Preparation method of peste des petits ruminants positive serum

Abstract

The invention relates to the technical field of biology, and discloses a preparation method of peste des petits ruminants positive serum, which comprises the following steps: culturing Vero cells, taking out basic seeds of the Vero cells from liquid nitrogen, quickly unfreezing in a water bath at 37 DEG C, centrifuging for 10 minutes at 1000r / min, discarding supernatant, preparing precipitated cells into cell suspension by using an MEM nutrient solution containing 10% of newborn calf serum, and performing freeze drying to obtain the peste des petits ruminants positive serum. Placing the cells in an incubator with 5% of CO2 at 37 DEG C for culturing for 3-4 days, and digesting, dispersing and subculturing after the cells grow to be full of a single layer; and breeding peste des petits ruminants virus seeds, and hydrating the peste des petits ruminants virus freeze-dried virus seeds by using a serum-free MEM nutrient solution according to the apparent loading quantity marked by a bottle label. According to the invention, the lyophilized peste des petits ruminant vaccine is used for primary immunization, a transfer factor, polyinosinic-polycytidylic acid, CpG and the like are added into a diluent, the peste des petits ruminant inactivated vaccine is used for secondary immunization, the level of the prepared peste des petits ruminant positive serum antibody can reach 1: 1280-1: 5120, and the titer is improved by 4-8 times.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing peste des petits ruminants positive serum. Background Art

[0002] Peste des petits ruminants, commonly known as sheep plague, is also known as pseudo-rinderpest of small ruminants, pneumococcal enteritis, and stomatitis pneumococcal complex. It is an acute viral infectious disease caused by the peste des petits ruminants virus. It mainly infects small ruminants and is characterized by fever, stomatitis, diarrhea, and pneumonia.

[0003] Peste des petits ruminants virus belongs to the genus Morbillivirus of the family Paramyxoviridae. It has similar physical, chemical and immunological properties to rinderpest virus. The virus is pleomorphic and usually rough and spherical. The virus particles are larger than those of rinderpest virus, and the nucleocapsid is a spiral hollow rod with characteristic subunits and an envelope. The virus can proliferate in fetal sheep kidney, testicular cells of fetal and newborn sheep, and Vero cells, and produce cytopathic effects (CPE) and form syncytia.

[0004] The incubation period of PPR is 4-5 days, with a maximum of 21 days. Natural disease is only seen in goats and sheep. Goats are severely ill, and sheep occasionally have severe cases. Some recovered goats develop aphthous lesions on their lips. The clinical symptoms of infected animals are similar to those of cattle with rinderpest. The acute body temperature can rise to 41°C and last for 3 to 5 days. Infected animals are restless, their back hair is dull, their mouth and nose are dry, and their appetite is reduced. They have mucopurulent nasal discharge and exhale foul-smelling gases. In the first 4 days of fever, the oral mucosa is congested, the buccal mucosa is progressively and extensively damaged, and salivates. Subsequently, necrotic lesions appear. Small, rough, red, superficial necrotic lesions appear in the oral mucosa, which later turn pink. The infected sites include the lower lip, lower gums, etc. In severe cases, necrotic lesions can be seen affecting the tooth pads, palate, cheeks, nipples, tongue, etc. In the later stage, bloody watery diarrhea, severe dehydration, weight loss, and a subsequent drop in body temperature occur. Coughing and abnormal breathing occur. The morbidity rate is as high as 100%. In severe outbreaks, the mortality rate is 100%. In mild outbreaks, the mortality rate does not exceed 50%. The disease is serious in young animals, with high morbidity and mortality. It is a Class I disease in my country. Positive serum is required for pathogen diagnosis and vaccine quality inspection of PPR, and positive serum can be extracted from vaccine-immunized animals. At present, the preparation method of PPR positive serum mainly relies on the following technical means: Naturally infected animal serum: Some research institutions or laboratories collect blood from animals naturally infected with PPRV and separate positive serum. However, this method has uncontrollable factors, such as large differences in antibody titers between individuals, low serum quality stability, and difficulty in ensuring safety, and there is a risk of spreading other pathogens.

[0005] Vaccine-immunized animal serum: Another common method is to immunize healthy animals with PPR vaccine (weak strain or inactivated vaccine), then collect blood and separate serum. Although this method can improve the uniformity of antibody titers to a certain extent, it still has the following problems: The immune effect of the vaccine is unstable: different batches of vaccines may cause fluctuations in serum antibody titers, affecting its reliability as a standard positive serum.

[0006] The immune response time is long: Traditional methods take a long time to obtain antibodies of sufficient titer, affecting the efficiency of serum preparation.

[0007] Single diluent formula: Existing technologies usually use a single PBS solution as a vaccine diluent without fully considering auxiliary components that can enhance immune responses, which may result in low or unstable antibody levels.

[0008] Therefore, we proposed a method for preparing PPR-positive serum to solve the above problems. Summary of the invention

[0009] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0010] In view of the problems existing in the above-mentioned existing methods for preparing PPR-positive serum, the present invention is proposed.

[0011] Therefore, the purpose of the present invention is to provide a method for preparing small ruminant peste des petits ruminants positive serum, which comprises the following steps: first immunization is performed with a small ruminant freeze-dried vaccine, transfer factor, poly I-C, CpG, etc. are added to the diluent, and second immunization is performed with a small ruminant inactivated vaccine. The antibody level of the prepared small ruminant positive serum can reach 1:1280-1:5120, and the titer is increased by 4-8 times.

[0012] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a positive serum of peste des petits ruminants, comprising the following steps: Step 1: Vero cell culture: take out the Vero cell basic seeds from liquid nitrogen, thaw quickly in a 37°C water bath, centrifuge at 1000r / min for 10 minutes, discard the supernatant, prepare the precipitated cells with MEM nutrient solution containing 10% newborn calf serum into a cell suspension, and culture in a 37°C, 5% CO2 incubator for 3 to 4 days. After the cells have grown into a monolayer, digest and disperse them for passage; Step 2: Propagation of PPR virus strains: hydrate the freeze-dried PPR virus strains with serum-free MEM nutrient solution according to the amount indicated on the bottle label, inoculate Vero cell monolayers at 1% to 3% of the weight of the cell growth solution, adsorb at 37°C for 1 hour, add MEM cell culture solution containing 2% newborn calf serum, and culture at 37°C for 4 to 6 days. When the cells show confluent cytopathic effects of more than 70%, move the cell bottle to -20°C, freeze and thaw repeatedly for 3 times, harvest the virus solution, aliquot and freeze at -20°C for storage; Step 3: Cultivation of PPR virus: add 1% of the weight of the virus solution propagated by the poisoned seed in step 2 to MEM cell maintenance solution containing 2% newborn calf serum, inoculate the Vero cell square bottle or spinner bottle that has grown into a good monolayer, and culture it statically at 37°C or rotate it at 8-10 rpm for 5-6 days. When the degree of cell cytopathic effect reaches more than 70%, move the cell bottle to -20°C, freeze and thaw it three times, harvest the virus solution, take samples for semi-finished product inspection, freeze and store the harvested virus solution at -20°C, and test the content of PPR virus; Step 4: Preparation of the finished product of PPR live vaccine: mix the PPR virus liquid and lyophilization protective agent in equal amounts and package them, and freeze-dry them in a vacuum; Preparation of PPR inactivated vaccine: add 0.1% formaldehyde solution to the PPR virus liquid that has passed the inspection, inactivate it at 37°C for 8 hours, add immune adjuvant at a ratio of 1:1, stir at a low speed for 5 minutes and then package them; Step 5: Preparation of PPR live vaccine diluent, respectively preparing PBS solutions of different components, including: preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% transfer factor to form transfer factor diluent; preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% poly I-C to form poly I-C diluent; preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% CpG to form CpG diluent. After the preparation of PPR live vaccine diluent, animals need to be immunized; Step 6: Blood collection and serum separation: Blood was collected from the jugular vein of the immune sheep and placed at 37°C for 60 minutes. After the blood coagulated, it was centrifuged at 3000 rpm for 15 minutes, and the serum was aspirated and stored at -20°C.

[0013] As a preferred embodiment of the method for preparing the PPR positive serum of the present invention, wherein: in the step 3, the virus content, specificity and purity are detected, and the virus content should be not less than 10 5.5 TCID 50 The specificity should comply with the regulations and there should be no contamination by bacteria, mold, mycoplasma and exogenous viruses.

[0014] As a preferred embodiment of the method for preparing the PPR positive serum of the present invention, the specific operation of the PPR virus content detection in step 3 is as follows: the virus solution is serially diluted 10 times with serum-free MEM cell culture medium, and 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Five dilutions were inoculated into 96-well cell culture plates. Each dilution was inoculated into 5 wells, 0.1 ml per well, and 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 / ml was added to each well. At the same time, a normal cell control was set up. The cells were cultured in a 37°C, 5% CO2 incubator for 6 days. During this period, the cell lesions were observed daily. According to the number of wells with cell lesions, the TCID was calculated according to the Reed-Muench method. 50 The virus content per 1ml should not be less than 10 5.5 TCID 50 .

[0015] As a preferred embodiment of the method for preparing the positive serum of PPR of the present invention, the step 6 also includes the preparation of the positive serum, which specifically includes the following steps: S1. Inject 2 ml of PPR virus solution subcutaneously into the neck of goats with negative PPR antibodies. Perform a second immunization with the same dose 14 days later. Take blood from the jugular vein 14 days after the second immunization to test PPR antibodies. For those with antibodies above 1:320, take blood and separate serum, which is frozen or freeze-dried for future use. S2. Dilute 1 bottle of PPR live vaccine with 2 ml transfer factor diluent, inject subcutaneously into the neck of PPR antibody negative goats, and inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization 14 days later. Draw blood from the jugular vein 14 days after the second immunization to detect PPR antibody. Draw blood from the goats with antibody above 1:640 to separate serum, and freeze or freeze-dry for storage; S3. Dilute 1 bottle of PPR live vaccine with 2 ml of poly Ion-cell diluent and inject subcutaneously into the neck of PPR antibody-negative goats. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to detect PPR antibody. If the antibody is above 1:640, collect blood to separate serum, and freeze or freeze-dry for storage. S4. Dilute 1 bottle of PPR live vaccine with 2 ml CpG diluent and inject subcutaneously into the neck of goats with negative PPR antibodies. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to test for PPR antibodies. If the antibody level is above 1:640, collect blood to separate the serum, and freeze or freeze-dry it for later use.

[0016] As a preferred scheme for the preparation method of the peste des petits ruminants positive serum of the present invention, it is found through positive serum experiments that transfer factor, poly I-C and CpG can all improve the immune effect of small ruminant vaccines, and the immune effect of inactivated vaccines is much higher than that of live vaccines.

[0017] As a preferred embodiment of the method for preparing the positive serum of PPR of the present invention, the serum neutralizing antibodies need to be detected in step 6, and the specific detection steps are as follows: A1. Dilute the immune sheep serum and positive control serum with serum-free MEM cell culture medium at 1:5, and then perform a 2-fold serial dilution; dilute the negative serum at 1:2, 1:4, and 1:8; A2. Add serum of each dilution to a 96-well cell culture plate, inoculate 5 wells of each dilution, 0.1 ml per well, and set up a virus regression control; add 100 TCID 50 / 0.1ml virus working solution 0.1ml, add 0.2ml serum-free MEM cell culture medium to the cell control well, add 0.1ml serum-free MEM cell culture medium to the virus control well, and act at 37℃ for 1 hour; A3. Add 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 cells / ml and culture in a 37°C, 5% CO2 incubator for 6 days. Observe cell lesions daily during this period.

[0018] As a preferred embodiment of the method for preparing the positive serum of PPR of the present invention, the test conditions for the detection of serum neutralizing antibodies are as follows: 10TCID 50 The virus control and negative serum control wells above should all show cell fusion cytopathic effects unique to PPR virus; 1TCID 50 Some wells of the virus control showed cytopathic effects; 0.1TCID 50 Virus regression control, positive serum control, cell wells with dilution below 1:160 and cell control wells should show no cytopathic effects.

[0019] As a preferred embodiment of the method for preparing the positive serum for PPR described in the present invention, the neutralizing antibody test of the serum is determined by recording the number of cytopathic wells at each dilution of the tested serum, and the highest dilution of serum with no cytopathic effect in all five test wells is taken as the neutralizing antibody titer.

[0020] The beneficial effects of the present invention are as follows: by using a conventional method, i.e., using a small ruminant virus liquid for primary and secondary immunizations, the prepared small ruminant positive serum antibody level can reach 1:320-1:640; while by using the improved method, i.e., using a small ruminant freeze-dried vaccine for primary immunization, adding transfer factor, poly I-C, CpG, etc. in the diluent, and using a small ruminant inactivated vaccine for secondary immunization, the prepared small ruminant positive serum antibody level can reach 1:1280-1:5120, and the titer is increased by 4-8 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them: Figure 1 The present invention is a schematic diagram of the steps of the method for preparing the positive serum of Peste des petits ruminants. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0023] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0024] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0025] Reference Figure 1 , provides a method for preparing a positive serum for PPR, comprising the following steps: Step 1: Vero cell culture: take out the Vero cell basic seeds from liquid nitrogen, thaw quickly in a 37°C water bath, centrifuge at 1000r / min for 10 minutes, discard the supernatant, prepare the precipitated cells with MEM nutrient solution containing 10% newborn calf serum into a cell suspension, and culture in a 37°C, 5% CO2 incubator for 3 to 4 days. After the cells have grown into a monolayer, digest and disperse them for passage; Step 2: Propagation of PPR virus strains: hydrate the freeze-dried PPR virus strains with serum-free MEM nutrient solution according to the volume indicated on the bottle label, inoculate Vero cell monolayers at 1% to 3% of the cell growth solution, adsorb at 37°C for 1 hour, add MEM cell culture solution containing 2% newborn calf serum, and culture at 37°C for 4 to 6 days. When the cells show confluent cytopathic effects of more than 70%, move the cell bottle to -20°C, freeze and thaw repeatedly for 3 times, harvest the virus solution, aliquot and freeze at -20°C for storage; Step 3: Cultivation of PPR virus: add 1% of the virus solution propagated by the poisoned seed in step 2 to MEM cell maintenance solution containing 2% of newborn calf serum, inoculate the Vero cell square bottle or spinner bottle that has grown into a good monolayer, and culture it statically at 37°C or rotate it at 8-10 rpm for 5-6 days. When the degree of cell cytopathic effect reaches more than 70%, move the cell bottle to -20°C, freeze and thaw it three times, harvest the virus solution, take samples for semi-finished product inspection, freeze and store the harvested virus solution at -20°C, and test the PPR virus content; Step 4: Preparation of the finished product of PPR live vaccine: mix the PPR virus liquid and lyophilization protective agent in equal amounts and package them, and freeze-dry them in a vacuum; Preparation of PPR inactivated vaccine: add 0.1% formaldehyde solution to the PPR virus liquid that has passed the inspection, inactivate it at 37°C for 8 hours, add immune adjuvant at a ratio of 1:1, stir at a low speed for 5 minutes and then package them; Step 5: Preparation of PPR live vaccine diluent, respectively preparing PBS solutions of different components, including: preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% transfer factor to form transfer factor diluent; preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% poly I-C to form poly I-C diluent; preparing PBS solution, sterilizing and cooling, aseptically adding 0.1% CpG to form CpG diluent. After the preparation of PPR live vaccine diluent, animals need to be immunized; Step 6: Blood collection and serum separation: Blood was collected from the jugular vein of the immune sheep and placed at 37°C for 60 minutes. After the blood coagulated, it was centrifuged at 3000 rpm for 15 minutes, and the serum was aspirated and stored at -20°C.

[0026] Among them, in step 3, the virus content, specificity and purity are tested, and the virus content should not be less than 10 5.5 TCID 50 The specificity should comply with the regulations and there should be no contamination by bacteria, mold, mycoplasma and exogenous viruses.

[0027] Among them, the specific operation of step 3 is to detect the content of PPR virus: dilute the virus solution in 10-fold series with serum-free MEM cell culture medium, and take 10 -3 , 10 -4 , 10-5 , 10 -6 , 10 -7 Five dilutions were inoculated into 96-well cell culture plates. Each dilution was inoculated into 5 wells, 0.1 ml per well, and 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 / ml was added to each well. At the same time, a normal cell control was set up. The cells were cultured in a 37°C, 5% CO2 incubator for 6 days. During this period, the cell lesions were observed daily. According to the number of wells with cell lesions, the TCID was calculated according to the Reed-Muench method. 50 The virus content per 1ml should not be less than 10 5.5 TCID 50 .

[0028] Among them, the production of PPR virus liquid is shown in Table 1:

[0029] The production status of PPR freeze-dried vaccine is shown in Table 2:

[0030] The production status of PPR inactivated vaccine is shown in Table 3:

[0031] Furthermore, in step 5, the PPR live vaccine diluent is prepared and then used to immunize an animal, and step 6 also includes the preparation of positive serum, which specifically includes the following steps: S1. Inject 2 ml of PPR virus solution subcutaneously into the neck of goats with negative PPR antibodies. Perform a second immunization with the same dose 14 days later. Take blood from the jugular vein 14 days after the second immunization to test PPR antibodies. For those with antibodies above 1:320, take blood and separate serum, which is frozen or freeze-dried for future use. S2. Dilute 1 bottle of PPR live vaccine with 2 ml transfer factor diluent, inject subcutaneously into the neck of PPR antibody negative goats, and inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization 14 days later. Draw blood from the jugular vein 14 days after the second immunization to detect PPR antibody. Draw blood from the goats with antibody above 1:640 to separate serum, and freeze or freeze-dry for storage; S3. Dilute 1 bottle of PPR live vaccine with 2 ml of poly Ion-cell diluent and inject subcutaneously into the neck of PPR antibody-negative goats. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to detect PPR antibody. If the antibody is above 1:640, collect blood to separate serum, and freeze or freeze-dry for storage. S4. Dilute 1 bottle of PPR live vaccine with 2 ml CpG diluent and inject subcutaneously into the neck of goats with negative PPR antibodies. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to test for PPR antibodies. If the antibody level is above 1:640, collect blood to separate the serum, and freeze or freeze-dry it for later use.

[0032] Examples of serum antibody levels after immunization with different methods are shown in Table 4: Table 4

[0033] Among them, in step six, serum neutralizing antibodies need to be tested. The specific testing steps are as follows: A1. Dilute the immune sheep serum and positive control serum with serum-free MEM cell culture medium at 1:5, and then perform a 2-fold serial dilution; dilute the negative serum at 1:2, 1:4, and 1:8; A2. Add serum of each dilution to a 96-well cell culture plate, inoculate 5 wells of each dilution, 0.1 ml per well, and set up a virus regression control; add 100 TCID 50 / 0.1ml virus working solution 0.1ml, add 0.2ml serum-free MEM cell culture medium to the cell control well, add 0.1ml serum-free MEM cell culture medium to the virus control well, and act at 37℃ for 1 hour; A3. Add 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 cells / ml and culture in a 37°C, 5% CO2 incubator for 6 days. Observe cell lesions daily during this period.

[0034] Furthermore, the test conditions for serum neutralizing antibody detection are: 10TCID 50 The virus control and negative serum control wells above should all show cell fusion cytopathic effects unique to PPR virus; 1TCID 50 Some wells of the virus control showed cytopathic effects; 0.1TCID 50 Virus regression control, positive serum control, cell wells with dilution below 1:160 and cell control wells should show no cytopathic effects.

[0035] Furthermore, the determination of serum neutralizing antibody detection is as follows: the number of cytopathic wells at each dilution of the tested serum is recorded, and the highest serum dilution at which no cytopathic effect is observed in all five test wells is taken as the neutralizing antibody titer.

[0036] in conclusion: Using the conventional method, i.e. using small ruminant virus liquid for the first and second immunizations, the prepared small ruminant positive serum antibody level can reach 1:320-1:640; while using the improved method, i.e. using small ruminant freeze-dried vaccine for the first immunization, adding transfer factor, poly I-C, CpG, etc. to the diluent, and using small ruminant inactivated vaccine for the second immunization, the prepared small ruminant positive serum antibody level can reach 1:1280-1:5120, and the titer is increased by 4-8 times.

[0037] Through experiments, it was found that transfer factor, poly I-C and CpG can all improve the immune effect of small ruminant vaccines, and the immune effect of inactivated vaccines is far higher than that of live vaccines.

[0038] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a positive serum for Peste des Petits Ruminants, characterized in that: The following steps are involved: Step 1: Vero cell culture: Take out the Vero cell basic seeds from liquid nitrogen, thaw quickly in a 37°C water bath, centrifuge at 1000r / min for 10 minutes, discard the supernatant, prepare the precipitated cells with MEM nutrient solution containing 10% newborn calf serum into a cell suspension, and culture in a 37°C, 5% CO2 incubator for 3 to 4 days. After the cells have grown into a monolayer, digest and disperse them for passage; Step 2: Propagation of PPR virus strains: hydrate the freeze-dried PPR virus strains with serum-free MEM nutrient solution according to the amount indicated on the bottle label, inoculate Vero cell monolayers at 1% to 3% of the weight of the cell growth solution, adsorb at 37°C for 1 hour, add MEM cell culture solution containing 2% newborn calf serum, and culture at 37°C for 4 to 6 days. When the cells show more than 70% confluent cytopathic effect, move the cell bottle to -20°C, freeze and thaw repeatedly for 3 times, harvest the virus solution, aliquot and freeze at -20°C for storage; Step 3: Cultivation of PPR virus: add 1% of the weight of the virus solution propagated by the poisoned seed in step 2 to MEM cell maintenance solution containing 2% newborn calf serum, inoculate the Vero cell square bottle or spinner bottle that has grown into a good monolayer, and culture it statically at 37°C or rotating at 8-10 rpm for 5-6 days. When the degree of cell pathogenesis reaches more than 70%, move the cell bottle to -20°C, freeze and thaw repeatedly for 3 times, harvest the virus solution, take samples for semi-finished product inspection, freeze and store the harvested virus solution at -20°C, and test the content of PPR virus; Step 4: Preparation of the finished product of PPR live vaccine: Mix the PPR virus liquid and lyophilization protective agent in equal amounts and package them, and freeze-dry them in a vacuum; Preparation of PPR inactivated vaccine: Add 0.1% formaldehyde solution to the PPR virus liquid that has passed the inspection, inactivate it at 37°C for 8 hours, add immune adjuvant at a ratio of 1:1, stir at a low speed for 5 minutes, and then package them; Step 5: preparing the PPR live vaccine diluent, respectively preparing PBS solutions with different components, including: preparing the PBS solution, sterilizing and cooling it, and aseptically adding 0.1% transfer factor to form a transfer factor diluent; Prepare PBS solution, sterilize and cool it, aseptically add 0.1% poly I-C to form poly I-C diluent; prepare PBS solution, sterilize and cool it, aseptically add 0.1% CpG to form CpG diluent, and the prepared PPR live vaccine diluent needs to be used to immunize animals; Step 6: Blood collection and serum separation: Blood was collected from the jugular vein of the immune sheep and placed at 37°C for 60 minutes. After the blood coagulated, it was centrifuged at 3000 rpm for 15 minutes, and the serum was aspirated and stored at -20°C.

2. The method for preparing the peste des petits ruminants positive serum according to claim 1, characterized in that: In step 3, the virus content, specificity and purity are tested, and the virus content should not be less than 10 5.5 TCID 50 The specificity should comply with the regulations and there should be no contamination by bacteria, mold, mycoplasma and exogenous viruses.

3. The method for preparing the PPR-positive serum according to claim 2, characterized in that: The specific operation of the step 3 is to dilute the virus solution in 10-fold series with serum-free MEM cell culture medium, and take 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Five dilutions were inoculated into 96-well cell culture plates. Each dilution was inoculated into 5 wells, 0.1 ml per well, and 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 / ml was added to each well. At the same time, a normal cell control was set up. The cells were cultured in a 37°C, 5% CO2 incubator for 6 days. During this period, the cell lesions were observed daily. According to the number of wells with cell lesions, the TCID was calculated according to the Reed-Muench method. 50 The virus content per 1ml should not be less than 10 5.5 TCID 50 .

4. The method for preparing the peste des petits ruminants positive serum according to claim 1, characterized in that: The step six also includes the preparation of positive serum, which specifically includes the following steps: S1. Inject 2 ml of PPR virus solution subcutaneously into the neck of goats with negative PPR antibodies. Perform a second immunization with the same dose 14 days later. Take blood from the jugular vein 14 days after the second immunization to test PPR antibodies. For those with antibodies above 1:320, take blood and separate serum, which is frozen or freeze-dried for future use. S2. Dilute 1 bottle of PPR live vaccine with 2 ml transfer factor diluent, inject subcutaneously into the neck of PPR antibody negative goats, and inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization 14 days later. Draw blood from the jugular vein 14 days after the second immunization to detect PPR antibody. Draw blood from the goats with antibody above 1:640 to separate serum, and freeze or freeze-dry for storage; S3. Dilute 1 bottle of PPR live vaccine with 2 ml of poly Ion-cell diluent and inject subcutaneously into the neck of PPR antibody-negative goats. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to detect PPR antibody. If the antibody is above 1:640, collect blood to separate serum, and freeze or freeze-dry for storage. S4. Dilute 1 bottle of PPR live vaccine with 2 ml CpG diluent and inject subcutaneously into the neck of goats with negative PPR antibodies. 14 days later, inject 5 ml of PPR inactivated vaccine into the neck muscle for the second immunization. 14 days after the second immunization, collect blood from the jugular vein to test for PPR antibodies. If the antibody level is above 1:640, collect blood to separate the serum, and freeze or freeze-dry it for later use.

5. The method for preparing the PPR-positive serum according to claim 4, characterized in that: Through positive serum experiments, it was found that transfer factor, poly I-C and CpG can all improve the immune effect of small ruminant vaccines, and the immune effect of inactivated vaccines is much higher than that of live vaccines.

6. The method for preparing the PPR-positive serum according to claim 1, characterized in that: In step 6, serum neutralizing antibodies need to be tested, and the specific testing steps are as follows: A1. Dilute the immune sheep serum and positive control serum with serum-free MEM cell culture medium at 1:5, and then perform a 2-fold serial dilution; dilute the negative serum at 1:2, 1:4, and 1:8; A2. Add serum of each dilution to a 96-well cell culture plate, inoculate 5 wells of each dilution, 0.1 ml per well, and set up a virus regression control; add 100 TCID 50 / 0.1ml virus working solution 0.1ml, add 0.2ml serum-free MEM cell culture medium to the cell control well, add 0.1ml serum-free MEM cell culture medium to the virus control well, and act at 37℃ for 1 hour; A3. Add 0.1 ml of Vero cell suspension with a concentration of 200,000 to 300,000 cells / ml and culture in a 37°C, 5% CO2 incubator for 6 days. Observe cell lesions daily during this period.

7. The method for preparing the PPR-positive serum according to claim 6, characterized in that: The test conditions for serum neutralizing antibody detection are as follows: 10TCID 50 The virus control and negative serum control wells above should all show cell fusion cytopathic effects unique to PPR virus; 1TCID 50 Some wells of the virus control showed cytopathic effects; 0.1TCID 50 Virus regression control, positive serum control, cell wells with dilution below 1:160 and cell control wells should show no cytopathic effects.

8. The method for preparing the PPR-positive serum according to claim 7, characterized in that: Determination of the serum neutralizing antibody test: record the number of cytopathic wells at each dilution of the tested serum, and take the highest serum dilution with no cytopathic effect in all 5 test wells as the neutralizing antibody titer.

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