Live vaccine heat-resistant protective agent as well as preparation method and application thereof

Abstract

The invention discloses a live vaccine heat-resistant protective agent as well as a preparation method and application thereof. The live vaccine heat-resistant protective agent is prepared from the following components in percentage by mass volume: 3%-5% of glucose, 1%-3% of thiourea, 5%-8% of lactose, 0.5%-1% of ascorbic acid, 1%-3% of glycine, 0.5%-1% of poloxamer 188, 1%-2% of povidone K30, 3%-6% of tertiary butanol and the balance of water for injection. The peste des petits ruminant vaccine freeze-drying agent is simple in component, easy in raw material obtaining and simple in preparation method, can effectively reduce the loss of various virus antigens in the freeze-drying process when the peste des petits ruminant live vaccine is prepared, and can ensure the immune efficacy and long-term stable preservation of the peste des petits ruminant live vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of heat-resistant protective agents for live vaccines in the field of biomedicine, and specifically relates to a heat-resistant protective agent for live vaccines and a preparation method and application thereof. Background Art

[0002] Peste des petits ruminants is an acute, highly contagious disease caused by the Peste des petits ruminants virus. It is also known as sheep plague, etc. Clinically, it is characterized by high fever, necrotizing stomatitis, diarrhea and pneumonia. The morbidity and mortality rates can be as high as 100% and 90%. The disease is mainly transmitted through direct contact, and the virus is present in the secretions and excrement of infected animals. The virus can also be transmitted through the air, especially in intensive breeding environments. In addition, contaminated feed and drinking water are also potential transmission routes. There is only one serotype of Peste des petits ruminants virus, which is divided into 4 groups, of which 3 groups originate from Africa and one group originates from Asia. It belongs to the Paramyxoviridae family, a member of the Morbillivirus genus, and is in the same genus as rinderpest virus, canine distemper virus, and measles virus. By summarizing and analyzing the epidemic characteristics of the disease, it can be found that the spread of Peste des petits ruminants is not affected by the season and can occur at any time. The incubation period is 2 to 6 days, and can be up to 21 days. It mainly infects sheep. Goats are more susceptible than sheep, and small and medium-sized goat breeds are more susceptible. Camels, cattle and pigs can also be infected with this virus, but they do not show clinical symptoms. One to two days after infection, the oral mucosa, gums, palate, lips, cheeks and tongue mucosa become necrotic, bleeding and drooling, and the oral mucosa is completely covered with cheesy substances. Similar changes can also be seen in the nasal mucosa and vaginal mucosa.

[0003] In recent years, with the rapid development of animal husbandry in my country, the density of breeding has increased, the types of diseases have increased and the outbreaks have been frequent, and the corresponding animal epidemic prevention model has also undergone fundamental changes. Epidemic prevention quality has become one of the bottlenecks restricting this change, and vaccine quality is a key issue. Long-term production practice has found that there is a significant difference in the efficacy of most vaccines produced in my country in the past and imported vaccines, and animals cannot produce effective immune protection after using some domestic vaccines. In order to make up for this defect, clinical veterinarians have to guide the vaccination of several times, dozens of times or even hundreds of times the dosage of the vaccine. Doing so will not only fail to guarantee the quality of epidemic prevention, but also cause side effects due to the introduction of too many other components into the animal's body. Therefore, finding the key factors affecting vaccine quality, improving production processes, and improving production levels have become the focus of animal epidemic prevention work. This study makes the storage, transportation, and use of PPR live vaccines more convenient and economical.

[0004] Freeze-dried live vaccines account for a considerable proportion of veterinary vaccines. According to different production processes and storage conditions, they can be divided into live vaccines with conventional protectants and live vaccines with heat-resistant protectants. Live vaccines with heat-resistant protectants have better protective properties than traditional protectants, making the storage, transportation and use of live vaccines more convenient and economical. Therefore, the production of live vaccines with heat-resistant protectants has become the mainstream. Heat-resistant protectants have immune activity but no pharmacological activity, which is the key to affecting the quality, potency and stability of live veterinary vaccines. The protective agent of the live vaccine of PPR currently used in the industry is composed of whey protein, sucrose and sodium glutamate, and its protective performance is not very ideal. Therefore, it is necessary to develop a new heat-resistant protective agent for the live vaccine of PPR. Summary of the invention

[0005] In view of the defects existing in the prior art, the present invention aims to propose a heat-resistant protective agent for the live vaccine of PPR, ensuring that it has good protective efficacy on the live vaccine of PPR, so that the live vaccine products of PPR can be stored for a long time and have good quality, potency and stability after long-term storage, which can meet the needs of market users, has good economic and social benefits, and has broad application prospects.

[0006] The first aspect of the present invention provides a live vaccine heat-resistant protective agent, which is composed of the following components in mass volume percentage: 3% to 5% glucose, 1% to 3% thiourea, 5% to 8% lactose, 0.5% to 1% ascorbic acid, 1% to 3% glycine, 0.5% to 1% poloxamer 188, 1% to 2% povidone K30, 3% to 6% tert-butanol, and the balance is water for injection.

[0007] Furthermore, in the first aspect, each component is dissolved in water for injection according to the ratio, and the heat-resistant protective agent for live vaccine is obtained after sterilization.

[0008] In the second aspect, the present invention further proposes an application of the live vaccine heat-resistant protective agent in the preparation of a live vaccine of peste des petits ruminants.

[0009] In a third aspect, the present invention also proposes a live vaccine of peste des petits ruminants prepared by using a live vaccine heat-resistant protective agent.

[0010] Based on the third aspect, the present invention further proposes a method for preparing a live vaccine of peste des petits ruminants, comprising the steps of: T1) mixing peste des petits ruminants virus venom with the live vaccine heat-resistant protective agent to obtain a vaccine stock solution; T2) freeze-drying the vaccine stock solution obtained in step T1) to obtain a live vaccine of peste des petits ruminants.

[0011] Preferably, the PPR live vaccine virus content in the PPR live vaccine is ≥10 3.5 TCID50 / ml.

[0012] Preferably, the content of PPR virus antigen in the PPR virus venom is 10 4.00 ~10 6.00 TCID 50 / ml.

[0013] Preferably, in step T1), the peste des petits ruminants virus venom and the heat-resistant protective agent are mixed in a volume ratio of 2:1 to 3:1.

[0014] Preferably, the mixing volume ratio of the peste des petits ruminants virus venom and the heat-resistant protective agent is 2:1.

[0015] Furthermore, the freeze-drying method in step T2) includes: (1) Pre-freezing stage: cool the stock vaccine solution to -10°C within 20 minutes, keep it at -10°C for 30 minutes, cool it to -40°C~-45°C within 20 minutes to 40 minutes, keep it at -45°C for 4 hours to 6 hours, and enter the vacuum stage after the sample is completely frozen; (2) Vacuuming stage: reduce the vacuum degree to 100mT~120mT within 20min and enter the sublimation drying stage; (3) Sublimation drying stage: within 2 hours, the plate temperature is raised from -45°C to -30°C and maintained for 4 hours to 6 hours; within 20 minutes, the plate temperature is raised from -30°C to -10°C and maintained for 8 hours to 10 hours; within 20 minutes, the plate temperature is raised from -10°C to -3°C and maintained for 8 hours to 10 hours, and then the analytical drying stage is entered; (4) Drying stage: Raise the temperature of the board layer to 28°C~30°C within 1h~3h and keep it warm for 6h~8h.

[0016] In summary, the heat-resistant protective agent provided by the present invention has simple ingredients, readily available raw materials, and a simple preparation method. When preparing a live vaccine of peste des petits ruminants, the loss of various viral antigens during freeze drying can be effectively reduced, and the immune efficacy and long-term stable storage of the live vaccine of peste des petits ruminants can be ensured. Using it as a heat-resistant freeze-drying protective agent for the live vaccine of peste des petits ruminants can significantly reduce the damage to the activity of the virus caused by various physical and chemical factors in the freeze drying of the product. The vaccine product prepared using it has a small drop in vaccine titer after heat resistance, and the efficacy of the vaccine is more stable and safe. At the same time, the present invention also optimizes the freeze-drying curve in the preparation of live vaccines, which can ensure the efficacy level of the live vaccine of peste des petits ruminants, and has a good heat-resistant protective effect, meets the needs of market users, has good economic and social benefits, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is a photo of the PPR live vaccine prepared with protective agent 4 in Example after packaging and freeze-drying. DETAILED DESCRIPTION

[0018] The present invention aims to provide a heat-resistant protective agent for a PPR live vaccine with good heat-resistant protective effect, so as to ensure that the PPR live vaccine can be stably stored for a long time and maintain its good immune efficacy level and safety. A PPR live vaccine with good immune efficacy, high safety and long-term stable storage is also provided based on the heat-resistant protective agent.

[0019] The present invention will be described in detail through the following specific embodiments.

[0020] The methods used in the following examples are conventional methods unless otherwise specified. The methods for obtaining various biological materials described in the examples are only to provide an experimental method for obtaining them to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0021] The embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be used as a limitation to the contents of the present invention.

[0022] Chemical substances used in the following examples include dextran and thiourea purchased from Sinopharm; lactose and ascorbic acid purchased from Tianjin Kemeng Chemical Company; glycine and poloxamer 188 purchased from Sigma; povidone K30 purchased from Nanjing Songguan Biotechnology Co., Ltd.; and tert-butyl alcohol purchased from Tianjin Damao Chemical Reagent Factory.

[0023] Example 1: Preparation of heat-resistant protective agent for PPR live vaccine This embodiment aims to prepare a heat-resistant protective agent for a live vaccine of peste des petits ruminants. The function of the heat-resistant protective agent is to protect the effective ingredients of the vaccine during the freeze-drying process and the storage stage after freeze-drying to ensure the immune effect of the vaccine. The preparation method of the heat-resistant protective agent specifically includes the following steps: 1.1. Preparation of the first solution: According to the composition contents of the heat-resistant protective agents (numbered 1, 2, 3, 4, and 5, respectively) shown in Table 1 below (where % represents the mass volume percentage content, i.e., g / 100 ml, the same below), accurately weigh the required amount of thiourea in quantitative water for injection, heat in a 60°C water bath and stir continuously until completely dissolved, weigh dextran and add it to the solution until completely dissolved, then accurately weigh lactose and add it to the solution, heat in a 60°C water bath and stir continuously to dissolve it, to obtain a first solution; 1.2. Preparation of the second solution: According to the component contents of each heat-resistant protective agent described in Table 1 below, accurately weigh povidone K30 in a quantitative amount of injection water, place in a 60°C water bath, and shake continuously until completely dissolved, then accurately weigh the required amount of poloxamer 188 in the solution to completely dissolve it, then accurately weigh ascorbic acid, glycine and tert-butanol, respectively, add them to the solution, place in a 60°C water bath, and shake continuously to dissolve them, and adjust the pH to 7.1-7.3 with 7.5% sodium bicarbonate solution to obtain a second solution; 1.3. After the first solution is fixed to volume, it is sterilized in an autoclave (116°C high pressure for 20-30 minutes), and after the second solution is fixed to volume, it is filtered and sterilized (0.22μm filter membrane), and the first solution and the second solution are mixed in a ratio of 1:1 to obtain the heat-resistant protective agent (named as protective agent 1, protective agent 2, protective agent 3, protective agent 4 and protective agent 5 respectively), and stored at 37°C for use.

[0024] Note: When preparing the heat-resistant protective agent, dissolve each reagent separately and gradually increase the amount of solute during the single dissolution to avoid oversaturation and incomplete dissolution.

[0025] Example 2: Preparation of Peste des Petits Ruminants Live Vaccine This embodiment uses the protective agent 1, protective agent 2, protective agent 3, protective agent 4 and protective agent 5 prepared in the above embodiment 1 as heat-resistant protective agents for live vaccines to prepare a live vaccine of PPR. The preparation method of the live vaccine of PPR includes the following steps: 2.1. Preparation of PPR virus venom 2.1.1. Preparation of PPR virus venom Fluid propagation of PPR virus: Take a well-grown Vero cell monolayer (provided by Jinyu Baoling Biological Pharmaceutical Co., Ltd.), discard the growth medium, digest the Vero cells that have grown a monolayer with 0.25% trypsin digestion solution, disperse them at a ratio of 1:3 to 1:4, and expand them at 37°C. Inoculate the expanded cultured Vero cells into a spinner bottle, culture them at 37°C for 48 to 72 hours at a speed of 8 to 9 rpm. When the spinner bottle cells grow into a good monolayer, discard the cell culture medium, and transfer the virus content ≥10 3.0 TCID 50 / ml of PPR virus seed was inoculated with PPR virus Clone 9 strain at a ratio of 1% to 3% of the maintenance solution, and adsorbed at 37°C for 1 hour. Then, MEM cell culture medium containing 2% bovine serum was added and cultured at 37°C for 4 to 6 days. When more than 80% of the cell pathological changes appeared, the virus solution was harvested and stored below -40°C for inspection.

[0026] 2.1.2. The final viral content determination is shown in Table 2 2.2. The content of PPR virus in each dose of freeze-dried PPR live vaccine is ≥10 4.0 TCID 50 / ml, calculate the amount of PPR virus venom for vaccine preparation; 2.3. Take the virus antigen solution in step 2.2 above and mix them evenly with the five heat-resistant protective agents (protective agent 1, protective agent 2, protective agent 3, protective agent 4 and protective agent 5) obtained in Example 1 at a volume ratio of 2:1 and 3:1 respectively. The specific operation is to uniformly add the vaccine virus antigen stock solution into the storage bottle containing the heat-resistant protective agent, and slowly mix it manually for about 15 minutes (to avoid generating too much foam), and then the vaccine stock solution can be obtained; 2.4. Use a pipette to dispense the vaccine stock solution obtained in step 2.3 into 3 ml (300 doses) / bottle, i.e., dispense into 3 ml / bottle, and then freeze-dry to prepare the PPR live vaccine (lyophilized preparation); the freeze-drying curve of freeze-drying is calculated based on freeze-drying 3 ml of vaccine stock solution: (1) Pre-freezing speed: The process selects a slow freezing rate to cool down the freeze-drying box after the product enters the box.

[0027] (2) Minimum pre-freezing temperature: Based on the eutectic point temperature of the mixed vaccine, which is -23°C, the minimum pre-freezing temperature should be lower than this temperature.

[0028] (3) Prefreezing time: Prefreezing stage: cool the stock vaccine solution from room temperature to -10°C within 20 minutes, keep it at -10°C for 30 minutes, cool it to -40°C~-45°C within 20 minutes to 40 minutes, keep it at -45°C for 4 hours to 6 hours, and enter the vacuum stage after the sample is completely frozen; (4) Vacuuming time: After pre-freezing, the pressure reduction stage begins immediately, and the vacuum degree is reduced to 100mT~120mT within 20 minutes until the end.

[0029] (5) Sublimation drying stage: When the vacuum degree drops to 120mT, heating begins. At this time, the sublimation drying stage begins. Within 2 hours, the plate temperature is raised from -45℃ to -30℃ and maintained for 4h~6h, within 20min, the plate temperature is raised from -30℃ to -10℃ and maintained for 8h~10h, within 20min, the plate temperature is raised from -10℃ to -3℃ and maintained for 8h~10h, and then the analytical drying stage is entered to make the frozen product in the bottle gradually sublime from top to bottom and finally take shape.

[0030] (6) Drying stage: When the product has completed sublimation drying, it has formed a preliminary shape. At this time, the vacuum degree needs to be increased. The vacuum degree is set to 10mT, thereby improving the heat transfer in the freeze dryer and increasing the convection heat transfer of the gas. The plate temperature is raised to an allowable temperature of 28°C within 1h to 3h and maintained for 6h to 8h. (7) Maximum allowable temperature of the product: The maximum allowable temperature of the board layer is 28°C, which is determined based on the product. Due to the heat difference transferred, the board layer temperature is often slightly lower than the maximum allowable temperature of the product.

[0031] (8) Total freeze-drying time: Based on the performance of the freeze dryer, vaccine characteristics and loading volume, the entire process of pre-freezing time, sublimation drying stage and analytical drying time is determined to be approximately 35 h to 47 h.

[0032] The virus content of each PPR live vaccine prepared in this embodiment before and after freeze-drying was tested, and the test results are shown in Tables 3 and 4. The vaccine products prepared in this embodiment were also tested for routine items according to the method recorded in the inspection procedures of the "Regulations of the People's Republic of China for Veterinary Biological Products", as shown in Tables 3 and 4.

[0033] From the test results in Table 3 and Table 4 above, it can be seen that the virus content (titer) of the PPR live vaccine prepared with Protectant 4 and Protectant 5 decreased slightly before and after freeze-drying, with a maximum decrease of only 10 0.25 TCID 50 / ml, the minimum virus content is not lost, so protective agent 4 and protective agent 5 can effectively protect the various viral antigens in the PPR live vaccine from being degraded during the freeze drying process, and obtain a vaccine product with a higher titer level. Relative to protective agent 4 and protective agent 5, protective agent 3 does not contain tert-butyl alcohol, and the virus titers of the PPR live vaccine before and after freeze drying are more reduced, because tert-butyl alcohol can form needle crystals when the vaccine is frozen, and leaves a tubular channel after the ice crystals sublime, thereby making the water vapor flow resistance greatly reduced in the animal vaccine freeze drying process, and the sublimation rate is significantly improved. Therefore, the present invention adds tert-butyl alcohol in animal vaccines to reduce the drying layer resistance, thereby accelerating the drying process and shortening the drying time. Meanwhile, tert-butyl alcohol can also make the product have a high surface area, good rehydration, and effectively improve the stability of the drug solution and the lyophilized product. Compared with protective agent 4 and protective agent 5, protective agent 1 does not contain glycine, and the virus titers of the PPR live vaccine also decreased significantly before and after freeze-drying, while glycine can inhibit the oxidation and aggregation of macromolecular substances in the protective agent; protective agent 2 does not contain poloxamer 188, which is another excipient used as a protein stabilizer in protein preparations. Because it does not have an ester bond, there is no risk of hydrolysis. It is mainly degraded in the solution in an oxidized form to form oxidized polyoxyethylene (POE) and polypropylene oxide (PPO), which can play a certain protective role against viruses. It can be seen that the appropriate proportions of the components in the heat-resistant protective agent provided by the present invention can protect the virus antigens in the PPR live vaccine from being degraded, and obtain a vaccine product with a higher titer level. According to the test results of Table 4, it can be seen that the PPR live vaccine obtained using protective agent 4 and protective agent 5 is light yellow or white spongy loose clumps after freeze-drying, and is easy to detach from the bottle wall ( Figure 1 The photo shows the freeze-dried products of live vaccines obtained after packaging (4 bottles) and freeze-drying using protective agent 4. The water content is below 2.0%, and it can be quickly dissolved in water. Therefore, the prepared vaccine products have a better freeze-dried appearance, lower water content and good solubility characteristics. Compared with protective agent 5, protective agent 4 has a better heat-resistant protection effect. The virus content of the vaccine before and after freeze-drying decreased by only 10% at most. 0.25 TCID 50 / ml, the minimum virus content is not lost, and a higher potency level of PPR live vaccine is obtained.

[0034] This embodiment also carried out an aging resistance test on the live vaccine of PPR prepared with protective agent 4 as the heat-resistant protective agent of the vaccine according to the method recorded in the inspection procedure document of "Regulations on Veterinary Biological Products of the People's Republic of China", which specifically included: placing the live vaccine of PPR at 37°C for 10 days and storing it at 2-8°C for 24 months, and detecting the changes in the content of each virus in the vaccine product. The test results are shown in Table 5 below.

[0035] From the test results in Table 5 above, it can be seen that the virus titers of the PPR live vaccine prepared with Protectant 4 as the heat-resistant protective agent did not drop by more than 10% after being stored at 37°C for 7 days, regardless of whether the ratio of antigen to protective agent was 2:1 or 3:1. 1.00 TCID 50 / ml, and the largest decrease is only 10 0.25 TCID 50 / ml; the virus titers of the vaccine after being stored at 2-8°C for 24 months did not drop by more than 10 1.00 TCID 50 / ml, and the largest decrease is only 10 0.50 TCID 50 / ml. When protective agent 5 is used as a heat-resistant protective agent to prepare a live vaccine of PPR, it also has a good heat-resistant protective effect as protective agent 4. Therefore, the heat-resistant protective agent provided by the present invention can ensure that the live vaccine of PPR using the same can be stably stored for a long time, and effectively inhibit the decrease of the titer of each virus therein, thereby ensuring the immune efficacy of each virus antigen.

[0036] Example 3: Preparation of heat-resistant protective agent for PPR live vaccine In this example, heat-resistant protective agents for PPR live vaccine were prepared according to the same method as steps 1.1-1.3 in Example 1, and the compositions and contents of heat-resistant protective agents listed in Table 6 below, which were named Protective Agent 6, Protective Agent 7 and Protective Agent 8 respectively. PPR live vaccine was prepared using Protective Agent 6, Protective Agent 7 and Protective Agent 8 according to the same method as steps 2.3-2.4 in Example 2, wherein antigen: protective agent = 2:1. Subsequently, the decrease in virus titers before and after freeze-drying of the PPR live vaccine prepared using Protective Agent 6, Protective Agent 7 and Protective Agent 8, as well as the routine items of the finished product, were tested according to the same method as in Example 2, and the test results are shown in Table 7 below.

[0037] From the results in Table 6 and Table 7 above, it can be seen that the virus content (titer) of the PPR live vaccine prepared using Protectant 6 to Protectant 8 decreased less before and after freeze-drying, with a maximum decrease of only 10 0.25 TCID 50 / ml, the minimum virus content is not lost, so protective agent 6-protectant 8 can effectively protect the various viral antigens in the PPR live vaccine from being degraded during the freeze-drying process, and obtain a vaccine product with a higher potency level. According to the test results in Table 7, it can also be seen that the PPR live vaccine obtained using protective agents 6-protectant 8 is light yellow or white spongy loose clumps after freeze-drying, and is easy to detach from the bottle wall. The water content is below 2.0%, and it can be quickly dissolved in water. Therefore, the prepared vaccine product has a better freeze-dried appearance, lower moisture content and good solubility characteristics. Therefore, the heat-resistant protective agent provided by the present invention can effectively protect the viral antigens of the PPR live vaccine using it from being degraded during the freeze-drying process, thereby ensuring the immune efficacy of the vaccine, and at the same time obtaining a PPR live vaccine product with a good freeze-dried appearance, lower moisture content and good solubility characteristics.

[0038] Example 4: Optimization of freeze-drying curve for preparing PPR live vaccine This embodiment uses the protective agent 4 obtained in Example 1 as a heat-resistant protective agent, and the vaccine virus antigen stock solution obtained in step 2.2 of Example 2 and the protective agent 4 are mixed in a volume ratio of 2:1 in the same method as step 2.3 in Example 2 to obtain a vaccine stock solution; the vaccine stock solution is then packaged into 3 ml / bottle and freeze-dried, wherein the inventors designed a variety of freeze-drying curves to screen and determine the ideal freeze-drying curve suitable for the small ruminant plague live vaccine. Based on the protective agent 4 obtained in Example 1 as a heat-resistant protective agent, two freeze-drying curves are listed below: freeze-drying curve 1 and freeze-drying curve 2.

[0039] Freeze-drying curve 1: the same as the freeze-drying curve used to prepare the PPR live vaccine in Example 2; the PPR live vaccine obtained based on the freeze-drying curve 1 is named live vaccine 1; Freeze-drying curve 2 (as a control freeze-drying curve): The freeze drying curve of the freeze drying is calculated based on freeze drying 3 ml of vaccine stock solution: (1) Pre-freezing stage: cool the vaccine stock solution to -40℃~-45℃ within 1 hour, maintain it at -45℃ for 4h~6h, and enter the vacuum stage after the sample is completely frozen; (2) Vacuuming stage: reduce the vacuum degree to 100mT~120mT within 20min and enter the sublimation drying stage; (3) Sublimation drying stage: within 2 hours, the plate temperature is raised from -45°C to -20°C and maintained for 6 hours to 8 hours; within 20 minutes, the plate temperature is raised from -20°C to -15°C and maintained for 8 hours to 10 hours; within 20 minutes, the plate temperature is raised from -15°C to -2°C and maintained for 4 hours to 6 hours, and then it enters the analytical drying stage; (4) Drying stage: Raise the board temperature to 28°C within 1 to 2 hours and keep it warm for 6 to 8 hours.

[0040] The PPR live vaccine obtained based on the freeze-drying curve 2 was named live vaccine 2.

[0041] According to the same method as in Example 2, the decrease in virus titer of live vaccine 1 and live vaccine 2 before and after freeze-drying and the routine items of the finished vaccine products were tested. The test results are shown in Table 8 below.

[0042] According to the test results in Table 8 above, the virus titer of PPR virus in PPR live vaccine 1 decreased by 10% after freeze-drying. 0.25 TCID 50 / ml, and the physical properties, solubility, and residual water content of the vaccine after freeze-drying were all good. The virus titer of the PPR live vaccine virus in live vaccine 2 decreased by 10% after freeze-drying. 0.75 TCID 50 / ml, and the physical properties after freeze-drying are slightly worse, the bottom of the vaccine melts slightly, shrinks, and a small amount of particles appear after dissolution. Among them, the virus titer of the live vaccine 1 of PPR is relatively large compared with that of the live vaccine 2. Therefore, the freeze-drying curve 1 of the present invention can not only reduce the loss of virus titer of the live vaccine of PPR during freeze-drying, but also improve product quality.

[0043] Example 5: Safety test of PPR live vaccine In this example, the PPR live vaccine (named as live vaccine 3 and live vaccine 4, respectively) prepared with protective agent 7 as heat-resistant protective agent and antigen and protective agent volume ratio of 2:1 and 3:1 in the above example 3 was used as the experimental group, and sterile saline was used as the control group. Five healthy susceptible goats about 1 year old (PPR virus neutralizing body titer less than 1:4) were subcutaneously inoculated in the neck, 1 ml per goat. After inoculation, the body temperature was measured once a day and clinical symptoms were observed for 14 consecutive days. As shown in Table 9 below, there was no abnormal reaction in the experimental goats in each group, indicating that the PPR live vaccine prepared by the present invention has good safety.

[0044] In summary, the heat-resistant protective agent provided by the present invention has simple ingredients, readily available raw materials, and a simple preparation method. When preparing a live vaccine of peste des petits ruminants, the loss of various viral antigens during freeze drying can be effectively reduced, and the immune efficacy and long-term stable storage of the live vaccine of peste des petits ruminants can be ensured. Using it as a heat-resistant freeze-drying protective agent for the live vaccine of peste des petits ruminants can significantly reduce the damage to the activity of the virus caused by various physical and chemical factors in the freeze drying of the product. The vaccine product prepared using it has a small drop in vaccine titer after heat resistance, and the efficacy of the vaccine is more stable and safe. At the same time, the present invention also optimizes the freeze-drying curve in the preparation of live vaccines, which can ensure the efficacy level of the live vaccine of peste des petits ruminants, and has a good heat-resistant protective effect, meets the needs of market users, has good economic and social benefits, and has broad application prospects.

[0045] 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 heat-resistant protective agent for live vaccines, characterized in that: It is composed of the following components in mass volume percentage: 3% to 5% glucose, 1% to 3% thiourea, 5% to 8% lactose, 0.5% to 1% ascorbic acid, 1% to 3% glycine, 0.5% to 1% poloxamer 188, 1% to 2% povidone K30, 3% to 6% tert-butanol, and the balance is water for injection.

2. A method for preparing a heat-resistant protective agent for live vaccines as claimed in claim 1, characterized in that: Dissolve each component in water for injection according to the ratio, and obtain the heat-resistant protective agent for live vaccine after sterilization.

3. Use of the heat-resistant protective agent for live vaccines as claimed in claim 1 in the preparation of live vaccines against peste des petits ruminants.

4. A live vaccine of peste des petits ruminants prepared from the live vaccine heat-resistant protective agent according to claim 1.

5. A method for preparing a live vaccine of peste des petits ruminants as claimed in claim 4, characterized in that: Includes steps: T1) mixing peste des petits ruminants virus venom with the heat-resistant protective agent for live vaccines according to claim 1 to obtain a vaccine stock solution; T2) freeze-drying the vaccine stock solution obtained in step T1) to obtain a live vaccine of peste des petits ruminants.

6. The preparation method according to claim 5, characterized in that: The PPR live vaccine virus content in the PPR live vaccine is ≥10 3.5 TCID 50 / ml.

7. The preparation method according to claim 5, characterized in that: The content of PPR virus antigen in the PPR virus venom is 10 4.00 ~10 6.00 TCID 50 / ml.

8. The preparation method according to claim 5, characterized in that: In step T1), the peste des petits ruminants virus venom and the heat-resistant protective agent are mixed in a volume ratio of 2:1 to 3:

1.

9. The preparation method according to claim 8, characterized in that: The mixing volume ratio of the peste des petits ruminants virus venom and the heat-resistant protective agent is 2:

1.

10. The preparation method according to claim 5, characterized in that: The freeze drying method in step T2) includes: (1) Pre-freezing stage: cool the stock vaccine solution to -10°C within 20 minutes, keep it at -10°C for 30 minutes, cool it to -40°C~-45°C within 20 minutes to 40 minutes, keep it at -45°C for 4 hours to 6 hours, and enter the vacuum stage after the sample is completely frozen; (2) Vacuuming stage: reduce the vacuum degree to 100mT~120mT within 20min and enter the sublimation drying stage; (3) Sublimation drying stage: within 2 hours, the plate temperature is raised from -45°C to -30°C and maintained for 4 hours to 6 hours; within 20 minutes, the plate temperature is raised from -30°C to -10°C and maintained for 8 hours to 10 hours; within 20 minutes, the plate temperature is raised from -10°C to -3°C and maintained for 8 hours to 10 hours, and then the analytical drying stage is entered; (4) Drying stage: Raise the temperature of the board layer to 28°C~30°C within 1h~3h and keep it warm for 6h~8h.

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