Human embryo lung fibroblast strain and method for using human embryo lung fibroblast strain for producing hand-foot-mouth viral vaccine

A technology of fibroblasts and human embryonic lungs, applied to embryonic cells, animal cells, antiviral agents, etc., can solve problems such as low yield, complicated process, and human hazards, so as to reduce production costs, reduce culture time, and save costs The effect of spending

Inactive Publication Date: 2013-02-06
云南沃森生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the disadvantage is low yield and high cell generation, which limits the improvement of virus yield and further use
[0017] Similarly, the currently used monkey kidney Vero cells also have a high algebra, which limits the production of viruses. More importantly, the residual heterologous protein of monkey kidney Vero is potentially harmful to the hu

Method used

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  • Human embryo lung fibroblast strain and method for using human embryo lung fibroblast strain for producing hand-foot-mouth viral vaccine
  • Human embryo lung fibroblast strain and method for using human embryo lung fibroblast strain for producing hand-foot-mouth viral vaccine
  • Human embryo lung fibroblast strain and method for using human embryo lung fibroblast strain for producing hand-foot-mouth viral vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The preparation of embodiment 1 reagent and culture medium

[0074] 1.1 Cell basal culture medium: 1000ml contains the following liquids

[0075]

[0076] 1.2 Complete cell culture medium (also called medium containing 10% serum): 1000ml contains the following liquids

[0077]

[0078] The volume of bovine serum and MEM volume of cell culture medium with various serum contents changes, and the others remain unchanged, such as medium containing 5% NBS, 1000ml contains the following liquid

[0079]

[0080] 1.3 Cell cryopreservation solution: 1000ml contains the following liquids

[0081]

[0082] 1.4 Cell / virus maintenance solution: 1000ml contains the following liquids

[0083] MEM 970ml

[0084] 7.5%NaHCO 3 20ml

[0085] 3% L-Glutamine 10ml

[0086] 1.5 0.01M PBS solution: 1000ml contains the following liquids

[0087]

Embodiment 2

[0088] The preparation of embodiment 2 Walvax-2 cell lines

[0089] 2.1 Acquisition of experimental materials: After obtaining the approval of the national health management department and the ethics committee, investigate the age, occupation and health status of the donated pregnant couple, the three generations of the parents of the fetus, and select no history of tumor diseases, no congenital abnormalities and genetics Fetuses with a family history of defects. After obtaining the consent of the parents of the fetus and their relatives, sign the "Donor Consent".

[0090] 2.2 Primary culture: send the embryo to the laboratory in time after induction of water sac, take out the lung tissue under aseptic conditions, tear off the membrane on the surface of the lung tissue, and cut it into 1-2mm 3 The tissue pieces were washed with PBS for 3 to 4 times; the tissue pieces were transferred into the Erlenmeyer flask; digested with trypsin for 30 minutes, neutralized with serum, cent...

Embodiment 3

[0095] Embodiment 3: the preparation of bivalent hand-foot-mouth virus inactivated vaccine

[0096] 3.1 Preparation of bivalent HFMD inactivated vaccine from cell culture flask sample:

[0097] A) Recover Walvax-2 cells from the working library in a T75 cell culture flask, add complete cell culture solution containing 10% bovine serum, pH is between 6.8 and 7.4, and culture at 37°C in a closed manner. After the cells grow into a single layer, wash with PBS, digest with trypsin, discard the trypsin, add the above nutrient solution, and subculture once every 3 days at a seeding ratio of 1:2, and obtain 4 T225 flask cells until logarithmic growth Expect.

[0098] B) Remove the old culture medium, add PBS to wash the cells, add EV71 or COX.A16 virus infection solution at 0.01MOI respectively, let it absorb for 1 hour, add 150ml of virus maintenance solution to cover the cell surface, and culture the virus-infected cells for 3-5 days respectively sky.

[0099] C) Harvest when th...

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Abstract

The invention provides a novel human embryo lung diploid fibroblast strain Walvax-2, CCTCCC201055. The cell strain is sensitive to main epidemic disease viral strains-coxsackievirus 16-type COX.A16 in a group A and enterovirus 71-type EV71 second-strain virus of a hand-foot-mouth disease, and virus output is high. The invention further provides a method for preparing a divalent hand-foot-mouth virus inactivated vaccine and application of the human embryo lung diploid fibroblast strain in preparation of the hand-foot-mouth virus inactivated vaccine. The divalent inactivated vaccine produced by the human embryo lung diploid fibroblast strain can effectively prevent hand-foot-mouth disease.

Description

[0001] Field of the invention: [0002] The invention belongs to the technical field of cell and virus culture. Specifically, the invention relates to a human embryonic lung diploid fibroblast cell line, the application of the cell line in the preparation of bivalent hand-foot-mouth virus inactivated vaccine, and the production of hand-foot-mouth virus with the cell line Method for bivalent inactivated vaccine of oral virus. Background technique: [0003] Viral vaccines can effectively prevent various human infectious diseases caused by viruses. Such as: rabies, meningitis, hepatitis B, leprosy mumps, polio, hand, foot and mouth and other epidemic infectious diseases. The cell substrates for the production of viral vaccines are mainly human embryonic lung diploid cells (hereinafter referred to as "diploid cells"), passaged cells and primary cells. As the culture substrate, the quality of the cell line directly affects the quality and yield of the vaccine, especially the safe...

Claims

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Application Information

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IPC IPC(8): C12N5/073A61K39/295A61K39/125A61P31/14
CPCY02A50/30
Inventor 黄镇何丽芳张翊马波王丽丽赵琼英张杨武雯俊陈敏王馨唐业峰
Owner 云南沃森生物技术股份有限公司
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