Preparation method and product of H9N2 subtype avian influenza inactivated vaccine

An inactivated vaccine and bird flu technology, applied in the field of bioengineering, can solve problems such as biological safety hazards and difficulty in the reproduction of bird flu virus cells, and achieve the effects of avoiding biological safety hazards, meeting the requirements of immune production, and simple and fast production methods

Active Publication Date: 2010-09-01
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to solve the problem that the avian influenza virus cells are difficult to reproduce, and the current avian influenza production uses a large number of chicken embryos, which often leads to biological safety hazards. A method and product for safe and continuous closed cell culture virus production are provided. The production of the vaccine can no longer rely on chicken embryo reproduction and reduce biological safety hazards. At the same time, the cell culture method can also produce high-titer viruses to meet the requirements of immune production. In the event of an epidemic, the vaccine can be quickly provided

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  • Preparation method and product of H9N2 subtype avian influenza inactivated vaccine
  • Preparation method and product of H9N2 subtype avian influenza inactivated vaccine
  • Preparation method and product of H9N2 subtype avian influenza inactivated vaccine

Examples

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Comparison scheme
Effect test

Embodiment 1

[0033] This example illustrates the screening method of the virus-adapted cell line and the final determination method of the virus-adapted cell line of the H9N2 subtype avian influenza isolate JY strain of the present invention.

[0034] 1. Screening

[0035] The H9N2 subtype avian influenza isolate JY strain described in the present invention is purchased from the Poultry Research Institute of the Chinese Academy of Agricultural Sciences, and the strain code is: type A avian influenza virus (AIV) A / Chicken / Jiangsu / JY / 99 (H9N2) strain, It is called JY strain for short.

[0036] The virus-adapted cells to be selected for screening according to the present invention are:

[0037] MDCK (canine kidney cells) was purchased from the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University;

[0038] VERO (African green monkey kidney cells) were purchased from the School of Pharmacy, Shanghai Jiao Tong University.

[00...

Embodiment 2

[0082] This example illustrates the domestication method of the virus-adapted cell line MDCK cells determined in Example 1.

[0083] Adapt the virus to the cell line MDCK cells for carrier-free culture, change the characteristics of its adherent growth, and make it suitable for the growth environment of full suspension culture:

[0084] Digest the MDCK cells cultured by 2 to 3 passages after resuscitation with trypsin, press 2×10 5 The cells / mL density was added to the Erlenmeyer shaker flask, cultured on a shaker with F-DMEM medium containing 8-10% serum, pH 7.5-7.1, and after 40-48 hours, the cell density reached 1-2×10 6 Cells / mL was divided into flasks for passage, and the cell morphology was observed at the same time. For each passage, shake flasks with good cell morphology were selected for passage in separate flasks, cultured on a shaker according to the above method, continuous passage for 43 passages, aliquoted, and frozen in liquid nitrogen.

Embodiment 3

[0086] Methods for primary expansion and continuous culture of virus-adapted cells.

[0087] (1) Primary expansion culture:

[0088] The domesticated MDCK cells were inoculated into 5L cell culture bioreactor with 1~5×10 5 cells / mL cells, cultured in suspension until expanded to 0.3~1×10 7 cells / mL, realize primary cell expansion culture;

[0089] Wherein, the medium is: F-DMEM medium containing 6-10% serum;

[0090] The culture conditions described are: dissolved oxygen 20-60%, PH value 7.0-7.4, temperature 35-38°C, primary flow acceleration rate 6L / day, stable post-flow acceleration rate 3L / day;

[0091] (2) Continuous culture:

[0092] After the primary expansion culture is completed, add fresh medium and pump out the cell suspension in the above cell culture bioreactor to maintain the stability of the bioreactor and ensure that the cells are expanded to 0.3-1 in the residence time of the reactor. ×10 7 cells / mL, realize continuous cell culture;

[0093] Wherein, the...

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Abstract

The invention relates to a preparation method and a product of an H9N2 subtype avian influenza inactivated vaccine. The technical points of the invention mainly relate to the screening, the determination and the domestication of a virus-adapted cell line, the primary amplification cultivation and the continuous cultivation of a virus-adapted cell, the preparation of virus fluid by virus-inoculated culture and the preparation of final inactivated vaccine products. Firstly, the invention avoids the virus propagating method using a large amount of chick embryos in the avian influenza production at present, thereby avoiding the problem of biological potential safety hazards, and overcoming the problem that the mass production of vaccines is enslaved to the supply of the chick embryos; secondly, the invention provides a safe, continuous and closed cell culture virus production method, is used for the preparation of the H9N2 subtype avian influenza inactivated vaccine, enables the use of the cell culture method, and can simultaneously produce high-titer viruses to meet the requirements for the immunological production; and finally, the vaccine production method of the invention is simple and fast, thereby realizing the fast vaccine supply at the epidemic situation.

Description

technical field [0001] The invention relates to a preparation method and product of an H9N2 subtype avian influenza inactivated vaccine, belonging to the technical field of bioengineering. Background technique [0002] Generally, chicken embryo propagation and cell culture methods are mainly used for the propagation of avian-derived viruses. However, because avian influenza virus cannot be directly produced and reproduced in cells, the chicken embryo propagation method is still used. The virus is inoculated into the matrix of chicken embryos and grown in chicken embryos. In the process, the virus replicates to achieve the purpose of mass production of viruses, which is a method similar to the reproduction of viruses under natural biological environmental conditions. The chicken embryo breeding method is simple and easy, without the need to process and domesticate the virus, and the isolated virus can be directly inoculated into chicken embryos. However, a large number of ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16
Inventor 李玉和沈明君范娟季明潘杰
Owner 扬州优邦生物药品有限公司
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