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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  • 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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Example Embodiment

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

[0034] Select SPF-grade white Laihang chicken breeding eggs that are produced within a week, have normal shape, uniform eggshell thickness, no cracks, and thick protein (Source: Xinxing Dahuanong Poultry Egg Co., Ltd., Address: Lezhu Town, Xinxing County, Guangdong Province, China The SPF field at the back of the Genwen Group headquarters, the same below), placed in an incubator at 37-39°C and a relative humidity of 40-80% for incubation, and use the egg inspection lamp to observe whether it is a fertilized egg and its vitality. Use chicken embryos that are 9 to 11 days old, have normal embryonic development, and can see clear blood vessels and activities. Soak it with 0.2% (m / v) neocervical solution for 5 minutes and then remove it, place the air chamber upward on the egg tray, and then disinfect with 2% (m / v) iodine tincture and 75% (v / v) alcohol before transferring to ultra-...

Example Embodiment

[0038] Example 1

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

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

[0041] Use PBS (pH 7.4) to dilute RV CTN-1V5 (from the China Institute for Food and Drug Control, the 5th generation virus of CTN-1 strain of vero cells) with a 10-fold serial dilution, and then follow the ratio of 1:100 to 1:1000 Proportionally inoculate vero monolayer cells (from China Institute for Food and Drug Control, 121 generation), add cell maintenance solution (based on 199 medium, add final concentration of 10% (v / v) after adsorption at 37°C for 60 minutes) Bovine serum, pH 7.2~8.0), placed at 37℃, 5% CO 2 Cultivat...

Example Embodiment

[0065] Example 2

[0066] According to the conditions of step 3 of Example 1, it was continuously transmitted to 28 generations to obtain 28 generations of virus species of RV CTNCEC25 strain. If the passages are continued according to the above conditions, it is found that the virus titer does not continue to rise with the increase of passages (the titer hovers around 6.0-6.5lgFFU / ml), so in order to further increase the titer of the obtained strain, The method 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 above 7.0lgFFU / ml. The optimized process parameters are as follows:

[0067] Determine the medium formula: Based on the 199 medium, supplement the appropriate amount of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), bovine serum and human albumin to make HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) The final content is 20mmol / L, t...

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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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