Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent and preparation method thereof

Abstract

The invention discloses a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent and a preparation method thereof, and belongs to the technical field of biological products. The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises component A and component B; component A comprises the following raw materials: sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan, lactose and water; component B comprises the following raw materials: coptis root extract, astragalus extract, phellodendron extract, gardenia extract and water. The invention also discloses a preparation method of the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent. The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent prepared by the invention solves the problem that the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine is inactivated or degraded after gastric digestion, and immune protection cannot be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and specifically relates to a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent and a preparation method thereof. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDV), and porcine rotavirus disease is a contagious disease caused by porcine rotavirus (RV). The main harm of both diseases is that they cause diarrhea in newborn piglets, which leads to dehydration and death of piglets. After the disease occurs, the piglets become dead pigs, which greatly reduces the feed-to-meat ratio, thus causing huge economic losses to farmers. At present, these two diseases often present as mixed infections, causing higher morbidity and mortality rates in farms, and bring huge direct and indirect economic losses to farms every year.

[0003] Currently, there are no specific treatment drugs for PEDV and RV. The most economical and effective method is to use vaccines for immunization and prevention. The onset of disease in piglets is mostly within half a month after birth. The immune system of piglets within half a month after birth is not yet mature. At this time, the effect of immunizing piglets against PEDV and RV is minimal. Therefore, piglets need to obtain maternal antibodies through colostrum to obtain passive immune protection against PEDV and RV. The most important immune factors in colostrum are IgG and IgA. Among them, IgA mainly protects the intestines and other mucosal tissues of piglets from invasion by pathogenic microorganisms, and is the most critical protective factor in the prevention and control of PEDV and RV. The gastrointestinal immunity of sows is related to the protection of newborn piglets. This is the intestinal-mammary-sIgA axis system. The essence of this axis system is that pathogenic microorganisms infect intestinal epithelial cells, resulting in the production of lymphoplasmablasts capable of synthesizing sIgA in the mesenteric lymph nodes. These lymphoplasmablasts can return to the intestinal lamina propria or transfer to the mammary gland to exert their effects. The lymphocytes in the basal layer of the mammary epithelium of immunized sows can continuously secrete sIgA and are not easily degraded by enzymes in milk and the intestines, thus providing lasting immune protection to piglets.

[0004] Therefore, when traditional inactivated vaccines or live vaccines are used for injection immunization for the prevention and control of PEDV and RV, it is impossible to provide piglets with secretory sIgA with protective effects. Clinical application results also show that oral immunization with moderately virulent live viruses can effectively prevent and control PEDV and RV. However, due to the low pH value in the stomach, active viruses (immunogens) are easily inactivated or degraded after being ingested by sows, making it impossible for them to reach the small intestine and exert effective immune protection. Summary of the invention

[0005] The purpose of the present invention is to provide a porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine diluent and a preparation method thereof. The porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine diluent solves the problem that the oral porcine epidemic diarrhea and porcine rotavirus bivalent live vaccine is inactivated or degraded after gastric digestion, and the immune protection effect cannot be achieved.

[0006] The technical solution adopted to achieve the above purpose is to provide a porcine epidemic diarrhea and porcine rotavirus two-in-one oral live vaccine diluent, including component A and component B; the mass ratio of component A to component B is 1.5-2.5:1;

[0007] Component A includes the following raw materials by weight: 7.5-8.5 parts of sodium chloride, 0.1-0.3 parts of potassium chloride, 1.3-1.5 parts of sodium dihydrogen phosphate, 0.2-0.3 parts of potassium dihydrogen phosphate, 1-3 parts of sodium alginate, 1-3 parts of chitosan, 0.5-2 parts of lactose and 900-1100 parts of water;

[0008] Component B comprises the following raw materials in parts by weight: 30-90 parts of coptis root extract, 20-60 parts of astragalus root extract, 20-60 parts of phellodendron amurense extract, 30-90 parts of gardenia jasminoides extract and 900-1100 parts of water.

[0009] The beneficial effect of the technical solution of the present invention is that the sodium alginate and chitosan in the component A of the diluent of the present invention can form a stable embedding structure together, and lactose is used as a stabilizer to enhance the stability of the embedding structure. Sodium alginate and chitosan have good biocompatibility, biodegradability and biological activity, wherein chitosan also has certain gel properties and adsorption properties; when the porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine is diluted with the diluent of the present invention, since porcine epidemic diarrhea virus and porcine rotavirus are negatively charged, the chitosan molecules with positive charges can effectively capture the vaccine antigens through electrostatic interaction, and while fixing the antigen virus, they are wrapped in the embedding structure formed by sodium alginate and chitosan through cross-linking and other methods; when the oral vaccine enters the stomach, the alkaline chitosan can neutralize gastric acid, form a viscous colloidal liquid in the gastric juice, and attach to the stomach wall to form a protective film to reduce the erosion of gastric acid. At this time, the viscous colloidal liquid of sodium alginate and chitosan further forms a protective gel covering layer, which can work together with lactose that is not easily decomposed by gastric acid to slow down or prevent the degradation or inactivation of the antigen virus under gastric acid conditions. Sodium alginate and chitosan can also effectively ensure the activity of antigenic viruses and control their release rate in the small intestine, achieving continuous and stable immune stimulation; lactose also has the functions of regulating gastric acid, promoting gastrointestinal motility and digestive gland secretion, and the combined effect with sodium alginate and chitosan can further enhance the virus's resistance to gastric acid; in addition, lactose is slightly sweet, which can improve the palatability of oral vaccines, so as to better complete the oral immunization of animals;

[0010] At the same time, the coptis root extract, astragalus extract, phellodendron extract and gardenia extract in component B are co-encapsulated with the antigen virus in the embedding structure formed by component A to reach the small intestine. Coptis root and phellodendron extract can stimulate the proliferation and differentiation of immune cells. By mixing with astragalus extract, they can effectively enhance the phagocytic and killing ability of immune cells. Finally, supplemented with gardenia extract, it can regulate the conduction of immune cells, making the immune response more accurate and efficient. This comprehensive synergistic enhancement effect can significantly enhance the ability of immune cells to recognize and clear viral antigens, so as to further enhance the immune protection effect of the vaccine. At the same time, coptis root The extract, astragalus extract, phellodendron extract and gardenia extract work together to enhance the stability and solubility of vaccine antigens, increase the exposure of vaccines in the small intestine, and enhance the interaction between vaccines and immune cells to enhance the immune response of vaccines; in addition, coptis extract, astragalus extract, phellodendron extract and gardenia extract can also play an antioxidant and anti-inflammatory role, which can reduce the inflammatory response and tissue damage that may occur after oral vaccination, and protect immune cells from damage by reducing the release of inflammatory factors and the generation of oxidative stress, making the immune response more lasting and stable, ultimately playing a highly effective immune protection role.

[0011] Preferably, the mass ratio of component A to component B is 2:1.

[0012] Preferably, component A includes the following raw materials in parts by mass: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 1-2 parts of sodium alginate, 1-2 parts of chitosan, 0.5-1 part of lactose and 1000 parts of water.

[0013] More preferably, component A includes the following raw materials in parts by mass: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 2 parts of sodium alginate, 2 parts of chitosan, 1 part of lactose and 1000 parts of water.

[0014] Preferably, component B includes the following raw materials in parts by weight: 90 parts of Coptis chinensis extract, 60 parts of Astragalus membranaceus extract, 60 parts of Phellodendron amurense extract, 90 parts of Gardenia jasminoides extract and 1000 parts of water.

[0015] The present invention also provides a method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, comprising the following steps:

[0016] (1) Mixing the raw materials in component A and adjusting the pH to 6.0-7.0, and then sterilizing to obtain component A;

[0017] (2) mixing and sterilizing the raw materials in component B to obtain component B;

[0018] (3) Mix component A and component B according to the mass ratio to obtain.

[0019] Preferably, in step (1), the pH is adjusted to 6.0.

[0020] Preferably, the sterilization temperature in step (1) and step (2) is 110-120° C., and the sterilization time is 15-20 min.

[0021] More preferably, the sterilization temperature in step (1) and step (2) is both 116° C., and the sterilization time is both 15 min.

[0022] The present invention also provides a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine, comprising a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine stock solution and the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent.

[0023] More preferably, the volume ratio of the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine concentrate to the above-mentioned porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent is 1:5-25.

[0024] The present invention has the following beneficial effects:

[0025] (1) The sodium alginate, chitosan and lactose in the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent prepared by the present invention are all biodegradable substances with no toxic side effects and good safety, which can achieve the embedding of antigen viruses and effectively prevent the inactivation or degradation of porcine epidemic diarrhea virus and porcine rotavirus under the acidic conditions of the stomach, so that they can be released in a targeted manner in the small intestine, and at the same time have a sustained release effect to achieve continuous and stable immune stimulation; the coptis root extract, astragalus extract, phellodendron extract and gardenia extract added at the same time are all traditional Chinese medicine extracts, which can produce a synergistic effect to further enhance the immune effect of the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine.

[0026] (2) The preparation method of the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent of the present invention is simple and does not require complicated processing. At the same time, the materials are relatively cheap and easy to obtain, and are suitable for large-scale industrial production. DETAILED DESCRIPTION

[0027] The features and properties of the present invention are further described in detail below in conjunction with the examples. The extracts of Coptis chinensis, Astragalus membranaceus, Phellodendron amurense and Gardenia jasminoides are all granules, and are provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0028] Example 1

[0029] Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, including component A and component B; the mass ratio of component A to component B is 2:1;

[0030] Component A includes the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 2 parts of sodium alginate, 2 parts of chitosan, 1 part of lactose and 1000 parts of water for injection;

[0031] Component B includes the following raw materials in parts by weight: 90 parts of Coptis chinensis extract, 60 parts of Astragalus membranaceus extract, 60 parts of Phellodendron chinense extract, 90 parts of Gardenia jasminoides extract and 1000 parts of water for injection.

[0032] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0033] (1) Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose and dissolve them in 800 parts of water for injection, adjust the pH of the solution to 6.0 with dilute hydrochloric acid solution, add the remaining parts by weight of water for injection, mix and sterilize in a high pressure steam sterilizer at 116° C. for 15 minutes, and cool to obtain component A;

[0034] (2) Weigh the extract of Coptis chinensis, Astragalus membranaceus, Phellodendron amurense and Gardenia jasminoides, dissolve them in 1000 parts of water for injection, sterilize them in a high pressure steam sterilizer at 116° C. for 15 min, and obtain component B after cooling;

[0035] (3) Mix component A and component B in a mass ratio of 2:1 to obtain the product.

[0036] Example 2

[0037] Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, including component A and component B; the mass ratio of component A to component B is 2:1;

[0038] Component A includes the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 1 part of sodium alginate, 1 part of chitosan, 0.5 parts of lactose and 1000 parts of water for injection;

[0039] Component B includes the following raw materials in parts by weight: 30 parts of Coptis chinensis extract, 20 parts of Astragalus membranaceus extract, 20 parts of Phellodendron amurense extract, 30 parts of Gardenia jasminoides extract and 1000 parts of water for injection.

[0040] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0041] (1) Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose and dissolve them in 800 parts of water for injection, adjust the pH of the solution to 6.0 with dilute hydrochloric acid solution, add the remaining parts by weight of water for injection, mix and sterilize in a high pressure steam sterilizer at 116° C. for 15 minutes, and cool to obtain component A;

[0042] (2) Weigh the extract of Coptis chinensis, Astragalus membranaceus, Phellodendron amurense and Gardenia jasminoides, dissolve them in 1000 parts of water for injection, sterilize them in a high pressure steam sterilizer at 116° C. for 15 min, and obtain component B after cooling;

[0043] (3) Mix component A and component B in a mass ratio of 2:1 to obtain the product.

[0044] Example 3

[0045] Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, comprising component A and component B; the mass ratio of component A to component B is 2.5:1;

[0046] Component A includes the following raw materials in parts by weight: 8.5 parts of sodium chloride, 0.3 parts of potassium chloride, 1.5 parts of sodium dihydrogen phosphate, 0.3 parts of potassium dihydrogen phosphate, 3 parts of sodium alginate, 3 parts of chitosan, 2 parts of lactose and 1100 parts of water for injection;

[0047] Component B includes the following raw materials in parts by weight: 90 parts of Coptis chinensis extract, 60 parts of Astragalus membranaceus extract, 60 parts of Phellodendron amurense extract, 90 parts of Gardenia jasminoides extract and 1100 parts of water for injection.

[0048] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0049] (1) Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose and dissolve them in 800 parts of water for injection, adjust the pH of the solution to 6.5 with 1.0 M NaOH solution, add the remaining parts of water for injection, mix and sterilize in a high pressure steam sterilizer at 110° C. for 20 min, and cool to obtain component A;

[0050] (2) Weigh the extract of Coptis chinensis, Astragalus membranaceus, Phellodendron amurense and Gardenia jasminoides, dissolve them in 1100 parts of water for injection of component B, sterilize them in a high-pressure steam sterilizer at 110° C. for 20 min, and cool them to obtain component B;

[0051] (3) Mix component A and component B in a mass ratio of 2.5:1 to obtain the product.

[0052] Example 4

[0053] Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, comprising component A and component B; the mass ratio of component A to component B is 1.5:1;

[0054] Component A includes the following raw materials in parts by weight: 7.5 parts of sodium chloride, 0.1 parts of potassium chloride, 1.3 parts of sodium dihydrogen phosphate, 0.2 parts of potassium dihydrogen phosphate, 1 part of sodium alginate, 1 part of chitosan, 0.5 parts of lactose and 900 parts of water for injection;

[0055] Component B includes the following raw materials in parts by weight: 30 parts of Coptis chinensis extract, 20 parts of Astragalus membranaceus extract, 20 parts of Phellodendron amurense extract, 30 parts of Gardenia jasminoides extract and 900 parts of water for injection.

[0056] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0057] (1) Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose and dissolve them in 700 parts of water for injection, adjust the pH of the solution to 7.0 with 1.0 M NaOH solution, add the remaining parts of water for injection, mix and sterilize in a high pressure steam sterilizer at 120° C. for 15 min, and cool to obtain component A;

[0058] (2) Weigh the extract of Coptis chinensis, Astragalus membranaceus, Phellodendron amurense and Gardenia jasminoides, dissolve them in 900 parts of water for injection, sterilize them in a high-pressure steam sterilizer at 120° C. for 15 min, and cool them to obtain component B;

[0059] (3) Mix component A and component B in a mass ratio of 1.5:1 to obtain the product.

[0060] Comparative Example 1

[0061] The diluent of porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine includes the following raw materials in parts by mass: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 2 parts of sodium alginate, 2 parts of chitosan, 1 part of lactose and 1000 parts of water for injection.

[0062] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0063] Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose, dissolve in 800 parts of water for injection, adjust the pH of the solution to 6.0 with dilute hydrochloric acid solution, add the remaining parts by mass of water for injection, mix, place in a high-pressure steam sterilizer at 116°C for sterilization for 15 minutes, and obtain after cooling.

[0064] Comparative Example 2

[0065] The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following raw materials in parts by weight: 90 parts of coptis root extract, 60 parts of astragalus extract, 60 parts of phellodendron extract, 90 parts of gardenia extract and 1000 parts of water for injection.

[0066] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0067] Weigh the coptis root extract, astragalus root extract, phellodendron amurense extract and gardenia jasmine extract, dissolve them in 1000 parts of water for injection, sterilize them in a high-pressure steam sterilizer at 116° C. for 15 minutes, and obtain the product after cooling.

[0068] Comparative Example 3

[0069] Porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, including component A and component B; the mass ratio of component A to component B is 2:1;

[0070] Component A includes the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 2 parts of sodium alginate, 2 parts of chitosan, 1 part of lactose and 1000 parts of water for injection;

[0071] Component B includes the following raw materials in parts by weight: 90 parts of coptis root extract, 60 parts of astragalus extract and 1000 parts of water for injection.

[0072] The method for preparing a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent comprises the following steps:

[0073] (1) Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium alginate, chitosan and lactose and dissolve them in 800 parts of water for injection, adjust the pH of the solution to 6.0 with dilute hydrochloric acid solution, add the remaining parts by weight of water for injection, mix and sterilize in a high pressure steam sterilizer at 116° C. for 15 minutes, and cool to obtain component A;

[0074] (2) Weigh the coptis root extract and the astragalus root extract, dissolve them in 1000 parts of water for injection, sterilize them in a high pressure steam sterilizer at 116° C. for 15 min, and cool them to obtain component B;

[0075] (3) Mix component A and component B in a mass ratio of 2:1 to obtain the product.

[0076] Experimental example: Evaluation of immune effect

[0077] 1. Preparation of Porcine Epidemic Diarrhea and Porcine Rotavirus Combined Oral Live Vaccine

[0078] 1.1 Preparation of porcine epidemic diarrhea virus (PEDV) antigen solution

[0079] Vero cells were cultured using a bioreactor and microcarrier suspension culture technology. After the Vero cells grew a full monolayer, the cells were washed with PBS and replaced with a maintenance solution containing 0.01 MOI PEDV seed virus and 5 μg / mL pancreatic enzyme for culture. When more than 50% of the cells on the microcarriers fell off, the virus solution was harvested, and the microcarriers were removed by repeated freezing and thawing three times. The supernatant was collected and filtered using a 1 μm filter membrane to remove cell debris, and then washed and filtered using a 100 kD membrane package as a PEDV antigen solution. The antigen solution was tested for virus content, sterility, mycoplasma, and exogenous virus, and could only be used for subsequent freeze-drying after all tests met the requirements.

[0080] 1.2 Preparation of porcine rotavirus (RV) antigen solution

[0081] Marc-145 cells were cultured using a bioreactor and full suspension culture technology. After the Marc-145 cells grew into a full monolayer, 0.01MOI RV seed virus and 2μg / mL pancreatic enzyme maintenance solution were added for culture. When the cell activity dropped below 40%, the cells were frozen and thawed three times, and then filtered with a 1μm filter membrane to remove cell debris. The cells were then washed and filtered with a 100kD membrane package and used as RV antigen liquid. The antigen liquid was tested for virus content, sterility, mycoplasma and exogenous virus, and could only be used for subsequent freeze-drying after all tests met the requirements.

[0082] 1.3 Freeze-drying of Porcine Epidemic Diarrhea and Porcine Rotavirus Bivalent Oral Live Vaccine

[0083] (1) Preparation of lyoprotectant: Weigh 400 g sucrose and 80 g gelatin and dissolve them in 800 mL water for injection. After fully dissolved, dilute to 1000 mL and sterilize in a high pressure steam sterilizer at 121°C for 30 min.

[0084] (2) Preparation of virus antigen solution: Use 0.01 mol / L PBS to dilute the PEDV virus solution and RV virus solution to a virus content of 10 7.3 TCID 50 / mL; the two diluted virus solutions were mixed in equal volumes to form a virus antigen solution;

[0085] (3) Freeze-drying of live vaccines: Mix the viral antigen solution and the freeze-drying protective agent in a ratio of 7:1, dispense into 2 mL / bottle, and freeze-dry using a vacuum freeze dryer;

[0086] (4) Inspection after freeze-drying: The live vaccine after freeze-drying shall be subjected to virus content test, sterility test, mycoplasma and exogenous virus test, etc. If all the above tests meet the requirements, the freeze-dried vaccine can be obtained.

[0087] 2. Piglet immune effect

[0088] 2.1 Preparation of phosphate buffered saline (PBS buffer)

[0089] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.41 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in 800 mL of water for injection, adjust the pH value to 7.0 after full dissolution, make the volume to 1000 mL, place it in a 116°C high-pressure steam sterilizer for sterilization for 15 min, and set aside after cooling.

[0090] 2.2 Dilution of live vaccines

[0091] The same batch of porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine was diluted using the diluents prepared in Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 and PBS buffer, respectively. During the dilution, 10 mL and 50 mL were used to dilute a bottle of freeze-dried vaccine, respectively, for immunization of piglets.

[0092] 2.3 Evaluation of piglet immunization effect

[0093] (1) Piglet immunization: 180 healthy susceptible piglets (PEDV antigen and antibody negative, RV antigen and antibody negative) aged 14 to 21 days were randomly divided into 10 groups, with 10 pigs in each group. They were orally immunized with different diluent formulas and two different volumes of diluents, with each pig orally receiving 2 mL of the vaccine. At the same time, a control group that only took the diluent orally and a control group that took PBS orally were set up. The specific grouping and immunization status are shown in Table 1.

[0094] Table 1 Grouping and immunization of experimental piglets

[0095]

[0096] (2) Antibody determination of immunized piglets: Blood was collected from piglets on the 14th, 21st and 28th day after oral immunization, and the serum was separated for antibody determination, and the PEDV and RV antibodies in the serum were determined respectively; the results are shown in Tables 2-3. It can be seen from Tables 2-3 that after the PEDV+RV oral dual live vaccine was diluted with diluents of different formulas and different volumes of diluents, the antibodies of the two viruses in the piglets began to turn positive on the 14th day after immunization, and the antibody positive rate reached a high level on the 28th day after immunization. The antibody positive rate at each time point of the live vaccine group diluted with different volumes of diluents in Example 1 was the highest, followed by the live vaccine group diluted in Example 2; the antibody positive rates of the live vaccine groups in Comparative Example 1 and Comparative Example 3 were basically the same, which was better than the live vaccine group diluted in Comparative Example 2.

[0097] Table 2 PEDV antibody titer determination results

[0098]

[0099]

[0100] Table 3 RV antibody titer determination results

[0101]

[0102]

[0103] (3) Protection test of immune piglets against virus: On the 28th day after oral immunization, 5 piglets in each group were randomly selected to be challenged with PEDV with a dose of 10 4.0 The remaining 5 piglets were challenged with RV strong virus at a dose of 10 4.0 MID; piglets challenged with different strong toxins were kept and managed separately in different areas. After PEDV challenge, the piglets were observed for 7 consecutive days, and anal swabs were collected from all piglets on the 4th day after challenge for PEDV fluorescence quantitative PCR detection of virus excretion; since most of the older piglets did not cause typical clinical symptoms after being infected with RV strong toxin alone, anal swabs were collected before immunization, on the 1st day, the 4th day and the 7th day for fluorescence quantitative PCR detection of virus excretion; the test results are shown in Tables 4 and 5. As can be seen from Table 4, after the use of PEDV strong virus for attack, the incidence rate of the live vaccine group diluted with the diluent prepared in Example 1 and Example 2 was the lowest, and no virus was detected in the anal swabs 4 days after the attack; the incidence rate and toxin excretion of the oral immunization groups of the diluted live vaccines of Comparative Example 1 and Comparative Example 3 after the attack were basically the same, and both were better than the diluted live vaccine group of Comparative Example 2; As can be seen from Table 5, after the use of RV strong virus for attack, the positive fluorescence detection of anal swabs and the toxin excretion after oral immunization with the diluted live vaccines of Examples 1 and Example 2 1 to 7 days after the attack were the lowest; the positive fluorescence detection of anal swabs and the toxin excretion of the oral immunization groups of the diluted live vaccines of Comparative Example 1 and Comparative Example 3 1 to 7 days after the attack were basically the same, and both were better than the diluted live vaccine group of Comparative Example 2.

[0104] Table 4 PEDV challenge protection test results

[0105]

[0106]

[0107] Table 5 RV challenge protection test results

[0108]

[0109]

[0110] 3. Clinical immune effect of pregnant sows

[0111] In a pig farm threatened by PEDV and RV infection, pregnant sows were immunized with PEDV+RV bivalent live vaccine and the diluents prepared in Example 1 and Comparative Example 3, respectively. The sows were observed to see whether they showed clinical symptoms such as loss of appetite, vomiting, diarrhea, etc. from immunization to one day before delivery, and the survival rate of the piglets born within 14 days of age was counted. The specific immunization and grouping conditions are shown in Table 6.

[0112] Table 6 Clinical immune status of pregnant sows

[0113]

[0114] The results of the evaluation of the clinical immunization effect of pregnant sows are shown in Table 7. It can be seen from Table 7 that in the pig farms threatened by PEDV and RV infection, the PEDV+RV bivalent live vaccine diluted by the diluent prepared in Example 1, the diluent prepared in Example 1, and the diluent prepared in Comparative Example 3 were used to dilute the PEDV+RV bivalent live vaccine to immunize pregnant sows, observe the clinical symptoms, and count the survival rate of piglets within 14 days of age; the results showed that the proportion of sows without clinical symptoms after oral inoculation with the porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine diluted in Example 1 was 93.06%, and the survival rate of piglets born by them within 14 days of age was 84.12%; and the sows immunized with the diluent of Example 1 did not show clinical symptoms The proportion of sows with clinical symptoms was 40%, and the proportion of piglets born within 14 days of age was 33.04%; the proportion of sows without clinical symptoms after oral administration of porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine diluted with the diluent of comparative example 3 was 50%, and the proportion of piglets born within 14 days of age was 49.30%; the proportion of sows without clinical symptoms in the control group (taking only PBS buffer) was 30%, and the proportion of piglets born within 14 days of age was 16.59%; the results showed that after oral immunization of pregnant sows with PEDV+RV bivalent live vaccine diluted with the diluent of Example 1, the proportion of sows with clinical symptoms was significantly lower than that of other groups, followed by the live vaccine group diluted with the diluent of comparative example 3 and oral administration of the diluent of Example 1 alone. For the diluent + bivalent live vaccine oral immunization group, clinical symptoms appeared in sows mainly within 1 to 3 weeks after immunization, and clinical symptoms appeared in piglets mainly in piglets born by pregnant sows within 1 to 4 weeks after immunization. The main reason is that it takes a certain amount of time for the vaccine to produce effective protection after immunization.

[0115] Table 7 Evaluation results of clinical immune effect of pregnant sows

[0116] Group quantity Proportion of sows without clinical symptoms Healthy and alive percentage of piglets at 14 days of age Immunization group 1 72 heads 93.06％(67 / 72) 84.12％(694 / 825) Immunization group 2 20 heads 40％(8 / 20) 33.04％(70 / 227) Immunization group 3 20 heads 50％(10 / 20) 49.30％(138 / 219) Control group 20 heads 30％(6 / 20) 16.59％(34 / 205)

[0117] In summary, the porcine epidemic diarrhea and porcine rotavirus bivalent oral live vaccine was diluted using diluents of different formulations for laboratory evaluation and clinical evaluation. The results showed that the antibody level and protection effect after comprehensive immunization were ranked as follows: Example 1 dilution = Example 2 dilution > Comparative Example 1 dilution = Comparative Example 3 dilution > Comparative Example 2 dilution > PBS buffer, that is, the dilution component A containing alginate, chitosan, and lactose components is best when used in combination with component B containing four traditional Chinese medicine components, while the best effect cannot be achieved when component A or component B is used alone or when the number of traditional Chinese medicine components is reduced.

[0118] The diluents prepared in Example 1 and Comparative Example 3 were respectively diluted and used to orally immunize sows threatened by epidemic diseases with the two-in-one oral live vaccine for porcine epidemic diarrhea and porcine rotavirus. The results showed that the diluent prepared in Example 1 had the best protective effect on both the sows themselves and the piglets they produced; while the effect of Comparative Example 3 with reduced Chinese medicine ingredients was lower than that of the dilution live vaccine group in Example 1, and the protective effect of the sows and piglets in the oral dilution group of Example 1 alone was higher than that of the control group without any treatment, which was basically consistent with the laboratory evaluation results. The test results show that component B in the diluent formula has a promoting effect on enhancing the non-specific immunity of sows and the protective effect of their piglets, and the combination of component A and component B in the diluent formula can prevent the two-in-one oral live vaccine for porcine epidemic diarrhea and porcine rotavirus from being degraded or inactivated by gastric acid, and maximize its immune protection effect.

[0119] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the scope of the present invention.

Claims

1. A porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent, characterized in that: It comprises component A and component B; the mass ratio of component A to component B is 2:1; The component A comprises the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 1-2 parts of sodium alginate, 1-2 parts of chitosan, 0.5-1 parts of lactose and 1000 parts of water; The B component includes the following raw materials in parts by weight: 30-90 parts of coptis chinensis extract, 20-60 parts of astragalus extract, 20-60 parts of phellodendron amurense extract, 30-90 parts of gardenia jasminoides extract and 900-1100 parts of water; coptis chinensis extract, astragalus extract, phellodendron amurense extract and gardenia jasminoides extract are all granules, provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

2. The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to claim 1, characterized in that: The component A includes the following raw materials in parts by mass: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 1.41 parts of sodium dihydrogen phosphate, 0.24 parts of potassium dihydrogen phosphate, 2 parts of sodium alginate, 2 parts of chitosan, 1 part of lactose and 1000 parts of water.

3. The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to claim 1, characterized in that: The B component includes the following raw materials in parts by weight: 90 parts of Coptis chinensis extract, 60 parts of Astragalus membranaceus extract, 60 parts of Phellodendron amurense extract, 90 parts of Gardenia jasminoides extract and 1000 parts of water.

4. The method for preparing the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Mix the raw materials in component A and adjust the pH to 6.0-7.0, then sterilize to obtain component A; (2) mixing and sterilizing the raw materials in component B to obtain component B; (3) Mix component A and component B according to the mass ratio to obtain.

5. The method for preparing the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to claim 4, characterized in that: In the step (1), the pH is adjusted to 6.

0.

6. The method for preparing the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to claim 4, characterized in that: In the steps (1) and (2), the sterilization temperature is 110-120° C. and the sterilization time is 15-20 min.

7. The method for preparing the porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine diluent according to claim 6, characterized in that: In the steps (1) and (2), the sterilization temperature is 116° C. and the sterilization time is 15 min.

8. A porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine, characterized in that: The invention comprises a porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine stock solution and the diluent according to any one of claims 1 to 3.

9. The porcine epidemic diarrhea and porcine rotavirus combined oral live vaccine according to claim 8, characterized in that: The volume ratio of the epidemic diarrhea and porcine rotavirus combined oral live vaccine stock solution to the diluent according to any one of claims 1 to 3 is 1:5 to 25.

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