Diluent for porcine pseudorabies live vaccine

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a diluent for a porcine pseudorabies live vaccine. The diluent for the porcine pseudorabies live vaccine is prepared by stirring and dissolving sodium dihydrogen phosphate, dipotassium phosphate, calcium chloride, sodium chloride, a traditional Chinese medicine extract, a stabilizer and water, and performing high-pressure steam sterilization, the traditional Chinese medicine extract is prepared by taking poria cocos and honeysuckle as traditional Chinese medicine raw materials and further reacting with chitosan and tannic acid, and the stabilizer is prepared by mixing gelatin, cyclodextrin and bovine serum albumin according to a mass ratio. When the diluent prepared by the method is used for diluting the porcine pseudorabies live vaccine, the stability and the activity of vaccine antigens can be effectively improved, the activity and the function of immune cells are enhanced, so that the immune effect of the vaccine is enhanced, and the prepared diluent for the porcine pseudorabies live vaccine solves the problem that a traditional live vaccine diluent does not have an immune enhancement effect.

Description

Technical Field

[0001] The present application relates to the field of biological medicine technology, and more specifically, it relates to a diluent for live porcine pseudorabies vaccine. Background Art

[0002] Porcine pseudorabies is an acute and highly contagious disease of pigs caused by porcine pseudorabies virus (PRV). This disease is one of the major infectious diseases that endanger the global pig industry. This disease is usually treated by prevention, preventing virus input, injecting vaccines and adopting PR purification measures. Among them, the vaccines for injection are: inactivated porcine pseudorabies vaccine, live pseudorabies vaccine, live porcine pseudorabies vaccine.

[0003] The live porcine pseudorabies vaccine is made from an artificially attenuated strain of porcine pseudorabies virus. After vaccination, the virus can proliferate to a limited extent in pigs, stimulating the body's immune system to produce an immune response, including the production of specific antibodies and the activation of cellular immunity, so that pigs can obtain immunity to porcine pseudorabies virus. When pigs are exposed to the virus again, the immune system can quickly recognize and eliminate the virus, protecting pigs from getting sick. When using the live vaccine, it needs to be diluted with a diluent. Vaccination is the main measure to prevent porcine pseudorabies, but there are many factors causing immune fluctuations during the immunization process. Using diluents with different components also plays an important role in the immune effect. Traditional live vaccine diluents are mostly phosphate buffer solutions or physiological saline, without immune enhancement effects. Therefore, the present application proposes a diluent for live porcine pseudorabies vaccine, which can significantly enhance the stability and immune effect of live porcine pseudorabies vaccine. Summary of the Invention

[0004] In order to solve the problem that traditional live vaccine diluents in the prior art do not have immune enhancement effects, the present application provides a diluent for live porcine pseudorabies vaccine.

[0005] In the first aspect, the present application provides a preparation method of a diluent for live porcine pseudorabies vaccine, which specifically includes the following steps:

[0006] Stir and dissolve sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water, and sterilize by high-pressure steam to obtain the diluent for live porcine pseudorabies vaccine.

[0007] Preferably, the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water is 7-9:13-15:3-5:8-10:10-12:3-5:1000.

[0008] Preferably, the Chinese herbal medicine extract is prepared by the following method:

[0009] S1. Stir and mix Poria cocos and honeysuckle flowers, crush and sieve them, add an ethanol solution, stir and mix, and then add cellulase for enzymatic hydrolysis to obtain a functional solution;

[0010] S2. Stir and mix chitosan with a dilute acetic acid solution for 1 - 2 h to form a chitosan solution;

[0011] S3. Stir and mix the functional solution with the chitosan solution, then add tannic acid, control the temperature at 30 - 35 °C, continue to stir and mix, and spray dry to obtain a traditional Chinese medicine extract.

[0012] Preferably, in the step S1, the mass ratio of Poria cocos, honeysuckle flowers, ethanol solution, and cellulase is 11 - 15:6 - 8:50:0.03 - 0.05.

[0013] Preferably, the mass concentration of the ethanol solution in the step S1 is 60% - 70%.

[0014] Preferably, the enzymatic hydrolysis temperature in the step S1 is 40 - 50 °C, and the time is 1 - 3 h.

[0015] Preferably, in the step S2, the mass ratio of chitosan to the dilute acetic acid solution is 1 - 3:50.

[0016] Preferably, the mass concentration of the dilute acetic acid solution in the step S2 is 1% - 3%.

[0017] Preferably, in the step S3, the mass ratio of the functional solution, chitosan solution, and tannic acid is 10 - 20:5 - 10:1 - 3.

[0018] Preferably, the stabilizer is composed of gelatin, cyclodextrin, and bovine serum albumin in a mass ratio of 3 - 7:2 - 4:2.

[0019] In a second aspect, the present application provides a diluent for a live porcine pseudorabies vaccine, which is prepared by the above - described method.

[0020] In summary, the present application has the following beneficial effects:

[0021] 1. The present application adopts a reasonable raw material combination and preparation process. By adding a traditional Chinese medicine extract and a stabilizer, the diluent for the live porcine pseudorabies vaccine prepared has good stability and immune enhancement effects, can significantly improve the immune effect of the live porcine pseudorabies vaccine, effectively stimulate the immune response, and enhance the immune effect.

[0022] 2. The Chinese medicine extract in this application is prepared by extracting active ingredients from Poria cocos and Lonicera japonica, and further reacting with chitosan and tannic acid. Poria cocos contains pachyman, which can enhance the phagocytic ability of macrophages, activate the complement system, promote the transformation of lymphocytes, and regulate the production of immune cytokines. Components such as chlorogenic acid in Lonicera japonica can improve the phagocytic ability of white blood cells. The two can produce a synergistic effect, more comprehensively enhancing the activity and function of immune cells, as well as regulating the secretion and balance of immune factors, making the immune response of the body more precise and effective. Chitosan can form a uniform protective film on the surface of the Chinese medicine extract, improving the stability and efficacy persistence of the active ingredients of Chinese medicine. On the one hand, the addition of tannic acid can produce a cross-linking effect with chitosan to form a stable network structure, further improving the stability of the Chinese medicine extract. On the other hand, tannic acid can form a complex with the antigen protein in the vaccine, stabilizing the antigen structure, improving the immunogenicity of the vaccine, and activating antigen-presenting cells, thereby improving the immune effect of the vaccine.

[0023] 3. This application uses a compound of gelatin, cyclodextrin, and bovine serum albumin as a stabilizer. On the one hand, gelatin can form a colloidal solution to wrap the antigen, and bovine serum albumin can form a protective film on the surface of the antigen. The two work synergistically to reduce the mutual collision and aggregation of antigen molecules, improving the physical stability of the antigen. On the other hand, bovine serum albumin contains amino acid residues with antioxidant effects, and certain components in gelatin also have certain antioxidant properties. Cyclodextrin can reduce the contact between substances that cause oxidation reactions and the antigen. The compound of the three can effectively improve the antioxidant ability of the antigen in the diluent and enhance its chemical stability. The compound of gelatin, cyclodextrin, and bovine serum albumin can synergistically improve the stability of the antigen, thereby improving the immune effect of the vaccine. Detailed implementation mode

[0024] The following further elaborates on this application in conjunction with examples.

[0025] Examples 1-5 provide a diluent for live pseudorabies vaccine for pigs.

[0026] In the examples and comparative examples of this application, the Poria cocos (specification: diced Poria cocos, purchased from Yunnan Guangkuntang Agricultural Technology Co., Ltd.), honeysuckle (brand: Jiang Zheng, purchased from Henan Jiangzheng Chinese Herbal Medicine Co., Ltd.), cellulase (CAS: 9012-54-8, purchased from Shaanxi Runfeng Biotechnology Co., Ltd.), chitosan (CAS: 9012-76-4, purchased from Shandong Haiyihua Biotechnology Co., Ltd.), tannic acid (CAS: 1401-55-4, purchased from Zhangjiajie Jiurui Biotechnology Co., Ltd.), gelatin (CAS: 9000-70-8, purchased from Nanjing Songguan Biotechnology Co., Ltd.), cyclodextrin (CAS: 68168-23-0, purchased from Zhejiang Yicun Biotechnology Co., Ltd.), bovine serum albumin (CAS: 9048-46-8, purchased from Hubei Chang'ao Pharmaceutical Co., Ltd.) were used.

[0027] Example 1

[0028] A preparation method of a diluent for porcine pseudorabies live vaccine specifically includes the following steps:

[0029] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water being 7:13:3:8:10:3:1000, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer (composed of gelatin, cyclodextrin and bovine serum albumin mixed in a mass ratio of 3:2:2) and water were stirred at 600 r / min for 1 h, and then sterilized by high-pressure steam. The sterilization temperature was 100 °C and the sterilization time was 10 min to obtain the diluent for porcine pseudorabies live vaccine.

[0030] The Chinese herbal medicine extract was prepared by the following method:

[0031] S1. According to the mass ratio of Poria cocos, honeysuckle, ethanol solution and cellulase being 11:6:50:0.03, Poria cocos and honeysuckle were stirred and mixed at 600 r / min for 30 min, then jointly pulverized and passed through a 60-mesh sieve. A 60% ethanol solution was added, and the mixture was stirred and mixed at 600 r / min for 40 min. Then cellulase was added for enzymatic hydrolysis. The enzymatic hydrolysis temperature was 40 °C and the time was 1 h to obtain a functional solution;

[0032] S2. According to the mass ratio of chitosan to dilute acetic acid solution being 1:50, chitosan and a 1% dilute acetic acid solution were stirred and mixed at 400 r / min for 1 h to form a chitosan solution;

[0033] S3. Mix the functional solution and chitosan solution at a mass ratio of 10:5:1 by stirring at 500 r / min for 30 min. Then add tannic acid, control the temperature at 30 °C, continue to stir and mix at 400 r / min for 1 h, and perform spray drying with an inlet air temperature of 140 °C, a feeding peristaltic rotation speed of 6 s / time, and a spray pressure of 0.1 Mpa to obtain the traditional Chinese medicine extract.

[0034] Example 2

[0035] A preparation method of a diluent for live porcine pseudorabies vaccine specifically includes the following steps:

[0036] Mix sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer (composed of gelatin, cyclodextrin and bovine serum albumin mixed at a mass ratio of 4:2.5:2) and water at a mass ratio of 7.5:13.5:3.5:8.5:10.5:3.5:1000 by stirring at 600 r / min for 1.2 h, and perform high-pressure steam sterilization at a sterilization temperature of 105 °C and a sterilization time of 12 min to obtain the diluent for live porcine pseudorabies vaccine.

[0037] The traditional Chinese medicine extract is prepared by the following method:

[0038] S1. Mix Poria cocos and honeysuckle at a mass ratio of 12:6.5:50:0.035 by stirring at 600 r / min for 30 min, jointly pulverize and pass through a 65-mesh sieve, add an ethanol solution with a mass concentration of 62%, stir and mix at 600 r / min for 40 min, and then add cellulase for enzymatic hydrolysis at an enzymatic hydrolysis temperature of 42 °C and a time of 1.5 h to obtain the functional solution;

[0039] S2. Mix chitosan and dilute acetic acid solution at a mass ratio of 1.5:50 by stirring at 400 r / min for 1.2 h to form a chitosan solution;

[0040] S3. Mix the functional solution and chitosan solution at a mass ratio of 12:6:1.5 by stirring at 500 r / min for 35 min. Then add tannic acid, control the temperature at 31 °C, continue to stir and mix at 400 r / min for 1 h, and perform spray drying with an inlet air temperature of 145 °C, a feeding peristaltic rotation speed of 6 s / time, and a spray pressure of 0.1 Mpa to obtain the traditional Chinese medicine extract.

[0041] Example 3

[0042] A preparation method of a diluent for live porcine pseudorabies vaccine specifically includes the following steps:

[0043] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer and water being 8:14:4:9:11:4:1000, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer (composed of gelatin, cyclodextrin and bovine serum albumin mixed according to the mass ratio of 5:3:2) and water are stirred at 600 r / min for 1.5 h, and then sterilized by high-pressure steam. The sterilization temperature is 110 °C and the sterilization time is 15 min to obtain the diluent for live porcine pseudorabies vaccine.

[0044] The traditional Chinese medicine extract is prepared by the following method:

[0045] S1. According to the mass ratio of Poria cocos, honeysuckle, ethanol solution and cellulase being 13:7:50:0.04, Poria cocos and honeysuckle are stirred and mixed at 600 r / min for 30 min, then co-ground through a 70-mesh sieve, added with an ethanol solution with a mass concentration of 65%, stirred and mixed at 600 r / min for 40 min, and then cellulase is added for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45 °C and the time is 2 h to obtain a functional solution;

[0046] S2. According to the mass ratio of chitosan to dilute acetic acid solution being 2:50, chitosan and a dilute acetic acid solution with a mass concentration of 2% are stirred and mixed at 400 r / min for 1.5 h to form a chitosan solution;

[0047] S3. According to the mass ratio of the functional solution, chitosan solution and tannic acid being 15:7:2, the functional solution and the chitosan solution are stirred and mixed at 500 r / min for 40 min, then tannic acid is added, the temperature is controlled at 32 °C, and stirring and mixing continue at 400 r / min for 1 h, followed by spray drying. The inlet air temperature is 150 °C, the feeding peristaltic rotation speed is 6 s / time, and the spray pressure is 0.1 Mpa to obtain the traditional Chinese medicine extract.

[0048] Example 4

[0049] A preparation method of a diluent for live porcine pseudorabies vaccine specifically includes the following steps:

[0050] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer and water being 8.5:14.5:4.5:9.5:11.5:4.5:1000, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer (composed of gelatin, cyclodextrin and bovine serum albumin mixed according to the mass ratio of 6:3.5:2) and water are stirred at 600 r / min for 1.8 h, and then sterilized by high-pressure steam. The sterilization temperature is 115 °C and the sterilization time is 18 min to obtain the diluent for live porcine pseudorabies vaccine.

[0051] The traditional Chinese medicine extract is prepared by the following method:

[0052] S1. According to the mass ratio of Poria cocos, honeysuckle, ethanol solution and cellulase being 14:7.5:50:0.045, mix Poria cocos and honeysuckle by stirring at 600 r / min for 30 min, co-crush them through a 75-mesh sieve, add an ethanol solution with a mass concentration of 68%, stir and mix at 600 r / min for 40 min, then add cellulase for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 48 °C and the time is 2.5 h to obtain a functional solution;

[0053] S2. According to the mass ratio of chitosan to dilute acetic acid solution being 2.5:50, mix chitosan and a dilute acetic acid solution with a mass concentration of 2.5% by stirring at 400 r / min for 1.7 h to form a chitosan solution;

[0054] S3. According to the mass ratio of the functional solution, chitosan solution and tannic acid being 18:9:2.5, mix the functional solution and chitosan solution by stirring at 500 r / min for 45 min, then add tannic acid, control the temperature at 34 °C, continue to stir and mix at 400 r / min for 1 h, and perform spray drying. The inlet air temperature is 155 °C, the feeding peristaltic rotation speed is 6 s / time, and the spray pressure is 0.1 Mpa to obtain the traditional Chinese medicine extract.

[0055] Example 5

[0056] A preparation method of a diluent for porcine pseudorabies live vaccine specifically includes the following steps:

[0057] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer and water being 9:15:5:10:12:5:1000, mix sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer (composed of gelatin, cyclodextrin and bovine serum albumin mixed according to the mass ratio of 7:4:2) and water by stirring at 600 r / min for 2 h, and perform high-pressure steam sterilization. The sterilization temperature is 120 °C and the sterilization time is 20 min to obtain the diluent for porcine pseudorabies live vaccine.

[0058] The traditional Chinese medicine extract is prepared by the following method:

[0059] S1. According to the mass ratio of Poria cocos, honeysuckle, ethanol solution and cellulase being 15:8:50:0.05, mix Poria cocos and honeysuckle by stirring at 600 r / min for 30 min, co-crush them through an 80-mesh sieve, add an ethanol solution with a mass concentration of 70%, stir and mix at 600 r / min for 40 min, then add cellulase for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 50 °C and the time is 3 h to obtain a functional solution;

[0060] S2. Mix chitosan and a 3% w / v dilute acetic acid solution at a mass ratio of 3:50 with stirring at 400 r / min for 2 h to form a chitosan solution;

[0061] S3. Mix the functional solution and the chitosan solution at a mass ratio of 20:10:3 with stirring at 500 r / min for 50 min, then add tannic acid, control the temperature at 35 °C, continue stirring and mixing at 400 r / min for 1 h, and perform spray drying with an inlet air temperature of 160 °C, a feeding peristaltic rotation speed of 6 s / time, and a spray pressure of 0.1 Mpa to obtain the Chinese medicine extract.

[0062] Comparative Example 1

[0063] Comparative Example 1 is the same as Example 1, except that the Chinese medicine raw material is replaced with Poria cocos instead of Lonicera japonica, as shown below:

[0064] The Chinese medicine extract is prepared by the following method:

[0065] S1. Crush Poria cocos through a 60-mesh sieve, add a 60% w / v ethanol solution, mix with stirring at 600 r / min for 40 min, then add cellulase for enzymatic hydrolysis at an enzymatic hydrolysis temperature of 40 °C for 1 h to obtain a functional solution, with the mass ratio of Poria cocos, ethanol solution, and cellulase being 17:50:0.03;

[0066] S2. Mix chitosan and a 1% w / v dilute acetic acid solution at a mass ratio of 1:50 with stirring at 400 r / min for 1 h to form a chitosan solution;

[0067] S3. Mix the functional solution and the chitosan solution at a mass ratio of 10:5:1 with stirring at 500 r / min for 30 min, then add tannic acid, control the temperature at 30 °C, continue stirring and mixing at 400 r / min for 1 h, and perform spray drying with an inlet air temperature of 140 °C, a feeding peristaltic rotation speed of 6 s / time, and a spray pressure of 0.1 Mpa to obtain the Chinese medicine extract.

[0068] Comparative Example 2

[0069] Comparative Example 2 is the same as Example 1, except that the Chinese medicine raw material is replaced with Lonicera japonica instead of Poria cocos, as shown below:

[0070] The Chinese medicine extract is prepared by the following method:

[0071] S1. According to the mass ratio of honeysuckle, ethanol solution and cellulase being 17:50:0.03, crush the honeysuckle and sieve it through a 60-mesh sieve, add an ethanol solution with a mass concentration of 60%, stir and mix at 600 r / min for 40 min, then add cellulase for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40 °C and the time is 1 h to obtain a functional solution;

[0072] S2. According to the mass ratio of chitosan to dilute acetic acid solution being 1:50, stir and mix chitosan with a dilute acetic acid solution with a mass concentration of 1% at 400 r / min for 1 h to form a chitosan solution;

[0073] S3. According to the mass ratio of the functional solution, chitosan solution and tannic acid being 10:5:1, stir and mix the functional solution and chitosan solution at 500 r / min for 30 min, then add tannic acid, control the temperature at 30 °C, continue to stir and mix at 400 r / min for 1 h, and perform spray drying. The inlet air temperature is 140 °C, the feeding peristaltic rotation speed is 6 s / time, and the spray pressure is 0.1 Mpa to obtain a traditional Chinese medicine extract.

[0074] Comparative Example 3

[0075] Comparative Example 3 is the same as Example 1, except that tannic acid is not added during the preparation of the traditional Chinese medicine extract, as shown below:

[0076] The traditional Chinese medicine extract is prepared by the following method:

[0077] S1. According to the mass ratio of poria cocos, honeysuckle, ethanol solution and cellulase being 11:6:50:0.03, stir and mix poria cocos and honeysuckle at 600 r / min for 30 min, jointly crush and sieve through a 60-mesh sieve, add an ethanol solution with a mass concentration of 60%, stir and mix at 600 r / min for 40 min, then add cellulase for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40 °C and the time is 1 h to obtain a functional solution;

[0078] S2. According to the mass ratio of chitosan to dilute acetic acid solution being 1:50, stir and mix chitosan with a dilute acetic acid solution with a mass concentration of 1% at 400 r / min for 1 h to form a chitosan solution;

[0079] S3. According to the mass ratio of the functional solution, chitosan solution and tannic acid being 10:5:1, stir and mix the functional solution and chitosan solution at 500 r / min for 30 min, and perform spray drying. The inlet air temperature is 140 °C, the feeding peristaltic rotation speed is 6 s / time, and the spray pressure is 0.1 Mpa to obtain a traditional Chinese medicine extract.

[0080] Comparative Example 4

[0081] Comparative Example 4 is the same as Example 1, except that gelatin is not added to the stabilizer, as shown below:

[0082] A preparation method of a diluent for live pseudorabies vaccine for pigs, specifically including the following steps:

[0083] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water being 7:13:3:8:10:3:1000, mix sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer (composed of cyclodextrin and bovine serum albumin mixed in a mass ratio of 2:2) and water and stir at 600 r / min for 1 h, then perform high-pressure steam sterilization, with the sterilization temperature being 100 °C and the sterilization time being 10 min, to obtain the diluent for live pseudorabies vaccine for pigs.

[0084] Comparative Example 5

[0085] Comparative Example 5 is the same as Example 1, with the only difference being that cyclodextrin is not added to the stabilizer, specifically as follows:

[0086] A preparation method of a diluent for live pseudorabies vaccine for pigs, specifically including the following steps:

[0087] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water being 7:13:3:8:10:3:1000, mix sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer (composed of gelatin and bovine serum albumin mixed in a mass ratio of 3:2) and water and stir at 600 r / min for 1 h, then perform high-pressure steam sterilization, with the sterilization temperature being 100 °C and the sterilization time being 10 min, to obtain the diluent for live pseudorabies vaccine for pigs.

[0088] Comparative Example 6

[0089] Comparative Example 6 is the same as Example 1, with the only difference being that bovine serum albumin is not added to the stabilizer, specifically as follows:

[0090] A preparation method of a diluent for live pseudorabies vaccine for pigs, specifically including the following steps:

[0091] According to the mass ratio of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer and water being 7:13:3:8:10:3:1000, mix sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal medicine extract, stabilizer (composed of gelatin and cyclodextrin mixed in a mass ratio of 3:2) and water and stir at 600 r / min for 1 h, then perform high-pressure steam sterilization, with the sterilization temperature being 100 °C and the sterilization time being 10 min, to obtain the diluent for live pseudorabies vaccine for pigs.

[0092] Performance detection

[0093] Immunization test of live pseudorabies vaccine for pigs diluted with the diluent for live pseudorabies vaccine for pigs.

[0094] The dilution solutions prepared in Examples 1-5 and Comparative Examples 1-6 and PBS buffer were respectively used to dilute the same batch of live porcine pseudorabies vaccine. When diluting, 10 mL of the dilution solution was used to dilute one vial of freeze-dried vaccine for immunizing piglets.

[0095] 120 healthy and susceptible piglets aged 14-21 days (negative for PRV antigen and antibody) were selected and randomly divided into 12 groups with 10 piglets in each group. The vaccines diluted with the dilution solutions prepared in Examples 1-5, Comparative Examples 1-6 and PBS buffer were orally administered respectively, and 2 mL was administered to each piglet.

[0096] 1. Antibody determination of immunized piglets

[0097] Blood samples were collected on the 14th day, 21st day and 28th day after oral immunization of piglets. After separating the serum, antibody determination was carried out to detect PRV antibodies in the serum. The results are shown in Table 1.

[0098] 2. Challenge protection test of immunized piglets

[0099] On the 28th day after oral immunization of piglets, PRV strong virus was used to challenge the experimental piglets, and the challenge dose was 10 4.0 MID. After PRV challenge, the piglets were continuously observed for 7 days, and anal swabs of all piglets were collected on the 1st day, 4th day and 7th day after challenge for fluorescence quantitative PCR detection of the virus excretion amount of PRV. The results are shown in Table 2.

[0100] Table 1

[0101]

[0102]

[0103] Table 2

[0104]

[0105] As can be seen from the data in Table 1 and Table 2, when piglets were orally administered the live porcine pseudorabies vaccine diluted with the dilution solutions prepared in Examples 1-5 and Comparative Examples 1-6, antibody positivity appeared on the 14th day, and the positive rate of PRV antibodies in the groups of Examples 1-5 was higher than that in the groups of Comparative Examples 1-6; the incidence rate of the group of live vaccine diluted with the dilution solution prepared in Examples 1-5 was the lowest, and no virus was detected in the anal swabs on the 4th day after challenge. Different degrees of illness occurred in the piglets in the groups of Comparative Examples 1-6, indicating that the raw materials and preparation process of the traditional Chinese medicine extract and the composition of the stabilizer will all affect the stability and immune effect of the dilution solution. The dilution solution for live porcine pseudorabies vaccine prepared in this application has an immune enhancing effect and can significantly enhance the immune effect of the vaccine.

[0106] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment that do not contribute creatively as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for preparing a diluent for porcine pseudorabies live vaccine, characterized in that: The specific steps include the following: Sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, Chinese herbal extract, stabilizer and water are stirred and dissolved, and sterilized by high-pressure steam to obtain a diluent for live pseudorabies vaccine of pigs.

2. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 1, characterized in that: The mass ratio of the sodium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride, traditional Chinese medicine extract, stabilizer and water is 7-9:13-15:3-5:8-10:10-12:3-5:1000.

3. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 1, characterized in that: The Chinese medicine extract is prepared by the following method: S1. Stir and mix Poria cocos and honeysuckle, crush and sieve, add ethanol solution, stir and mix, and then add cellulase for enzymolysis to obtain a functional solution; S2, stirring and mixing chitosan and dilute acetic acid solution for 1-2 hours to form a chitosan solution; S3. Stir and mix the functional solution and chitosan solution, then add tannic acid, control the temperature to 30-35° C., continue stirring and mixing, and spray-dry to obtain a Chinese medicine extract.

4. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 3, characterized in that: In the step S1, the mass ratio of Poria cocos, honeysuckle, ethanol solution and cellulase is 11-15:6-8:50:0.03-0.

05.

5. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 3, characterized in that: The mass concentration of the ethanol solution in step S1 is 60%-70%.

6. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 3, characterized in that: In step S1, the enzymatic hydrolysis temperature is 40-50° C. and the time is 1-3 h.

7. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 3, characterized in that: In step S2, the mass ratio of chitosan to dilute acetic acid solution is 1-3:

50.

8. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 3, characterized in that: In the step S3, the mass ratio of the functional solution, the chitosan solution and the tannic acid is 10-20:5-10:1-3.

9. The method for preparing a diluent for porcine pseudorabies live vaccine according to claim 1, characterized in that: The stabilizer is composed of gelatin, cyclodextrin and bovine serum albumin mixed in a mass ratio of 3-7:2-4:

2.

10. A diluent for porcine pseudorabies live vaccine, characterized in that: The vaccine is prepared by the method for preparing a diluent for live pseudorabies vaccine of pigs as described in any one of claims 1 to 9.

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