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A kind of canine adenovirus type I inactivated vaccine and preparation method thereof

A technology of canine adenovirus and inactivated vaccines, applied in biochemical equipment and methods, viruses, vaccines, etc., to achieve good neutralizing antibody titers and excellent immune effects

Active Publication Date: 2020-11-27
HENGYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems caused by the use of serum in the preparation process of canine adenovirus type Ⅰ virus vaccine and improve the safety of the vaccine, the present invention aims to provide an inactivated canine adenovirus type Ⅰ vaccine and its preparation method, which adopts a serum-free process Cultivation enhances the biosafety of vaccine raw materials

Method used

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  • A kind of canine adenovirus type I inactivated vaccine and preparation method thereof
  • A kind of canine adenovirus type I inactivated vaccine and preparation method thereof
  • A kind of canine adenovirus type I inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Serum-free culture process acclimatization of canine kidney cells (MDCK)

[0043] (1) Wash the canine kidney cells (MDCK) growing into a monolayer with pH 7.4 phosphate buffered saline solution twice;

[0044] (2) Add 0.25% trypsin for digestion, and discard the trypsin solution when the cells become round;

[0045] (3) Add MEM medium with a final concentration of 5% fetal bovine serum (v / v), add tylosin at a final concentration of 2-10 µg / mL, shake well, and place in a closed culture at 37°C for 24 hours;

[0046] (4) Set the culture temperature to 38.5°C and continue the airtight culture for 72 hours;

[0047] (5) The cells are covered with a monolayer, inoculated at a ratio of 1:10, and subcultured, and subcultured for 3 generations according to steps (1)-(4) to obtain seed cells;

[0048] (6) Wash the canine kidney cells (MDCK) growing into a monolayer twice with pH 7.4 phosphate buffered saline solution;

[0049] (7) Add 0.25% trypsin for digestion, a...

Embodiment 2

[0059] Example 2: Effects of various added factors on the proliferation characteristics of canine adenovirus type Ⅰ in serum-free culture

[0060] (1) Test material: Canine adenovirus type Ⅰ (CAV-Ⅰ) strain is CAV-ZY 180711, which I isolated and identified in 2018. For details, please refer to my published papers: Yi Cheng, Deng Li, Tang Qinghai et al., "Isolation and identification of a type I canine adenovirus", "China Animal Quarantine", Volume 36, Issue 2, 2019, pages 82-87, here, the applicant and the inventor guarantee that the The biological material can be released to the public within 20 years.

[0061] (2) Experimental design: Based on my previous research, I found that EDTA-Na 2 , taurine and cortisol respectively have a certain promoting effect on the proliferation of canine adenovirus type Ⅰ on MDCK cells. Therefore, in order to better carry out serum-free culture of canine adenovirus type Ⅰ, EDTA-Na 2 , taurine and cortisol are prepared into pro-virus proliferat...

Embodiment 3

[0069] Example 3: Proliferation characteristics of canine adenovirus type Ⅰ in serum-free culture

[0070] (1) Passage the serum-free acclimated MDCK cells obtained in Example 1 in the same way. When the confluence of the cells is about 90%, pour out all the culture medium in the spinner bottle, and add 0.9% (w / v) Wash the cells with 100 mL of NaCl washing solution, discard the washing solution, and repeat the washing once more.

[0071] (2) Add the serum-free MEM medium obtained in Example 2-1 and its diluted canine adenovirus type 1 with a dilution factor of 1000 times, so that the virus multiplicity of infection is 1 TCID 50 , cultured in a spinner bottle with a rotating speed of 1 revolution / 5min, a culture temperature of 38.5°C, and airtight culture. Make 12 replicate samples, collect 3 replicate samples respectively at 48h, 72h, 96h and 120h after culture for TCID 50 determination.

[0072] (3) Serum-containing culture of canine adenovirus type Ⅰ was set up as a contr...

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Abstract

The invention relates to a canine adenovirus I-type inactivated vaccine and a preparation method thereof. The canine adenovirus I-type inactivated vaccine is prepared by a serum-free culture process,the immunogenicity of the canine adenovirus I-type inactivated vaccine is equivalent to that of a canine adenovirus I-type vaccine prepared by a serum-containing culture process, and the vaccine prepared by a canine adenovirus I-type cultured by the serum-free culture process can achieve better neutralizing antibody titer in an early immunity period as compared with a vaccine prepared by the serum-containing culture process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a canine adenovirus type I inactivated vaccine and a preparation method thereof. Background technique [0002] Canine adenovirus (CAV) is the most pathogenic virus in the mammalian adenovirus genus. As one of the pets that have the closest contact with humans and spend the longest time with humans, dogs are inseparable from human daily life, and their health status is often closely related to the health status of humans. Therefore, it is important to pay attention to the health of pet dogs It has important public health implications for animal welfare and human health. [0003] At present, the main method for the prevention and treatment of canine infectious hepatitis in my country is vaccination. Commercialized vaccines are all attenuated vaccines, that is, to obtain and prepare the vaccines, it is necessary to prepare high-quality virus fluids in vitro by cell culture. In the ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/235A61P31/20C12N7/06
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/20C12N7/00C12N2710/10334C12N2710/10363
Inventor 唐青海杨海易程王芳宇杨灿何丽芳刘会敬唐娇玉易诚曹丽敏唐斯萍
Owner HENGYANG NORMAL UNIV
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