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Method for improving Japanese encephalitis virus titer

A Japanese encephalitis virus, Japanese encephalitis technology, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the problems of affecting immunogenicity, clinical immune failure, and the titer is not very good.

Active Publication Date: 2015-06-03
HUNAN SINOLAND BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But on the other hand, the potency of Japanese encephalitis virus in the process of proliferation is still low due to the influence of trypsinization conditions, culture conditions, and the biological characteristics of the virus itself during the preparation of hamster kidney primary cells. Not very good, the difference between each batch is relatively large, and the production cycle is long, so it affects immunogenicity and even leads to clinical immune failure

Method used

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  • Method for improving Japanese encephalitis virus titer
  • Method for improving Japanese encephalitis virus titer
  • Method for improving Japanese encephalitis virus titer

Examples

Experimental program
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Effect test

Embodiment approach

[0026] According to the method for preparing Japanese encephalitis vaccine of the present invention, since the freezing and thawing operation is omitted in the process of multiplying Japanese encephalitis virus, the titer of the harvested virus liquid is significantly improved, so it can be used to pass The Japanese encephalitis virus vaccine with high potency, stability and good immunogenicity was prepared by mixing vaccines. As an embodiment, the Japanese encephalitis vaccine is an inactivated vaccine and / or a live attenuated vaccine.

[0027] To sum up, the method of the present invention omits the freezing and thawing operation in the conventional method, and directly harvests the virus liquid without freezing and thawing after the cells reach the required lesion level, and mixes the virus liquid to prepare the Japanese encephalitis vaccine. In this way, on the one hand, higher quality and more stable Japanese encephalitis virus can be produced by significantly increasing ...

preparation Embodiment 1

[0031]Treatment of hamster kidneys: The collected 10-14-day-old hamster kidneys (commercially purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) were treated with 500 ml of Chinese medicine containing 400 units / ml double antibody (commercially purchased from GIBCO brand). Wash twice with Kirschner's solution (commercially purchased from Gibco brand). One 10,000-mL cell bottle is suitable for the kidney tissue of 20 hamsters. Mince the tissue with scissors and cut evenly. Then rinse the tissue in the beaker repeatedly with Hank's solution containing 400 units / ml double antibody until the supernatant in the beaker is clear, and finally fully precipitate, and pour off the supernatant.

[0032] Trypsin digestion: use 7.5 mass% NaHCO3 to adjust the pH of trypsin to 7.2-7.4, and then pour the above precipitated tissue into the digestion bottle (aseptic operation). Add 0.25 mass% trypsin (commercially purchased from GIBCO brand) according to the volume ratio of try...

preparation Embodiment 2

[0037] Prepare according to the method identical with Preparation Example 1, difference is:

[0038] In the trypsin digestion step, the volume ratio of trypsin:hamster kidney tissue was 2.5:1, and the digestion was performed at 7°C for 12 hours.

[0039] In the step of cell dispersion culture, the volume ratio of Ruhan liquid and RPMI1640 is 1:0.9, and the cell concentration for culture is 2×10 8 number of cells / L.

[0040] During virus inoculation and harvesting, after inoculating JE seed virus at 0.8% by volume of the maintenance liquid, culture it at 37°C, and harvest the virus liquid when the cytopathic pathology reaches 100%.

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Abstract

The invention provides a method for proliferation of a Japanese encephalitis virus. The method for proliferation of the Japanese encephalitis virus comprises the steps of culturing cells, inoculating Japanese encephalitis viruses to the cells which are provided with mono-layers in an overgrowing mode, and directly obtaining a virus solution until a rate of a cytopathic effect reaches to 75%-95% after inoculation. The invention further provides a preparation method of Japanese encephalitis vaccines. The method for proliferation of the Japanese encephalitis virus and the preparation method of the Japanese encephalitis vaccines can remarkably improve a Japanese encephalitis virus titer, and meanwhile shorten production periods, and can prepare the Japanese encephalitis vaccines which are better, stable, and good in immunogenicity.

Description

technical field [0001] The invention relates to a method for multiplying Japanese encephalitis virus and preparing Japanese encephalitis virus vaccine. In particular, it relates to a method capable of significantly increasing the titer of Japanese encephalitis virus. Background technique [0002] Epidemic encephalitis B (Japanese encephalitis B, referred to as "JE"), also known as Japanese encephalitis (JE), is caused by Japanese encephalitis virus (Japanese encephalitis virus, JEV, referred to as "JE virus") ) caused by mosquito-borne acute infectious diseases. This disease can seriously invade and infect the central nervous system, causing clinical symptoms and presenting neurological symptoms. At the same time, the disease is a natural focal disease, which can infect not only humans, but also other animals such as horses and pigs. In recent years, the outbreak of the disease has spread from Asia to Australia and other regions. Mosquitoes are the main vector of Japanes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/12A61P31/14
CPCY02A50/30
Inventor 张桂红陶柏辉徐雷王和平黄岚芬
Owner HUNAN SINOLAND BIOLOGICAL PHARMA
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