A live vaccine heat-resistant protective agent, and a preparation method and application thereof

Abstract

The application discloses a live vaccine heat-resistant protective agent and a preparation method and application thereof, which is composed of the following components in mass volume percentage: 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 tert-butyl alcohol, and the balance is water for injection. The application has simple components, easily available raw materials and simple preparation method, can effectively reduce the loss of virus antigens in the freeze-drying process when the live vaccine for small ruminant pestivirus is prepared, and can ensure the immunization efficacy and long-term stable storage of the live vaccine for small ruminant pestivirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of heat-resistant protective agent for live vaccine in the field of biological medicine, and particularly relates to a heat-resistant protective agent for live vaccine and a preparation method and application thereof. BACKGROUND

[0002] Peste des petits ruminants (PPR) is an acute and virulent infectious disease caused by Peste des petits ruminants virus. It is also known as sheep plague, etc. The clinical symptoms are high fever, necrotic stomatitis, diarrhea and pneumonia. The morbidity and mortality can be as high as 100% and 90%, respectively. The disease is mainly transmitted through direct contact, and the virus exists in the secretions and excretions of infected animals. The virus can also be transmitted through the air, especially in densely populated environments. In addition, contaminated feed and drinking water are also potential transmission routes. Peste des petits ruminants virus has only one serotype and is divided into four groups, three of which are derived from Africa and one from Asia. It belongs to the Paramyxoviridae family and is a member of the Morbillivirus genus, which is the same as the Rinderpest virus, Canine distemper virus and Measles virus. By summarizing and analyzing the characteristics of the disease, it can be found that the transmission and prevalence of Peste des petits ruminants are not affected by seasons and can occur at any time. The incubation period is 2-6 days, with a maximum of 21 days. It mainly infects sheep, and goats are more susceptible than sheep. Small breeds of goats are more susceptible, and camels, cattle and pigs can also be infected with the virus, but do not show clinical symptoms. One to two days after infection, the oral mucosa, gums, palate, lips, cheeks and tongue mucosa necrotize, bleed, salivate, and the oral mucosa is completely attached by a cheese-like substance. Similar changes can also be seen in the nasal mucosa and vaginal mucosa.

[0003] In recent years, with the rapid development of China's animal husbandry, the breeding density has increased, the types of diseases have increased and the outbreaks have occurred frequently. The animal epidemic prevention mode has also undergone fundamental changes. The quality of epidemic prevention has become one of the bottlenecks restricting this change, and the quality of vaccine is the key problem. Long-term production practice has found that there is a significant difference in efficacy between most domestic vaccines and imported vaccines. After using some domestic vaccines, animals cannot produce effective immune protection. In order to make up for this deficiency, clinical veterinarians have to guide the inoculation of several times, tens of times or even hundreds of times the dosage of vaccines. However, this not only cannot guarantee the quality of epidemic prevention, but also can cause side effects due to the introduction of too many other components into the animal body. Therefore, finding the key factor affecting the quality of vaccine, improving the production process and improving the production level have become the focus of animal epidemic prevention work. This study makes the live vaccine for Peste des petits ruminants more convenient and economical in storage, transportation and use.

[0004] Freeze-dried live vaccine accounts for a considerable proportion in veterinary vaccines, and can be divided into conventional protective agent live vaccine and heat-resistant protective agent live vaccine according to different production processes and storage conditions. The heat-resistant protective agent live vaccine has better protective performance than the traditional protective agent, so that the storage, transportation and use of the live vaccine are more convenient and economical. Therefore, the heat-resistant protective agent for producing live vaccine has become the mainstream. The heat-resistant protective agent has immunological activity but no pharmacological activity, and is the key to affect the quality, potency and stability of the veterinary live vaccine. At present, the protective agent for the live vaccine of small ruminant pestivirus used in the industry is composed of whey protein, sucrose and sodium glutamate, and the protective performance is not very ideal. Therefore, it is necessary to develop a new heat-resistant protective agent for the live vaccine of small ruminant pestivirus. SUMMARY

[0005] In view of the defects in the prior art, the present application aims to provide a heat-resistant protective agent for the live vaccine of small ruminant pestivirus, which ensures good protective efficacy for the live vaccine of small ruminant pestivirus, so that the live vaccine product of small ruminant pestivirus can be stored for a long time and has good quality, potency and stability after long-term storage, can meet the needs of market users, has good economic and social benefits, and has a wide application prospect.

[0006] The first aspect of the present application provides a heat-resistant protective agent for live vaccine, which is composed of the following components in mass / volume percentage: 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 tert-butyl alcohol, and the balance is water for injection.

[0007] Further, 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 application further provides an application of the heat-resistant protective agent for live vaccine in preparing the live vaccine of small ruminant pestivirus.

[0009] In the third aspect, the present application further provides a live vaccine of small ruminant pestivirus prepared from the heat-resistant protective agent for live vaccine.

[0010] Based on the third aspect, the present application further provides a preparation method of the live vaccine of small ruminant pestivirus, which comprises the following steps:

[0011] T1) uniformly mixing the virus liquid of small ruminant pestivirus with the heat-resistant protective agent for live vaccine to obtain a vaccine stock solution;

[0012] T2) freeze-drying the vaccine stock solution obtained in step T1) to obtain the live vaccine of small ruminant pestivirus.

[0013] Preferably, the content of live PPR vaccine virus in the live PPR vaccine is ≥10 3.5 TCID 50 / ml.

[0014] Preferably, the content of PPR virus antigen in the PPR virus toxin is 10 4.00 ~10 6.00 TCID 50 / ml.

[0015] More preferably, the PPR virus toxin and the heat-resistant protective agent are mixed in a volume ratio of 2:1~3:1 in step T1).

[0016] Preferably, the volume ratio of the PPR virus toxin and the heat-resistant protective agent is 2:1.

[0017] Further, the freeze-drying method in step T2) includes:

[0018] (1) Pre-freezing stage: the vaccine stock solution is cooled to -10℃ within 20 min, kept at -10℃ for 30 min, cooled to -40℃~-45℃ within 20 min~40 min, kept at -45℃ for 4h~6h, and then transferred to the vacuum stage after the sample is completely frozen;

[0019] (2) Vacuum stage: the vacuum degree is reduced to 100mT~120mT within 20 min, and then transferred to the sublimation drying stage;

[0020] (3) Sublimation drying stage: the plate layer temperature is raised from -45℃ to -30℃ within 2h and maintained for 4h~6h, the plate layer temperature is raised from -30℃ to -10℃ within 20 min and maintained for 8h~10h, the plate layer temperature is raised from -10℃ to -3℃ within 20 min and maintained for 8h~10h, and then transferred to the desorption drying stage;

[0021] (4) Desorption drying stage: the plate layer temperature is raised to 28℃~30℃ within 1h~3h, and maintained for 6h~8h.

[0022] In summary, the heat-resistant protective agent component provided by the present application is simple, raw materials are easy to obtain, and the preparation method is simple. When formulating peste des petits ruminants live vaccine, the loss of each virus antigen during freeze-drying process can be effectively reduced, the immunopotency and long-term stable storage of the peste des petits ruminants live vaccine can be ensured, the use of the heat-resistant freeze-drying protective agent as the heat-resistant freeze-drying protective agent of the peste des petits ruminants live vaccine can significantly reduce the damage of various physical and chemical factors in the freeze-drying process to the virus activity, the vaccine titer decreases less after heat resistance, the potency of the vaccine is more stable, and the safety is good. Meanwhile, the freeze-drying curve in the preparation of the live vaccine is optimized, the potency level of the peste des petits ruminants live vaccine can be ensured, the heat-resistant protective effect is good, the needs of market users can be met, good economic benefits and social benefits are obtained, and the application prospect is wide. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A picture of the sub-packaged freeze-dried peste des petits ruminants live vaccine prepared by using the protective agent 4 in the examples. DETAILED DESCRIPTION

[0024] The present application aims to provide a heat-resistant protective agent for peste des petits ruminants live vaccine with good heat-resistant protective effect, so as to ensure that the peste des petits ruminants live vaccine can be stably stored for a long time and maintain good immunopotency level and safety. Based on the heat-resistant protective agent, a peste des petits ruminants live vaccine with good immunopotency, high safety and long-term stable storage is also provided.

[0025] The present application is described in detail through the following specific embodiments.

[0026] In the following examples, the methods used are conventional methods unless otherwise specified. The obtaining routes of various biological materials described in the examples are only provided as a route for obtaining experiments to achieve the specific purposes disclosed, and should not be regarded as a limitation on the source of biological materials of the present application. In fact, the source of the biological materials used is extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the hints in the examples.

[0027] The examples are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given. The examples will help to understand the present application, but should not be regarded as a limitation on the content of the present application.

[0028] The chemical substances used in the following examples are as follows: dextran, thiourea, purchased from a national pharmaceutical company; lactose, ascorbic acid, purchased from Tianjin Kelong Chemical Company; glycine, poloxamer 188, purchased from Sigma Company; povidone K30, purchased from Nanjing Songguan Biological Technology Co., Ltd.; tert-butyl alcohol, purchased from Tianjin Damao Chemical Reagent Factory.

[0029] Example 1: Preparation of heat-resistant protective agent for peste des petits ruminants live vaccine

[0030] The embodiment aims to prepare a heat-resistant protective agent for small ruminant pestivirus live vaccine, which protects the effective components of the vaccine during the freeze-drying process and the storage stage after freeze-drying to ensure the immunization effect of the vaccine, and the preparation method of the heat-resistant protective agent comprises the following steps:

[0031] 1.1, preparation of the first solution: according to the component content (where % represents the mass percentage content, i.e. g / 100 ml, the same below) of the heat-resistant protective agent (its number is 1, 2, 3, 4, 5) shown in the following Table 1, the required amount of thiourea is precisely weighed in a quantitative injection water, heated in a 60°C water bath during stirring until completely dissolved, then dextran is weighed and added to the solution until completely dissolved, then lactose is precisely weighed and added to the solution, heated in a 60°C water bath without stopping stirring until dissolved, and the first solution is obtained;

[0032] 1.2, preparation of the second solution: according to the component content of each heat-resistant protective agent described in the following Table 1, povidone K30 is precisely weighed in a quantitative injection water, placed in a 60°C water bath, and shaken without stopping until completely dissolved, then the required amount of poloxamer 188 is precisely weighed and added to the solution to completely dissolve, then ascorbic acid, glycine and tert-butyl alcohol are precisely weighed and added to the solution, placed in a 60°C water bath, shaken without stopping to dissolve, and the PH is adjusted to 7.1~7.3 using 7.5% sodium bicarbonate solution, and the second solution is obtained;

[0033] 1.3, the first solution is sterilized in an autoclave (116°C high pressure for 20~30min) after being made to a constant volume, and the second solution is sterilized by filtration (0.22μm filter membrane) after being made to a constant volume, 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 standby.

[0034] Note: When preparing the heat-resistant protective agent, each reagent is dissolved separately, and the amount of solute is gradually increased during the single dissolution to avoid over-saturation and incomplete dissolution.

[0035]

[0036] Example 2: Preparation of small ruminant pestivirus live vaccine

[0037] The 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 the heat-resistant protective agent of the live vaccine to prepare the small ruminant pestivirus live vaccine, and the preparation method of the small ruminant pestivirus live vaccine comprises the following steps:

[0038] 2.1, preparation of small ruminant pestivirus virus solution

[0039] 2.1.1, Preparation of Peste des petits ruminants virus virus fluid

[0040] Peste des petits ruminants virus fluid propagation: take well-grown Vero cell monolayer, discard the growth solution, digest the well-grown Vero cell monolayer with 0.25% trypsin digestion solution, disperse at a ratio of 1:3 to 1:4, and culture at 37°C. The expanded Vero cells are inoculated into a rotating bottle and cultured at 37°C for 48 to 72 hours at a speed of 8 to 9 revolutions per hour. The cell culture solution is discarded, and the Peste des petits ruminants virus strain with a virus content of ≥10 3.0 TCID 50 / ml is inoculated into the Peste des petits ruminants virus Clone 9 strain at a ratio of 1% to 3% of the maintenance solution, adsorbed at 37°C for 1 hour, and then 2% bovine serum-containing MEM cell culture solution is added and cultured at 37°C for 4 to 6 days. When more than 80% of the cells show cytopathic effect, the virus fluid is harvested and stored at -40°C or below for testing.

[0041] 2.1.2, The final virus content is shown in Table 2

[0042]

[0043] 2.2, The Peste des petits ruminants virus content in each head of the Peste des petits ruminants live vaccine after freeze-drying is ≥10 4.0 TCID 50 / ml, and the vaccine dose of the Peste des petits ruminants virus fluid is calculated;

[0044] 2.3, The virus antigen solution obtained in step 2.2 is mixed 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 at a constant speed, and manually mix slowly for about 15 minutes (to avoid excessive foam), to obtain the vaccine stock solution;

[0045] 2.4, The vaccine stock solution obtained in step 2.3 is divided into 3ml (300 head portions) per bottle using a pipette, i.e. 3ml per bottle, and then freeze-dried to obtain the Peste des petits ruminants live vaccine (freeze-dried preparation). The freeze-drying curve for freeze-drying 3ml of vaccine stock solution is as follows:

[0046] (1) Pre-freezing speed: the slow freezing rate is selected after the product is put into the freeze-drying box.

[0047] (2) The minimum temperature of pre-freezing: The minimum temperature of pre-freezing is lower than the co-crystal point temperature of the mixed vaccine, which is -23℃.

[0048] (3) The pre-freezing time: In the pre-freezing stage, the vaccine stock solution is cooled from room temperature to -10℃ within 20 min, kept at -10℃ for 30 min, cooled to -40℃-45℃ within 20 min-40 min, kept at -45℃ for 4 h-6 h, and then transferred to the vacuum stage after the sample is completely frozen.

[0049] (4) The vacuum time: The pre-freezing is immediately followed by the pressure reduction stage, and the vacuum degree is reduced to 100 mT-120 mT within 20 min until the end.

[0050] (5) The sublimation drying stage: The heating is started when the vacuum degree is reduced to 120 mT, and the sublimation drying stage is started at this time. The shelf temperature is increased from -45℃ to -30℃ within 2 h and maintained for 4 h-6 h, the shelf temperature is increased from -30℃ to -10℃ within 20 min and maintained for 8 h-10 h, and the shelf temperature is increased from -10℃ to -3℃ within 20 min and maintained for 8 h-10 h, and then the product is transferred to the resolution drying stage, so that the frozen product in the bottle is gradually sublimated from top to bottom, and finally shaped.

[0051] (6) The resolution drying stage: The product has formed a preliminary shape when the sublimation drying is completed, and at this time, the vacuum degree is increased, and the vacuum degree is set to 10 mT, so as to improve the heat transfer in the freeze-drying box and increase the convective heat transfer of the gas, and the shelf temperature is increased to 28℃ within 1 h-3 h and maintained for 6 h-8 h.

[0052] (7) The maximum allowable temperature of the product: The maximum allowable temperature of the shelf is 28℃ according to the product. Due to the difference in heat transfer, the shelf temperature is usually slightly lower than the maximum allowable temperature of the product.

[0053] (8) The total freeze-drying time: According to the performance of the freeze-dryer, the characteristics of the vaccine, and the loading amount, the total time of the pre-freezing time, the sublimation drying stage, and the resolution drying stage is determined to be about 35 h-47 h.

[0054] The virus content of each Peste des petits ruminants live vaccine prepared in this example before and after freeze-drying was detected, and the detection results are shown in Tables 3 and 4. The vaccine product prepared in this example was also detected for routine items according to the method recorded in the inspection procedure document of the Regulations for Veterinary Biological Products of the People's Republic of China, as shown in Tables 3-4.

[0055]

[0056]

[0057] From the detection results of Table 3 and Table 4, it can be seen that the virus content (titer) of the small ruminant pestivirus live vaccine prepared by using the protective agent 4 and the protective agent 5 is less decreased before and after freeze-drying, and the maximum decrease is only 10 0.25 TCID 50 / ml, and the minimum virus content is not lost, so the protective agent 4 and the protective agent 5 can effectively protect the virus antigens in the small ruminant pestivirus live vaccine from being degraded during freeze-drying, and a vaccine product with a higher titer level is obtained. Compared with the protective agent 4 and the protective agent 5, the protective agent 3 does not contain tert-butyl alcohol, and the virus titer of the small ruminant pestivirus live vaccine is more decreased before and after freeze-drying. Since tert-butyl alcohol can form needle-shaped crystals when the vaccine is frozen, and tubular channels are left after the sublimation of ice crystals, so that the water vapor flow resistance during the freeze-drying process of the animal vaccine is greatly reduced, and the sublimation rate is significantly improved. Therefore, the tert-butyl alcohol is added to the animal vaccine in the present application to reduce the drying layer resistance, thereby accelerating the drying process and shortening the drying time. At the same time, the 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 freeze-dried product. Compared with the protective agent 4 and the protective agent 5, the protective agent 1 does not contain glycine, and the virus titer of the small ruminant pestivirus live vaccine is also more decreased before and after freeze-drying. Glycine in the protective agent can inhibit the oxidation and aggregation of macromolecular substances; the protective agent 2 does not contain poloxamer 188, and poloxamer 188 is another excipient used as a protein stabilizer in protein preparations. Since it does not have an ester bond, it will not have the risk of hydrolysis. It mainly degrades in the form of oxidation in the solution, forming oxidized polyoxyethylene (POE) and polypropylene oxide (PPO), which can protect the virus to a certain extent. It can be seen that the components in the heat-resistant protective agent provided by the present application can protect the virus antigens in the small ruminant pestivirus live vaccine from being degraded, and a vaccine product with a higher titer level is obtained. According to the detection results of Table 4, it can be seen that the small ruminant pestivirus live vaccine obtained by using the protective agent 4 and the protective agent 5 after being divided (4 bottles) and freeze-dried is a light yellow or white sponge-like loose mass, and is easy to separate from the bottle wall Figure 1 (shown in the photo of the live vaccine freeze-dried product obtained after being divided (4 bottles) and freeze-dried by using the protective agent 4), and the water content is below 2.0%, which can be quickly dissolved in water, so that the prepared vaccine product has a better freeze-dried appearance, a lower water content and good dissolution characteristics. Compared with the protective agent 5, the heat-resistant protection effect of the protective agent 4 is better, and the virus content of the vaccine before and after freeze-drying is decreased by only 10 0.25 TCID 50 / ml, and the minimum virus content is not lost, so the protective agent 4 and the protective agent 5 can effectively protect the virus antigens in the small ruminant pestivirus live vaccine from being degraded during freeze-drying, and a vaccine product with a higher titer level is obtained. Compared with the protective agent 4 and the protective agent 5, the protective agent 3 does not contain tert-butyl alcohol, and the virus titer of the small ruminant pestivirus live vaccine is more decreased before and after freeze-drying. Since tert-butyl alcohol can form needle-shaped crystals when the vaccine is frozen, and tubular channels are left after the sublimation of ice crystals, so that the water vapor flow resistance during the freeze-drying process of the animal vaccine is greatly reduced, and the sublimation rate is significantly improved. Therefore, the tert-butyl alcohol is added to the animal vaccine in the present application to reduce the drying layer resistance, thereby accelerating the drying process and shortening the drying time. At the same time, the 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 freeze-dried product. Compared with the protective agent 4 and the protective agent 5, the protective agent 1 does not contain glycine, and the virus titer of the small ruminant pestivirus live vaccine is also more decreased before and after freeze-drying. Glycine in the protective agent can inhibit the oxidation and aggregation of macromolecular substances; the protective agent 2 does not contain poloxamer 188, and poloxamer 188 is another excipient used as a protein stabilizer in protein preparations. Since it does not have an ester bond, it will not have the risk of hydrolysis. It mainly degrades in the form of oxidation in the solution, forming oxidized polyoxyethylene (POE) and polypropylene oxide (PPO), which can protect the virus to a certain extent. It can be seen that the components in the heat-resistant protective agent provided by the present application can protect the virus antigens in the small ruminant pestivirus live vaccine from being degraded, and a vaccine product with a higher titer level is obtained. According to the detection results of Table 4, it can be seen that the small ruminant pestivirus live vaccine obtained by using the protective agent 4 and the protective agent 5 after being divided (4 bottles) and freeze-dried is a light yellow or white sponge-like loose mass, and is easy to separate from the bottle wall

[0058] The embodiment also carries out aging resistance test on the live vaccine for Peste des petits ruminants prepared by using the protective agent 4 as the heat resistance protective agent according to the method recorded in the inspection regulation document of the Regulations of the People's Republic of China on Veterinary Biological Products, wherein the specific method is that the live vaccine for Peste des petits ruminants is placed at 37 DEG C for 10 days, and is stored at 2-8 DEG C for 24 months, the change of the virus content in the vaccine product is detected, and the detection results are shown in the following table 5.

[0059] From the detection results in the above table 5, it can be known that the live vaccine for Peste des petits ruminants prepared by using the protective agent 4 as the heat resistance protective agent, whether the antigen: protective agent is 2:1 or 3:1, the virus titer of the vaccine after being stored at 37 DEG C for 7 days is not more than 10 1.00 TCID 50 / ml, and the most decreased one is only 10 0.25 TCID 50 / ml; the virus titer of the vaccine after being stored at 2-8 DEG C for 24 months is also not more than 10 1.00 TCID 50 / ml, and the most decreased one is only 10 0.50 TCID 50 / ml. When the protective agent 5 is used as the heat resistance protective agent to prepare the live vaccine for Peste des petits ruminants, the protective agent also has the good heat resistance protective effect as the protective agent 4. Therefore, the heat resistance protective agent provided by the application can ensure that the live vaccine for Peste des petits ruminants using the same can be stably stored for a long time, and effectively inhibit the decrease of the virus titer, so as to ensure the immunization efficiency of the virus antigen.

[0060] Example 3: Preparation of heat resistance protective agent for live vaccine for Peste des petits ruminants

[0061] The embodiment is prepared according to the same method of steps 1.1-1.3 in the embodiment 1, according to the composition and content of the heat resistance protective agent listed in the following table 6, and is named as the protective agent 6, the protective agent 7 and the protective agent 8. The live vaccine for Peste des petits ruminants is prepared by using the protective agent 6, the protective agent 7 and the protective agent 8 according to the same method of steps 2.3-2.4 in the embodiment 2, wherein the antigen: protective agent = 2:1. Subsequently, the decrease of the virus titer before and after the freeze-drying of the live vaccine for Peste des petits ruminants prepared by using the protective agent 6, the protective agent 7 and the protective agent 8, and the detection of the routine items of the finished product are carried out according to the same method of the embodiment 2, and the detection results are shown in the following table 7.

[0062]

[0063]

[0064] From the results of Table 6 and Table 7, it can be seen that the virus content (titer) of the live vaccine of small ruminant pestivirus prepared using the protective agents 6-8 decreases less before and after freeze-drying, and the maximum decrease is only 10 0.25 TCID 50 / ml, and the minimum virus content is not lost, so the protective agents 6-8 can effectively protect the virus antigens in the live vaccine of small ruminant pestivirus from degradation during freeze-drying, and obtain a vaccine product with a higher titer level. According to the detection results in Table 7, it can also be seen that the live vaccine of small ruminant pestivirus obtained using the protective agents 6-8 is light yellow or white after freeze-drying, and is easy to separate from the bottle wall, and the water content is below 2.0%, which can be quickly dissolved in water, so the prepared vaccine product has a better freeze-drying appearance, lower water content and good dissolution characteristics. Therefore, the heat-resistant protective agent provided by the present application can effectively protect the virus antigens in the live vaccine of small ruminant pestivirus using the same from degradation during freeze-drying, thereby ensuring the immunization efficacy of the vaccine, and at the same time obtaining a live vaccine product of small ruminant pestivirus with good freeze-drying appearance, lower water content and good dissolution characteristics.

[0065] Example 4: Optimization of freeze-drying curve for preparing live vaccine of small ruminant pestivirus

[0066] In this example, the protective agent 4 obtained in Example 1 is used as a heat-resistant protective agent, and the vaccine virus antigen stock obtained in step 2.2 of Example 2 is mixed with the protective agent 4 in a volume ratio of 2:1 to obtain a vaccine stock, according to the same method as step 2.3 in Example 2. Then, the vaccine stock is divided into 3 ml per bottle and subjected to freeze-drying treatment. In this example, the inventors designed multiple freeze-drying curves to screen and determine the ideal freeze-drying curve suitable for the live vaccine of small ruminant pestivirus. Based on the protective agent 4 obtained in Example 1 as a heat-resistant protective agent, two freeze-drying curves are listed as follows: freeze-drying curve 1 and freeze-drying curve 2.

[0067] Freeze-drying curve 1: the same as the freeze-drying curve used for preparing the live vaccine of small ruminant pestivirus in Example 2; the live vaccine of small ruminant pestivirus obtained based on the freeze-drying curve 1 is named as live vaccine 1;

[0068] Freeze-drying curve 2 (as a control freeze-drying curve):

[0069] The freeze-drying curve for freeze-drying 3 ml of vaccine stock is as follows:

[0070] (1) Pre-freezing stage: the vaccine stock is cooled to -40°C to -45°C within 1 h, and maintained at -45°C for 4 h to 6 h. After the sample is completely frozen, it is transferred to the vacuum stage;

[0071] (2) Vacuum stage: the vacuum degree is reduced to 100 mT to 120 mT within 20 min, and then transferred to the sublimation drying stage.

[0072] (3) Sublimation drying stage: the temperature of the plate layer is raised from -45℃ to -20℃ within 2h and maintained for 6h-8h, the temperature of the plate layer is raised from -20℃ to -15℃ within 20min and maintained for 8h-10h, the temperature of the plate layer is raised from -15℃ to -2℃ within 20min and maintained for 4h-6h, and then the plate layer is transferred to the resolution drying stage;

[0073] (4) Resolution drying stage: the temperature of the plate layer is raised to 28℃ within 1h-2h and maintained for 6h-8h.

[0074] The live vaccine 2 based on the freeze-drying curve 2 is obtained.

[0075] According to the same method in Example 2, the virus titer reduction of the live vaccine 1 and the live vaccine 2 before and after freeze-drying and the routine items of the vaccine finished product are detected, and the detection results are shown in Table 8.

[0076]

[0077] According to the detection results in Table 8, the virus titer of the PPRV in the live vaccine 1 is reduced by 10 0.25 TCID 50 / ml after freeze-drying, and the physical properties, solubility and residual moisture of the vaccine after freeze-drying are good. The virus titer of the PPRV in the live vaccine 2 is reduced by 10 0.75 TCID 50 / ml after freeze-drying, and the physical properties after freeze-drying are slightly poor, the bottom of the vaccine is slightly melted, and a small amount of particles appear after dissolution. The virus titer reduction of the live vaccine 1 is relatively more than that of the live vaccine 2. Therefore, the freeze-drying curve 1 adopted by the present application can not only reduce the virus titer loss of the PPRV during freeze-drying, but also improve the product quality.

[0078] Example 5: Safety test of PPRV live vaccine

[0079] In this example, the PPRV live vaccines obtained by using the protective agent 7 as the heat-resistant protective agent and by preparing the antigen and the protective agent in a volume ratio of 2:1 and 3:1 in Example 3 (named as live vaccine 3 and live vaccine 4, respectively) are taken as the experimental groups, and sterile physiological saline is taken as the control group, and 5 healthy susceptible goats (PPRV neutralization body titer less than 1:4) of about 1 year old are inoculated subcutaneously in the neck with 1ml each. The body temperature is measured once a day after inoculation, and the clinical symptoms are observed for 14 days, as shown in Table 9, and no abnormal reaction is observed in the experimental goats in each group, indicating that the PPRV live vaccine prepared by the present application has good safety.

[0080]

[0081] In summary, the heat-resistant protective agent component provided by the present application is simple, raw materials are easy to obtain, and the preparation method is simple. When formulating peste des petits ruminants live vaccine, the loss of each virus antigen in the freeze-drying process can be effectively reduced, the immunization efficacy and long-term stable storage of the peste des petits ruminants live vaccine can be ensured, the use of the heat-resistant freeze-drying protective agent as the peste des petits ruminants live vaccine can significantly reduce the damage of various physical and chemical factors in the freeze-drying process to the virus activity, the vaccine product prepared by using the heat-resistant freeze-drying protective agent has less vaccine titer drop after heat resistance, the efficacy of the vaccine is more stable, and the safety is good. Meanwhile, the freeze-drying curve in the preparation of the live vaccine is optimized, the efficacy level of the peste des petits ruminants live vaccine can be ensured, the heat-resistant protective effect is good, the needs of market users can be met, good economic benefits and social benefits are obtained, and the application prospect is wide.

[0082] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the present application is described in detail with reference to the foregoing embodiments, for those skilled in the art, the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A live vaccine heat resistance protecting agent, characterized by, It is composed of the following mass volume percentage of components: 3% glucose, 2% thiourea, 7% lactose, 0.7% ascorbic acid, 3% glycine, 1% poloxamer 188, 2% povidone K30, 5% tert-butyl alcohol, the balance is water for injection.

2. A method for preparing a heat-resistant protective agent for a live vaccine according to claim 1, characterized by: According to the proportion, each component is dissolved in water for injection, and after sterilization, a live vaccine heat-resistant protective agent is obtained.

3. The use of a live vaccine heat-resistant protective agent as claimed in claim 1 in the preparation of a live Peste des petits ruminants vaccine.

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

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

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

7. The preparation method according to claim 5, characterized in that The content of the peste des petits ruminants virus antigen in the peste des petits ruminants virus solution is 10 4.00 ~ 10 6.00 TCID 50 / ml.

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

1.

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

1.

10. The method of claim 5, wherein, The freeze-drying method in step T2) includes: (1) pre-freezing stage: the vaccine stock solution is cooled to -10℃ within 20min, kept at -10℃ for 30min, cooled to -40℃ to -45℃ within 20min to 40min, kept at -45℃ for 4h to 6h, and then transferred to the vacuum stage after the sample is completely frozen; (2) vacuum stage: the vacuum degree is reduced to 100mT to 120mT within 20min, and then transferred to the sublimation drying stage; (3) sublimation drying stage: the plate layer temperature is raised from -45℃ to -30℃ within 2h and maintained for 4h to 6h, the plate layer temperature is raised from -30℃ to -10℃ within 20min and maintained for 8h to 10h, the plate layer temperature is raised from -10℃ to -3℃ within 20min and maintained for 8h to 10h, and then transferred to the desorption drying stage; (4) desorption drying stage: the plate layer temperature is raised to 28℃ to 30℃ within 1h to 3h, and maintained for 6h to 8h.

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