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Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development

A technology of rabies virus and high immunity, which is applied in the direction of antiviral agents, viruses/bacteriophages, and medical preparations containing active ingredients, etc., which can solve the problem of low immunogenicity, adverse immune reactions of immunized people, and insufficient protection of vaccine immunity to achieve high immunogenicity, good immune protection effect and high safety

Active Publication Date: 2013-06-12
广州瑞贝斯药业有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For people who are severely exposed to rabies virus, if the immunogenicity of the virus strain used for vaccine production is low, the ordinary antigen dose is not enough to ensure the immune protection effect of the vaccine, and further increasing the antigen dose may lead to serious adverse events in the immune population. immune response

Method used

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  • Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development
  • Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development
  • Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Virus strain G 0 preparation of

[0041] Suspend the dry powder of lyophilized rabies virus fixed strain 4aG in water for injection, dilute it 10 times with maintenance solution A (M199 medium containing 0.5% bovine blood albumin, pH7.0-8.0), and inoculate guinea pigs weighing 45-50g in the brain cavity , 0.1ml / rat; 96 hours later, the brains were aseptically removed, and the three brains were combined, and 12ml of maintenance solution A was added, and the homogenizer was used to make a homogenate; high-speed centrifugation (10000×G, 30 minutes, 4°C) to remove the precipitate; The supernatant was placed on a sucrose layer with a density gradient of 30-60% for ultracentrifugation (80000-100000×G, 240 minutes, 4°C), and the solutions were collected in groups, and the sucrose content and antigen content of each component solution were measured; Combine the components with a sucrose concentration of 45-55%, put them into a dialysis bag with a molecular cut-off of 30K, dial...

Embodiment 2

[0043] Virus strain GV 1 preparation of

[0044] For Vero cells adherently cultured for 24-72 hours, digest with trypsin and use maintenance solution B (M199 medium with 0.01%-0.1% trypsin, 0.1-1mM EDTA, 0.5-1% bovine serum albumin, pH7.0- 8.0) Made with a density of 4×10 6 The cell suspension of cells / ml; Add the G prepared in Example 1 in an amount of 1:1-1:100 by virus: cells 0 Virus suspension, place at 37°C, shake regularly; inoculate cell culture flasks after 30 minutes, the inoculum size is 5×10 5 -2×10 6 cells / ml, add culture solution A (M199 medium containing 0.5% bovine albumin, pH7.0-8.0), and culture at 37°C; remove culture solution A after 48 hours and add maintenance solution B (containing 0.5 % bovine serum albumin M199 medium, pH7.8), adherent culture under the condition of 30-34°C; harvest the culture medium after 144 hours, carry out sucrose density gradient ultracentrifugation purification and concentrated virus according to the same method as in Example...

Embodiment 3

[0046] Virus strain GNV 1 preparation of

[0047] The GV prepared in embodiment 2 1 Virus suspension, inoculated nude mice with a body weight of 8-10g in the brain cavity, and the inoculated dose was 20ul / only, and the brain was aseptically taken after 72-96 hours, and the virus suspension was prepared according to the same method as in Example 1, numbered GNV 1 . The virus titer determined by the mouse brain cavity method was 8.02 LogLD 50 / ml.

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Abstract

The invention relates to seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development, which belongs to the field of biotechnology. The method comprises the following steps of: (1) augmentation and purification of rat brain virus strain; (2) culturing of the rat brain virus adaptive to Vero cell; (3) augmentation of the virus in nude rat brain tissue by Vero cell culture; (4) alternate transfer of the virus in nude rat brain tissue and Vero cell; (5) continuous transfer of the virus in Vero cell; (6) monoclonal purification, screening, storage and strain culturing of the virus; and (7) transfer stability analysis of the bred virus strain, wherein the obtained optional virus strain is applied as a virus strain for vaccine production. The new rabies virus fixed strain bred by the method can be augmented at high titer in the Vero cell; the augmented virus has higher immunogenicity than the present rabies virus fixed strain; stabilities of biology, genetics and other characteristics are still maintained after a plurality of times of continuous transfer in Vero cell, so the method provided by the invention can be used for large-scale industrial production of rabies vaccine with better immunoprotection effect and higher safety.

Description

technical field [0001] The invention relates to a method for breeding a fixed strain of rabies virus and its application, in particular to the breeding of a fixed strain of highly immunogenic rabies virus and its application in the development of rabies vaccine. Background technique [0002] Rabies is a serious and fatal epidemic. Once clinical symptoms appear after infection with rabies virus, the prognosis for survival is extremely poor, and death is almost inevitable. At present, more than 2.5 billion people in the world live in rabies endemic areas and are threatened by the disease, causing more than 50,000 deaths every year, more than half of whom are children under the age of 15, who are high-risk groups of rabies infection. China is also an area with a high incidence of rabies, and the death rate of rabies has long ranked first among all kinds of legal infectious diseases in China. [0003] Since there is no effective treatment for rabies that can be successfully ap...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/205A61P31/14C12R1/93
Inventor 艾文沈名锋刘钿莲
Owner 广州瑞贝斯药业有限公司
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