Heat-resistant protective agent as well as preparation method and application thereof

Abstract

The invention discloses a heat-resistant protective agent as well as a preparation method and application thereof, and belongs to the technical field of biology. The heat-resistant protective agent comprises a liquid A and a liquid B, the liquid A comprises the following components in percentage by weight: 15%-20% of cane sugar, 5%-10% of polyvinylpyrrolidone, 1%-2% of sorbitol and the balance of water; the liquid B comprises the following components in percentage by weight: 1%-2% of glutamine, 5%-10% of sodium glutamate, 1%-2% of glycine, 0.1%-0.5% of saponin and the balance of water; the volume ratio of the solution A to the solution B is (0.5-2): (0.5-2). The heat-resistant protective agent can remarkably prolong the storage life of the vaccine, improve the stability of the vaccine in the storage process and improve the immune effect of the vaccine, and has the dual effects of heat resistance and immune enhancement. The heat-resistant protective agent provided by the invention provides a new method for ensuring the stability of vaccines in the transportation process, and provides technical support for the application of vaccines in remote areas.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a heat-resistant protective agent, a preparation method thereof, and an application thereof. Background Art

[0002] With the large-scale and intensive development of the livestock industry and the continuous improvement of the demand for animal disease prevention and control, vaccines, as the core tool for preventing diseases, have a continuously growing market demand. However, the effectiveness of vaccines is often restricted by storage and transportation conditions, and the problem of vaccine inactivation due to high temperature caused by imperfect cold chain logistics facilities is particularly prominent. Against this background, heat-resistant protective agents for vaccines, as a key technical solution, have gradually become the focus of industry attention.

[0003] Traditional vaccines are sensitive to temperature and usually need to be stored frozen at -15°C. However, in practical applications, insufficient power supply and lack of cold chain equipment in some areas result in vaccines being exposed to high-temperature environments, with reduced potency or even inactivation. Heat-resistant protective agents can significantly improve the stability of vaccines in high-temperature environments, extend the shelf life, and reduce storage costs by adding stabilizers or improving the formulation process. However, some heat-resistant protective agents have problems such as high cost and complex processes, and need to be customized for different vaccines, and a universal solution has not yet been formed.

[0004] Maternal antibodies are a type of specific antibodies that newborns obtain from their mothers through the placenta, colostrum, etc. It can protect piglets from various pathogen infections within a certain period of time. However, high levels of maternal antibodies in piglets can specifically neutralize vaccines, seriously interfering with the immune effect of vaccines. Therefore, it is particularly important to develop a heat-resistant protective agent with heat resistance and immune enhancement effects. Summary of the Invention

[0005] The purpose of the present invention is to provide a heat-resistant protective agent, a preparation method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. The heat-resistant protective agent of the present invention can significantly increase the storage period of vaccines, improve the stability of vaccines during storage, and also improve the immune effect of vaccines, having the dual effects of heat resistance and immune enhancement. The heat-resistant protective agent of the present invention provides a new method for ensuring the stability of vaccines during transportation and provides technical support for the application of vaccines in remote areas.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a heat-resistant protective agent, comprising liquid A and liquid B;

[0008] The liquid A comprises raw materials in the following mass percentages: 15% - 20% of sucrose, 5% - 10% of polyvinylpyrrolidone, and 1% - 2% of sorbitol, with the balance being water;

[0009] The liquid B comprises raw materials in the following mass percentages: 1%-2% glutamine, 5%-10% sodium glutamate, 1%-2% glycine, and 0.1%-0.5% saponin, with the balance being water;

[0010] The volume ratio of the liquid A to the liquid B is (0.5 - 2):(0.5 - 2).

[0011] Optionally, the volume ratio of the liquid A to the liquid B is 1:1.

[0012] The present invention also provides a preparation method of the above heat-resistant protectant, comprising the following steps:

[0013] (1) Dissolve sucrose, polyvinylpyrrolidone, and sorbitol in water to obtain the liquid A;

[0014] (2) Dissolve glutamine, sodium glutamate, glycine, and saponin in water to obtain the liquid B;

[0015] (3) Mix the liquid A and the liquid B to obtain the heat-resistant protectant.

[0016] The present invention also provides the application of the above heat-resistant protectant in the preparation of virus vaccines.

[0017] Optionally, the virus vaccines include classical swine fever virus vaccine, porcine pseudorabies virus vaccine, or porcine reproductive and respiratory syndrome virus vaccine.

[0018] The present invention also provides the application of the above heat-resistant protectant in extending the shelf life of vaccines.

[0019] The present invention also provides the application of the above heat-resistant protectant in the preparation of preparations capable of improving the immune effect of vaccines.

[0020] The present invention also provides a preparation method of a virus vaccine. After mixing the above heat-resistant protectant and virus liquid according to a volume ratio of 1:1, perform freeze-drying to obtain the virus vaccine.

[0021] Optionally, the virus includes classical swine fever virus, porcine pseudorabies virus, or porcine reproductive and respiratory syndrome virus.

[0022] The present invention discloses the following technical effects:

[0023] The heat-resistant protectant of the present invention is added with the surfactant saponin, which is used in combination with polyvinylpyrrolidone, glutamine, etc., reducing the freeze-drying loss during the freeze-drying process, being able to scavenge free radicals during storage, and improving the stability of the product during storage. Saponin also has an immune-enhancing function, can stimulate the cellular immunity and humoral immunity of the body, and can enhance the immune effect of the vaccine. The heat-resistant protectant of the present invention does not contain gelatin and protein, the protein content of the protectant itself is low, reducing the occurrence probability of immune side reactions. By reasonably matching and using various raw materials, the present invention significantly improves the heat resistance and immune effect of the vaccine product, can be used for the preparation of various virus vaccines, and has a wide application range. The classical swine fever live vaccine, porcine pseudorabies live vaccine and porcine reproductive and respiratory syndrome live vaccine prepared by using the heat-resistant protectant of the present invention can be stored at 2 - 8°C for 36 months from the original storage at below -15°C for 24 months, and the storage period at 37°C can reach 15 days, realizing cold storage during the storage and transportation process, not being afraid of temperature changes during transportation, and significantly extending the storage period and quality. Detailed Description of the Invention

[0024] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0028] Regarding the terms "comprising", "including", "having", "containing", etc. used in this text, they are all open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] Solution A: Dissolve 20% sucrose, 10% polyvinylpyrrolidone, and 2% sorbitol in injection water by mass percentage, make up the volume to 1000 mL with injection water, and sterilize at 116°C under high pressure for 30 min.

[0031] Solution B: Dissolve 2% glutamine, 10% sodium glutamate, 2% glycine, and 0.5% saponin in injection water by mass percentage, make up the volume to 1000 mL with injection water, and filter and sterilize with a 0.22 μm microporous membrane.

[0032] Mix Solution A and Solution B in a volume ratio of 1:1 to obtain heat-resistant protectant 1.

[0033] Example 2

[0034] Solution A: Dissolve 15% sucrose, 8% polyvinylpyrrolidone, and 1% sorbitol in injection water by mass percentage, make up the volume to 1000 mL with injection water, and sterilize at 116°C under high pressure for 30 min.

[0035] Solution B: Dissolve 1% glutamine, 10% sodium glutamate, 1% glycine, and 0.3% saponin in injection water by mass percentage, make up the volume to 1000 mL with injection water, and filter and sterilize with a 0.22 μm microporous membrane.

[0036] Mix Solution A and Solution B in a volume ratio of 1:1 to obtain heat-resistant protectant 2.

[0037] Comparative Example 1

[0038] Based on Example 1, in this comparative example, replace 0.5% saponin in Solution B with 0.5% vitamin C, and keep the other components and the preparation process the same as in Example 1 to obtain heat-resistant protectant 3.

[0039] Test Example 1

[0040] Mix the classical swine fever virus solution (provided by Reipur (Baoding) Biopharmaceutical Co., Ltd.) with heat-resistant protectants 1 - 3 in a volume ratio of 1:1, dispense into bottles at 2.0 mL / bottle, and then freeze-dry with a freeze-vacuum dryer to obtain the test vaccines.

[0041] Meanwhile, a control group was established. The formula of the control vaccine protectant was 10 g of gelatin, 40 g of sucrose, and distilled water was added to 100 mL, and it was sterilized at 116 °C for 40 minutes. The vaccine was prepared according to the volume ratio of protectant:virus solution:PBS = 0.3:1:1.1, and it was subpackaged at 2.4 mL / bottle, and then freeze-dried by a freeze-vacuum dryer to obtain the control vaccine.

[0042] (1) Vaccine stability determination

[0043] ① Detection of stability under refrigerated storage: Samples were taken at 0, 6, 12, 24, and 36 months after freeze-drying when stored at 2 - 8 °C, the virus content of the vaccine was measured, 3 bottles each time, and the average value was taken. The measurement results are shown in Table 1, unit: TCID 50 / head dose.

[0044] Table 1 Results of the stability of each group of vaccines under refrigerated storage

[0045] Group 0 months 6 months 12 months 24 months 36 months Heat-resistant protective agent live vaccine 1 <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.5 > Heat-resistant protective agent live vaccine 2 <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.5 > Heat-resistant protective agent live vaccine 3 <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.5 > <![CDATA[10 4.1 > <![CDATA[10 3.5 > Control vaccine <![CDATA[10 4.5 > <![CDATA[10 3.3 > <![CDATA[10 2.9 > <![CDATA[<10 2.5 > <![CDATA[<10 2.5 >

[0046] As can be seen from Table 1, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 2 - 8 °C for 24 months, the virus content in the vaccine did not change. When stored for 36 months, there was still a relatively high virus content in the vaccine, and the degree of decrease was not obvious. In contrast, for the vaccine prepared with heat-resistant protectant 3, when stored for 6 months, the virus content in the vaccine was stable, but a downward trend began to appear when stored for 12 months. When stored for 36 months, the virus content was as low as 10 3.5 ; for the control vaccine, when stored for 6 months, a rapid downward trend in the virus content had already occurred. When stored for 24 months, the virus content was already lower than 10 2.5 . This result indicates that the heat-resistant protectant of the present invention can significantly extend the storage period of the vaccine through the reasonable combination of its components.

[0047] ② Detection of stability under 37 °C storage: Take 20 bottles, and samples were taken at 0, 7, 10, and 15 days when stored at 37 °C to measure the virus content, 3 bottles each time. The measurement results are shown in Table 2, unit: TCID 50 / head dose.

[0048] Table 2 Results of the stability of each group of vaccines under 37 °C storage

[0049] Group 0d 7d 10d 15d Heat-resistant protective agent live vaccine 1 <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.5 > <![CDATA[10 4.5 > Heat-resistant protective agent live vaccine 2 <![CDATA[10 4.8 > <![CDATA[10 4.8 > <![CDATA[10 4.5 > <![CDATA[10 4.5 > Heat-resistant protective agent live vaccine 3 <![CDATA[10 4.8 > <![CDATA[10 4.5 > <![CDATA[10 3.9 > <![CDATA[10 3.5 > Control vaccine <![CDATA[10 4.5 > <![CDATA[10 3.3 > <![CDATA[<10 2.5 > <![CDATA[<10 2.5 >

[0050] As can be seen from Table 2, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 37°C for 7 days, the virus content in the vaccines remained unchanged. When stored for 15 days, there was still a relatively high virus content in the vaccines, and the degree of decrease was not obvious. In contrast, for the vaccine prepared using heat-resistant protectant 3, a trend of decreasing virus content was already observed when stored for 7 days; for the control vaccine, a rapid decrease in virus content was already evident when stored for 7 days, and when stored for 10 days, the virus content was already lower than 10 2.5 . This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the storage period of the vaccine can be significantly extended, and the stability and effectiveness of the vaccine in an abnormal storage environment at 37°C can be improved, ensuring the stability of the vaccine when temperature fluctuations occur during transportation and storage.

[0051] (2) Immunization effect

[0052] A total of 100 35-day-old piglets with maternally-derived antibodies were randomly divided into 5 groups, with 20 piglets in each group. One group served as the blank control, and the other 4 groups were intramuscularly injected with the experimental vaccine and the control vaccine in the neck, at a dose of 1 dose per pig. Blood samples were collected at 0 month, 1 month, and 2 months after immunization, and the sera were separated. The antibody titers were detected using an IDEXX classical swine fever virus antibody detection kit (a blocking rate of ≥40% was considered antibody-positive). The antibody levels in pigs after vaccine immunization are shown in Table 3.

[0053] Table 3 Immunization effects of vaccines in each group

[0054]

[0055] As can be seen from Table 3, maternally-derived antibodies were present in the animals in the blank control group, and the antibody decline rate was fast, and the antibodies became negative after 1 month. For the animals immunized with the vaccines prepared using heat-resistant protectant 1 or 2, 1 month after immunization, the antibodies in the animals remained at about 60%, and 2 months after immunization, the antibodies remained at about 80%, which were much higher than those of the vaccines prepared using heat-resistant protectant 3 and the control vaccine. This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the immunization effect of the vaccine can be significantly improved.

[0056] Experimental Example 2

[0057] Porcine pseudorabies virus solution (provided by Ruipu (Baoding) Biopharmaceutical Co., Ltd.) was respectively mixed with heat-resistant protectants 1-3 in a volume ratio of 1:1, and then dispensed into bottles at 2.0 mL per bottle, and then freeze-dried using a freeze-vacuum dryer to obtain the experimental vaccines.

[0058] Meanwhile, a control group was established. The formulation of the control vaccine protectant was 10 g of gelatin, 40 g of sucrose, and distilled water was added to 100 mL. It was sterilized at 116 °C for 40 minutes. The vaccine was prepared according to the volume ratio of protectant:virus solution:PBS = 0.3:1:1.1, and sub-packaged at 2.4 mL / bottle, and then freeze-dried by a freeze-vacuum dryer to obtain the control vaccine.

[0059] (1) Vaccine stability determination

[0060] ① Detection of stability under refrigerated storage: Samples were taken at 0, 6, 12, 24, and 36 months after freeze-drying and stored at 2 - 8 °C to measure the virus content, 3 bottles each time. The measurement results are shown in Table 4, unit: TCID 50 / head dose.

[0061] Table 4 Results of the stability of vaccines in each group under refrigerated storage

[0062]

[0063]

[0064] As can be seen from Table 4, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 2 - 8 °C for 24 months, the virus content in the vaccines remained unchanged. When stored for 36 months, there was still a relatively high virus content in the vaccines, and the degree of decrease was not obvious. In contrast, for the vaccine prepared with heat-resistant protectant 3, when stored for 6 months, the virus content in the vaccine was stable, but a downward trend began to appear when stored for 12 months. When stored for 36 months, the virus content was as low as 10 5.5 ; for the control vaccine, when stored for 6 months, the virus content had already shown a rapid downward trend. When stored for 36 months, the virus content was only 10 2.3 . This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the effect of significantly extending the storage period of the vaccine can be achieved.

[0065] ② Detection of stability under 37 °C storage: Take 20 bottles, and samples were taken at 0, 7, 10, and 15 days after placing at 37 °C to measure the virus content, 3 bottles each time. The measurement results are shown in Table 5, unit: TCID 50 / head dose.

[0066] Table 5 Results of the stability of vaccines in each group under 37 °C storage

[0067] Group 0d 7d 10d 15d Heat-resistant protective agent live vaccine 1 <![CDATA[10 6.7 > <![CDATA[10 6.7 > <![CDATA[10 6.5 > <![CDATA[10 6.5 > Heat-resistant protective agent live vaccine 2 <![CDATA[10 6.7 > <![CDATA[10 6.7 > <![CDATA[10 6.5 > <![CDATA[10 6.5 > Heat-resistant protective agent live vaccine 3 <![CDATA[10 6.7 > <![CDATA[10 6.3 > <![CDATA[10 5.9 > <![CDATA[10 5.3 > Control vaccine <![CDATA[10 6.5 > <![CDATA[10 5.3 > <![CDATA[10 4.5 > <![CDATA[10 2.5 >

[0068] As can be seen from Table 5, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 37°C for 7 days, the virus content in the vaccines remained unchanged. When stored for 15 days, there was still a relatively high virus content in the vaccines, and the degree of decrease was not obvious. In contrast, for the vaccine prepared using heat-resistant protectant 3, a trend of decreasing virus content was already observed on the 7th day of storage; for the control vaccine, a rapid decrease in virus content was already observed on the 7th day of storage, and when stored for 15 days, the virus content was only 10 2.5 . This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the storage period of the vaccine can be significantly extended, and the stability and effectiveness of the vaccine in an abnormal storage environment at 37°C can be improved, ensuring the stability of the vaccine when there are temperature fluctuations during transportation and storage.

[0069] (2) Immune effect

[0070] One hundred 50-day-old maternally antibody-positive pigs were selected and randomly divided into 5 groups, with 20 pigs in each group. One group was used as the blank control, and the other 4 groups were intramuscularly injected with the test vaccine and the control vaccine in the neck, 1 dose per pig. The second immunization was carried out 4 weeks after the first immunization, 1 dose per pig. Blood was collected at 0 month after immunization, and 2 weeks and 4 weeks after the second immunization, the serum was separated, and the neutralizing antibody was measured. The neutralizing antibody levels of pigs after vaccine immunization are shown in Table 6.

[0071] Table 6 Immune effects of vaccines in each group

[0072]

[0073] As can be seen from Table 6, maternally derived antibodies were present in the animals in the blank control group, and the neutralizing antibody titer decreased rapidly. The neutralizing antibodies were all negative 2 weeks after the second immunization. For the animals immunized with the vaccines prepared using heat-resistant protectant 1 or 2, the neutralizing antibody titers in the animals were higher, and the immune effect was maintained for a longer time. In contrast, the neutralizing antibodies produced by the vaccines prepared using heat-resistant protectant 3 and the control vaccine in the animals were lower, and the neutralizing antibody decreased rapidly. This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the immune effect of the vaccine can be significantly improved.

[0074] Test Example 3

[0075] Porcine reproductive and respiratory syndrome virus liquid (provided by Ruipu (Baoding) Biopharmaceutical Co., Ltd.) was respectively mixed with heat-resistant protectants 1 - 3 in a volume ratio of 1:1, and then sub-packed at 2.0 mL per bottle, and then freeze-dried by a freeze-vacuum dryer to obtain the test vaccines.

[0076] Meanwhile, a control group was established. The formula of the control vaccine protectant was 10 g of gelatin, 40 g of sucrose, and distilled water was added to 100 mL, and it was sterilized at 116 °C for 40 minutes. The vaccine was prepared according to the volume ratio of protectant:virus solution:PBS = 0.3:1:1.1, and it was subpackaged at 2.4 mL / bottle, and then freeze-dried by a freeze-vacuum dryer to obtain the control vaccine.

[0077] (1) Vaccine stability determination

[0078] ① Detection of stability under refrigerated storage: Samples were taken at 0, 6, 12, 24, and 36 months after freeze-drying and placed at 2 - 8 °C to measure the virus content, 3 bottles each time. The measurement results are shown in Table 7, unit: TCID 50 / head dose.

[0079] Table 7 Results of the stability of each group of vaccines under refrigerated storage

[0080]

[0081]

[0082] As can be seen from Table 7, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 2 - 8 °C for 24 months, the virus content in the vaccines remained unchanged. When stored for 36 months, there was still a relatively high virus content in the vaccines, and the degree of decrease was not obvious. In contrast, for the vaccine prepared with heat-resistant protectant 3, when stored for 6 months, the virus content in the vaccine was stable, but when stored for 12 months, a downward trend began to appear. When stored for 36 months, the virus content was as low as 10 4.9 ; for the control vaccine, when stored for 6 months, the virus content had already shown a rapid downward trend. When stored for 24 months, the virus content was only 10 3.1 . This result indicates that through the reasonable combination of various components of the heat-resistant protectant of the present invention, the effect of significantly extending the storage period of the vaccine can be achieved.

[0083] ② Detection of stability under 37 °C storage: Take 20 bottles, and samples were taken at 0, 7, 10, and 15 days after placing at 37 °C to measure the virus content, 3 bottles each time. The measurement results are shown in Table 8, unit: TCID 50 / head dose.

[0084] Table 8 Results of the stability of each group of vaccines under 37 °C storage

[0085] Group 0d 7d 10d 15d Heat-resistant protective agent live vaccine 1 <![CDATA[10 6.1 > <![CDATA[10 6.1 > <![CDATA[10 5.7 > <![CDATA[10 5.5 > Heat-resistant protective agent live vaccine 2 <![CDATA[10 6.1 > <![CDATA[10 6.1 > <![CDATA[10 5.7 > <![CDATA[10 5.5 > Heat-resistant protective agent live vaccine 3 <![CDATA[10 6.1 > <![CDATA[10 5.7 > <![CDATA[10 5.3 > <![CDATA[10 4.9 > Control vaccine <![CDATA[10 5.9 > <![CDATA[10 5.1 > <![CDATA[10 4.3 > <![CDATA[10 3.3 >

[0086] As can be seen from Table 8, for the vaccines prepared using heat-resistant protectant 1 or 2, when stored at 37°C (room temperature) for 7 days, the virus content in the vaccines remained unchanged. When stored for 15 days, there was still a relatively high virus content in the vaccines, and the degree of decrease was not obvious. In contrast, for the vaccine prepared using heat-resistant protectant 3, a trend of decreasing virus content was already observed on the 7th day of storage; for the control vaccine, a rapid decrease in virus content was already observed on the 7th day of storage, and when stored for 15 days, the virus content was only 10 3.3 . This result indicates that through the reasonable combination of various components, the heat-resistant protectant of the present invention can significantly extend the shelf life of the vaccine, and improve the stability and effectiveness of the vaccine in an abnormal storage environment at 37°C, ensuring the stability of the vaccine when temperature fluctuations occur during transportation and storage.

[0087] (2) Immunization effect

[0088] Select 100 14-day-old maternally antibody-positive pigs, randomly divide them into 5 groups with 20 pigs per group. One group serves as the blank control, and the other 4 groups are respectively intramuscularly injected with the test vaccine and the control vaccine in the neck, 1 dose per pig. Blood is collected at 0 month, 1 month, and 2 months after immunization, serum is separated, and the antibody titer is detected using the IDEXX Porcine Reproductive and Respiratory Syndrome Virus Antibody Detection Kit (S / P≥0.4 is antibody positive). The antibody levels of pigs after vaccine immunization are shown in Table 9.

[0089] Table 9 Immunization effects of vaccines in each group

[0090]

[0091]

[0092] As can be seen from Table 9, maternally derived antibodies were present in the animals in the blank control group, and the antibody decline rate was fast, and the antibodies were all negative 1 month after the first immunization. For the animals immunized with the vaccines prepared using heat-resistant protectant 1 or 2, the antibodies continued to rise after immunization, and reached about 2.0 2 months after immunization, which was higher than the vaccines prepared using heat-resistant protectant 3 and the control vaccine. This result indicates that through the reasonable combination of various components, the heat-resistant protectant of the present invention can significantly improve the immunization effect of the vaccine.

[0093] Example 3

[0094] Solution A: Dissolve 20% sucrose, 5% polyvinylpyrrolidone, and 2% sorbitol in injection water according to mass percentage, make up the volume to 1000 mL with injection water, and sterilize at 116°C under high pressure for 30 min;

[0095] Solution B: Dissolve 2% glutamine, 10% sodium glutamate, 2% glycine, and 0.5% saponin by mass percentage in injection water, make up the volume to 1000 mL with injection water, and sterilize by filtration through a 0.22 μm microporous membrane;

[0096] Mix Solution A and Solution B in a volume ratio of 1:1 to obtain a heat-resistant protective agent.

[0097] Example 4

[0098] Solution A: Dissolve 20% sucrose, 10% polyvinylpyrrolidone, and 2% sorbitol by mass percentage in injection water, make up the volume to 1000 mL with injection water, and autoclave at 116 °C for 30 min;

[0099] Solution B: Dissolve 2% glutamine, 5% sodium glutamate, 2% glycine, and 0.5% saponin by mass percentage in injection water, make up the volume to 1000 mL with injection water, and sterilize by filtration through a 0.22 μm microporous membrane;

[0100] Mix Solution A and Solution B in a volume ratio of 1:1 to obtain a heat-resistant protective agent.

[0101] Example 5

[0102] Solution A: Dissolve 20% sucrose, 10% polyvinylpyrrolidone, and 2% sorbitol by mass percentage in injection water, make up the volume to 1000 mL with injection water, and autoclave at 116 °C for 30 min;

[0103] Solution B: Dissolve 2% glutamine, 10% sodium glutamate, 2% glycine, and 0.1% saponin by mass percentage in injection water, make up the volume to 1000 mL with injection water, and sterilize by filtration through a 0.22 μm microporous membrane;

[0104] Mix Solution A and Solution B in a volume ratio of 1:1 to obtain a heat-resistant protective agent.

[0105] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A heat-resistant protective agent, characterized in that, It includes liquid A and liquid B; The liquid A includes raw materials with the following mass percentages: 15%-20% of sucrose, 5%-10% of polyvinylpyrrolidone, and 1%-2% of sorbitol, with the balance being water; The liquid B includes raw materials with the following mass percentages: 1%-2% of glutamine, 5%-10% of sodium glutamate, 1%-2% of glycine, and 0.1%-0.5% of saponin, with the balance being water; The volume ratio of the liquid A to the liquid B is (0.5-2):(0.5-2).

2. The heat-resistant protective agent according to claim 1, characterized in that, The volume ratio of the liquid A to the liquid B is 1:

1.

3. A method for preparing the heat-resistant protective agent according to claim 1 or 2, characterized in that, It includes the following steps: (1) Dissolve sucrose, polyvinylpyrrolidone, and sorbitol in water to obtain liquid A; (2) Dissolve glutamine, sodium glutamate, glycine, and saponin in water to obtain liquid B; (3) Mix the liquid A and the liquid B to obtain the heat-resistant protectant.

4. Use of the heat-resistant protectant according to claim 1 or 2 in the preparation of a virus vaccine.

5. The application according to claim 4, wherein The virus vaccine includes a classical swine fever virus vaccine, a porcine pseudorabies virus vaccine, or a porcine reproductive and respiratory syndrome virus vaccine.

6. Use of the heat-resistant protectant according to claim 1 or 2 in extending the shelf life of a vaccine.

7. Use of the heat-resistant protectant according to claim 1 or 2 in the preparation of a preparation capable of improving the immune effect of a vaccine.

8. A method for preparing a viral vaccine, characterized in that, After mixing the heat-resistant protectant according to claim 1 or 2 with the virus liquid in a volume ratio of 1:1, freeze-dry to obtain the virus vaccine.

9. The preparation method according to claim 8, wherein The virus includes a classical swine fever virus, a porcine pseudorabies virus, or a porcine reproductive and respiratory syndrome virus.

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