CN111686245B - Inactivated vaccine for rabbit hemorrhagic disease virus type 2 and preparation method thereof - Google Patents
Inactivated vaccine for rabbit hemorrhagic disease virus type 2 and preparation method thereof Download PDFInfo
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Abstract
The invention provides a rabbit hemorrhagic disease virus type 2 inactivated vaccine and a preparation method thereof, belonging to the technical field of vaccine preparation, wherein the preparation method comprises the following steps: 1) Inoculating a rabbit hemorrhagic disease virus type 2 strain SC2020 to susceptible rabbits; 2) Collecting the viscera tissues of the rabbits which die within 24-96 hours after inoculation and have typical histopathological changes; 3) The organ tissues and physiological saline were mixed at a ratio of 1g: (4-6) mixing in a ratio of mL, homogenizing, and filtering to obtain a tissue homogenate; 4) Mixing the tissue homogenate with physiological saline until the ratio of the organ tissue to the physiological saline is 1g: (18-20) mL to obtain a vaccine antigen, and inactivating the vaccine antigen to obtain the inactivated vaccine for rabbit hemorrhagic disease virus type 2. The inactivated vaccine for rabbit hemorrhagic disease virus type 2 has high safety, good immune effect and simple and convenient process.
Description
Technical Field
The invention belongs to the technical field of vaccine preparation, and particularly relates to a rabbit hemorrhagic disease virus type 2 inactivated vaccine and a preparation method thereof.
Background
Rabbit Hemorrhagic Disease (RHD) is an acute, highly contagious, highly lethal disease caused by Rabbit Hemorrhagic Disease Virus (RHDV). The classical RHDV only infects rabbits, the rabbits with the age of more than 2 months are susceptible, the death rate reaches 90 percent, the rabbits usually die 48 to 72 hours after infection, the dead rabbits are characterized by blood stasis, swelling and bleeding of parenchymal organs such as a respiratory system and liver, spleen, kidney, heart and the like, and the young rabbits are not easy to feel. In 2010, a new strain of RHDV, designated RHDV2, was first discovered in france. The rabbits with different ages of days are susceptible to RHDV2, and the disease death rate of the infected rabbits can reach 90%. The RHDV2 has similar symptoms and pathological autopsy changes when infected dead rabbits with the classical RHDV strains, but has great difference in genetic characteristics and antigenicity, and vaccines prepared from the classical strains cannot effectively protect the RHDV2 infection.
In 4 months in 2020, acute death cases occur in a certain rabbit farm in Sichuan, and the clinical symptoms and pathological changes of dead rabbits are very similar to those of rabbit hemorrhagic disease. The rabbit disease team of the institute of veterinary medicine of agricultural sciences of Jiangsu province determines that the pathogen is RHDV2 through a red blood cell agglutination test, an RT-PCR analysis, a sequence determination and a virus replication test, and the RHDV2 serotype strain is discovered in China for the first time.
Because the RHDV2 can not be protected by the genetic engineering vaccine prepared by the classical strain and related to the classical strain, the inactivated vaccine for rabbit hemorrhagic disease type 2 is developed to meet the disease prevention and control requirements of the rabbit industry, and is very important for preventing and controlling rabbit hemorrhagic disease type 2.
Disclosure of Invention
In view of the above, the invention aims to provide a rabbit hemorrhagic disease virus type 2 inactivated vaccine and a preparation method thereof, wherein the inactivated vaccine has high safety, good immune effect and simple and convenient process.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of a rabbit hemorrhagic disease virus type 2 inactivated vaccine, which comprises the following steps:
1) Inoculating a rabbit hemorrhagic disease virus type 2 strain SC2020 to susceptible rabbits;
2) Collecting viscera tissues of the rabbits which die within 24-96 hours after inoculation and have typical histopathological changes;
3) The organ tissues and physiological saline were mixed at a ratio of 1g: (4-6) mixing in a ratio of mL, homogenizing, and filtering to obtain a tissue homogenate;
4) Mixing the tissue homogenate with physiological saline until the ratio of the organ tissue to the physiological saline is 1g: (18-20) mL to obtain a vaccine antigen, and inactivating the vaccine antigen to obtain the inactivated vaccine for rabbit hemorrhagic disease virus type 2.
Preferably, the inoculation amount of the inoculation in the step 1) is 0.8-1.2 mL/seed; the months age of the susceptible rabbit is 2-4 months.
Preferably, the organ tissue in step 2) includes one of liver, spleen and kidney.
Preferably, the ratio of the organ tissues to the physiological saline in step 3) is 1g:5mL.
Preferably, the filtration in step 3) is mesh screen filtration; the mesh number of the mesh screen is 80-120 meshes.
Preferably, the inactivation of the vaccine antigen in step 4) comprises the steps of: mixing the vaccine antigen with formaldehyde, and inactivating the mixture for 20 to 28 hours at the temperature of between 35 and 40 ℃; the volume fraction of the formaldehyde in the system is 0.3-0.5%.
Preferably, the inactivation process is performed by shaking every 2-3 h, and the shaking time is 2-4 min.
The invention provides a rabbit hemorrhagic disease virus type 2 inactivated vaccine prepared by the preparation method.
Preferably, the storage temperature of the inactivated rabbit hemorrhagic disease virus type 2 vaccine is 2-8 ℃.
Preferably, the minimum immune dose of the inactivated rabbit hemorrhagic disease virus type 2 vaccine is 0.25 mL/mouse.
The invention has the beneficial effects that: the invention provides a rabbit hemorrhagic disease virus type 2 inactivated vaccine and a preparation method thereof, which is obtained by inoculating susceptible rabbits with a rabbit hemorrhagic disease virus type 2 strain SC2020, collecting the viscera tissues of the rabbits which die after inoculation and have typical histopathological changes, mixing the viscera tissues with normal saline, homogenizing, filtering and inactivating; the inactivated rabbit hemorrhagic disease virus type 2 vaccine has high safety, good immune effect and simple and convenient process.
Detailed Description
The invention provides a preparation method of a rabbit hemorrhagic disease virus type 2 inactivated vaccine, which comprises the following steps: 1) Inoculating a rabbit hemorrhagic disease virus type 2 strain SC2020 to susceptible rabbits; 2) Collecting the viscera tissues of the rabbits which die within 24-96 hours after inoculation and have typical histopathological changes; 3) The organ tissues were mixed with physiological saline at a ratio of 1g: (4-6) mixing in a ratio of mL, homogenizing, and filtering to obtain a tissue homogenate; 4) Mixing the tissue homogenate with physiological saline until the ratio of the organ tissues to the physiological saline is 1g: (18-20) mL to obtain a vaccine antigen, and inactivating the vaccine antigen to obtain the inactivated vaccine for rabbit hemorrhagic disease virus type 2.
In the present invention, a susceptible rabbit is inoculated with rabbit hemorrhagic disease virus type 2 strain SC 2020. In the invention, the rabbit hemorrhagic disease virus type 2 strain SC2020 is isolated from Sichuan China, and the GenBank accession number is: MT383749. In the invention, the rabbit hemorrhagic disease virus type 2 strain SC2020 HAs strong toxicity, fast death and high HA titer of dead rabbit livers. In the present invention, the inoculation amount of the inoculation is preferably 0.8 to 1.2 mL/piece, more preferably 1 mL/piece; the month age of the susceptible rabbit is 2-4 months; the inoculation is preferably subcutaneous inoculation.
After the inoculation, the invention collects the viscera tissues of the rabbits which die within 24 to 96 hours after the inoculation and have typical histopathological changes. In the present invention, the organ tissue includes one of liver, spleen and kidney; in the practice of the present invention, the liver, spleen and kidney are preferably weighed after removal of connective tissue. In the present invention, after weighing, the organ tissues are preferably minced, and the operation and parameters of the mincing are not particularly limited, and can be performed by the conventional operation in the art.
After obtaining the organ tissues, the present invention mixes the organ tissues with physiological saline in an amount of 1g: (4-6) mL, homogenizing and filtering to obtain tissue homogenate. In the present invention, the ratio of the organ tissue to the physiological saline is preferably 1g:5mL; in the present invention, the ratio of the organ tissue to the physiological saline is not limited to g: mL, the same magnification or reduction can be used, for example mg: μ L. In the present invention, the homogenization is preferably carried out using a triturator, and the temperature of the homogenization is preferably 3 to 5 ℃, more preferably 4 ℃. In the present invention, the filtration is preferably mesh screen filtration; the mesh number of the mesh screen is preferably 80 to 120 mesh, more preferably 100 mesh.
In the present invention, the tissue homogenate is mixed with a physiological saline until the ratio of the organ tissue to the physiological saline is 1g: (18-20) mL to obtain the vaccine antigen. In the present invention, the physiological saline is preferably sterile physiological saline, and the method for sterilizing the physiological saline in the present invention is not particularly limited, and a method for sterilizing physiological saline that is conventional in the art may be used. In the present invention, the ratio of the organ tissue to the physiological saline in the vaccine antigen is preferably 1g.
After the vaccine antigen is obtained, the vaccine antigen is inactivated, and the inactivated rabbit hemorrhagic disease virus type 2 vaccine is obtained. In the present invention, inactivating the vaccine antigen preferably comprises the steps of: mixing the vaccine antigen with formaldehyde for inactivation for 20-28 h at 35-40 ℃; in the present invention, the volume fraction of formaldehyde in the system is preferably 0.3% to 0.5%, more preferably 0.4%. In the inactivation process, the inactivation is preferably performed once every 2 to 3 hours, and the time of shaking for each time is preferably 2 to 4min, and more preferably 3min; the shaking function is that the formaldehyde is fully contacted with the vaccine antigen, and the complete inactivation of the antigen is ensured.
The invention also provides the inactivated vaccine of rabbit hemorrhagic disease virus type 2 prepared by the preparation method. In the invention, the inactivated rabbit hemorrhagic disease virus type 2 vaccine is preferably quantitatively subpackaged, plugged, sealed and labeled. In the invention, the storage temperature of the inactivated vaccine for rabbit hemorrhagic disease virus type 2 is preferably 2-8 ℃; the minimum immune dose of the rabbit hemorrhagic disease virus type 2 inactivated vaccine is preferably 0.25 mL/rabbit.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Strain screening
Respectively removing connective tissues from liver samples diagnosed by 2 commercial rabbits and young rabbits in different areas, adding physiological saline for mashing, and filtering to prepare tissue homogenate, wherein the homogenate contains the mashed liver weight (g): volume of physiological saline (ml) 1: and 9, repeatedly freezing and thawing for 3 times. Centrifuging at 8000rpm for 30min, collecting supernatant, sterilizing and filtering with 0.22 μm filter, numbering JT1, JT2, ZY1, ZY2, respectively, and subcutaneously inoculating to healthy and susceptible rabbits of 2-4 months age, 3 rabbits per group, 1 mL. After inoculation, dead rabbits were dissected, liver tissues were aseptically collected and placed in a sterile container, and the volume ratio of physiological saline 1:9 dilution homogenate, repeated freeze-thaw 3 times, hemagglutination titer was determined, and the results are given in table 1 below.
TABLE 1 detection of vaccinated dead rabbits
According to HA titer and death time of the attacking rabbit, selecting a ZY2 strain with fast death causing and high HA titer of dead rabbit liver as a strain for vaccine preparation, and naming the strain as SC2020 (GenBank accession number MT383749; published documents: weihoujun, hubo, model Shiyu and the like; separation identification and sequence analysis of rabbit hemorrhagic disease virus type 2 [ J ]. Jiangsu agricultural science 2020, 36 (2): 404-409.) as an original generation; the dead rabbit liver homogenate was designated SC2020/04-F1 and was separately cryopreserved.
Example 2
Subculturing of strains
SC2020/04-F1 was centrifuged at 8000rpm for 30min, and the supernatant was sterile filtered through a 0.22 μm filter and labeled as R2-F1. R2-F1 is inoculated subcutaneously to 1mL of 3 healthy and susceptible rabbits with the age of 2-4 months. Performing a autopsy on the dead rabbits after inoculation, aseptically collecting rabbit livers which die within 24-96 hours after the inoculation and die for no more than 1 hour and have histopathological changes as virus seeds, placing the virus seeds in an aseptic container, removing connective tissues on the virus seeds, and performing sterilization treatment on the rabbit livers by using a physiological saline solution 1:9 diluting the homogenate, repeating freezing and thawing for 3 times, marking as SC2020/04-F2, and freezing and preserving. The strain is inoculated to healthy susceptible rabbits to be continuously passaged to the 8 th generation according to the method, the hemagglutination titer is determined, and the strains are respectively frozen and preserved, and the result is shown in the following table 2.
TABLE 2 hemagglutination titer results for each strain generation
| The number of virus strains | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 |
| Hemagglutination potency (log 2) | 7 | 8 | 8 | 7 | 8 | 8 | 8 | 8 |
Example 3
Vaccine preparation
The 8 th generation virus of rabbit hemorrhagic disease virus type 2 is inoculated subcutaneously to 1mL of 3 susceptible rabbits with 2-4 months age. The rabbit liver, spleen and kidney which die within 24-96 hours after inoculation and have typical histopathological changes are aseptically collected, the connective tissues of the rabbit liver, spleen and kidney are removed, and the ratio of the connective tissues to the cell surface is 1g: adding physiological saline into the 5mL of the mixture, mashing the mixture, filtering the mixture by using a 100-mesh screen to prepare tissue homogenate, and then fixing the volume to ensure that the tissue homogenate contains mashed tissue weight (g): volume of physiological saline (ml) 1:19, taking the tissue homogenate as a vaccine antigen, adding formaldehyde with the final concentration of 0.4%, mixing uniformly, replacing the bottle, inactivating at 37 ℃ for 24h, shaking once every 2h, and shaking for 3min each time. After 24h, the inactivation test of the antigen is carried out (see example 4), after the test is qualified, shaking up, quantitatively subpackaging, plugging and sealing are carried out, the label is 202002, and the antigen is stored for later use at 2-8 ℃.
Example 4
Inactivation test for antigens
Fully shaking the inactivated antigen in the example 3, taking 3 healthy susceptible rabbits with the age of 35-42 days, injecting 2.0mL of the antigen per rabbit subcutaneously in the neck, and continuously observing for 3 days. The results show that all the test rabbits are healthy and alive, and the antigen inactivation is qualified.
Example 5
Sterility testing of vaccines
The vaccine of the embodiment 3 is fully shaken up, 2 pieces of TG tubules are respectively inoculated with the vaccine to be detected, 0.2mL of each of the 2 pieces is inoculated into 2 pieces of TG tubules, each of the 0.2mL of each of the 2 pieces is cultured at 35-37 ℃,1 piece of TG tubules is cultured at 23-25 ℃, another 0.2mL of the TG tubules is inoculated into 1 piece of TSB tubule, the TG tubules are cultured at 23-25 ℃, the culture is carried out for 7 days, the TG tubules are all aseptically grown, and the vaccine is aseptically tested to be qualified.
Example 6
Safety testing of vaccines
The vaccine of example 3 was thoroughly shaken, and 5 healthy rabbits of 35 to 42 days old were used as a test group, and 2.0mL of the vaccine was injected subcutaneously into the neck. Meanwhile, 5 rabbits in the same condition were used as controls without vaccination, and were continuously observed for 14 days to record mental status, diet, feces, local and systemic reactions, etc. of the two groups. The results show that all the tested rabbits are healthy, the mental state, diet and feces are normal, the absorption of the injection part is good, and no lump exists, which indicates that the vaccine has good safety, and the results are shown in the following table 3.
TABLE 3 safety test results for vaccines
Example 7
Potency testing of vaccines
The vaccine of example 3 was shaken well, and 5 healthy rabbits of 35-42 days old were injected with 1.0mL of vaccine each, while 5 rabbits of the control group were set up. After 14 days of immunization, 5 immunized rabbits are taken, and together with 5 rabbits in a control group, 1.0mL of rabbit hemorrhagic disease virus type 2R 2-F1 virus seeds are subcutaneously injected into the neck of each rabbit, the continuous observation is carried out for 7 days, the number of dead and protected animals is recorded, and the dead rabbits are subjected to autopsy, and the results are shown in the following table 4. The results showed that the control rabbits all died and the immunized rabbits all became healthy. The vaccine efficacy test is qualified.
Table 4 efficacy test results for vaccines
Example 8
Minimum immune dose testing of vaccines
The vaccine of example 3 was used and shaken well, and healthy susceptible rabbits of 35 to 42 days old were subcutaneously injected into the neck at 1.0mL, 0.5mL, 0.25mL, and 0.125mL, respectively, while control rabbits were established at 5 rabbits/group. 14 days after immunization, 1.0 mL/rabbit of rabbit hemorrhagic disease virus type 2R 2-F1 virus seeds are injected subcutaneously into the neck of each of the immunized rabbits and the control rabbits, the observation is continuously carried out for 7 days, the number of dead and protected animals is recorded, and the dead rabbits are subjected to autopsy, and the results are shown in the following table 5.
TABLE 5 minimum immunization dose test results for vaccines
The results show that: when the vaccine immunization is 0.25 mL/dose or more, the immune rabbit can be protected against the virulent attack of the rabbit hemorrhagic disease virus type 2. The results show that 0.25 mL/minimum immunization dose of the inactivated vaccine.
Example 9
Immunization generation phase of vaccine
The vaccine of example 3 was shaken well, 20 healthy, susceptible rabbits of 35-42 days old were each injected with 1.0mL of vaccine, and 20 rabbits of the control group were set up at the same time. After 3, 7, 10 and 14 days of immunization, 5 immunized rabbits are respectively taken, and together with 5 control rabbits, 1.0mL of rabbit hemorrhagic disease virus type 2R 2-F1 virus seeds are subcutaneously injected into the neck of each rabbit, and after 7 days of continuous observation, the number of dead and protected animals is recorded, and the dead rabbits are subjected to autopsy. The results show that: when the vaccine is immunized by 1 mL/vaccine, the rabbit hemorrhagic disease virus type 2 virulent strain can be completely protected 7 days after immunization (see table 6).
TABLE 6 immunization generation phase test results for vaccines
The embodiment shows that the rabbit hemorrhagic disease type 2 inactivated vaccine prepared by the invention is feasible and can generate good immune protection effect on healthy and susceptible rabbits.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (7)
1. A preparation method of a rabbit hemorrhagic disease virus type 2 inactivated vaccine is characterized by comprising the following steps:
1) Inoculating a rabbit hemorrhagic disease virus type 2 strain SC2020 to susceptible rabbits;
2) Collecting the viscera tissues of the rabbits which die within 24-96 hours after inoculation and have typical histopathological changes;
3) Mixing the organ tissues with normal saline according to the proportion of 1g (4-6) mL, homogenizing and filtering to obtain tissue homogenate;
4) Mixing the tissue homogenate with normal saline until the ratio of the organ tissues to the normal saline is 1g (18-20) mL to obtain a vaccine antigen, and inactivating the vaccine antigen to obtain the rabbit hemorrhagic disease virus type 2 inactivated vaccine;
the inoculation amount of the inoculation in the step 1) is 0.8-1.2 mL/inoculation; the month age of the susceptible rabbit is 2-4 months;
the organ tissues in the step 2) comprise one of liver, spleen and kidney;
inactivating the vaccine antigen in step 4) comprises the following steps: mixing the vaccine antigen with formaldehyde, and inactivating the mixture for 20 to 28 hours at the temperature of between 35 and 40 ℃; the volume fraction of the formaldehyde in the system is 0.3-0.5%.
2. The method according to claim 1, wherein the ratio of the organ tissue of step 3) to the physiological saline is 1g.
3. The method of claim 2, wherein the filtration in step 3) is mesh filtration; the mesh number of the mesh screen is 80-120 meshes.
4. The preparation method according to claim 1, wherein the inactivation is performed by shaking every 2-3 h for 2-4 min.
5. The inactivated rabbit hemorrhagic disease virus type 2 vaccine prepared by the preparation method of any one of claims 1 to 4.
6. The inactivated vaccine for rabbit hemorrhagic disease virus of type 2 according to claim 5, wherein the preservation temperature of the inactivated vaccine for rabbit hemorrhagic disease virus of type 2 is 2 to 8 ℃.
7. The inactivated rabbit hemorrhagic disease virus vaccine of type 2 according to claim 5, wherein the minimum immune dose of the inactivated rabbit hemorrhagic disease virus vaccine of type 2 is 0.25 mL/mouse.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104998258A (en) * | 2015-08-06 | 2015-10-28 | 山东华宏生物工程有限公司 | Method for preparing propolis inactivated vaccine applicable to rabbit viral haemorrhagic syndrome |
| CN108588034A (en) * | 2018-03-22 | 2018-09-28 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of rabbit hemorrhagic disease virus of variation and its application in preparing inactivated vaccine |
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|---|---|---|---|---|
| CN104998258A (en) * | 2015-08-06 | 2015-10-28 | 山东华宏生物工程有限公司 | Method for preparing propolis inactivated vaccine applicable to rabbit viral haemorrhagic syndrome |
| CN108588034A (en) * | 2018-03-22 | 2018-09-28 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of rabbit hemorrhagic disease virus of variation and its application in preparing inactivated vaccine |
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| 兔出血症病毒2型的分离鉴定与序列分析;魏后军等;《江苏农业学报》;20200430;第36卷(第2期);第407-408页 * |
| 兔出血症病毒新毒株ＲHDV2的流行与控制(综述);王芳等;《江苏农业科学》;20161020;第44卷(第9期);全文 * |
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