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Raccoon parvovirus enteritis and canine distemper dual inactivated vaccine and preparation method thereof

A dual inactivated vaccine and parvovirus technology, which is applied in the field of veterinary biological products, can solve the problems of poor immunogenicity of inactivated vaccines, increase the workload of farmers, and strong virulence of live vaccines, etc., to achieve safety and Good immunogenicity, no need for a large number of production personnel, and the effect of automated culture methods

Active Publication Date: 2022-08-02
QILU ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no special canine distemper and parvovirus vaccine for raccoon dogs on the market, and many farmers use alternative vaccines, which cannot achieve better immune protection effect. Therefore, secondary immunization of raccoon dogs is often required, which not only increases the cost, It also increases the workload of the farmers, and at the same time causes a greater stress response in the raccoon dogs
Moreover, most canine distemper vaccines are live vaccines, and there is a risk of strong virulence; the vaccines prepared for parvovirus enteritis are mainly inactivated whole virus vaccines or live vaccines. Inactivated vaccines have poor immunogenicity, and live vaccines have virulence. The risk of returning to strength

Method used

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  • Raccoon parvovirus enteritis and canine distemper dual inactivated vaccine and preparation method thereof
  • Raccoon parvovirus enteritis and canine distemper dual inactivated vaccine and preparation method thereof
  • Raccoon parvovirus enteritis and canine distemper dual inactivated vaccine and preparation method thereof

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Experimental program
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preparation example Construction

[0074] 1) Preparation of VP2 protein of raccoon dog parvovirus: select vigorously growing Sf9 cells to inoculate a cell culture tank for full suspension culture, and the inoculation density is 1.5×10 6 ~2.0×10 6 cells / ml, cultured at 27°C, when the density of Sf9 cells in the cell culture tank reached 1.5×10 6 ~3.0×10 6Cells / ml, inoculate the recombinant baculovirus RDPV-SN1 strain at MOI=0.2~2.0, culture at 27°C for 72~96h, harvest the cell culture, add 2% BEI solution to it, make the final concentration of BEI 0.1%, After mixing, it was inactivated at 37 °C for 48 hours, then 50% sodium thiosulfate solution was added to the cell culture to make the final concentration 0.2%, and the inactivation was terminated after stirring for 1 hour; after sterility and inactivation tests, it was obtained Raccoon dog parvovirus VP2 protein, hemagglutination titer to 1% porcine red blood cells ≥ 1:2048, stored at 2-8°C for future use;

[0075] 2) Preparation of inactivated antigen of can...

Embodiment 1

[0086] Example 1——Research on suspension culture process of recombinant baculovirus RDPV-SN1 strain

[0087] 1. Research on the culture technology of the recombinant baculovirus RDPV-SN1 strain in the flask

[0088] (1) Effect of cell density on hemagglutination titer of expressed protein

[0089] When the Sf9 cell density is 1.5 × 10 6 cells / ml, 2.0×10 6 cells / ml, 2.5×10 6 cells / ml, 3.0×10 6 When cells / ml, the recombinant baculovirus RDPV-SN1 strain was inoculated at MOI=0.5, and samples were taken at 60h, 72h, 84h, 96h, 108h, and 120h after inoculation to determine the hemagglutination titer of the expressed protein.

[0090] Results The recombinant baculovirus RDPV-SN1 strain was inoculated into Sf9 cells with different cell densities, and the Sf9 cell density was 1.5×10 when inoculated. 6 , 2.0×10 6 , 2.5×10 6 , 3.0×10 6 There was no significant difference in the hemagglutination titer of the expressed protein at 72h after inoculation, and the hemagglutination tite...

Embodiment 2

[0111] Embodiment 2——The research on suspension culture technology of canine distemper virus CDV-11 strain

[0112] 1. Study on the culture technology of canine distemper virus CDV-11 strain 7L cell culture tank

[0113] (1) Microcarrier preparation and sterilization 2+ , Mg 2+ PBS (0.1 mol / L, pH 7.2, the same below) was washed 2 to 3 times, and PBS was added to sterilize at 121 °C for 30 min. After sterilization, discard the PBS and add MEM cell culture medium containing 8% newborn bovine serum at 36.5°C, containing 5% CO. 2 Place in the incubator for 4h.

[0114] (2) Vero cell microcarrier suspension culture Select Vero spinner flask cells with good morphology and vigorous growth, digest with EDTA-trypsin dispersion to prepare cell suspension, inoculate a stirring cell culture flask, rotate speed 30r / min, and cell density is 4.5×10 5 cells / ml, the microcarrier concentration was 4 g / L, and the culture temperature was 36.5°C to 37.5°C.

[0115] (3) Influence of culture t...

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Abstract

The invention relates to a dual inactivated vaccine of raccoon parvovirus enteritis and canine distemper and a preparation method thereof. The active ingredients of the dual inactivated vaccine include the raccoon parvovirus VP2 protein obtained by transfecting Sf9 cells with the recombinant baculovirus RDPV‑SN1 strain and the inactivated antigen of canine distemper virus. The present invention aims at the risk that the virulence of the raccoon dog distemper and parvovirus dual live vaccines will return to strong, and the dual inactivated vaccines used in the market generally have the problem of low immune efficacy after the raccoon parvovirus is inactivated. A dual inactivated vaccine prepared from raccoon parvovirus VP2 protein and canine distemper virus inactivated antigen with high virus content. The dual vaccine can be used in the prevention of raccoon parvovirus enteritis and canine distemper at the same time, and has the advantages of good immunogenicity, high safety, strong controllability, high cultivation efficiency and automation, and large-scale production. Advantages, can achieve the purpose of "one needle, two defenses", has broad application prospects.

Description

technical field [0001] The invention relates to a dual inactivated vaccine of raccoon parvovirus enteritis and canine distemper and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Raccoon parvovirus enteritis is an acute and highly contagious infectious disease caused by raccoon parvovirus of the family Parvoviridae. At present, raccoon dog parvovirus enteritis widely exists in various countries in the world. The disease mainly causes enteritis of raccoon dogs, and the mortality rate can reach 30% to 60%. [0003] Canine distemper is an acute, febrile, highly contagious infectious disease caused by Morbillivirus (Canine Distemper virus, CDV) of the family Paramyxoviridae. Young animals mostly have an acute and fatal course. Animals can become chronic and persistent infections. The clinical symptoms are mainly characterized by biphasic body temperature rise, accompanied by catarrhal pneumonia, and gastroent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/23A61K39/175A61P31/20A61P31/14C07K14/015C12N15/866C12N15/35
CPCA61K39/12A61P31/20A61P31/14C07K14/005C12N15/86A61K2039/552A61K2039/70A61K2039/5252A61K2039/55505C12N2750/14334C12N2760/18434
Inventor 董海曼李雯宋晓飞葛平萍秦旭伟李营贾爱琴王蕾徐龙涛
Owner QILU ANIMAL HEALTH PROD
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