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Method for producing influenza viruses in large scale by using bioreactor

A bioreactor, influenza virus technology, applied in the fields of biochemical equipment and methods, microorganisms, viruses/phages, etc., can solve the problems of not being able to adjust in real time, unable to ensure the optimal culture state of cells, etc., to achieve no contamination by exogenous factors, The effect of real-time controllable cell culture conditions and easy large-scale production

Active Publication Date: 2011-02-23
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] (1) It overcomes the shortcomings of the roller bottle cell culture, which cannot adjust the culture conditions such as nutrients, pH and dissolved oxygen in the culture medium in real time, and cannot ensure that the cells are in the best culture state;

Method used

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  • Method for producing influenza viruses in large scale by using bioreactor
  • Method for producing influenza viruses in large scale by using bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Influenza virus: H9N2 subtype influenza virus HN strain (see literature: Avian influenza virus (H9N2 subtype, HL strain) and avian influenza virus (H9 subtype) in some provinces in China The antigenic correlation and cross-immunity research, 2007 Proceedings of the Annual Meeting of the Society of Animal Husbandry and Veterinary Medicine, 2007, 35-38), the applicant holds this strain and is willing to distribute it to the public within 20 years from the date of application in accordance with the relevant provisions of the Patent Law.

[0063] Bioreactor and carrier: Tidecell tidal bioreactor (the volume of carrier tank is 20L) and "BioNOC II" carrier were purchased from CESCO Bioengineering Company in Taiwan.

[0064] Passage cell line: MDCK (NBL-2) (this cell line was deposited with ATCC under the accession number CCL-34, see http: / / www.atcc.org for details).

[0065] Serum-free cell culture medium: DMEM medium (manufactured by Invitrogen, product number 12800-82, batc...

Embodiment 2

[0088] Influenza virus: H1N1 subtype influenza virus (refer to Chinese patent CN1644686A for the strain, and the strain is deposited in the China General Microorganism Collection and Management Center under the deposit number CGMCC No. 1014).

[0089] Serum-free cell culture medium: MEM medium (manufactured by GIBCO, product number 61100-087, batch number 678277) was prepared according to the product instructions, and the pH value was adjusted to 7.2.

[0090] Cell culture medium containing 8% (V / V) serum: use MEM medium (produced by GIBCO, Cat. No. 61100-087, Lot No. 678277) according to the product instructions, and add 8% (V / V) newborn bovine serum ( Manufactured by GIBCO, Cat. No. 16010-14, Lot No. 693745), pH adjusted to 7.2.

[0091] Serum-free cell culture medium containing TPCK trypsin: use MEM medium (produced by GIBCO, product number 61100-087, batch number 678277) to prepare according to the product instructions, and add TPCK trypsin (produced by Sigma-Aldrich compa...

Embodiment 3

[0098] Influenza virus: H5N3 subtype influenza virus (refer to Chinese patent CN1884498A for the strain, and the strain is deposited in the China General Microorganism Collection and Management Center under the deposit number CGMCC No. 1339).

[0099] Serum-free cell culture medium containing TPCK trypsin: use DMEM medium (manufactured by Invitrogen, product number 12800-82, batch number 309313) to prepare according to the product instructions, and add TPCK trypsin (manufactured by Sigma-Aldrich company, product number T3053) to the end. The concentration was 1 mg / L, and the pH value was adjusted to 7.2.

[0100] Other reagents and materials used in this example are the same as those in Example 1.

[0101] The steps, methods and parameters of each culture stage adopted in this example are the same as those in Example 1.

[0102] Finally, 50L of virus liquid was harvested. After testing, the HA was 1:1024, and the virus content was 10 8.5 TCID 50 / ml.

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Abstract

The invention provides a method for producing influenza viruses in large scale by using a bioreactor, which comprises the following steps of: (1) inoculating passage cell suspension into a vector tank, and setting the parameters of the bioreactor to perform a cell adsorption and culture process, wherein a serum culture medium is used in the cell adsorption and culture process; (2) when cells are grown to proper density, rinsing the cells to remove serum components, inoculating influenza virus liquid into the vector tank, and setting the parameters of the bioreactor to perform a virus adsorption and propagation process, wherein a serum-free culture medium containing TPCK (tosyl-L-phenylalanine chloromethyl ketone) pancreatin is used in the virus adsorption and propagation process; and (3) harvesting the virus liquid. The method has the advantage that: the high-titer virus liquid can be obtained by adopting the tide type bioreactor and using the passage cells for propagating the influenza viruses. The method overcomes the defects of the traditional transfer bottle culture and other bioreactors, and can continuously produce high-titer viruses in large scale.

Description

technical field [0001] The present invention relates to a method for producing influenza virus. More specifically, the present invention relates to a method for large-scale production of influenza virus using a tidal bioreactor. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family, and can be divided into three types (genus) A (A), B (B) and C (C) according to the serological reaction of its internal proteins (mainly NP protein and M1 protein). . Influenza B and C viruses primarily infect humans and, rarely, seals and pigs, but have never been isolated in birds. Influenza A virus can be further divided into 16 HA subtypes and 9 NA subtypes according to the serological reaction of glycoproteins HA and NA. Most influenza virus subtypes composed of 16 HA subtypes and 9 NA subtypes mainly exist in birds and animals. Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and H5, H7, and H9 are the most harmful to birds. subtype strains. A...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12N7/00
Inventor 张许科孙进忠乔荣岑
Owner PU LIKE BIO ENG
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