potency. Therefore, its protective performance is better than that of traditional protective agents, making the storage, transportation, and use of live vaccines more convenient and economical. With the rapid development of large-scale breeding, epidemic prevention quality is a key issue. The cold chain transportation and storage temperature requirements of live vaccines are very high. Poor control directly affects the quality of vaccines. The research and development of heat-resistant protective agents is particularly important. Summary of the invention

[0003] The purpose of the present invention is to solve the problem that the quality of the existing porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus (G5 type) triple live vaccine (attenuated Chinese strain + attenuated CV777 strain + NX strain) is affected by poor cold chain transportation control, and to provide a vaccine freeze-dried heat-resistant protective agent and its preparation method and application.

[0004] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0005] The first object of the present invention is to provide a vaccine freeze-dried heat-resistant protective agent, which is used for porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus triple live vaccine, and is composed of the following components in weight percentage:

[0006]

[0007] The balance is water for injection.

[0008] Preferably, the vaccine freeze-dried heat-resistant protective agent is composed of the following components in weight percentage:

[0009]

[0010]

[0011] The balance is water for injection.

[0012] The second object of the present invention is to provide a method for preparing the above-mentioned vaccine freeze-dried heat-resistant protective agent, comprising the following steps:

[0013] S1. Take gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran, mannitol and water for injection in percentage by weight, dissolve gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran and mannitol in boiling water for injection accounting for 3 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, add water for injection until the solution volume is 4 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared after all the solutions are dissolved, and then sterilize at 116° C. for 25 minutes to obtain protective agent A;

[0014] S2, taking glycine, sodium glutamate, and L-arginine by weight percentage, dissolving glycine, sodium glutamate, and L-arginine in a room temperature injection water accounting for 1 / 10 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, and after being completely dissolved, adding injection water until the solution volume is 1 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared; then filtering with a 0.22 micron sterilizing filter element, and collecting the filtrate as the protective agent B;

[0015] S3. Cool the protective agent A to room temperature and then fully mix it with the protective agent B to prepare a freeze-dried heat-resistant protective agent for the vaccine.

[0016] The third object of the present invention is to provide an application of a vaccine freeze-dried heat-resistant protective agent in the preparation of a triple live freeze-dried vaccine of porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus, comprising: taking the vaccine freeze-dried heat-resistant protective agent and the triple live vaccine antigens of porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus in a volume ratio of 2:3, stirring and mixing them thoroughly and controlling the temperature, packaging the mixed liquid after the temperature of the mixed liquid rises to 20°C, and obtaining a triple live freeze-dried vaccine of porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus through vacuum freeze-drying.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The components of the vaccine freeze-dried heat-resistant protective agent of the present invention interact with each other, thereby improving the temperature tolerance of the triple live vaccine of porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus during the freeze-drying process and reducing the loss of efficacy. The virus loss rate before and after freeze-drying does not exceed 0.2 titers.

[0019] The triple live vaccine of porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus (attenuated Chinese strain + attenuated CV777 strain + NX strain) combined with the vaccine of the present invention has a high degree of aging resistance after being freeze-dried with a heat-resistant protective agent. It has good physical properties after being placed at 37°C for 15 days, and the virus titer of each bottle of vaccine only decreases by 0.2 to 0.3 titers. It can be stored for 36 months at 2 to 8°C without a significant decrease in titer, which shows that the present invention solves the problem that the quality of conventional protective agent live vaccines is affected by poor cold chain transportation control.

[0020] The vaccine freeze-dried heat-resistant protective agent of the present invention has low raw material cost, simple preparation method and can realize industrial large-scale production. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0022] The porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus triple live vaccine used in the following examples is a porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine (attenuated Chinese strain + attenuated CV777 strain + NX strain) antigen, which is produced by Jilin Zhengye Biological Products Co., Ltd. The remaining components or raw materials are not particularly limited and can be directly purchased from the market. Their specific sources are no longer recorded in this example.

[0023] Example 1

[0024] A vaccine freeze-dried heat-resistant protective agent, used for porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus (G5 type) triple live vaccine, composed of the following components in weight percentage:

[0025] Gelatin 0.8%, brain heart infusion 5.5%, trypsin 4.5%, trehalose 8%, sucrose 2.5%, sodium thiosulfate 2%, polyvinylpyrrolidone (PVPK30) 1%, dextran 0.5%, mannitol 0.4%, glycine 0.3%, sodium glutamate 0.05%, L-arginine 0.25%, and the balance is water for injection.

[0026] The preparation method of the above-mentioned vaccine freeze-dried heat-resistant protective agent is as follows:

[0027] S1. Take gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran, mannitol and water for injection in percentage by weight, dissolve gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran and mannitol in boiling water for injection accounting for 3 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, add water for injection until the solution volume is 4 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared after all the solutions are dissolved, and then sterilize at 116° C. for 25 minutes to obtain protective agent A;

[0028] S2, taking glycine, sodium glutamate, and L-arginine by weight percentage, dissolving glycine, sodium glutamate, and L-arginine in a room temperature injection water accounting for 1 / 10 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, and after being completely dissolved, adding injection water until the solution volume is 1 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared; then filtering with a 0.22 micron sterilizing filter element, and collecting the filtrate as the protective agent B;

[0029] S3. Cool the protective agent A to room temperature and then fully mix it with the protective agent B to prepare a freeze-dried heat-resistant protective agent for the vaccine.

[0030] Example 2

[0031] The difference from the above-mentioned embodiment 1 is that the vaccine freeze-dried heat-resistant protective agent is composed of the following components in weight percentage:

[0032] Gelatin 0.6%, brain heart infusion 6.5%, trypsin 3.5%, trehalose 6%, sucrose 3%, sodium thiosulfate 2.5%, polyvinylpyrrolidone (PVPK30) 0.6%, dextran 0.6%, mannitol 0.3%, glycine 0.4%, sodium glutamate 0.03%, L-arginine 0.2%, and the balance is water for injection.

[0033] Example 3

[0034] The difference from the above-mentioned embodiment 1 is that the vaccine freeze-dried heat-resistant protective agent is composed of the following components in weight percentage:

[0035] Gelatin 1%, brain heart infusion 4.5%, trypsin 3.5%, trehalose 10%, sucrose 2%, sodium thiosulfate 1.5%, polyvinylpyrrolidone (PVPK30) 1.2%, dextran 0.4%, mannitol 0.5%, glycine 0.2%, sodium glutamate 0.06%, L-arginine 0.3%, and the balance is water for injection.

[0036] Comparative Example

[0037] A conventional protective agent for a triple live vaccine of porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) (weakened Chinese strain + weakened CV777 strain + NX strain), comprising the following weight components: 10% gelatin, 40% sucrose, and the remainder water for injection. Preparation method: weigh 3 / 5 of the total volume of water for injection, dissolve gelatin and sucrose in sequence, dissolve the next reagent after the previous reagent is completely dissolved, and after all the reagents are dissolved, add the remainder water for injection, sterilize at 116°C for 20 minutes, and obtain a comparative protective agent.

[0038] Example 4

[0039] The above-mentioned vaccine freeze-dried heat-resistant protective agent is mainly used for freeze-dried storage of porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine (weakened Chinese strain + weakened CV777 strain + NX strain). The vaccine freeze-dried heat-resistant protective agent prepared in the above-mentioned Examples 1, 2, and 3 and the porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine (weakened Chinese strain + weakened CV777 strain + NX strain) antigens are respectively taken in a volume ratio of 2:3 Stir and mix thoroughly and control the temperature. After the temperature of the mixed liquid rises to 20°C, package it in 3.5 ml / bottle. For the comparative example, conventional protective agent and porcine infectious gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine (attenuated Chinese strain + attenuated CV777 strain + NX strain) antigens are mixed in a ratio of 1:7 and packaged in 2.4 ml / bottle. Then, vacuum freeze-dry it immediately to obtain porcine infectious gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live freeze-dried vaccine.

[0040] The freeze-dried vaccine prepared in Example 4 was taken as samples for the following tests:

[0041] ⑴ Appearance: Light yellow sponge-like loose lumps, easy to separate from the bottle wall, and quickly dissolve after adding diluent.

[0042] ⑵ The sterility test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all samples showed sterile growth.

[0043] ⑶ Mycoplasma test was carried out according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and no mycoplasma growth was found in any samples.

[0044] (4) The exogenous virus test was carried out in accordance with the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and all samples were free of exogenous virus contamination.

[0045] ⑸ Identification test: Dilute 1 dose of vaccine (1 ml) 1000 times with RPMI 1640 maintenance solution or DMEM maintenance solution, and mix with a mixture of three specific positive sera of porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) (the neutralizing antibody titer of each positive serum should not be less than 1:128, and the ratio is 1:1:1), place at 37℃ for 1 hour, and inoculate 4 bottles of ST, Vero, and Marc-145 cell culture bottles (specification is 25mL) that have grown into a good monolayer, 0.6mL / bottle. At the same time, set up 4 bottles of ST, Vero, and Marc-145 normal cell controls; set up 4 bottles of three virus controls of attenuated Chinese strain, attenuated CV777 strain, and NX strain, and inoculate each bottle with 0.3ml of virus solution. Place in a 37℃, 5% incubator for 5-7 days, and observe and record CPE every day. There should be no CPE in the positive serum neutralizing virus group and the normal cell control group, while CPE should be present in the virus control group.

[0046] (6) Safety test: 4 healthy susceptible piglets aged 3 to 5 days were used, and 10 doses of vaccine were inoculated through the Houhai acupoint (the vaccine was diluted to 1 dose / ml with sterile saline according to the dose indicated on the bottle label), and observed for 14 consecutive days. No adverse reactions were observed in the test pigs.

[0047] ⑺Efficacy test

[0048] Virus content determination

[0049] ① Determination of the content of porcine transmissible gastroenteritis virus: Dilute the vaccine to 1 dose / ml with RPMI1640 maintenance solution, mix it with PEDV and PoRV specific positive serum mixture (neutralizing antibody titer is 1:32, ratio is 1:1) in equal amounts, place it in a 37℃ incubator for 1 hour, make 10-fold serial dilutions, and take 10 -2 , 10 -3 , 10 -4 , 10 -5 Four dilutions were inoculated into 96-well culture plates with well-grown ST cell monolayers (washed three times with RPMI 1640 maintenance solution without trypsin before inoculation), 8 wells were inoculated with each dilution, 10 μl / well, 90 μl / well of RPMI1640 maintenance solution was added, and the plates were cultured in a 37°C, 5% CO2 incubator for 5-7 days. CPE was observed and recorded daily, and TCID was calculated according to the Reed-Muench method. 50 , the virus content of the first dose should be ≥10 5.0 TCID 50 .

[0050] ② Determination of porcine epidemic diarrhea virus content: Dilute the vaccine to 1 dose / ml with RPMI1640 maintenance solution, mix it with TGEV and PoRV specific positive serum mixture (neutralizing antibody titer is 1:32, ratio is 1:1) in equal amounts, place it in a 37℃ incubator for 1 hour, make 10-fold serial dilutions, and take 10 -2 , 10 -3 , 10 -4 , 10 -5 Four dilutions were inoculated into 96-well culture plates with well-grown Vero cell monolayers (washed three times with RPMI 1640 maintenance medium II without trypsin before inoculation), 8 wells were inoculated with each dilution, 10 μl / well, 90 μl / well of RPMI1640 maintenance medium was added, and the plates were cultured in a 37°C, 5% CO2 incubator for 5-7 days. The cytopathic effect (CPE) was observed and recorded daily, and the TCID was calculated according to the Reed-Muench method. 50 , the virus content of the first dose should be ≥10 5.0 TCID 50 .

[0051] ③ Determination of porcine rotavirus content: Dilute the vaccine to 1 aliquot / ml with DMEM maintenance solution, add trypsin with a final concentration of 20 μg / ml, place in a 37°C incubator for 1.5 hours, mix with TGEV and PEDV specific positive serum mixture (neutralizing antibody titer is 1:32, ratio is 1:1) in equal amounts, place in a 37°C incubator for 1 hour, make 10-fold serial dilutions, and take 10 -2 , 10 -3 , 10 -4 , 10 -5 Four dilutions were inoculated into 96-well culture plates with well-grown Marc-145 cell monolayers (washed three times with 0.01 mol / L PBS, pH 7.2 before inoculation). Each dilution was inoculated into 8 wells, 10 μl / well, and 90 μl of DMEM maintenance solution was added. The plates were cultured in a 37°C, 5% CO2 incubator for 5 to 7 days. The CPE was observed and recorded daily, and the TCID was calculated according to the Reed-Muench method. 50 , the virus content of the first dose should be ≥10 5.0 TCID 50 .

[0052] ⑻ The residual moisture content of each sample was ≤3.5% as determined in the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine.

[0053] ⑼The vacuum degree was measured according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and the vacuum degree of each sample was qualified.

[0054] ⑽The nucleic acid test for African swine fever virus shall be carried out according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and the nucleic acid of African swine fever virus should be negative.

[0055] The virus content of the freeze-dried vaccine prepared using the heat-resistant protective agent for freeze-dried vaccines prepared in Example 1 and the freeze-dried vaccine prepared using the conventional protective agent in the comparative example under different conditions and for different storage times, the unit is lgTCID 50 / First portion.

[0056] ⑴ The changes in the virus titer of porcine infectious gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine (attenuated Chinese strain + attenuated CV777 strain + NX strain) before and after freeze-drying are shown in Table 1.

[0057] Table 1

[0058]

[0059] From the results in Table 1, it can be seen that the protective performance of the heat-resistant protectant vaccine of the present invention during the freeze-drying process is slightly better than that of the conventional protectant vaccine. The freeze-drying losses of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus and porcine rotavirus freeze-dried viruses were all within 0.2 titers after three repeated tests.

[0060] ⑵ Aging resistance test and shelf life test of porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus (G5 type) triple live vaccine (weakened Chinese strain + weakened CV777 strain + NX strain)

[0061] The freeze-dried vaccine prepared using the heat-resistant protective agent for freeze-drying the vaccine prepared in Example 1 and the freeze-dried vaccine prepared using the conventional protective agent in the comparative example were stored at 37°C and 2-8°C, respectively. Several bottles were taken out regularly to detect changes in virus content. The results are shown in Tables 2 and 3.

[0062] Table 2

[0063]

[0064]

[0065] Table 3

[0066]

[0067]

[0068]

[0069] As can be seen from Table 2, the conventional protective agent vaccine was heat-resistant at 37°C for 15 days through 3 repeated experimental data, and the virus content was reduced by 2.3 to 2.5 titers. The freeze-dried vaccine prepared using the vaccine freeze-dried heat-resistant protective agent prepared in Example 1 had a loss of less than 0.3 titers.

[0070] As can be seen from Table 3, the conventional protective agent vaccine was repeated three times and the virus content was reduced by 1.8 to 2.3 titers when stored at 2 to 8°C for 36 months. The freeze-dried vaccine prepared using the vaccine freeze-dried heat-resistant protective agent prepared in Example 1 had a loss of less than 0.3 titers.

[0071] In summary, the freeze-dried heat-resistant protective agent of the vaccine of the present invention can effectively improve the shelf life of the triple live vaccine (attenuated Chinese strain + attenuated CV777 strain + NX strain) for infectious gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type).

[0072] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A vaccine freeze-dried heat-resistant protective agent, characterized in that: The vaccine freeze-dried heat-resistant protective agent is used for porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus triple live vaccine, and is composed of the following components by weight percentage: Gelatin 0.8%; Brain heart infusion 5.5%; Tryptone 4.5%; Trehalose 8%; Sucrose 2.5%; Sodium thiosulfate 2%; Polyvinylpyrrolidone K30 1%; Dextran 0.5%; Mannitol 0.4%; Glycine 0.3%; Monosodium glutamate 0.05%; L-arginine 0.25%; The balance is water for injection.

2. A method for preparing a freeze-dried heat-resistant protective agent for vaccines as claimed in claim 1, characterized in that: The following steps are involved: S1. Take gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran, mannitol and water for injection in percentage by weight, dissolve gelatin, brain heart infusion, trypsin, trehalose, sucrose, sodium thiosulfate, polyvinyl pyrrolidone K30, dextran and mannitol in boiling water for injection accounting for 3 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, add water for injection until the solution volume is 4 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared after all the solutions are dissolved, and then sterilize at 116° C. for 25 minutes to obtain protective agent A; S2, taking glycine, sodium glutamate and L-arginine by weight percentage, dissolving glycine, sodium glutamate and L-arginine in a room temperature injection water accounting for 1 / 10 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared in sequence, and after being completely dissolved, adding injection water until the solution volume is 1 / 5 of the total volume of the freeze-dried heat-resistant protective agent for the vaccine to be prepared; then filtering with a 0.22 micron sterilizing filter element, collecting the filtrate as the protective agent B; S3. Cool the protective agent A to room temperature and then mix it thoroughly with the protective agent B to prepare a freeze-dried heat-resistant protective agent for the vaccine.

3. A use of the vaccine freeze-dried heat-resistant protective agent as claimed in claim 1 in the preparation of a triple live freeze-dried vaccine for porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus, characterized in that: include: Take the vaccine freeze-dried heat-resistant protective agent and the porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus triple live vaccine antigens in a volume ratio of 2:3, stir and mix them thoroughly and control the temperature. After the temperature of the mixed solution rises to 20°C, package them and obtain the porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus triple live freeze-dried vaccine through vacuum freeze-drying.

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