A combined immunization method of an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and a live rabbit-derived classical swine fever virus (CSFV) vaccine

Abstract

The present invention discloses a combined immunization method of a porcine reproductive and respiratory syndrome (PRRS) inactivated vaccine and a live rabbit-derived classical swine fever (CSF) vaccine. 1) Prepare the PRRS inactivated vaccine; 2) Prepare a freeze-dried live rabbit-derived CSF vaccine containing anti-PRRSV specific splenic lymph node transfer factor at the nanoscale; 3) After warming the above two vaccines to room temperature, dissolve the live rabbit-derived CSF vaccine with the PRRS inactivated vaccine to obtain a mixed vaccine, and then immunize piglets with the mixed vaccine. The present invention uses a nanoscale live rabbit-derived CSF vaccine containing anti-PRRSV specific splenic lymph node transfer factor and a PRRS inactivated vaccine for combined immunization. After the nanoscale live rabbit-derived CSF vaccine enters the body, it can quickly and abundantly provide splenic lymph factors containing anti-PRRS in a short time, effectively stimulating the cellular immune response of the body and promoting the cellular immune response. The present invention can prevent two diseases with one injection, reduce the number of immunizations, reduce the immune stress of the pig herd, and improve the immunization effect.

Description

Technical Field

[0001] The present invention belongs to the field of biological products, and particularly relates to a combined immunization method of an inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine and a live rabbit-derived classical swine fever (CSF) vaccine. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a highly contagious and highly lethal infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). At present, the prevention and control measures for this disease mainly include "biosecurity control, feeding environment management, vaccination, and drug health care", among which vaccination is a relatively effective prevention and control measure. There are a variety of vaccine researches in China, including inactivated vaccines, attenuated vaccines, and vaccines at the in-depth molecular level. At present, the attenuated live PRRS vaccine occupies the mainstream market because the live vaccine produces antibodies quickly and can produce a short-term control effect on PRRS in the initial stage. However, after immunization with the attenuated vaccine, viremia can be induced for about one month, allowing the vaccine virus to be contaminated and spread. Blue ear disease will exhibit an antibody-dependent enhancement effect against this virus, and the infection of PRRSV becomes more severe as the concentration of non-neutralizing antibodies in the body increases. At the same time, the PRRS virus itself is prone to mutation and immune escape. Long-term and high-intensity immunization with live PRRS vaccines may promote the mutation of wild PRRS virus, even accelerate recombination, and the reversion of virulence, etc., increasing the diversity of virus strains. The inactivated vaccine has the advantages of safety, no risk of spreading the virus and causing new epidemic sources, and no reversion of virulence, etc., fully meeting the future needs of pig disease prevention and control in China's pig industry.

[0003] Classical swine fever is a contagious disease caused by Classical swine fever virus (CSFV), clinically characterized by high fever, bleeding, and high mortality. This disease seriously endangers the healthy and stable development of China's pig industry. At present, there is no specific drug for the treatment of this disease, and immunoprophylaxis and timely culling of infected pigs are the best choices for preventing the disease. Most current vaccines are attenuated CSF vaccines, which can be divided into cell-derived and rabbit-derived. The live rabbit-derived CSF vaccine, also known as the CSF spleen and lymph vaccine, is prepared by inoculating CSFV into healthy rabbits and harvesting and grinding the spleens and mesenteric lymph nodes of rabbits showing typical fever. After this vaccine enters the pig body, it can not only stimulate the body to produce antibodies through CSFV to play a humoral immune role, but also the transfer factor in the spleen and lymph tissue can stimulate the body to produce a cellular immune response.

[0004] At present, classical swine fever vaccine and porcine reproductive and respiratory syndrome (PRRS) vaccine are used to immunize pig herds separately according to the immunization program in clinical practice, or the two vaccines are immunized simultaneously by separate-point injection, that is, two or more needles. There are disadvantages such as great immune stress in pig herds and high working intensity for veterinarians in pig farms. To exert its immune protection, PRRS vaccine requires not only humoral immunity but also cellular immunity. At present, inactivated PRRS vaccines lack the ability to stimulate the body to produce specific cellular immunity. In addition, the existing classical swine fever spleen and lymph vaccine is freeze-dried after being formulated by grinding tissue technology, and the particles are not fine and uniform enough, so the spleen and lymph factors cannot be rapidly released in large quantities in a short time after the vaccine is injected into the body. Summary of the Invention

[0005] The purpose of the present invention is to provide a combined immunization method for inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine and live rabbit-derived classical swine fever vaccine. The technical idea of the present invention is as follows: Cellular immunity plays an extremely important role in the prevention and control of PRRS. Like all other viral diseases, cellular immunity plays an important role in antiviral infection, and it is of great significance for virus clearance and disease prevention. The cellular immune response caused by PRRSV infection usually appears relatively late, generally detectable 4 weeks after infection, and can only be maintained for about 3 weeks. Spleen and lymph transfer factor is a good adjuvant for enhancing the body's cellular immune response. To make up for the cellular immune blank period that appears after PRRS vaccine immunization, the present invention uses a nano-level live rabbit-derived classical swine fever vaccine containing anti-PRRSV specific spleen and lymph transfer factor (hereinafter referred to as live rabbit-derived classical swine fever vaccine) and inactivated PRRSV vaccine for combined immunization. After the nano-level live rabbit-derived classical swine fever vaccine enters the body, it can rapidly and abundantly provide spleen and lymph factors containing anti-PRRSV in a short time, which can effectively stimulate the body's cellular immune response and promote cellular immune response. At the same time, the first immunization times recommended for the two selected vaccines in piglets in clinical practice are similar.

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

[0007] A combined immunization method for inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine and live rabbit-derived classical swine fever vaccine, comprising the following steps:

[0008] 1) Preparation of inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine

[0009] Inoculate the PRRSV vaccine seed virus at 0.5-2% of the volume of the cell growth maintenance medium into Marc-145 cells grown to a confluent monolayer, culture for 48-60 h to harvest PRRSV antigen, and the titer of PRRSV antigen before inactivation ≥ 10 8.5 TCID 50 / ml, the harvested PRRSV antigen is inactivated with an inactivator (non - formaldehyde), and the volume ratio of the inactivator to the antigen is 1:2000 - 1:5000, thereby obtaining the inactivated PRRSV antigen. After passing the inactivation test and sterility test, the inactivated PRRSV antigen is used as the aqueous phase and mixed and emulsified with the oil phase (adjuvant) at a weight ratio of 1:1 - 1:9 to prepare the inactivated vaccine against porcine reproductive and respiratory syndrome. The dosage form of this vaccine can be, but is not limited to, water - in - oil or water - in - oil - in - water dosage forms.

[0010] 2) Preparation of live vaccine against classical swine fever from rabbits

[0011] The inactivated PRRSV antigen obtained in step 1) and the attenuated classical swine fever virus adapted to rabbits are simultaneously inoculated into healthy large rabbits. Select the rabbits showing typical febrile reactions, slaughter them within 24 hours after the body temperature drops and thereafter, aseptically collect the spleens and lymph nodes of the rabbits, and mix and grind them with a solvent (such as physiological saline, PBS solution, milk - sucrose protectant, etc.) at a weight ratio of 1:0.5 - 1:2, homogenize, and filter through a 100 - mesh sieve to remove fascia, fat, etc., to obtain spleen - lymph tissue. Then, this spleen - lymph tissue is prepared into a nanoscale spleen - lymph homogenate by a continuous - flow high - pressure homogenizer with online temperature control at 4°C. Then, the nanoscale spleen - lymph homogenate is freeze - dried with milk - sucrose freeze - drying protectant at a volume ratio of 1:1 - 1:3 (requiring that the finished product per dose contains ≥0.01 g of spleen - lymph tissue), which is the freeze - dried live vaccine against classical swine fever from rabbits containing nano - scale spleen - lymph transfer factor specific to PRRSV.

[0012] 3) Combined immunization method

[0013] 3 - 1) Mixing of vaccines

[0014] Before combined immunization, warm up the inactivated vaccine against porcine reproductive and respiratory syndrome and the live vaccine against classical swine fever from rabbits. After warming up to room temperature, dissolve 1 dose (2 ml) of the inactivated vaccine against porcine reproductive and respiratory syndrome in 1 dose of the live vaccine against classical swine fever from rabbits to obtain the mixed vaccine;

[0015] 3 - 2) Immunization program

[0016] Immunize piglets at 28 - 35 days of age with the mixed vaccine. After the mixed vaccine is fully mixed, immunize at a dose of 2 ml / head. 21 - 30 days after the first combined immunization, boost immunize once according to step 3 - 1). The combined immunization requires that the two vaccines be mixed and used immediately.

[0017] It should be noted that

[0018] In step 1), the dosage form of the inactivated vaccine against porcine reproductive and respiratory syndrome is not limited to water - in - oil, water - in - oil - in - water and other dosage forms, as long as it is a dosage form that can dissolve the freeze - dried vaccine.

[0019] In step 2), when preparing the live classical swine fever vaccine of rabbit origin, in addition to the above method (inactivated PRRSV antigen and live classical swine fever virus are directly inoculated into adult rabbits), the following method can also be adopted: Adult rabbits are pre-inoculated with inactivated PRRSV antigen 1 to 2 times at an interval of 14 days, and then inactivated PRRSV antigen and attenuated classical swine fever virus of rabbit origin are inoculated into adult rabbits.

[0020] 3) The combined immunization method provided by the present invention is not limited to inactivated PRRS vaccine and live classical swine fever vaccine, and other vaccines can also be combined immunized by a similar method.

[0021] The present invention has the following beneficial effects:

[0022] 1. Combined immunization reduces immune stress

[0023] Normal immunization program for classical swine fever vaccine in piglets: The first immunization is at 25 - 30 days old; the booster immunization is at 60 - 70 days old; while the normal immunization program for PRRS vaccine in piglets: The first immunization is at 28 - 35 days old; the booster immunization is at 56 - 63 days old. Above, when the two vaccines are immunized separately or simultaneously, they are injected at different points, that is, two or more needles.

[0024] The technical solution of the present invention directly dilutes the live classical swine fever vaccine of rabbit origin with inactivated PRRS vaccine of pig origin for combined immunization. The live classical swine fever vaccine containing specific splenic lymph transfer factor against PRRSV is equivalent to an immune enhancer of inactivated PRRS vaccine. On the basis of achieving one - needle dual - prevention, it can also reduce the immune stress of the pig group, effectively improve the clinical immune effect of inactivated PRRS vaccine, and reduce the workload of veterinarians.

[0025] 2. Preparation of nano - level live classical swine fever vaccine of rabbit origin

[0026] The traditional classical swine fever spleen - lymph vaccine is a spleen - lymph tissue vaccine containing live classical swine fever virus prepared by a grinding process, with uneven particle sizes. After the vaccine is injected into the body, the spleen - lymph factors cannot be rapidly and massively released in a short time.

[0027] The present invention makes the live classical swine fever vaccine of rabbit origin into nano - level, enabling the spleen - lymph tissue to rapidly and massively release spleen - lymph factors in the body, and activating the cellular immune response of the body.

[0028] 3. Enhancement of immune effect

[0029] The existing inactivated PRRS vaccine of pig origin lacks a specific enhancer to stimulate the body to produce cellular immune response. However, the present invention prepares a live classical swine fever vaccine of rabbit origin containing specific splenic lymph transfer factor against PRRSV, which can improve the specific cellular immune response of inactivated PRRS vaccine and become an immune enhancer of inactivated PRRS vaccine of pig origin. Description of the Drawings

[0030] Figure 1 It is a trend chart of lymphocyte activity of each experimental group. Detailed implementation mode

[0031] Example 1

[0032] Comparison of different immunization methods

[0033] 1) Porcine reproductive and respiratory syndrome (PRRS) inactivated vaccine

[0034] The PRRSV vaccine seed virus was inoculated into Marc-145 cells grown to a confluent monolayer at 0.5-2% of the volume of the cell growth maintenance fluid, cultured for 48-60 h, and the PRRSV antigen was harvested. The titer of the PRRSV antigen before inactivation was ≥10 8.5 TCID 50 / ml. The harvested PRRSV antigen was inactivated with an inactivator (non-formaldehyde), and the volume ratio of the inactivator to the PRRSV antigen was 1:2000-1:5000, thereby obtaining the inactivated PRRSV antigen. After passing the inactivation test and sterility test, the inactivated PRRSV antigen was used as the aqueous phase and mixed and emulsified with the oil phase (adjuvant) at a weight ratio of 1:1-1:9 to prepare the PRRS inactivated vaccine. The dosage form of this vaccine can be, but is not limited to, water-in-oil or water-in-oil-in-water dosage form.

[0035] 2) Preparation of live classical swine fever vaccine from rabbits

[0036] The inactivated PRRSV antigen in step 1) and the attenuated classical swine fever virus were simultaneously inoculated into healthy adult rabbits. Rabbits showing a typical febrile reaction were selected and sacrificed within 24 hours after the body temperature dropped. The spleen and lymph nodes of the rabbits were aseptically collected, and they were mixed and ground with a solvent (normal saline) at a weight ratio of 1:0.5-1:2, homogenized, and filtered through a 100-mesh sieve to remove fascia, fat, etc. to obtain spleen-lymph tissue. Then, this spleen-lymph tissue was prepared into nanoscale spleen-lymph homogenate using an ultra-high pressure continuous flow homogenizer with on-line temperature control at 4°C. The nanoscale spleen-lymph homogenate was then mixed with milk-sucrose freeze-drying protectant at a volume ratio of 1:1-1:3 (requiring that each dose of the finished product contains ≥0.01 g of spleen-lymph tissue), which is the freeze-dried live classical swine fever vaccine from rabbits containing anti-PRRSV specific spleen-lymph transfer factor at the nanoscale.

[0037] 3) Immunization method

[0038] 3-1) Grouping and immunization program

[0039] Healthy and susceptible piglets aged 28-35 days were randomly divided into four groups, A, B, C, and D, with 10 piglets in each group.

[0040] Group A: Combined immunization group of PRRS inactivated vaccine and live classical swine fever vaccine from rabbits. The immunization method for each piglet was: Dissolve 1 dose (2 ml) of PRRS inactivated vaccine with 1 dose of live classical swine fever vaccine from rabbits, fully mix them, and then immunize the piglets;

[0041] Group B: Inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine immunization group, only immunized with inactivated PRRS vaccine, immunization dose 2 ml / head;

[0042] Group C: Live rabbit-derived classical swine fever (CSF) vaccine immunization group, only immunized with live rabbit-derived CSF vaccine, immunization dose 1 dose / head;

[0043] Group D (control group): Injected with normal saline, dose 2 ml / head;

[0044] The immunization program is shown in Table 1:

[0045] Table 1 Immunization procedures and doses of each experimental group

[0046]

[0047] 3-2) Determination of lymphocyte immune activity

[0048] Blood samples were collected from each group before immunization, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, and 28 days after immunization to isolate lymphocytes, and the activity of lymphocytes in each experimental group was compared by the E-rosette test method. The results are shown in Table 2. Figure 1 as shown.

[0049] Table 2 Mean values of lymphocyte activity ratios at each time point in each experimental group (lymphocyte activity Ea% = E-positive cells / 200 lymphocytes)

[0050]

[0051] From Figure 1 it can be seen that the activity ratio of lymphocytes in Group A after immunization is better than that of other groups; Group C is better than Group B. It shows that the splenic lymphokines in the splenic lymphoid tissue can activate peripheral blood lymphocytes after entering the body, improving the cellular immune level of the body. The activated lymphocytes increase rapidly 5-7 days after immunization and tend to be stable 21 days after immunization, and show a downward trend 28 days after immunization, while the antibody conversion in the pig population generally occurs 21 days after immunization with the PRRS vaccine. Therefore, the activated lymphocytes decline after antibody conversion, which can play a role in filling the immune blank period of the PRRS vaccine.

[0052] 2-3) Antibody detection

[0053] 30 days after the first immunization, 7 days and 14 days after the second immunization, blood samples were collected from each experimental group to isolate serum, and the antibody levels of each group were detected using a kit. Among them, the IDEXX classical swine fever antibody detection kit was used to detect the classical swine fever antibody level, and it was positive when the blocking rate ≥ 40%; the HIPRA PRRS antibody detection kit was used to detect the PRRS antibody level, and it was positive when the IRPC > 20. The antibody positive rate results are shown in Table 3 and Table 4.

[0054] Table 3 Porcine reproductive and respiratory syndrome (PRRS) antibody positive rates in each experimental group 30 days after the first immunization and 7 days and 14 days after the second immunization (number of antibody-positive animals / total number of immunized animals)

[0055]

[0056] Table 4 Classical swine fever (CSF) antibody positive rates in each experimental group on the 30th day after the first immunization and on the 15th day after the second immunization (number of antibody-positive animals / total number of immunized animals)

[0057]

[0058] As can be seen from Table 3, the PRRS antibody positive rate of Group A (7 / 10) 7 days after the second immunization was higher than that of Group B (4 / 10), indicating that the rabbit-derived live CSF vaccine described in the present invention can improve the antibody level of the inactivated PRRS vaccine. As can be seen from Table 4, when the combined immunization of the inactivated PRRS vaccine and the rabbit-derived live CSF vaccine in Group A was compared with the rabbit-derived live CSF vaccine in Group C, the CSF antibody positive rate reached 10 / 10 positive on the 30th day after the first immunization. This shows that the inactivated PRRS vaccine does not affect the production of antibodies by the rabbit-derived live CSF vaccine.

[0059] 3 - 4) Vaccine protection rate (post-challenge autopsy observation)

[0060] At 14 days after the second immunization, challenge was carried out using a highly virulent PRRSV strain for testing, and the challenge dose was 1 ml / head. At 14 days after challenge, autopsy was performed to observe the pathological changes in the lungs and lymph nodes. The criteria for morbidity in the internal organs after challenge were as follows: the lungs showed a reddish-brown mottled appearance and did not collapse; the lymph nodes were moderately to severely enlarged.

[0061] Table 5 Protection of the lungs and lymph nodes in each experimental group after challenge

[0062] Grouping Vaccination Vaccine protection rate Group A Combined immunization of inactivated PRRS vaccine and live rabbit-derived classical swine fever vaccine 8 / 10 Group B Inactivated PRRS vaccine 6 / 10 Group C Live rabbit-derived classical swine fever vaccine 1 / 10 Group D (control group) Normal saline 0 / 10

[0063] As can be seen from Table 5, the challenge protection rate of the combined immunization group of the inactivated PRRS vaccine and the rabbit-derived live CSF vaccine in Group A was higher than that of the other three groups. This shows that the combined immunization of the inactivated PRRS vaccine and the rabbit-derived live CSF vaccine can improve the clinical protection effect of the inactivated PRRS vaccine.

[0064] In summary, in this example, a nano-level rabbit-derived live CSF vaccine containing anti-PRRSV specific splenic lymph node transfer factor was used for combined immunization with an inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine. After the nano-level rabbit-derived live CSF vaccine enters the body, it can rapidly and abundantly provide splenic lymph factors containing anti-PRRSV in a short time, effectively stimulating the cellular immune response of the body to promote cellular immune response and improving the clinical immunization effect of the inactivated PRRS vaccine. At the same time, the combined immunization of the inactivated PRRS vaccine and the rabbit-derived live CSF vaccine can prevent two diseases with one injection, reduce the number of immunizations, reduce the immune stress of the pig herd, and improve the immunization effect.

[0065] Example 2

[0066] A combined immunization method of a porcine reproductive and respiratory syndrome inactivated vaccine and a rabbit-derived classical swine fever live vaccine, comprising the following steps

[0067] 1) Preparation of the porcine reproductive and respiratory syndrome inactivated vaccine

[0068] Inoculate the PRRSV vaccine seed virus into Marc-145 cells grown to a confluent monolayer at a ratio of 0.5% of the volume of the cell growth maintenance medium, culture for 48 h, and harvest the PRRSV antigen. The titer of the PRRSV antigen before inactivation is ≥ 10 8.5 TCID 50 / ml. Inactivate the harvested PRRSV antigen with a non-formaldehyde inactivator, and the volume ratio of the inactivator to the antigen is 1:2000. Thus, the inactivated PRRSV antigen is obtained. After passing the inactivation test and sterility test, take the inactivated PRRSV antigen as the aqueous phase and mix and emulsify it with the oil phase (adjuvant) at a weight ratio of 1:1 to prepare the porcine reproductive and respiratory syndrome inactivated vaccine.

[0069] 2) Preparation of the rabbit-derived classical swine fever live vaccine

[0070] Simultaneously inoculate the inactivated PRRSV antigen obtained in step 1) and the classical swine fever lapinized attenuated virus into healthy large rabbits. Select the rabbits showing the typical fever reaction, slaughter them within 24 hours after the body temperature drops and thereafter, aseptically collect the spleens and lymph nodes of the rabbits, mix and grind them with the solvent (PBS solution) at a weight ratio of 1:0.5, homogenize, and filter through a 100-mesh sieve to remove fascia, fat, etc. to obtain the spleen-lymph tissue. Then, use a high-pressure continuous flow homogenizer with online temperature control at 4°C to prepare the spleen-lymph tissue into a nanoscale spleen-lymph homogenate. Then, lyophilize the nanoscale spleen-lymph homogenate and the milk-sucrose lyoprotectant at a volume ratio of 1:1 (it is required that the finished product per dose contains ≥ 0.01 g of spleen-lymph tissue), which is the freeze-dried vaccine of the rabbit-derived classical swine fever live vaccine containing the nanoscale spleen-lymph transfer factor specific to PRRSV.

[0071] 3) Combined immunization method

[0072] 3-1) Mixing of the vaccines

[0073] Before performing the combined immunization, warm up the porcine reproductive and respiratory syndrome inactivated vaccine and the rabbit-derived classical swine fever live vaccine. After warming up to room temperature, dissolve one dose (2 ml) of the porcine reproductive and respiratory syndrome inactivated vaccine with one dose of the rabbit-derived classical swine fever live vaccine to obtain the mixed vaccine;

[0074] 3-2) Immunization program

[0075] Immunize piglets aged 28 - 35 days with the mixed vaccine. After thoroughly mixing the mixed vaccine, immunize at a dose of 2 ml / head. 21 days after the first combined immunization, boost the immunization once according to step 3-1). The combined immunization requires that the two vaccines be mixed and used immediately.

[0076] Example 3

[0077] A combined immunization method of a porcine reproductive and respiratory syndrome inactivated vaccine and a rabbit-derived classical swine fever live vaccine, comprising the following steps

[0078] 1) Preparation of the porcine reproductive and respiratory syndrome inactivated vaccine

[0079] Inoculate the PRRSV vaccine seed virus at 2% of the volume of the cell growth maintenance fluid into Marc-145 cells grown to a confluent monolayer, culture for 60 h to harvest the PRRSV antigen, and the titer of the PRRSV antigen before inactivation ≥ 10 8.5 TCID 50 / ml. Inactivate the harvested PRRSV antigen with a non-formaldehyde inactivator, and the volume ratio of the inactivator to the antigen is 1:5000, thereby obtaining the inactivated PRRSV antigen. After passing the inactivation test and sterility test, take the inactivated PRRSV antigen as the aqueous phase and mix and emulsify it with the oil phase (adjuvant) at a weight ratio of 1:9 to prepare the porcine reproductive and respiratory syndrome inactivated vaccine.

[0080] 2) Preparation of the rabbit-derived classical swine fever live vaccine

[0081] Simultaneously inoculate the inactivated PRRSV antigen obtained in step 1) and the classical swine fever lapinized attenuated virus into healthy large rabbits. Select the rabbits showing the typical fever reaction, slaughter them within 24 hours after the body temperature drops and thereafter, aseptically collect the spleens and lymph nodes of the rabbits, mix and grind them with the solvent (milk sucrose protective agent) at a weight ratio of 1:2, homogenize, and filter through a 100-mesh sieve to remove fascia, fat, etc. to obtain the spleen-lymph tissue. Then, use a high-pressure continuous flow homogenizer with online temperature control at 4°C to prepare the spleen-lymph tissue into a nanoscale spleen-lymph homogenate. Then, freeze-dry the nanoscale spleen-lymph homogenate and the milk sucrose freeze-drying protective agent at a volume ratio of 1:3 (requiring that the finished product of 1 dose contains ≥ 0.01 g of spleen-lymph tissue), which is the freeze-dried vaccine of the rabbit-derived classical swine fever live vaccine containing the nanoscale spleen-lymph transfer factor specific for anti-PRRSV.

[0082] 3) Combined immunization method

[0083] 3-1) Mixing of the vaccines

[0084] Before implementing the combined immunization, warm up the porcine reproductive and respiratory syndrome inactivated vaccine and the rabbit-derived classical swine fever live vaccine. After warming up to room temperature, dissolve 1 dose of the rabbit-derived classical swine fever live vaccine with 1 dose (2 ml) of the porcine reproductive and respiratory syndrome inactivated vaccine to obtain the mixed vaccine;

[0085] 3-2) Immunization program

[0086] Immunize piglets aged 28 - 35 days with the mixed vaccine. After the mixed vaccine is fully mixed, immunize at a dose of 2 ml / head. 30 days after the first combined immunization, boost immunize once according to step 3-1). The combined immunization requires that the two vaccines be mixed and used immediately.

Claims

1. A preparation method of a mixed vaccine for combined immunization of an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and a live rabbit-derived classical swine fever virus (CSFV) vaccine, characterized in that: It includes the following steps: 1) Preparation of inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine Inoculate the PRRSV vaccine seed virus into Marc-145 cells grown to a confluent monolayer, culture for 48 - 60 h, harvest the PRRSV antigen, inactivate the harvested PRRSV antigen with an inactivating agent to obtain inactivated PRRSV antigen. After passing the inactivation test and sterility test, take the inactivated PRRSV antigen as the aqueous phase and mix and emulsify it with the oil phase at a weight ratio of 1:1 - 1:9 to prepare the inactivated porcine reproductive and respiratory syndrome vaccine; 2) Preparation of live rabbit-derived classical swine fever vaccine Simultaneously inoculate the inactivated PRRSV antigen obtained in step 1) and the lapinized attenuated classical swine fever virus into healthy adult rabbits. Select rabbits showing typical febrile reactions, slaughter them within 24 hours after the body temperature drops and thereafter. Sterilely collect the spleens and lymph nodes of the rabbits, mix and grind them with the solvent at a weight ratio of 1:0.5 - 1:2, homogenize, filter to obtain spleen and lymph tissue, then use a homogenizer to prepare the spleen and lymph tissue into nanoscale spleen and lymph homogenate, and then freeze-dry the nanoscale spleen and lymph homogenate with the freeze-drying protectant at a volume ratio of 1:1 - 1:3 to obtain the freeze-dried live rabbit-derived classical swine fever vaccine containing nanoscale spleen and lymph transfer factor specific to PRRSV; 3) Preparation method of combined vaccine Before implementing combined immunization, warm the inactivated porcine reproductive and respiratory syndrome vaccine and the live rabbit-derived classical swine fever vaccine. After warming to room temperature, dissolve 1 dose of the live rabbit-derived classical swine fever vaccine with 1 dose of the inactivated porcine reproductive and respiratory syndrome vaccine to obtain the combined vaccine.

2. The preparation method of a mixed vaccine for combined immunization of an inactivated porcine reproductive and respiratory syndrome virus vaccine and a live rabbit-derived classical swine fever virus vaccine according to claim 1, characterized in that: In step 1), the inoculation amount of the PRRSV vaccine seed virus is 0.5 - 2% of the volume of the cell growth maintenance medium.

3. The preparation method of a mixed vaccine for combined immunization of a porcine reproductive and respiratory syndrome inactivated vaccine and a rabbit-derived live classical swine fever vaccine according to claim 1, characterized in that: In step 1), the PRRSV antigen titer before inactivation ≥ 10 8.5 TCID 50 / ml.

4. A preparation method of a mixed vaccine for combined immunization of an inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine and a live rabbit-derived classical swine fever (CSF) vaccine according to claim 1, characterized in that: In step 1), the inactivating agent is a non-formaldehyde inactivating agent, and the volume ratio of the inactivating agent to the antigen is 1:2000 - 1:5000.

5. A method for preparing a mixed vaccine by combining an inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine and a live rabbit-derived classical swine fever (CSF) vaccine as claimed in claim 1, characterized in that: In step 2), the solvent is one of normal saline, PBS solution, and milk-sucrose protectant.

6. The preparation method of the mixed vaccine for combined immunization of a porcine reproductive and respiratory syndrome inactivated vaccine and a live rabbit-derived classical swine fever vaccine according to claim 1, wherein: In step 2), the freeze-drying protectant is milk-sucrose.

7. A preparation method of a mixed vaccine for combined immunization of an inactivated porcine reproductive and respiratory syndrome vaccine and a live rabbit-derived classical swine fever vaccine according to claim 1, characterized in that: In step 2), after the spleen and lymph homogenate is mixed and freeze-dried with the freeze-drying protectant, 1 dose of the finished product contains ≥0.01 g of spleen and lymph tissue.

8. The preparation method of a mixed vaccine for combined immunization of an inactivated porcine reproductive and respiratory syndrome vaccine and a live rabbit-derived classical swine fever vaccine according to claim 1, characterized in that: In step 3), dissolve 1 dose of the live rabbit-derived classical swine fever vaccine with 1 dose of the inactivated porcine reproductive and respiratory syndrome vaccine.

Images