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Large-scale culture method of H9N2 subtype avian influenza virus

A technology for large-scale cultivation of avian influenza virus, which is applied in the field of large-scale cultivation of H9N2 subtype avian influenza virus, can solve the problems that the cultured virus titer cannot meet the requirements of vaccine production, and achieve easy industrialized large-scale operation, easy standardization, and virus immunity Original good effect

Inactive Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solved the problem that the virus titer of H9N2 avian influenza virus cultured on MDCK cells cannot meet the requirements of vaccine production

Method used

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  • Large-scale culture method of H9N2 subtype avian influenza virus
  • Large-scale culture method of H9N2 subtype avian influenza virus
  • Large-scale culture method of H9N2 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Rapid adaptation of avian influenza virus A / Duck / Hubei / W1 / 2004 (H9N2) strain to MDCK cells:

[0037] 1) Take the MDCK cells with a dense monolayer, digest the cells and spread them into a 6-well cell culture plate on average, and wait until the cells are full of the monolayer, the chicken embryo allantoic fluid original virus venom (its HA titer>2 5 ) was diluted 10 times with DMEM medium, and 10 -3 、10 -4 、10 -5 、10 -6 Each dilution of venom was 100ul, supplemented to 3ml with 1.2ug / ml DMEM containing TPCK-trypsin, and the cell wells without virus were set up as negative controls.

[0038] 2) At 37°C, remove the virus liquid after virus adsorption for 1.5 hours, wash MDCK cells with DMEM for 3 times, add 1.2ug / ml DMEM 3ml containing TPCK-trypsin to each well, and observe every 6 hours after 24 hours For cytopathic changes, measure HA titer, observe cytopathic changes every 3 hours after 48 hours, measure HA titer, freeze and thaw the 6-well plate once at...

Embodiment 2

[0046] 7.5L Stirred Reactor → 40L Basket Fixed Bed Reactor Cultivation:

[0047] Bioreactor: 7.5L reactor, 40L basket fixed bed reactor from NBS Company in the United States

[0048] Carrier: cytodex-1 (GE Company), FibraCel Discs (NBS Company, USA)

[0049] Cell growth medium: DMEM (GIBICO) with volume ratio of 8% serum

[0050] 1) Cultivate seed cells in a 7.5L stirred reactor: take cytodex 144g at a concentration of 8g / L, hydrate with 1000ml PBS at room temperature for 24 hours and balance 3 times to make the pH7.2-7.6, and add it to the reactor together with PBS After sterilization, precipitate cytodex1, pump out PBS and replace it with 10% volume ratio of newborn bovine serum (Sijiqing) DMEM, adjust the reactor parameters so that the temperature is 35.5-37°C, pH 7.2, stirring 30rpm, DO (dissolved oxygen) is 30-80, inoculate MDCK cells after each parameter of the reactor is stable, the total amount of cells is 1.2*10 9 . After inoculation, the culture parameters were a...

Embodiment 3

[0056] Viral immunity evaluation:

[0057] 1) Inactivation and emulsification of cytotoxicity

[0058] Reactor-produced virus (HA titer 2 10 ) was inactivated by formaldehyde solution, and the inactivated virus was emulsified with the oil phase (ExxonMobil) at a volume ratio of 2:1 to prepare an oil adjuvant inactivated vaccine.

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Abstract

The invention discloses a large-scale culture method of an H9N2 subtype avian influenza virus. According to the large-scale culture method, the H9N2 virus is rapidly adapted to MDCK, the HA titer of the virus is 28, MDCK cells are cultured as seed cells by taking a 7.5L reactor as a seed cell tank and cytodex1 as a carrier, the seed cells are amplified to large-scale culture in a 40L fixed bed basket type stirring system reactor, and 80L of H9N2 viruses of which the hemagglutination titer (HA) is up to 210 can be obtained. The virus produced by using the method is high in titer and large in yield, the method is easy in standardization and is applicable to all H9 subtype influenza viruses, and the difficulty that the culture virus titer cannot meet vaccine production when the H9N2 avian influenza virus is cultured on the MDCK cells is solved.

Description

technical field [0001] The invention belongs to the technical field of animal and veterinary biology, and in particular relates to a method for large-scale cultivation of H9N2 subtype avian influenza virus. The method is simple and easy, and the rapid adaptation of the H9N2 virus on MDCK cells is carried out by combining microcarriers and sheet carriers for large-scale cultivation of the influenza virus. Background technique [0002] H9N2 subtype avian influenza (Avian influenza, AI) is one of the important poultry infectious diseases that continue to endanger the development of poultry industry in my country and the world. H9N2 subtype avian influenza is a low pathogenic avian influenza. In a typical outbreak, the onset is sudden, the infection rate is high, and the transmission range is large; in an atypical epidemic, there are no characteristic symptoms, which mainly cause egg production decline, brooding and chicken vaccination failure. The infection rate of the diseas...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
Inventor 金梅林李国红周明光徐高原洪灯左静徐晓娟喻文波王丹张盼陈绣镯陈焕春
Owner HUAZHONG AGRI UNIV
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