Composite live vaccine for porcine reproductive and respiratory syndrome

Abstract

The invention discloses a porcine reproductive and respiratory syndrome composite live vaccine, the composite live vaccine is composed of a porcine reproductive and respiratory syndrome virus antigen and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1: 1-1.5; the protective agent comprises 0.5 to 1.5 g of aluminum hydroxide gel, 0.5 to 1 g of composite vitamin, 0.5 to 1.5 g of cane sugar, 1.5 to 2.5 g of diethylaminoethyl glucose, 1 to 2 g of composite amino acid, 5 to 10 g of a polysaccharide composition, 5 to 10 g of gelatin and the balance of a phosphate buffer solution, totaling 100 g. According to the invention, the problems of energy waste and inconvenient storage caused by strict cold chain conditions required in the transportation and storage processes of conventional vaccines are effectively solved. The vaccine not only has good heat resistance and can effectively protect the activity of viruses, but also has the advantages of simple formula, low cost, suitability for mass production and the like, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to a porcine reproductive and respiratory syndrome composite live vaccine, belonging to the technical field of animal vaccines. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Pigs of different ages, breeds and genders can be infected. The disease is characterized by reproductive disorders in sows, respiratory symptoms in piglets, immunosuppression, high infection rate and high mortality. The disease has caused serious economic losses to the global pig industry.

[0003] At present, the common method for preventing and controlling PRRS is immunization. The commonly used PRRS vaccines in the world mainly include live attenuated vaccines and inactivated vaccines. The PRRSV live attenuated vaccine has obvious protective effects, but the frequent mutation of PRRSV has led to the vaccine having strong strain specificity, and there is a risk of gene recombination and virulence reversion. The live attenuated vaccine can detect strong anti-PRRSV antibody response in the early stage of immunization (7-9 days after infection), but the antibodies produced in the early stage are non-neutralizing antibodies. They not only cannot prevent the infection of the virus, but also cause the antibody-dependent enhancement of the virus. The PRRSV-specific neutralizing antibodies can only be detected 28 days after the virus infection, and they are always maintained at a low level. The cellular immune response induced by the live attenuated vaccine appears at 14 days, with low levels and slow growth. The PRRSV attenuated live vaccine can effectively resist the infection of homologous wild strains and reduce the incidence of clinical diseases, but its protective effect against heterologous wild strains is poor.

[0004] The attenuated porcine reproductive and respiratory syndrome vaccine has the advantages of good immune effect and long immune period. In addition, the attenuated vaccine can also be used to immunize sows to obtain passive immunity for piglets. Studies have shown that the use of live vaccines has a positive effect on the occurrence and severity of clinical diseases, the duration of viremia and detoxification. Immunity with live vaccines can induce cellular immunity, resist viremia, prevent the virus from multiplying in the lungs and causing damage to the respiratory and reproductive systems. However, the ability of humoral immunity induced by attenuated vaccines is low, and the level of neutralizing antibodies produced is low, which affects the body's ability to resist viral attacks. After immunization with live vaccines, PRRSV antibodies appear about 2 weeks after immunization and reach a peak four weeks after immunization, but these antibodies have no neutralizing activity. Normally, the production of PRRSV-specific neutralizing antibodies appears about four weeks after immunization, and its titer is low throughout the immunization period.

[0005] In recent years, a lot of research has been conducted on live vaccines, most of which have solved the problem of heat resistance of vaccines during transportation and use, but there has been little improvement in improving the effectiveness of live vaccines.

[0006] In view of the above problems, a porcine reproductive and respiratory syndrome live combination vaccine with good vaccine effect and high heat resistance is proposed. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a porcine reproductive and respiratory syndrome live composite vaccine.

[0008] In order to achieve the above-mentioned purpose, the present invention is implemented through the following technical scheme: a porcine reproductive and respiratory syndrome complex live vaccine, composed of porcine reproductive and respiratory syndrome virus antigen and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1-1.5.

[0009] Preferably, the protective agent includes 0.5-1.5g of aluminum hydroxide gel, 0.5-1g of complex vitamins, 0.5-1.5g of sucrose, 1.5-2.5g of diethylaminoethyl glucose, 1-2g of complex amino acids, 5-10g of polysaccharide composition, 5-10g of gelatin, and phosphate buffer supplemented to 100g.

[0010] Preferably, the protective agent includes 1-1.5g of aluminum hydroxide gel, 0.5-1g of vitamin C, 0.5-1g of sucrose, 1.5-2g of diethylaminoethyl glucose, 1-1.5g of complex amino acids, 5-8g of polysaccharide composition, 8-10g of gelatin, and phosphate buffer supplemented to 100g.

[0011] By adopting the above technical scheme, after the aluminum hydroxide gel adsorbs the antigen, the surface area of ​​the antigen is increased, and the configuration of the active gene is changed, thereby enhancing the immunogenicity of the antigen. Diethylaminoethyl glucose can be used as a targeting inducer to improve the recognition and presentation ability of antigen-presenting cells. It can not only enhance the recognition and presentation ability of the combined antigen porcine reproductive and respiratory syndrome virus (PRRSV), but also enhance the recognition and presentation ability of double-stranded nucleic acids, further improving the ability to induce cellular immunity. Sucrose has an osmotic effect, can inhibit the growth of harmful microorganisms, prolong the shelf life of the product, and has good water solubility. It can improve the survival rate of microorganisms, form a uniform suspension, play a moisture relief role, and prevent the denaturation of active components.

[0012] Preferably, the polysaccharide composition consists of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and liquorice polysaccharide.

[0013] Preferably, the mass ratio of the mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and liquorice polysaccharide is 1:1-1.5:0.5-1:1-2.

[0014] By adopting the above technical scheme, polysaccharides and sugar complexes are not only used as energy resources and constituent materials in organisms, but more importantly, they exist in all cell membrane structures and participate in various cell activities in life phenomena. Polysaccharides are important components of all living organisms and have the ability to scavenge free radicals, increase the activity of antioxidant enzymes and inhibit lipid peroxidation.

[0015] Preferably, the extraction method of mulberry leaf polysaccharide is specifically as follows:

[0016] S1: Crush the mulberry leaves to 50-100 mesh, add distilled water 20 times the mass of the mulberry leaves, and place in a 90℃ water bath for 2h. Collect the liquid, centrifuge, and take the supernatant;

[0017] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0018] S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

[0019] Preferably, the method for extracting liquorice polysaccharide is specifically as follows:

[0020] S1: Grind licorice into 50-80 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant;

[0021] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0022] S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

[0023] Preferably, the method for extracting okara polysaccharide is specifically as follows:

[0024] S1: drying the dregs to a moisture content of 10%-20%, and subjecting the dregs to extrusion spray treatment at 150-170°C and 200-300r / min;

[0025] S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant;

[0026] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0027] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

[0028] Preferably, the method for extracting oat polysaccharides is specifically as follows:

[0029] S1: crush oats into 20-50 mesh, add distilled water to adjust the water content of oats to 15%-25%, and perform extrusion spray treatment at 140-160℃ and 150-300r / min;

[0030] S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant;

[0031] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0032] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

[0033] Preferably, the complex amino acids consist of essential amino acids.

[0034] Beneficial effects of the present invention:

[0035] (1) The present invention effectively solves the problems of energy waste and storage inconvenience caused by the strict cold chain conditions required during the transportation and storage of conventional vaccines.

[0036] (2) The vaccine of the present invention not only has good heat resistance and can effectively protect the activity of the virus, but also has the advantages of simple formula, low cost, and suitability for mass production, thereby reducing production costs.

[0037] (3) The vaccine of the present invention can greatly improve the use effect of live vaccines, quickly produce neutralizing antibodies, reduce the immune gap period, reduce the incidence of pigs, and improve the body's ability to resist diseases. DETAILED DESCRIPTION

[0038] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0039] Example 1

[0040] A porcine reproductive and respiratory syndrome complex live vaccine consists of porcine reproductive and respiratory syndrome virus antigens and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.

[0041] In this embodiment, the protective agent includes 0.5g of aluminum hydroxide gel, 1g of complex vitamins, 1g of sucrose, 2g of diethylaminoethyl glucose, 1.5g of complex amino acids, 10g of polysaccharide composition, 6g of gelatin, and 78g of phosphate buffer.

[0042] In this embodiment, the polysaccharide composition is composed of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and licorice polysaccharide, and the mass ratio thereof is 1:1:0.5:2.

[0043] In this embodiment, the extraction method of mulberry leaf polysaccharide is specifically as follows:

[0044] S1: Crush the mulberry leaves into 50 mesh, add distilled water 20 times the mass of the mulberry leaves, and place in a 90℃ water bath for 2h. Collect the liquid, centrifuge, and take the supernatant;

[0045] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0046] S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

[0047] In this embodiment, the method for extracting licorice polysaccharide is specifically as follows:

[0048] S1: Grind licorice into 80 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant;

[0049] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0050] S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

[0051] In this embodiment, the method for extracting dregs polysaccharide is specifically as follows:

[0052] S1: drying the dregs to a moisture content of 10%, and subjecting the dregs to extrusion spray treatment at 150°C and 300r / min;

[0053] S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant;

[0054] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0055] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

[0056] In this embodiment, the method for extracting oat polysaccharides is specifically as follows:

[0057] S1: crush oats into 20 mesh, add distilled water to adjust the water content of oats to 25%, and perform extrusion spray treatment at 140℃ and 150r / min;

[0058] S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant;

[0059] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0060] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

[0061] In this embodiment, the complex amino acids consist of essential amino acids.

[0062] Example 2

[0063] A porcine reproductive and respiratory syndrome compound live vaccine consists of porcine reproductive and respiratory syndrome virus antigen and a protective agent, and the mass ratio of porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.5.

[0064] In this embodiment, the protective agent includes 1g of aluminum hydroxide gel, 0.8g of complex vitamins, 1g of sucrose, 1.5g of diethylaminoethyl glucose, 1g of complex amino acids, 8g of polysaccharide composition, 5g of gelatin, and 81.7g of phosphate buffer.

[0065] In this embodiment, the polysaccharide composition is composed of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and licorice polysaccharide, and the mass ratio thereof is 1:1.5:0.5:2.

[0066] In this embodiment, the extraction method of mulberry leaf polysaccharide is specifically as follows:

[0067] S1: Crush the mulberry leaves to 100 mesh, add distilled water 20 times the mass of the mulberry leaves, place in a 90℃ water bath for 2h, collect the liquid, centrifuge and take the supernatant;

[0068] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0069] S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

[0070] In this embodiment, the method for extracting licorice polysaccharide is specifically as follows:

[0071] S1: Grind licorice into 50 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant;

[0072] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0073] S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

[0074] In this embodiment, the method for extracting dregs polysaccharide is specifically as follows:

[0075] S1: drying the dregs to a moisture content of 20%, and subjecting the dregs to extrusion spray treatment at 160°C and 200r / min;

[0076] S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant;

[0077] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0078] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

[0079] In this embodiment, the method for extracting oat polysaccharides is specifically as follows:

[0080] S1: crush oats into 50 mesh, add distilled water to adjust the water content of oats to 20%, and perform extrusion spray treatment at 160℃ and 150r / min;

[0081] S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant;

[0082] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0083] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

[0084] The rest is the same as in Example 1.

[0085] Example 3

[0086] A porcine reproductive and respiratory syndrome compound live vaccine consists of porcine reproductive and respiratory syndrome virus antigens and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.25.

[0087] In this embodiment, the protective agent includes 1g of aluminum hydroxide gel, 0.5g of complex vitamins, 0.5g of sucrose, 2.5g of diethylaminoethyl glucose, 1g of complex amino acids, 5g of polysaccharide composition, 10g of gelatin, and 79.5g of phosphate buffer.

[0088] In this embodiment, the polysaccharide composition consists of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and licorice polysaccharide, and the mass ratio thereof is 1:1:0.5:1.

[0089] In this embodiment, the method for extracting licorice polysaccharide is specifically as follows:

[0090] S1: Grind licorice into 60 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant;

[0091] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0092] S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

[0093] In this embodiment, the method for extracting dregs polysaccharide is specifically as follows:

[0094] S1: drying the dregs to a moisture content of 15%, and subjecting the dregs to extrusion spray treatment at 170°C and 300r / min;

[0095] S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant;

[0096] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0097] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

[0098] The rest is the same as in Example 1.

[0099] Example 4

[0100] A porcine reproductive and respiratory syndrome compound live vaccine consists of porcine reproductive and respiratory syndrome virus antigens and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.4.

[0101] In this embodiment, the protective agent includes 1.5g of aluminum hydroxide gel, 0.5g of complex vitamins, 1.5g of sucrose, 1.5g of diethylaminoethyl glucose, 2g of complex amino acids, 6g of polysaccharide composition, 8g of gelatin, and 79g of phosphate buffer.

[0102] In this embodiment, the polysaccharide composition consists of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and liquorice polysaccharide, and the mass ratio thereof is 1:1:1:2.

[0103] In this embodiment, the extraction method of mulberry leaf polysaccharide is specifically as follows:

[0104] S1: Crush the mulberry leaves to 80 mesh, add distilled water 20 times the mass of the mulberry leaves, and place in a 90℃ water bath for 2h. Collect the liquid, centrifuge, and take the supernatant;

[0105] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0106] S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

[0107] In this embodiment, the method for extracting oat polysaccharides is specifically as follows:

[0108] S1: crush oats into 20 mesh, add distilled water to adjust the water content of oats to 15%, and perform extrusion spray treatment at 140℃ and 300r / min;

[0109] S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant;

[0110] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0111] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

[0112] The rest is the same as in Example 1.

[0113] Example 5

[0114] A porcine reproductive and respiratory syndrome compound live vaccine consists of porcine reproductive and respiratory syndrome virus antigen and a protective agent, and the mass ratio of porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.5.

[0115] In this embodiment, the protective agent includes 1.5g of aluminum hydroxide gel, 1g of complex vitamins, 1.5g of sucrose, 2g of diethylaminoethyl glucose, 2g of complex amino acids, 10g of polysaccharide composition, 5g of gelatin, and 77g of phosphate buffer.

[0116] In this embodiment, the polysaccharide composition consists of mulberry leaf polysaccharide, oat polysaccharide, okara polysaccharide and licorice polysaccharide, and the mass ratio thereof is 1:1:0.5:1.

[0117] The rest is the same as in Example 1.

[0118] Example 6

[0119] A porcine reproductive and respiratory syndrome compound live vaccine consists of porcine reproductive and respiratory syndrome virus antigens and a protective agent, and the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1.3.

[0120] In this embodiment, the protective agent includes 0.5g of aluminum hydroxide gel, 0.6g of complex vitamins, 0.5g of sucrose, 2.5g of diethylaminoethyl glucose, 1.5g of complex amino acids, 5g of polysaccharide composition, 10g of gelatin, and 79.4g of phosphate buffer.

[0121] In this embodiment, the extraction method of mulberry leaf polysaccharide is specifically as follows:

[0122] S1: Crush the mulberry leaves to 70 mesh, add distilled water 20 times the mass of the mulberry leaves, and place in a 90℃ water bath for 2h. Collect the liquid, centrifuge, and take the supernatant;

[0123] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0124] S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

[0125] In this embodiment, the method for extracting licorice polysaccharide is specifically as follows:

[0126] S1: Grind licorice into 70 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant;

[0127] S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected;

[0128] S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

[0129] In this embodiment, the method for extracting dregs polysaccharide is specifically as follows:

[0130] S1: drying the okara to a moisture content of 150%, and subjecting the okara to extrusion spray treatment at 165°C and 225 r / min;

[0131] S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant;

[0132] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0133] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

[0134] In this embodiment, the method for extracting oat polysaccharides is specifically as follows:

[0135] S1: crush oats into 40 mesh, add distilled water to adjust the water content of oats to 20%, and perform extrusion spray treatment at 150°C and 200r / min;

[0136] S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant;

[0137] S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate;

[0138] S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

[0139] The rest is the same as in Example 1.

[0140] Test Example 1 Safety Test

[0141] Thirty healthy weaned piglets aged 40-50 days were selected and randomly divided into 6 groups, with 5 piglets in each group. The vaccines prepared in Examples 1 to 6 of the present invention (1 mL / head) were injected into each group of piglets. After the injection, the piglets in each group were placed in the same management environment for feeding and management. The body temperature of the piglets was measured and recorded at regular intervals every day, and the experiment was completed after 14 days of continuous observation.

[0142] The results showed that during the test period, none of the piglets in each group had elevated body temperature (i.e., body temperature exceeded 40°C), and there were no obvious clinical symptoms, indicating that the vaccines in each group were safe.

[0143] Test Example 2 Heat Resistance Test

[0144] The vaccine prepared in Example 1 was stored at 37°C, 2-8°C, and -15°C, respectively, and the conventional vaccine was used as a control. The properties, vacuum degree, residual moisture, and virus content were measured on the 7th and 14th day. The results are shown in Table 1.

[0145] Table 1 Heat resistance test results

[0146]

[0147] As shown in Table 1, the vaccine of the present invention was stored at 37°C for 7 days, and the titer dropped by 0.2 titers, while the conventional vaccine dropped by 0.4 titers; after being stored for 14 days, the titer dropped by 0.3 titers, while the conventional vaccine dropped by 0.6 titers. It can be seen that the vaccine of the present invention has better heat resistance.

[0148] Test Example 3 Shelf Life Test

[0149] The vaccine prepared in Example 1 was stored at 2-8°C for a certain period of time, and the conventional vaccine was used as a control. The properties, vacuum degree, residual moisture and virus content were measured at 12 months, 18 months and 24 months. The results are shown in Table 2.

[0150] Table 2 Shelf life test results

[0151]

[0152] As shown in Table 2, the vaccine of the present invention was stored at 2-8°C for 12 months, and the titer of the conventional vaccine decreased by 0.2 titers, while the titer of the conventional vaccine decreased by 0.8 titers; the titer of the vaccine of the present invention remained unchanged after 18 months of storage, while the titer of the conventional vaccine decreased by 1.3 titers; the titer of the vaccine of the present invention decreased by 0.5 titers after 24 months of storage, while the titer of the conventional vaccine decreased by 1.5 titers. It can be seen that the vaccine of the present invention can be stored for a longer time than the conventional vaccine at 2-8°C, and can still maintain a good titer.

[0153] Test Example 4 Immune protection test

[0154] Fifteen healthy piglets aged 40-50 were randomly divided into three groups, each with 5 pigs. One group was injected with the vaccine prepared in Example 1 (1 mL / pig), one group was injected with conventional vaccine (1 mL / pig), and one group was injected with physiological saline as a control. They were isolated and raised under the same conditions. After 28 days, all pigs were challenged with a strong toxin for efficacy testing. Each pig was inoculated with 2 ml (≥10 3.8 TCID 50 / mL), measured body temperature every day, observed for 21 days, and recorded the number of morbidity and mortality of the test pigs. The specific results are shown in Table 3.

[0155] Table 3 Immunization results

[0156] Number of cases / head Healthy number / head Incidence rate / % Example 1 Vaccine prepared 0 5 0 Routine vaccines 0 5 0 Normal saline 5 0 100

[0157] The results in Table 3 show that the vaccine of the present invention can protect piglets against the attack of strong toxins.

[0158] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0159] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A porcine reproductive and respiratory syndrome combined live vaccine, characterized in that: The invention is composed of porcine reproductive and respiratory syndrome virus antigen and a protective agent, wherein the mass ratio of the porcine reproductive and respiratory syndrome virus antigen to the protective agent is 1:1-1.

5.

2. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 1, characterized in that: The protective agent comprises 0.5-1.5g of aluminum hydroxide gel, 0.5-1g of complex vitamins, 0.5-1.5g of sucrose, 1.5-2.5g of diethylaminoethyl glucose, 1-2g of complex amino acids, 5-10g of polysaccharide composition, 5-10g of gelatin, and phosphate buffer supplemented to 100g.

3. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 2, characterized in that: The protective agent comprises 1-1.5g of aluminum hydroxide gel, 0.5-1g of vitamin C, 0.5-1g of sucrose, 1.5-2g of diethylaminoethyl glucose, 1-1.5g of complex amino acids, 5-8g of polysaccharide composition, 8-10g of gelatin, and phosphate buffer supplemented to 100g.

4. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 3, characterized in that: The polysaccharide composition consists of mulberry leaf polysaccharide, oat polysaccharide, bean dregs polysaccharide and liquorice polysaccharide.

5. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 4, characterized in that: The mass ratio of the mulberry leaf polysaccharide, oat polysaccharide, bean dregs polysaccharide and liquorice polysaccharide is 1:1-1.5:0.5-1:1-2.

6. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 5, characterized in that: The extraction method of mulberry leaf polysaccharide is specifically as follows: S1: Crush the mulberry leaves to 50-100 mesh, add distilled water 20 times the mass of the mulberry leaves, and place in a 90℃ water bath for 2h. Collect the liquid, centrifuge, and take the supernatant; S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected; S3: Dissolve the precipitate obtained in step S2 with distilled water, filter through a 0.22 μm filter membrane, concentrate, and freeze-dry to obtain mulberry leaf polysaccharide.

7. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 6, characterized in that: The extraction method of the licorice polysaccharide is specifically as follows: S1: Grind licorice into 50-80 mesh, add distilled water 25 times the mass of licorice, and place in a water bath at 80℃ for 3h. Collect the liquid, centrifuge and take the supernatant; S2: The supernatant is subjected to alcohol precipitation, centrifuged, and the precipitate is collected; S3: dissolving the precipitate obtained in step S2 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain glycyrrhizic polysaccharide.

8. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 7, characterized in that: The extraction method of okara polysaccharide is specifically as follows: S1: drying the okara to a moisture content of 10%-20%, and subjecting the okara to extrusion spray treatment at 150-170°C and 200-300r / min; S2: Add 15 times the mass of distilled water to the extruded okara, place in a 75℃ water bath for 2h, centrifuge and take the supernatant; S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate; S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain okara polysaccharide.

9. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 8, characterized in that: The oat polysaccharide extraction method is specifically as follows: S1: crush oats into 20-50 mesh, add distilled water to adjust the water content of oats to 15%-25%, and perform extrusion spray treatment at 140-160℃ and 150-300r / min; S2: Add 20 times the mass of distilled water to the extruded oats, place in a water bath at 80°C for 2 hours, centrifuge, and collect the supernatant; S3: alcohol precipitation of the supernatant, centrifugation and collection of the precipitate; S4: dissolving the precipitate obtained in step S3 with distilled water, filtering through a 0.22 μm filter membrane, concentrating, and freeze-drying to obtain oat polysaccharides.

10. A porcine reproductive and respiratory syndrome combined live vaccine as claimed in claim 9, characterized in that: The complex amino acid consists of essential amino acids.

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