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Deer-derived bovine viral diarrhea inactivated vaccine and preparation method thereof

A technology of bovine viral diarrhea and inactivated vaccine, applied in the field of vaccine, can solve the problems of unsatisfactory immune effect of sika deer mucosal disease, miscarriage, stillbirth, etc.

Inactive Publication Date: 2020-09-25
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Viral diarrhea / mucosal diseases are spreading in the sika deer herd, but there is still no special vaccine for sika deer viral diarrhea / mucosal diseases in my country. In many cases, bovine-derived vaccines are used instead, and bovine-derived vaccines are used The immune effect of sika deer mucosal disease is not satisfactory
At the same time, it has been reported that immunization of pregnant female animals with attenuated BVDV vaccine may lead to miscarriage or stillbirth

Method used

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  • Deer-derived bovine viral diarrhea inactivated vaccine and preparation method thereof
  • Deer-derived bovine viral diarrhea inactivated vaccine and preparation method thereof
  • Deer-derived bovine viral diarrhea inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation and acquisition of deer-derived bovine viral diarrhea virus JL05 strain

[0026] The deer-derived bovine viral diarrhea virus (BVDV) JL05 strain used in the present invention is isolated from the spleen of suspected diseased sika deer. Inoculation of the isolated virus into MDBK cells can produce obvious cytopathic changes, which were detected by neutralization test and indirect immunofluorescence test using BVDV positive antibody; according to the full gene sequence of BVDV published by GenBank, specific primers were designed and synthesized as follows:

[0027] BU: 5'-TCGACGCTTTGGAGGACA-3';

[0028] BD: 5'-CCATGTGCCATGTACAG-3'.

[0029] The size of the target fragment is 186bp, which is used for RT-PCR detection of BVDV. The results showed that the isolated virus was BVDV, and the results of fluorescence identification were as follows: figure 1 As shown, the PCR identification results are as follows figure 2 shown.

[0030] According to the w...

Embodiment 2

[0033] Example 2 Passage and Plaque Cloning Purification of Deer-derived BVDV JL05 Strain

[0034] The isolated deer-derived BVDV JL05 strain was continuously passaged in vitro through MDBK cells to 30 passages. Plaque cloning and purifying viruses were carried out at intervals of 5 generations (ie F5, F10, F15, F20, and F25 generations were carried out for plaque cloning and purifying viruses). Take the deer-derived BVDVJL05 strain virus for 10-fold serial dilution, and the dilution range is 10 -2 ~10 -6 After the virus is diluted, inoculate a 6-well cell culture plate full of 80% monolayer MDBK cells, 1ml / well, discard the virus solution after 1 hour of adsorption, wash 3 times with serum-free DMEM, add 2×DMEM and sterilized agar solution Mixed solution, 3ml / well, cooled at room temperature and placed at 37°C, 5% CO 2 Cultivate in an incubator for 3-4 days, observe the plaques, pick a single plaque in a well with few plaques, save it and pass it down.

Embodiment 3

[0035] The preparation of embodiment 3 seedlings with antigen solution

[0036] 1. Preparation of deer-derived BVDV JL05 strain antigen by adherent culture cells in spinner bottles

[0037]Cultivate MDBK cells in spinner bottles. When the cells are 90-100% monolayer, they are digested and passaged with trypsin-EDTA cell dispersion solution, dispersed into new spinner bottles at a ratio of 1:3-1:5, and the cells grow The solution is DMEM containing 5-8% newborn bovine serum. When the cells grew to 80-90% monolayer, the deer-derived BVDV JL05 strain of the present invention was inoculated into MDBK cells with an infectious dose (MOI) of 0.01-0.05, and the cell maintenance solution was DMEM containing 3% horse serum, and continued After culturing for 60-84 hours, the virus liquid was harvested when the cytopathic effect reached 80-90%, and stored at -20°C for later use.

[0038] 2. Preparation of deer-derived BVDV JL05 strain antigen using microcarrier suspension culture techno...

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Abstract

The deer-derived bovine viral diarrhea inactivated vaccine and a preparation method thereof relate to the field of vaccine technology and fill in the blank of special vaccines for sika deer viral diarrhea / mucosal disease. The deer-derived bovine viral diarrhea virus JL05 strain of the present invention has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on January 11, 2018, and the preservation number is CGMCC No.15283. The present invention uses MDBK cells to passage the deer-derived BVDV JL05 strain virus and perform plaque cloning to purify the virus. After detection, the deer-derived BVDV JL05 strain virus content reaches 10 7.2 TCID 50 / ml or more, and free from bacteria, mold, mycoplasma and exogenous virus contamination, meeting the virus requirements for seedling production. The virus was inactivated by BEI, mixed with 206 adjuvant and emulsified to prepare a vaccine. The safety and efficacy tests proved that the vaccine was safe for sika deer, and the protection rate of the virus challenged could reach 100%.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to an inactivated vaccine for deer-derived bovine viral diarrhea and a preparation method thereof. Background technique [0002] Bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus in the family Flaviviridae, and has serological cross-reactions with the swine fever virus and the sheep border virus in the same genus. BVDV can cause infection in cattle, sheep, pigs and deer, and the clinical manifestations are fever, mucosal ulcers, persistent infection, diarrhea and reproductive disorders. Deer infected with BVDV can cause miscarriage in doe, stillbirth, weak fetus, deformed fetus, viremia offspring, intractable diarrhea and mucosal disease in deer, resulting in obstruction of feeding and nutrient absorption, and secondary infection, which seriously affects The development of young deer and the production performance of adult deer even cause a large number of dee...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/04A61K39/12A61P31/14
CPCA61K39/12A61K2039/5252C12N7/00C12N2770/24334C12N2770/24363
Inventor 冷雪杜锐时坤李健明宫庆龙孙志博郜艳雪徐宁栾美惠
Owner JILIN AGRICULTURAL UNIV
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