Positive serum for lumpy skin disease virus, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of veterinary biological products, and in particular to a positive serum for a lumpy skin disease virus, and a preparation method therefor and a use thereof. Healthy negative cattle are inoculated with an inactivated vaccine, the number of inoculations is greater than or equal to 2, and the dose of the inactivated vaccine inoculated each time is larger than or equal to 6 mL, so that a positive serum for a lumpy skin disease virus is finally prepared. The positive serum for a lumpy skin disease virus has a neutralizing antibody titer of ≥1:32, and can be fully applicable to each test item involved in the vaccine development process, so that the present invention solves the bottleneck problem of quality control in the development process of inactivated vaccines for lumpy skin diseases, fills the gap in the preparation process of positive serums for a lumpy skin disease virus, and has important significance for long-term development of veterinary biological product industry.

Description

Bovine lumpy skin disease virus positive serum and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, in particular to a bovine lumpy skin disease virus-positive serum and a preparation method and application thereof. Background Art

[0002] Lumpy skin disease (LSD) is a cattle epidemic caused by the lumpy skin disease virus (LSDV) of the Poxviridae family. Clinical manifestations include fever, skin edema, and local hard nodules or ulcers. It can seriously affect the production performance, milk production, and physical condition of cattle (damaging the hide, causing miscarriage and infertility, etc.), causing huge losses to the cattle industry. The development of a vaccine for this infectious disease is extremely urgent.

[0003] During the development of inactivated vaccines, specific serum is required to neutralize the vaccine toxin. Currently, there are no positive serums developed for the LSDV inactivated vaccine on the market. Furthermore, in current research, based on the characteristics of the LSDV virus and reference to existing techniques for preparing positive serum for various viruses, the titers of the positive serum obtained are generally low (less than or equal to 1:16), which cannot meet the testing requirements during vaccine development.

[0004] In view of this, the present invention is proposed.

[0005] Summary of the Invention

[0006] To address the above-mentioned technical problems, the present invention provides a bovine lumpy skin disease virus-positive serum, its preparation method, and its use. The bovine lumpy skin disease virus-positive serum has a neutralizing antibody titer of ≥1:32 and is fully applicable to various testing items involved in vaccine development. This addresses the quality control bottleneck that has plagued the development of inactivated bovine lumpy skin disease vaccines, fills a gap in the bovine lumpy skin disease virus-positive serum preparation process, and is of great significance to the long-term development of the veterinary biological products industry.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a bovine lumpy skin disease virus-positive serum, wherein the neutralizing antibody titer of the bovine lumpy skin disease virus-positive serum is ≥1:32; the bovine lumpy skin disease virus-positive serum is prepared by vaccinating healthy negative cows with inactivated vaccines, and the number of vaccinations is ≥2 times; the dose of inactivated vaccine for each vaccination is ≥6mL / head; preferably, the dose of inactivated vaccine for each vaccination is 8-12mL / head.

[0009] The titer of bovine lumpy skin disease virus-positive serum obtained by existing conventional methods is generally low (≤1:16), which cannot meet the testing requirements during vaccine development. The present invention uses an inactivated vaccine to vaccinate healthy negative cattle, with the number of vaccinations ≥2 times and the inactivated vaccine dose ≥6mL each time, to finally produce a bovine lumpy skin disease virus-positive serum. The neutralizing antibody titer of this bovine lumpy skin disease virus-positive serum is ≥1:32, which can meet the use requirements of various testing items involved in the vaccine development process.

[0010] In a second aspect, the present invention provides a method for preparing bovine lumpy skin disease virus-positive serum. The method uses an inactivated vaccine to vaccinate healthy, negative cattle to produce positive serum with a neutralizing antibody titer of 1:32 or higher. The vaccinations are performed 2 or more times, with a dose of 6 mL / head per vaccination. Preferably, the dose is 8-12 mL / head, and more preferably, 10 mL / head.

[0011] The production process of virus-positive serum must ensure the individual health of the immune animals, and by continuously producing positive serum, the production cost is reduced and economic losses are avoided. Therefore, when using the inactivated bovine nodular dermatitis virus vaccine to immunize healthy negative cattle, the art generally uses a dose of 2-4mL / head for immunization. The present invention uses an overdose injection method to immunize healthy negative cattle, and a relatively high titer of bovine nodular dermatitis virus-positive serum is obtained in a short period of time, which can meet the use requirements of various test items involved in the vaccine development process.

[0012] The present invention uses an inactivated vaccine to inoculate healthy negative cattle. The bovine lumpy skin disease virus content of the inactivated vaccine is preferably ≥0.5×10 6.0 TCID 50 / mL, more preferably ≥0.5×10 7.0 TCID 50 / mL, the inactivated vaccine with this specification can achieve better immune effect, which in turn helps to increase the neutralizing antibody titer of positive serum after immunization.

[0013] The present invention uses an inactivated vaccine to inoculate healthy negative cattle. The inactivated vaccine is preferably obtained by compounding an inactivated antigen stock solution with a viral adjuvant. The antigen stock solution is obtained by inoculating host cells with bovine nodular dermatitis virus for culture. The maintenance liquid formula used in the culture preferably includes 11801-211, V006201-010 and CM-VA001.

[0014] Among them, 11801-211, V006201-010 and CM-VA001 are maintenance solutions purchased from commercial channels. 11801-211 was purchased from Jianshun Biotechnology Co., Ltd.; V006201-010 was purchased from Shanghai Aopuma Biotechnology Co., Ltd.; and CM-VA001 was purchased from Inner Mongolia Jinyuankang Bioengineering Co., Ltd.

[0015] The culture of bovine lumpy skin disease virus is difficult. The virus content in the virus solution obtained by conventional culture is only 10 4.0 TCID 50 / mL, which is not effective when used for subsequent immunization. In the bovine lumpy skin disease virus culture process of the present invention, a bovine lumpy skin disease virus maintenance solution formula comprising 11801-211, V006201-010 and CM-VA001 is preferably used, which can significantly increase the bovine lumpy skin disease virus content in the virus solution.

[0016] In the special maintenance solution formula, 11801-211, V006201-010 and CM-VA001 are preferably prepared according to their respective labeled amounts and then mixed uniformly according to a specific volume ratio. In the present invention, the volume ratio of 11801-211, V006201-010 and CM-VA001 is preferably 1-2:1-2:5-9, more preferably 1.5:1.5:7. This preferred maintenance solution formula is used for bovine lumpy skin disease virus culture, which can increase the virus content in each 1mL of virus solution to 10 7.0 TCID 50 -10 7.5 TCID 50 .

[0017] In the present invention, the antigen stock solution is preferably concentrated and purified using a hollow fiber prior to inactivation. The hollow fiber preferably has a density of 400-600 kD, more preferably 450-550 kD, and even more preferably 500 kD. The antigen stock solution concentrated and purified using hollow fiber ultrafiltration is safer and can be used for superdose immunization of healthy negative cattle according to the present invention, further helping to ensure the health and safety of the immunized animals.

[0018] In the present invention, conventional commercially available viral adjuvants, host cells and inactivators can be used to prepare the inactivated vaccine for positive serum preparation of the present invention.

[0019] Preferably, the viral adjuvant is selected from 206 adjuvant; the host cells are selected from MDBK cells; and the inactivator used in the antigen stock solution is selected from β-propiolactone. Compared to other sources, the preferred 206 adjuvant of the present invention has a more pronounced effect on enhancing immune response, and the viral fluid obtained from MDBK cells has a higher viral content and is obtained more quickly. β-propiolactone effectively inactivates the virus while minimizing the impact on the antigen's properties.

[0020] In a more preferred embodiment provided by the present invention, the steps of preparing the inactivated vaccine include:

[0021] S1: Inoculate MDBK cells with bovine lumpy dermatitis virus and culture at 37°C for 5-8 days. Harvest the virus solution, freeze-thaw three times, and centrifuge at 8000 rpm for 30 minutes. Take the supernatant after centrifugation and concentrate it 7 times by 500KD hollow fiber ultrafiltration. Then wash it three times with sterile PBS to obtain the antigen concentrated filtrate.

[0022] S2: The antigen concentrated elution filtrate obtained in step S1 was inactivated with β-propiolactone solution at a ratio of 1:2000 (v / v) at 4°C for 24 hours, followed by incubation in a water bath at 37°C for 2 hours to obtain an inactivated antigen solution.

[0023] S3: Centrifuge the inactivated antigen solution prepared in step S2 at 8000 rpm for 20 minutes. Collect the supernatant as the aqueous phase. Separately, obtain qualified 206 adjuvant, sterilize it by autoclave, and cool it to 30°C to form the oil phase. The aqueous phase and the oil phase are then mixed in a 1:1 mass ratio and emulsified at 30-33°C for 30 minutes to obtain the inactivated vaccine.

[0024] Furthermore, the present invention uses inactivated vaccines to inoculate healthy negative cattle, and the healthy negative cattle are preferably 2-3 months old.

[0025] More preferably, the antigen and / or antibody test results of the healthy negative cow are negative; the antigen includes bovine lumpy skin disease virus; the antibodies include bovine lumpy skin disease virus antibodies, and also include at least one of the following: bovine foot-and-mouth disease virus antibodies, bovine viral diarrhea / mucosal disease virus antibodies, and bovine infectious rhinotracheitis virus antibodies.

[0026] Further preferably, the antigen and antibody test results of the healthy negative cow are negative; the antigen includes bovine lumpy skin disease virus; the antibodies include bovine lumpy skin disease virus antibodies, bovine foot-and-mouth disease virus antibodies, bovine viral diarrhea / mucosal disease virus antibodies and bovine infectious rhinotracheitis virus antibodies.

[0027] In the production process of positive serum, the immunized animal has a significant impact on the titer of the final product. The preferred healthy negative cattle of the present invention have a stronger immune response to bovine lumpy skin disease virus and can maintain a high antibody titer continuously and stably, resulting in a better quality positive serum.

[0028] In a more preferred embodiment provided by the present invention, the healthy negative cattle are selected from a number of healthy susceptible cattle that are just 3-5 days old. First, venous blood is collected from each of them. Then, the selected cattle are isolated and raised separately. At the same time, the collected blood samples are placed at room temperature for about 2 hours, transferred to 2-8°C and allowed to stand overnight, and then centrifuged at 4000r / min for 10 minutes. The upper serum is collected for antigen (bovine nodular skin disease virus) and antibody (bovine nodular skin disease virus antibody, bovine foot-and-mouth disease virus antibody, bovine viral diarrhea / mucosal disease virus antibody, infectious rhinotracheitis virus antibody) detection. The cattle with negative test results are immediately moved into an animal house that meets the requirements of veterinary GMP for long-term breeding for 2-3 months to obtain healthy negative cattle to be vaccinated.

[0029] Furthermore, the present invention uses an inactivated vaccine to inoculate healthy negative cattle, and the number of vaccinations is preferably two, and the time interval between the first and second vaccinations is preferably 14-28 days, more preferably 14-21 days, and more preferably 21 days; the bovine lumpy skin disease virus-positive serum is harvested on the Nth day after the second vaccination, and the value of N is preferably ≥7, more preferably, the value of N is preferably 14-21, and more preferably 21. The above preferred vaccination procedure helps to obtain higher titer positive serum, and the immunized animals can serve as a blood supply source for positive serum for a longer period of time.

[0030] In a more preferred embodiment provided by the present invention, after the screened negative cattle are raised to 2-3 months of age, 10 mL of the prepared bovine nodular dermatitis inactivated vaccine is injected intramuscularly to each cattle, which is recorded as the first immunization; 21 days after the first immunization, 10 mL of the vaccine is injected intramuscularly to each cattle by the same vaccination route, which is recorded as the second immunization; starting from 14 days after the second immunization, blood is collected every 7 days to detect the LSDV neutralizing antibody titer in the serum. When the antibody value is greater than 1:16, blood can be collected to separate the serum; when the antibody value is lower than 1:16, booster immunization is performed once every month according to the method and dosage of the second immunization until the antibody value is greater than 1:16, and then blood is collected again.

[0031] In the present invention, the blood collection method is preferably the jugular vein full closure blood collection method, and the specific operation is as follows:

[0032] M1: Place the test cow in a standing position, securing its hind legs and head to prevent it from moving or injuring anyone. Shave the hair on one side of the neck with electric scissors; then disinfect with iodine tincture, followed by deiodination with 75% alcohol. After local disinfection, use your dominant hand to locate the jugular vein and use your other hand to apply moderate pressure to the lower neck (neither too light nor too heavy). Once the jugular vein swells due to slowed blood flow, gently insert a special blood collection needle parallel to the jugular vein. The other end of the needle will be connected to a sterile, cleaned, and autoclaved silicone tube, and the other end of the silicone tube will be connected to a sterile blood collection bottle (bag). Once blood is drawn, immediately secure the needle and the upper half of the connected silicone tube to the neck with special tape to prevent the needle from slipping or dislocating. During blood collection, the cow should be frequently moved around or hammered on the heart to increase bleeding. This step yields positive whole blood.

[0033] M2: Place the positive whole blood obtained in M1 at room temperature for 2 hours, then transfer it to 2-8°C and place it at rest for 16-24 hours. The best way to place the blood sample is to allow the maximum area to contact the blood collection bottle (bag). Then, transfer the precipitated serum into a centrifuge tube in a Class II biosafety cabinet in a clean area (10,000-level area), centrifuge it at 6000r / min for 20 minutes, collect the supernatant, and obtain the positive serum.

[0034] M3: After mixing the positive serum obtained from M2 according to the cow number, place it in a 56℃ water bath for 30 minutes, filter and sterilize it using a 0.45μm and 0.22μm two-stage filter element, then take several small samples from each bottle for testing (1ml / bottle), and store the remaining serum at -80℃.

[0035] In the present invention, after blood collection, the blood sample is preferably tested. The tests preferably include a property test, a sterility test, a mycoplasma test, a neutralization titer assay, and a specificity test. The neutralizing antibody titer is determined using a cell neutralization assay, with the LSD neutralizing antibody titer for each vial of serum being no less than 1:16. The specificity test should be positive for bovine lumpy skin disease virus antibodies and negative for bovine foot-and-mouth disease virus, bovine viral diarrhea / mucosal disease virus, and bovine infectious rhinotracheitis virus antibodies.

[0036] In a third aspect, the present invention provides the bovine lumpy skin disease virus-positive serum, or the use of the bovine lumpy skin disease virus-positive serum obtained by the preparation method, wherein the use comprises at least one of the following methods:

[0037] (1) preparing reagents or kits for testing or identifying bovine lumpy skin disease virus;

[0038] (2) Used for exogenous virus detection or cell neutralization detection in the production of inactivated vaccines for bovine lumpy skin disease;

[0039] (3) Prepare bovine lumpy skin disease virus positive serum standard. Beneficial effects

[0040] The present invention provides a specific bovine lumpy skin disease virus-positive serum, its preparation method, and application. The bovine lumpy skin disease virus-positive serum has a neutralizing antibody titer of ≥1:32, making it fully applicable to various testing items involved in vaccine development, thereby resolving the quality control bottleneck that has plagued the development of inactivated bovine lumpy skin disease vaccines. The preparation method is particularly suitable for the preparation of bovine lumpy skin disease virus-positive serum, filling a gap in the industry for the preparation of this virus-positive serum and having important implications for the long-term development of the veterinary biological products industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0042] Figure 1 is an electrophoretogram of PCR detection of bovine lumpy skin disease virus antigen in Example 2 of the present invention. "M" represents DL2000 DNA Marker; "+" represents a positive control; "-" represents a negative control; lanes 1 to 20 represent the following cow numbers: 230006 / 5190 / 1659 / 5408 / 6108 / 5465 / 5902 / 6025 / 5331 / 5328 / 5985 / 5301 / 5311 / 5703 / 5237 / 6035 / 5115 / 5383 / 5401 / 5387, all of which were negative by PCR.

[0043] Figure 2 shows the pathological changes of MDBK cells after neutralization of LSDV with sera of different titers in Experimental Example 2 of the present invention. Panel A shows normal cell morphology after neutralization of LSDV; panel B shows pathological changes after non-neutralization of LSDV. DETAILED DESCRIPTION

[0044] The present invention provides a bovine nodular dermatitis virus-positive serum, and its preparation method and application. The positive serum is mainly used for quality control during the development or production of an inactivated vaccine for bovine nodular dermatitis. The positive serum uses an inactivated bovine nodular dermatitis vaccine as an immunogen, selects healthy susceptible cattle aged 2-3 months for multiple immunizations, and uses a fully enclosed mode to collect blood and obtain it after serum separation. The serum has a high neutralizing antibody titer (≥1:32), and the preparation process includes the preparation of an inactivated vaccine, screening of negative animals, immunization and blood collection to detect antibody levels, serum collection and separation, and serum testing.

[0045] Healthy negative cattle are isolated and housed in GMP animal facilities from birth to avoid infection with other pathogens. After multiple immunizations with overdose inactivated vaccines, positive serum is obtained from these healthy negative cattle. This positive serum is highly specific, sterile, free of mycoplasma, and free of foreign viral contamination. Neutralizing antibody titers are high (≥1:32).

[0046] The bovine lumpy skin disease virus positive serum prepared by the present invention can effectively neutralize LSDV. When the positive serum titer is greater than or equal to 1:32, it can neutralize 10 6.50 TCID 50 / mL or more LSDV; when the titer is diluted to 1∶16, it can still neutralize 10 6.50 TCID 50 This indicates that the bovine lumpy skin disease virus-specific positive serum prepared by the present invention can meet the requirements of the exogenous virus test of LSDV.

[0047] The bovine lumpy skin disease virus positive serum prepared by the invention can be used as a positive control serum in a cell neutralization test during the development of an inactivated vaccine for bovine lumpy skin disease.

[0048] The bovine lumpy skin disease virus positive serum preparation process of the present invention comprises the steps of preparing an inactivated vaccine, screening negative animals, immunizing and collecting blood to detect antibody levels, collecting and separating serum, and testing serum.

[0049] In some embodiments, the preparation of the inactivated vaccine of the present invention is achieved by the following steps:

[0050] S1: Inoculate MDBK cells with bovine lumpy dermatitis virus and culture at 37°C for 5-8 days. Harvest the virus solution, freeze-thaw three times, and centrifuge at 8000 rpm for 30 minutes. Take the supernatant after centrifugation and concentrate it 7 times by 500KD hollow fiber ultrafiltration. Then wash it three times with sterile PBS to obtain the antigen concentrated filtrate.

[0051] S2: The antigen concentrated elution filtrate obtained in step S1 was inactivated with β-propiolactone solution at a ratio of 1:2000 (v / v) at 4°C for 24 hours, followed by incubation in a water bath at 37°C for 2 hours to obtain an inactivated antigen solution.

[0052] S3: Centrifuge the inactivated antigen solution prepared in step S2 at 8000 rpm for 20 minutes. Collect the supernatant as the aqueous phase. Separately, obtain qualified 206 adjuvant, sterilize it by autoclave, and cool it to 30°C to form the oil phase. The aqueous phase and the oil phase are then mixed in a 1:1 mass ratio and emulsified at 30-33°C for 30 minutes to obtain the inactivated vaccine.

[0053] In some embodiments, the negative animal screening described in the present invention is achieved by:

[0054] Several healthy, susceptible cattle, 3-5 days old, were selected for venous blood sampling. The selected cattle were then isolated and housed separately. The collected blood samples were allowed to stand at room temperature for approximately 2 hours, then transferred to a 2-8°C refrigeration unit and allowed to rest overnight. The samples were then centrifuged at 4000 rpm for 10 minutes, and the upper serum was collected for antigen and antibody testing. Upon receipt of the results, the selected negative cattle were immediately moved to a long-term animal house that complies with veterinary GMP requirements. The animal house was rigorously disinfected before the experiment, and during the experiment, environmental hygiene and personnel flow were strictly controlled to prevent cross-contamination.

[0055] The antigen detection items described in the negative animal screening in the present invention are: bovine lumpy dermatitis virus antigen PCR detection, and the detection method is the ordinary PCR method described in GBT39602-2020; other exogenous virus detections required by the "Chinese Veterinary Pharmacopoeia", and the detection method refers to the method described in the "Veterinary Pharmacopoeia".

[0056] The antibody detection items described in the negative animal screening of the present invention include: bovine lumpy skin disease virus antibody detection, the detection method is the cell neutralization detection method described in the "Chinese Veterinary Pharmacopoeia", and the antibody value is required to be no higher than 1:4 to be judged as negative; bovine foot-and-mouth disease virus antibody detection, detected by ELISA method, purchased from Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; bovine viral diarrhea / mucosal disease virus antibody detection, detected by ELISA method, purchased from Beijing IDEXX Biotechnology Co., Ltd. (IDEXX, item number: P00645-5); bovine infectious rhinotracheitis virus antibody detection, detected by ELISA method, purchased from Beijing IDEXX Biotechnology Co., Ltd. (IDEXX, item number: 99-41459). The ELISA test is carried out according to the kit instructions. If the test result of any one of the test items is positive, the corresponding animal will not be used as the experimental animal of this study, and the serum sample can no longer be tested for other items.

[0057] In some embodiments, the immunization and blood collection for antibody level detection described herein are achieved by the following method: after screened negative cattle are raised to 2-3 months of age, 10 mL of the prepared inactivated bovine nodular dermatitis vaccine is injected intramuscularly into each cattle, marking it as the first vaccination; 21 days after the first vaccination, 10 mL of the same vaccination route is injected intramuscularly into each cattle, marking it as the second vaccination; starting 14 days after the second vaccination, blood is collected every 7 days to detect the LSDV neutralizing antibody titer in the serum. When the antibody value is greater than 1:16, blood can be collected and serum can be separated; when the antibody value is less than 1:16, booster immunization is performed once every month according to the method and dosage of the second vaccination. Blood collection is then resumed until the antibody value exceeds 1:16. Since the cattle are isolated and raised in a clean area from birth, they can effectively avoid infection from other pathogens. The prepared positive serum has strong specificity, and after vaccination, the titer produced is higher (compared to healthy susceptible cattle raised in a natural environment).

[0058] In some embodiments, the serum collection and separation described herein is performed by collecting bovine blood using the jugular vein blood collection method. The specific steps are:

[0059] Animal restraint: After the test cattle to be blooded are driven into the special corridor for restraining test cattle, their hind legs and heads are restrained with special ropes to prevent them from moving around and hurting people.

[0060] Blood collection: Shave one side of the neck with an electric razor. Disinfect with iodine tincture, then deiodine with 75% alcohol. After local disinfection, locate the jugular vein with your dominant hand and apply moderate pressure to the lower neck with your other hand (neither too light nor too heavy). Once the jugular vein swells due to slowed blood flow, gently insert a dedicated blood collection needle parallel to the jugular vein. The other end of the needle will be connected to a sterile, cleaned, and autoclaved silicone tube, which in turn will be connected to a sterile blood collection bottle (bag). Once blood is drawn, secure the needle and the upper half of the attached silicone tube to the neck with special tape to prevent the needle from slipping or becoming dislocated. During blood collection, the cow should be frequently moved around or hammered on the heart to increase bleeding. Each cow uses a separate set of blood collection equipment.

[0061] Preparation for serum separation: After blood collection, seal the blood collection bottle (bag) and the silicone tube at the blood collection end and let it stand at room temperature for 2 hours. The standing method should be such that the maximum area of ​​blood sample contacts the blood collection bottle (bag); then move it to 2-8℃ and let it stand for 16-24 hours. The standing method is: place the blood collection bottle (bag) at a high place, and the other end of the sterile silicone tube connected to the serum collection bottle is facing downward, so that all the precipitated serum flows into the serum collection bottle; then transfer the precipitated serum into a centrifuge tube in a Class II biosafety cabinet in a clean area (10,000-level area), centrifuge it at 6000r / min for 20 minutes, and take the supernatant for later use.

[0062] Serum testing and storage: After the separated serum is mixed, it is placed in a 56℃ water bath for 30 minutes, filtered and sterilized using a 0.45μm and 0.22μm two-stage filter cartridge, and then several small samples (1ml / bottle) are taken from each bottle for testing. The remaining serum is stored at -80℃.

[0063] During the preparation of positive serum, existing blood collection techniques for large animals (cattle) are highly traumatic and expensive. The blood collection method employed in the present invention effectively reduces trauma, maintains healthy growth of the experimental animals after blood collection, allows for multiple blood collections, and allows for use in other experiments, effectively reducing testing costs. Furthermore, the blood collection process is completely closed, helping to avoid contamination; the cattle remain in a normal standing position, allowing for a large blood volume to be collected.

[0064] In some embodiments, the serum testing of the present invention includes property testing, sterility testing, mycoplasma testing, neutralization titer determination, and specificity testing.

[0065] Properties: Randomly draw 5 tubes of serum and observe with the naked eye. It should be a light yellow transparent liquid.

[0066] Sterility test: Randomly draw 5 tubes of serum and test according to the appendix of the current "Chinese Veterinary Pharmacopoeia". There should be no sterile growth.

[0067] Mycoplasma test: Randomly draw 5 tubes of serum and test according to the appendix of the current "Chinese Veterinary Pharmacopoeia". There should be no mycoplasma growth.

[0068] Neutralizing antibody titer: Five tubes of serum were randomly selected and tested using the cell neutralization method. The LSD neutralizing antibody titer of each bottle of serum was not less than 1:16.

[0069] Specificity: The antibodies to bovine lumpy skin disease virus should be positive, and the antibodies to bovine foot-and-mouth disease virus, bovine viral diarrhea / mucosal disease virus, and bovine infectious rhinotracheitis virus should all be negative.

[0070] In some embodiments, the positive serum prepared by the present invention is used as a positive control serum in a cell neutralization test. The test method is as follows: first, the neutralization LSDV virus is diluted to 200 TCID in DMEM medium containing 2% newborn calf serum. 50 / 0.1ml; then, non-immune bovine (or guinea pig) serum and negative serum were diluted 1:2, 1:4, and 1:8 with DMEM culture medium containing 2% newborn calf serum. Positive serum and immune guinea pig serum were serially diluted 2-fold to 1:64 (or the dilution was increased appropriately according to the actual situation). The serum to be tested and the positive and negative sera at each dilution were added to a 96-well cell culture plate, with 4 wells of each dilution inoculated, and 50μl per well. In addition, 4 wells each of normal cell control and virus neutralization control were set up without sample addition; then, 200TCID were added to each serum to be tested, virus neutralization control, negative serum, and positive serum. 50 Add 50 μl / well of 0.1ml of neutralizing virus working solution. Add 50 μl / well of DMEM culture medium containing 2% newborn calf serum to each neutralizing virus control well, and add 100 μl / well of DMEM culture medium containing 2% newborn calf serum to the normal cell control well. After shaking to mix, incubate at 37°C for 1 hour, shaking 2-3 times during the process. Finally, add 100 μl / well of MDBK cell suspension containing 8-10% newborn calf serum at a concentration of approximately 200,000 cells / ml to all wells. Incubate at 37°C, 5% CO2 in a 5% CO2 incubator for 5-8 days. Observe cytopathic effects daily using an inverted microscope.

[0071] In some embodiments, the positive serum prepared by the present invention can still maintain its original titer after being stored at -80°C for one year, which can ensure the smooth progress of the test items using positive serum such as cell neutralization.

[0072] The present invention solves the bottleneck problem of the lack of standard positive serum in the quality control system (such as virus species identification and differentiation) that has plagued the development of inactivated vaccines for bovine nodular dermatitis. It is of great significance for improving the quality of inactivated vaccines for bovine nodular dermatitis and the level of epidemic prevention and control of bovine nodular dermatitis in my country.

[0073] The present invention adopts pure virus seeds with high virus content to prepare inactivated vaccines, thereby ensuring the purity and immune effect of positive serum.

[0074] The immune animals used in the present invention have been raised in GMP animal houses, which further ensures the purity of the positive serum.

[0075] The present invention adopts a multiple immunization method and regularly monitors the antibody level to ensure high titer of positive serum.

[0076] During the blood collection process, the present invention adopts a fully closed mode for blood collection. During the entire blood collection process, there is no contact with the outside world, which further reduces the risk of positive serum being contaminated with other pathogens (or microorganisms); at the same time, the prepared serum is comprehensively tested to ensure the quality of the positive serum.

[0077] The positive serum prepared by the present invention is suitable for testing bovine nodular dermatitis virus species and exogenous viruses in products, as well as identification testing and the like.

[0078] The following examples provide a detailed description of the technical solutions provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available.

[0079] Example 1

[0080] This embodiment provides a method for preparing bovine lumpy skin disease virus positive serum, which includes the steps of inactivated vaccine preparation, negative animal screening, vaccination and immunization, and serum separation.

[0081] The steps for preparing the inactivated vaccine are as follows:

[0082] (1) Antigen preparation: Bovine nodular dermatitis virus was inoculated into MDBK cells at 2-5% (v / v), and a special maintenance solution (11801-211, V006201-010 and CM-VA001 were mixed, first prepared according to the labeled amount of each maintenance solution, and then mixed evenly at a volume ratio of 1.5:1.5:7) was used. The cells were cultured at 37°C for 5-8 days and harvested when the cytopathic effect (CPE) reached 80% or more. The harvested virus solution was frozen and thawed three times, centrifuged at 8000 r / min for 30 minutes to remove cell debris, and samples were taken for TCID detection. 50 The virus content per 1ml should be ≥10 6.0 TCID 50 At the same time, the supernatant after centrifugation was concentrated 7 times by 500KD hollow fiber ultrafiltration, and then washed 3 times with sterile PBS to obtain the antigen concentrated filtrate.

[0083] Among them, taking the antigen preparation process of batch number LSD22001 as an example, the antigen production details are shown in Table 1; the virus content and sterility test results of the antigen liquid are shown in Table 2.

[0084] Table 1 Antigen production details

[0085] Table 2 Antigen solution virus content and sterility test results

[0086] (2) Antigen inactivation: β-propiolactone solution was added to the above-mentioned antigen concentrated filtrate at a ratio of 1:2000 (v / v), and inactivated at 4°C for 24 h (stirring evenly every 120 min), followed by incubation at 37°C in a water bath for 2 h to obtain an inactivated antigen solution.

[0087] Taking the antigen of batch number LSD22001 as an example, the inactivated antigen liquid was subjected to an inactivation test. The inactivation test results are shown in Table 3.

[0088] Table 3 Antigen liquid inactivation test results

[0089] (3) Vaccine preparation

[0090] Preparation of aqueous phase: Take the qualified inactivated antigen solution prepared in step (2), centrifuge it at 8000r / min for 20min, and take the supernatant as the aqueous phase.

[0091] Preparation of oil phase: Take the qualified 206 adjuvant, sterilize it by high pressure, and cool it to 30℃ as the oil phase.

[0092] Emulsification: Mix the water phase and the oil phase in a mass ratio of 1:1 (add the water phase to the oil phase) and emulsify at 30-33°C for 30 minutes. Aliquot the emulsified vaccine according to the indicated amount, label it, and store it at 2-8°C until ready for use.

[0093] (4) Inspection: The properties inspection, sterility inspection, safety inspection and efficacy inspection are carried out respectively. All inspection methods are carried out in accordance with the methods described in the Chinese Veterinary Pharmacopoeia.

[0094] Taking the vaccine prepared with inactivated antigen batch number LSDM22001 (batch number 22001) as an example, the test results are shown in Tables 4, 5 and 6.

[0095] Table 4 Inactivated vaccine properties, filling inspection, and sterility test results

[0096] Table 5 Safety test results

[0097] Table 6 Statistical results of cattle efficacy test (immune challenge method)

[0098] The test results show that this batch of vaccine has passed all tests and can be stored at 2-8℃ for future use.

[0099] Example 2

[0100] This embodiment provides a method for preparing bovine lumpy skin disease virus positive serum, which includes the steps of inactivated vaccine preparation, negative animal screening, vaccination and immunization, and serum separation.

[0101] Among them, the negative animal screening steps are as follows:

[0102] Select several healthy susceptible cattle that are 3-5 days old, draw blood from their veins, and then isolate and raise the selected cattle. Place the collected blood samples at room temperature for about 2 hours, move them to 2-8℃ and let them stand overnight, then centrifuge them at 4000r / min for 10 minutes, and draw the upper serum for antigen and antibody detection.

[0103] The antigen detection items are: bovine lumpy skin disease virus antigen PCR detection, and the detection method is the ordinary PCR method described in GBT39602-2020; other exogenous virus detection required by the "Chinese Pharmacopoeia", the detection method refers to the method described in the "Veterinary Pharmacopoeia".

[0104] Antibody testing included: bovine lumpy skin disease virus (LSDV) antibody testing, using the cell-based neutralization assay described in the Chinese Veterinary Pharmacopoeia; a negative antibody test was defined as an antibody value no higher than 1:4; foot-and-mouth disease virus (FMDV) antibody testing, using an ELISA assay purchased from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; bovine viral diarrhea / mucosal disease virus (BVDV) antibody testing, using an ELISA assay purchased from Beijing IDEXX Biotechnology Co., Ltd. (Cat. No. P00645-5); and bovine infectious rhinotracheitis virus (IBRV) antibody testing, using an ELISA assay purchased from Beijing IDEXX Biotechnology Co., Ltd. (Cat. No. 99-41459). ELISA testing was performed according to the kit instructions.

[0105] If any test result in the test items is positive, the corresponding animal will not be used as the experimental animal in this study, and the serum sample will no longer be tested for other items.

[0106] In this example, 20 cattle were screened from the cooperative ranch of Jinyu Baoling Biological Pharmaceutical Co., Ltd., and the serum test results are shown in Table 7.

[0107] Table 7 Negative serum test results

[0108] Two of the cows were selected for the experiment, numbered 23006 and 5328. They were then moved to an animal house that complies with veterinary GMP requirements (this experiment was conducted in the P10 animal house at Jinyu Baoling Biological Pharmaceutical Co., Ltd.). The animal house was rigorously disinfected before the experiment, and during the experiment, environmental hygiene and personnel flow in the animal house were strictly controlled to avoid cross-contamination.

[0109] Example 3

[0110] This embodiment provides a method for preparing bovine lumpy skin disease virus positive serum, which includes the steps of inactivated vaccine preparation, negative animal screening, vaccination and immunization, and serum separation.

[0111] The immunization procedures are as follows:

[0112] The bovine nodular dermatitis inactivated vaccine (batch number: 22001) prepared in Example 1 and the negative cattle screened in Example 2 (cow numbers: 23006 and 5328) were used. The screened negative cattle were raised to 2-3 months of age, and then 10 mL of the prepared bovine nodular dermatitis inactivated vaccine was injected intramuscularly into each cow, which was recorded as one immunization; 21 days after the first immunization, 10 mL was injected intramuscularly into each cow by the same vaccination route, which was recorded as the second immunization.

[0113] Starting 14 days after the second vaccination, blood samples were collected every 7 days to detect the LSDV neutralizing antibody titer in the serum. When the antibody value was greater than 1:16, blood samples were collected to separate the serum (when the antibody value was lower than 1:16, booster immunization was performed once every month according to the method and dosage of the second vaccination until the antibody value was greater than 1:16, and then blood samples were collected to separate the serum).

[0114] In this example, the antibody value test results 14 and 21 days after the second immunization are shown in Table 8.

[0115] Table 8 Neutralizing antibody detection results at different immunization times

[0116] The antibody values ​​of all blood samples were not less than 1:32. At the same time, the LSDV antigen was negative, meeting the technical requirements.

[0117] Example 4

[0118] This embodiment provides a method for preparing bovine lumpy skin disease virus positive serum, which includes the steps of inactivated vaccine preparation, negative animal screening, vaccination and immunization, and serum separation.

[0119] In this example, based on Example 3, 21 days after immunization, bovine blood was collected by jugular vein blood sampling, and then the serum was separated and tested.

[0120] The serum separation procedure is as follows:

[0121] After blood collection is completed, the blood collection bottle (bag) and the silicone tube at the blood collection end are sealed and allowed to stand at room temperature for 2 hours. The standing method should be such that the maximum area of ​​blood sample contacts the blood collection bottle (bag); then, it is moved to 2-8℃ and allowed to stand for 16-24 hours. The standing method is: the blood collection bottle (bag) is placed at a high place, and the other end of the sterile silicone tube connected to the serum collection bottle is facing downward, so that all the precipitated serum flows into the serum collection bottle; then, in a Class II biosafety cabinet in a clean area (10,000-level area), the precipitated serum is transferred into a centrifuge tube, centrifuged at 6000r / min for 20 minutes, and the supernatant is taken for later use.

[0122] In this example, the specific preparation of positive serum is shown in Table 9.

[0123] Table 9 Preparation of positive serum

[0124] After serum separation, it is tested and stored: the separated serum is mixed, placed in a 56°C water bath for 30 minutes, filtered and sterilized using a 0.45μm and 0.22μm two-stage filter element, and then several small samples (1ml / bottle) are taken from each bottle for testing, and the remaining serum is stored at -80°C.

[0125] The test items include property test, sterility test, mycoplasma test, neutralization potency test, and specificity test, as follows:

[0126] Properties: Randomly draw 5 tubes of serum and observe with naked eyes, it should be light yellow transparent liquid;

[0127] Sterility test: Randomly draw 5 tubes of serum and test them according to the appendix of the current Chinese Veterinary Pharmacopoeia. There should be no bacterial growth.

[0128] Mycoplasma test: Randomly draw 5 tubes of serum and test according to the appendix of the current Chinese Veterinary Pharmacopoeia. There should be no mycoplasma growth.

[0129] Neutralizing antibody titer determination: Randomly select 5 tubes of serum and use the cell neutralization method to test. The LSD neutralizing antibody titer of each bottle of serum should not be less than 1:16;

[0130] Specificity test: Bovine lumpy skin disease virus antibodies should be positive, and bovine foot-and-mouth disease virus, bovine viral diarrhea / mucosal disease virus, and bovine infectious rhinotracheitis virus antibodies should all be negative.

[0131] The serum that has passed the test will be packaged separately according to the cow number, labeled with the serum name, batch number, specifications, production date, etc., and stored at -80℃.

[0132] The test results of the two batches of positive sera prepared in this example are shown in Table 10.

[0133] Table 10 Positive serum test results

[0134] The neutralization titers of the two batches of sera were not less than 1:32, and both were light yellow transparent liquids; sterile and mycoplasma-free; no antibodies to bovine foot-and-mouth disease virus, bovine viral diarrhea / mucosal disease virus, and bovine infectious rhinotracheitis virus were detected, and the specificity was good.

[0135] Experimental Example 1

[0136] In this experimental example, two batches of positive sera prepared in Example 4 were selected to neutralize different concentrations of LSDV.

[0137] The results showed that when the titer of positive serum was greater than or equal to 1:32, it could neutralize 10 6.50 TCID 50 / mL or more LSDV; when the titer is diluted to 1∶16, it can still neutralize 10 6.50 TCID 50 / mL of LSDV. This shows that the bovine lumpy skin disease virus-specific positive serum prepared by the process of the present invention can meet the requirements of the exogenous virus test of LSDV toxin.

[0138] Experimental Example 2

[0139] This experimental example provides an application of bovine lumpy skin disease virus positive serum in cell neutralization test during the development of bovine lumpy skin disease inactivated vaccine: two batches of positive serum prepared in Example 4 were selected and used as positive serum control substances for cell neutralization test.

[0140] The test method is: first dilute the neutralizing LSDV virus to 200 TCID in DMEM medium containing 2% newborn calf serum. 50 / 0.1ml; then, non-immune bovine serum (non-immune bovine serum was used for testing in this experiment, and those skilled in the art can choose to replace it with non-immune guinea pig serum based on actual conditions) and negative serum were diluted 1:2, 1:4, and 1:8 with DMEM culture medium containing 2% newborn calf serum, and positive serum and immune guinea pig serum were serially diluted 2-fold to 1:64 (this experiment was diluted according to this standard, and those skilled in the art can appropriately increase the dilution based on actual conditions). The serum to be tested at each dilution, as well as the positive and negative sera, were added to a 96-well cell culture plate, and 4 wells were inoculated for each dilution, with 50μl per well. 4 wells each for normal cell control and neutralization virus control were set up, and no sample was added; then, 200TCID 50 / 0.1ml of neutralizing virus working solution (50μl / well). Add 50μl / well of DMEM culture medium containing 2% newborn calf serum to each neutralizing virus control well, and add 100μl / well of DMEM culture medium containing 2% newborn calf serum to the normal cell control well. After shaking to mix, incubate at 37°C for 1 hour, shaking 2-3 times during the process. Finally, add 100μl / well of MDBK cell suspension containing 8-10% newborn calf serum at a concentration of approximately 200,000 cells / ml to all wells. Incubate at 37°C, 5% CO2 in a 5% CO2 incubator for 5-8 days. Observe cytopathic effects daily using an inverted microscope.

[0141] The test results are shown in Table 11.

[0142] Table 11 Summary of the results of multiple cell neutralization tests

[0143] The results showed that after multiple tests and verification, the potency of the two batches of positive sera was able to maintain the original results (slight fluctuations were normal), and there were no problems such as cytotoxicity caused by the positive sera.

[0144] Experimental Example 3

[0145] In this experimental example, two batches of positive sera prepared in Example 4 were selected, and the titer was tested every 3 months after storage. Five sera were randomly selected for each test. The titer did not decrease after 12 months of testing.

[0146] The neutralizing antibody test results at different storage times are shown in Table 12.

[0147] Table 12 Neutralizing antibody detection results at different storage times

[0148] The results show that the bovine lumpy skin disease virus positive serum prepared by the present invention can ensure that the test items using positive serum such as cell neutralization are carried out smoothly.

[0149] Comparative Example 1

[0150] This comparative example provides a method for preparing bovine lumpy dermatitis virus-positive serum. Compared with Example 1, the only difference is the method for preparing the antigen.

[0151] The antigen preparation method in this comparative example is consistent with the preparation process of Example 1, except that the maintenance solutions used are one or more of 11801-211 (number: A), V006201-010 (number: V), and CM-VA001 (number: C), each of which is prepared according to the labeled amount of each maintenance solution and mixed at a certain volume ratio. The maintenance solution ratios used in this comparative example and the viral content test results of the antigen solutions prepared using the corresponding maintenance solutions are shown in Table 13.

[0152] Table 13 Neutralizing antibody detection results at different immunization times

[0153] In this comparative example, the antibody test results 14 and 21 days after the second vaccination are shown in Table 14.

[0154] Table 14 Neutralizing antibody detection results at different immunization times

[0155] The virus content in the antigen liquid prepared in this comparative example is far lower than the virus content requirement of the antigen liquid for negative cattle vaccination, and the titer of the positive serum finally prepared is ≤1:8, which does not meet the test requirements.

[0156] Comparative Example 2

[0157] This comparative example provides a method for preparing bovine lumpy dermatosis virus-positive serum. Compared with Example 3, the only difference is that 2-3 month old naturally raised cattle are selected for vaccination (purchased from pastures around Hohhot City, and LSDV antigen and antibody tests are both negative).

[0158] In this comparative example, the antibody value test results 14 and 21 days after the second immunization are shown in Table 15.

[0159] Table 15 Neutralizing antibody detection results at different immunization times

[0160] It can be seen that: in this comparative example, naturally raised cattle were selected for vaccination, and the antibody value test results after the second vaccination were significantly lower than those in Example 3.

[0161] Comparative Example 3

[0162] This comparative example provides a method for preparing bovine lumpy skin disease virus-positive serum. Compared with Example 3, the only difference is that 2-3 month old naturally raised cattle are selected for vaccination (purchased from pastures around Hohhot City, and LSDV antigen and antibody tests are both negative); the vaccination dose is 2 ml.

[0163] In this comparative example, the antibody value test results 14 and 21 days after the second vaccination are shown in Table 16.

[0164] Table 16 Neutralizing antibody detection results at different immunization times

[0165] It can be seen that: in this comparative example, naturally raised cattle were selected for vaccination, and the antibody value test results after the second vaccination were significantly lower than those in Example 3.

[0166] The above-described embodiments merely illustrate several implementation methods of the present invention, and are provided to facilitate a specific and detailed understanding of the technical solutions of the present invention. They should not be construed as limiting the scope of the invention patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the scope of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A bovine lumpy skin disease virus positive serum, characterized in that: The neutralizing antibody titer of the bovine nodular skin disease virus positive serum is ≥1:32; the bovine nodular skin disease virus positive serum is obtained by vaccinating healthy negative cows with inactivated vaccines, the number of vaccinations is ≥2 times; the dose of inactivated vaccine for each vaccination is ≥6mL / head.

2. A method for preparing bovine nodular skin disease virus positive serum, characterized in that: Healthy negative cattle were inoculated with inactivated vaccines to obtain positive serum with a neutralizing antibody titer of ≥1:32; the number of inoculations was ≥2 times; and the dose of inactivated vaccine for each inoculation was ≥6 mL / head.

3. The method for preparing bovine nodular dermatosis virus positive serum according to claim 2, characterized in that: The bovine nodular dermatosis virus content of the inactivated vaccine is ≥ 0.5×10 6.0 TCID 50 / mL.

4. The method for preparing bovine nodular dermatosis virus positive serum according to claim 3, characterized in that: The inactivated vaccine is obtained by compounding an inactivated antigen stock solution with a virus adjuvant; the antigen stock solution is obtained by inoculating a host cell culture with bovine nodular dermatosis virus; the maintenance solution formula used in the culture includes 11801-211, V006201-010 and CM-VA001.

5. The method for preparing bovine nodular dermatosis virus positive serum according to claim 4, characterized in that: The antigen stock solution is concentrated and purified by 400-600KD hollow fiber before inactivation; The viral adjuvant is selected from 206 adjuvant; The host cell is selected from MDBK cells; The inactivator used in the antigen stock solution is selected from β-propiolactone.

6. The method for preparing bovine nodular skin disease virus positive serum according to any one of claims 2 to 5, characterized in that: The healthy negative cattle are 2-3 months old.

7. The method for preparing bovine nodular skin disease virus positive serum according to any one of claims 2 to 5, characterized in that: The antigen and / or antibody test results of the healthy negative cattle are negative; The antigens include bovine lumpy skin disease virus; The antibodies include bovine lumpy skin disease virus antibodies, and also include at least one of the following: bovine foot-and-mouth disease virus antibodies, bovine viral diarrhea / mucosal disease virus antibodies, and bovine infectious rhinotracheitis virus antibodies.

8. The method for preparing bovine nodular skin disease virus positive serum according to claim 7, characterized in that: The antigen and antibody test results of the healthy negative cow are negative; the antibodies include bovine lumpy skin disease virus antibodies, bovine foot-and-mouth disease virus antibodies, bovine viral diarrhea / mucosal disease virus antibodies and bovine infectious rhinotracheitis virus antibodies.

9. The method for preparing bovine nodular skin disease virus positive serum according to any one of claims 2 to 5, characterized in that: The inoculation is performed twice; the time interval between the first inoculation and the second inoculation is 14-28 days; the harvest time of the bovine nodular skin disease virus positive serum is the Nth day after the second inoculation, N≥7.

10. The use of the bovine nodular skin disease virus-positive serum according to claim 1, or the bovine nodular skin disease virus-positive serum obtained by the preparation method according to any one of claims 2 to 9, wherein the use comprises at least one of the following methods: (1) preparing reagents or kits for testing or identifying bovine nodular skin disease virus; (2) Used for exogenous virus detection or cell neutralization detection in the production of inactivated vaccines for bovine nodular dermatitis; (3) Prepare bovine lumpy skin disease virus positive serum standard.

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