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Adapting method of rabies virus (RV) CTN-1 strain to primary chicken embryo fibroblast

A rabies virus, CTN-1 technology, applied in the field of rabies vaccine, to achieve good immune protection, good stability and immune protection effect

Active Publication Date: 2014-06-18
SHENZHEN WEIGUANG BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, there is no literature addressing the adaptation of RV CTN-1 strains to CEC

Method used

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  • Adapting method of rabies virus (RV) CTN-1 strain to primary chicken embryo fibroblast
  • Adapting method of rabies virus (RV) CTN-1 strain to primary chicken embryo fibroblast
  • Adapting method of rabies virus (RV) CTN-1 strain to primary chicken embryo fibroblast

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preparation example Construction

[0033] The preparation process of the CEC suspension involved in the present invention is as follows:

[0034] Select SPF-grade White Leghorn eggs that have been produced within one week, have normal shape, uniform eggshell thickness, no cracks, and thick protein (Source: Xinxing Dahuanong Poultry Egg Co., Ltd., Address: Lan, Lezhu Town, Xinxing County, Guangdong Province, China Root Wen’s Group Headquarters Houshan SPF field, the same below), put them in an incubator at 37-39°C and a relative humidity of 40-80% for incubation, and use an egg inspection lamp to observe whether they are fertilized eggs and their vitality. Choose 9-11 days old chicken embryos with normal development, visible blood vessels and activities. Soak in 0.2% (m / v) bromogeramine solution for 5 minutes, remove it, put the air chamber upwards on the egg tray, and then disinfect it with 2% (m / v) tincture of iodine and 75% (v / v) alcohol, then move it into the ultra-clean Taiwan. Take out the chicken embryo...

Embodiment 1

[0039] Taking the CTN-1V5 strain as the mother strain, through the comparison and screening of multiple passage ways and processes, it was found that the strains obtained by passage according to the following process routes can gradually adapt to the growth in chicken embryo cells. The chicken embryo cell-adapted strain of rabies virus It was named CTNCEC25 strain.

[0040] Step 1: subculture the CTN-1V5 strain on vero cells to increase the virus titer.

[0041] Use PBS (pH7.4) to make 10-fold serial dilutions of RV CTN-1V5 (from the China Institute for Food and Drug Control, the 5th generation virus of CTN-1 strain vero cells), and then follow the ratio of 1:100 to 1:1000. Proportionally inoculate vero monolayer cells (from China National Institutes for Food and Drug Control, passage 121), absorb at 37°C for 60 minutes, and then add cell maintenance solution (based on 199 medium, add 10% (v / v) of final concentration Bovine serum, pH7.2~8.0), placed at 37℃, 5%CO 2 Cultivate ...

Embodiment 2

[0066] According to the conditions of Step 3 of Example 1, it was continuously propagated to 28 generations, and the 28 generations of virus seeds of RV CTNCEC25 strain were obtained. If the above-mentioned conditions continue to be subcultured, it is found that the virus titer does not continue to rise with the increase in the number of passages (the titer hovers around 6.0-6.5 lgFFU / ml), so in order to further increase the titer of the obtained strain, The invention optimizes the process from the aspects of culture medium formula, culture temperature, MOI, pH value, etc., and the optimized process can increase the titer of the virus strain to more than 7.0 lgFFU / ml. The optimized process parameters are as follows:

[0067] Determine the medium formula: Based on the 199 medium, add appropriate amount of HEPES (4-hydroxyethylpiperazineethanesulfonic acid), bovine serum and human serum albumin to make HEPES (4-hydroxyethylpiperazineethanesulfonic acid) The final content of ser...

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Abstract

The invention provides an adapting method of a rabies virus (RV) CTN-1 strain to a primary chicken embryo fibroblast. The adapting method comprises the following steps of carrying out 10 continuous passages on RV CTN-1V5 in a vero cell to obtain a CTN-1V15 strain; carrying out one passage on a virus seed of the CTN-1V15 strain in a chicken embryo to obtain a first-generation virus of the RV CTN chicken embryo; carrying out passage on the first-generation virus of the RV CTN chicken embryo in the chicken embryo fibroblast to enable the first-generation virus of the RV CTN chicken embryo to gradually adapt to the chicken embryo fibroblast. The invention provides the adapting method of the RV CTN-1 strain to CEC (chicken embryo cardiomyocytes); by using the method, the CTN strain can be rapidly and efficiently multiplied in the CEC; moreover, the obtained RV CTN chicken embryo cell adapting strain can be stably multiplied on the CEC, and has favorable stability and immune protection. The invention also provides an inactivated vaccine prepared by using the RV CTN chicken embryo cell adapting strain, and the inactivated vaccine has favorable immune protection and can be used for producing refined and purified RV for people.

Description

technical field [0001] The invention relates to the field of rabies vaccines, in particular to a method for adapting rabies virus CTN-1 strain to primary chicken embryo fibroblasts. Background technique [0002] Rabies virus (RV) is a highly neurotropic virus. It is a non-segmented single-stranded negative-sense RNA virus in the Rhabdoviridae (Lyssavirus genus), which can cause zoonosis in the world Sexually transmitted diseases - rabies. It is reported that there are about 55,000 deaths due to rabies every year in the world, and the actual number of deaths should be significantly higher than the statistics (http: / / www.worldrabiesday.org / ). At present, except Japan, Britain, Hawaii and a few other countries and regions where no rabies occurs, the disease is distributed worldwide. Asia and Africa are the most severe areas of human rabies, accounting for 99% of the total number of deaths worldwide. Among countries that officially report rabies, India ranks first with more t...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/205A61P31/14
CPCA61K39/12A61K2039/5252A61P31/14C12N7/00C12N2760/20121C12N2760/20134C12N2760/20151
Inventor 郭采平王春华罗姗刘永娣容伟华李慧周维周兰贞田华张佩丁玉江黄伟荣吴开永张运佳王红冰王锦才吴恩应
Owner SHENZHEN WEIGUANG BIOLOGICAL PROD
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