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Method for preparing polyvalent vaccine of primary hamster kidney cells of flu

A technology for influenza and vaccines, applied in the biological field, can solve the problems of vaccine retrovirus contamination, slow adaptation of influenza virus, insufficient source of chicken embryos, etc., and achieve the advantages of large-scale production, sufficient kidney sources, and reduced production costs Effect

Inactive Publication Date: 2013-08-28
深圳市孚沃德生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses primary hamster kidney cells as the proliferation matrix of influenza virus, which solves the problems of insufficient source of chicken embryos and the vulnerability of vaccine to retrovirus contamination, and also solves the problems of slow adaptation of influenza virus in passaged cells and weak virus titer The problem

Method used

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  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu
  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu
  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu

Examples

Experimental program
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preparation example Construction

[0056] 1. Preparation of hamster kidney cells

[0057] Choose healthy golden hamsters that are 10 to 14 days old, kill them with drinking water and wash them for 1 to 2 times, and disinfect them with 1‰ of Neoceramide for 1 to 3 times, each for 3 to 8 minutes. In a sterile environment, use sterile scissors to dissect the hamster and take out the kidney. After cutting, add a digestive solution composed of 0.1% to 0.5% trypsin and 0.01% to 0.05% EDTA, and place it at 2 to 8°C. Digest for 15-20 hours, discard the digestion solution, add growth solution to disperse the cells, prepare to 1.0×10 7 ~1.0×10 8 Cells / ml of cell suspension. Take the primary cell suspension, inoculate it in 3L, 10L spinner flask or cell bioreactor according to the ratio of cell suspension: growth solution of 1:20~1:100, and then add the growth solution to make the cells The initial concentration is 1.0×10 5 ~5.0×10 6 Pcs / ml. The rotating flask has CO at 37℃ 2 Cell culture is carried out in the environment ...

Embodiment 1

[0062] Example 1: Preparation of virus seeds adapted to primary hamster kidney cells of influenza virus

[0063] Primal Toxic Seed Armor 1 (H 1 N 1 ) Type is IVR-116, which is the 8th generation chicken embryo allantoic fluid freeze-dried to preserve the virus species; 3 (H 3 Type N2) is NYMC X-15F, which is the 8th generation chicken embryo allantoic fluid freeze-dried and preserves the virus seed; Type B is B / Jiangsu / 10 / 2003, which is the 6th generation chicken embryo allantoic fluid freeze-dried and preserves the virus seed.

[0064] The above-mentioned three kinds of virus were unsealed in a dedicated sterile room, and adapted to passage 2 times on SPF chicken embryos. The final harvested viral allantoic fluid was subjected to sterility test and determination of hemagglutination titer (HA titer). The sterility test is qualified, the hemagglutination titer of A1 and A3 are both 1:640, and the hemagglutination titer of B is 1:320, so as to establish the main seed bank for product...

Embodiment 2

[0070] Example 2: Preparation of virus seeds adapted to primary hamster kidney cells of influenza virus

[0071] Primal Toxic Seed Armor 1 (H 1 N 1 ) The type is IVR-116, which is the 8th generation chicken embryo allantoic fluid freeze-dried preservation virus species: A 3 (H 3 N 2 ) Type is NYMC X-15F, which is the 8th generation chicken embryo allantoic fluid freeze-dried to preserve the virus species; Type B is B / Jiangsu / 10 / 2003 (recommended by WHO in 2004), which is the 6th generation chicken embryo allantoic fluid freeze-dried and preserved Poison seed.

[0072] The main generation seeds are the three types of influenza main generation seeds prepared in Example 1.

[0073] The dissection of the hamster kidney, the preparation of the cell suspension, and the inoculation of the glass bottle culture are the same as in Example 1.

[0074] When the cells in the culture flask reach 60% to 80% of the culture surface, change to maintenance solution I after rinsing, and press 10 3 Dilute...

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Abstract

The invention discloses a method for preparing a flu vaccine. The method comprises the following steps of: a) performing adaptability passage on the original viruses of flu viruses through unspecific pathogenic chick embryos to serve as main-generation seeds; b) preparing primary hamster kidney cells, and culturing by using a culture flask or a cell biological reactor; c) infecting the by using the main-generation seeds, and performing adaptability passage until viruses with the virus clotting titer of not lower than 1:640 are obtained and used as working seeds of the vaccine; d) infecting the primary hamster kidney cells by using the working seeds, performing virus amplification until vaccine monovalent stock solution with the virus clotting titer of not lower than 1:320 is obtained; ande) concentrating, inactivating and purifying the vaccine monovalent stock solution, mixing different types of monovalent virus solution, and sub-packing into finished products. The primary hamster kidney cells have adequate sources and are easy to produce on a large scale. The prepared vaccine does not contain reproducible deoxyribonucleic acid (DNA) which has tumorigenicity on human bodies or animal bodies and has high safety.

Description

[0001] This application is a divisional application of Chinese patent application CN200610112125.9. Technical field [0002] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing a primary hamster kidney cell vaccine for influenza and a prepared vaccine. Background technique [0003] Influenza, abbreviated as influenza, is a serious respiratory infection. In the influenza pandemic of 1918-1919, more people died of influenza virus infection than in World War II, which lasted for four years. Influenza is often characterized by repeated epidemics, sudden occurrences, rapid spread, small local epidemics, and large epidemics all over the world. The mortality rate of infants and the elderly is high, tens of thousands every year. At present, influenza is still an important issue affecting the public health of the world, and recently the highly pathogenic avian influenza has a great risk of invading humans. Therefore, vaccination is stil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/145C12N7/08A61P31/16
Inventor 李云英王玉清吴歧
Owner 深圳市孚沃德生物技术有限公司
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