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Bovine rotavirus, bovine coronavirus combined inactivated vaccine and preparation method thereof

A dual inactivated vaccine and bovine rotavirus technology, applied in biochemical equipment and methods, viruses, vaccines, etc., can solve problems such as difficult virus isolation

Active Publication Date: 2019-08-16
QILU ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty of BRV virus isolation, it needs to be treated with a certain concentration of proteolytic enzymes such as trypsin to effectively infect cells.

Method used

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  • Bovine rotavirus, bovine coronavirus combined inactivated vaccine and preparation method thereof
  • Bovine rotavirus, bovine coronavirus combined inactivated vaccine and preparation method thereof
  • Bovine rotavirus, bovine coronavirus combined inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1——preparation of bovine rotavirus, bovine coronavirus dual inactivated vaccine (JY strain+YQ strain)

[0065] 1. Preparation of BRV-JY strain and BCV-YQ strain antigens by spinner bottle attachment

[0066] (1) Cultivate cells: Resuscitate cells MA-104 and HCT-8 into cell flasks, grow for about 48 hours, digest with EDTA-trypsin cell dispersion, and passage according to the volume ratio of 1:4. Serum-free DMEM culture solution (pH value 7.0) was added for spinner bottle culture, and when the cells were 90%-100% full, they were used for further passage or virus inoculation.

[0067] (2) Preparation of antigen (virus solution) for seedling preparation: Take the above-mentioned MA-104 cells that have grown well, wash them 2 to 3 times with PBS, and inoculate the BRV-JY strain according to (V / V) 0.5%. Inoculation. Serum-free DMEM medium (pH 7.0) was added for cultivation. After 5 to 7 days, when 80% of the cells have CPE, place the spinner bottle at -20°C and...

Embodiment 2

[0084] Embodiment 2—the finished product inspection of bovine rotavirus, bovine coronavirus dual inactivated vaccine (JY strain+YQ strain)

[0085] 1. Sterility test: according to the appendix of the current "Chinese Veterinary Pharmacopoeia", it should meet the requirements. The above test results are shown in Table 2.

[0086] Table 2 Physical properties, sterility test results

[0087]

[0088] 2. Safety inspection:

[0089] (1) Substitute animal test: 7 healthy rabbits of 1.5 to 2.0 kg were selected for the test, including 5 in the immunization group and 2 in the control group. Each neck of the rabbits in the immunization group was subcutaneously injected with 1.0ml of vaccine (two-point injection, 0.5ml Each neck of control group rabbits subcutaneously injects 1.0ml of cell culture (two-point injection, 0.5ml / point), continuously observes for 7 days and measures body temperature at fixed point every afternoon, all are healthy and alive, and the injection site of immu...

Embodiment 3

[0105] Embodiment 3——Newborn calf antibody tracking test

[0106] Select 10 dry dairy cows with BRV, BCV antigen and antibody double negative (antigen negative means RT-PCR test is negative, antibody negative means serum neutralizing antibody titer ≤ 1:4), including 5 cows in the immunization group and 5 cows in the control group 2.0ml of the vaccine was injected intramuscularly into each neck of cattle in the immunized group, and 2.0ml of cell culture was injected intramuscularly into each neck of the cattle in the control group, and the immunization was boosted in the same way 21 days later. After the calving of the test cows, blood was collected from newborn calves that did not eat colostrum, and the titers of BRV and BCV neutralizing antibodies were detected. Blood was collected 60 days after the calf was born to detect BRV and BCV neutralizing antibody titers. For newborn calves calved by immunized cattle, the 4 / 5BRV neutralizing antibody titer was ≥1:64, and the 4 / 5BCV ...

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Abstract

The invention relates to a bovine rotavirus, a bovine coronavirus combined inactivated vaccine and a preparation method thereof. The active ingredient of the vaccine of the present invention comprisesinactivated antigen of bovine rotavirus and bovine coronavirus, and the antigenic component of the combined inactivated vaccine can be subjected to virus propagation through a continuous cell line (aiming at bovine rotavirus and bovine coronavirus) or microcarrier suspension culture (aiming at bovine rotavirus), and a seedling process and the like can be optimized. The vaccine safety, effectiveness test and antibody tracking results show that no adverse reaction is generated after immunization of cattle with the combined inactivated vaccine of the present invention, and no abortion is inducedafter immunization of pregnant cattle. Immunization of cattle produces immune protection, and newborn calves can obtain high levels of maternal antibodies through breast milk. The results show that the vaccine of the invention is safe and reliable and can be used for preventing the generation of yak diarrhea.

Description

technical field [0001] The invention relates to a dual inactivated vaccine of bovine rotavirus and bovine coronavirus and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Calf diarrhea is a disease that causes high mortality in newborn calves and can cause severe economic losses. Its pathogens mainly include bovine rotavirus (Bovine Rotavirus, BRV) and bovine coronavirus (Bovine Coronavirus, BCV). BRV belongs to the Reoviridae family and the genus Rotavirus. It is a double-stranded RNA virus. It mostly infects calves under 1 month old. The main clinical symptoms are watery diarrhea, depression, loss of appetite, severe dehydration and acidosis. . BCV belongs to the Coronaviridae family and the genus Coronavirus. It is a single-stranded positive-sense RNA virus that mainly encodes five proteins, including S, M, N, E, and HE. In addition to causing diarrhea in calves, it can also cause winter diarrhea and he...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61K39/215A61K39/15A61P31/14
CPCA61K39/12A61P31/14A61K2039/70A61K2039/552A61K2039/5252C12N2720/12334C12N2770/20034
Inventor 毕玉彧张娣高凤宋晓飞李营贾爱琴秦绪伟王蕾徐龙涛
Owner QILU ANIMAL HEALTH PROD
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