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Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells

A suspension culture and cell technology, applied in the field of medicine and biology, can solve the problems of failure to achieve full suspension culture of cells

Inactive Publication Date: 2012-05-16
BEIJING SKYWING TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] But the existing swine fever vaccine production technology that uses porcine testis cells or porcine kidney cells as cell substrates is all spinner bottle adherent culture or microcarrier suspension culture, and has not yet achieved full suspension culture of cells. In terms of production cost, production efficiency and There are great limitations in meeting market demand

Method used

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  • Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells
  • Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells
  • Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 Utilizes genetic modification suspension culture ST cell

[0067] 1) Stable transfection of ST cells with an expression vector containing the siat7e gene;

[0068] (1) Escherichia coli DH5α competent cells were transfected with a full-length human siat7e gene expression vector (Cat.No.EX-V1581-M03, Genecopoeia). Plasmid DNA was extracted using an endotoxin-free plasmid extraction kit (QIAGEN).

[0069] (2) Day 1: transfer 5×10 5 ST cells (provided by the China Veterinary Drug Administration) were incubated in a 6-well plate, and the cells were incubated for 24 hours at about 90% confluence.

[0070] (3) On the next day, take 10 μg of plasmid DNA and dilute it in 250 μL Opti-MEM medium, mix well; 50 μL liposomes are diluted in 250 μL Opti-MEM medium, mix gently, and let stand for 15 minutes.

[0071] (4) Mix the solutions prepared above, and let stand at room temperature for 20min; replace each well of the 6-well plate described in step (2) with 2 mL of O...

Embodiment 2-4

[0084] Except that the experimental conditions were changed according to Table 1, the same operations as in Example 1 were carried out.

[0085] Table 1

[0086]

[0087]

Embodiment 5

[0088] Embodiment 5 utilizes the method for the ST cell production swine fever vaccine of large-scale culture genetic modification suspension culture

[0089] A part of the cells verified by suspension culture (ST cells, Example 1) was frozen and stored, and the other part was continued to be cultured in the cell culture flask. When the cells grow normally in cell culture flasks and their viability is greater than 90%, they are inoculated into shake flasks. Expand cultured cells in shake flasks, press 3×10 5 The cells / mL cell density were inoculated into a 650L bioreactor (Beijing Qingda Tianyi Technology Co., Ltd.), and the cell culture conditions were as follows:

[0090] Working volume: 300L

[0091] Cell culture temperature: 37°C

[0092] pH: 7.0

[0093] Dissolved oxygen: 40%;

[0094] Cell culture medium: modified MEM cell culture medium MD611 (Beijing Qingda Tianyi Technology Co., Ltd.) + 5% serum

[0095] Cultured for 4 days, when the cell density was 3×10 6 Cells...

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Abstract

The invention provides a method for suspension culture of subculture cells, which comprises the following steps that: (1) expression vectors containing gene sequences, capable of weakening the cell adhesion property, are used for stably transfecting pig testis cells or pig kidney cells; (2) a stable transfection cell clone is screened and separated; (3) the gene expression in the stable transfection cell clone is detected; and (4) the stable transfection cell clone is subjected to suspension culture. The method further relates to a method for producing hog cholera vaccine by using the pig testicle cells or the pig kidney cells obtained through the suspension culture.

Description

technical field [0001] The invention relates to the field of medicine and biology, in particular to a method for suspending culture of passaged cells, the passaged cells include pig testicular cells or pig kidney cells; and further relates to a method for using the cells to produce a swine fever vaccine. Background technique [0002] In vitro cell culture methods include adherent culture and suspension culture, wherein suspension culture can be further divided into microcarrier suspension culture and full suspension culture. Compared with adherent culture, suspension culture has the following advantages: (1) it can continuously expand the production capacity; (2) it is conducive to the full contact of nutrients and gases in the cell culture medium, and it is easy to control the culture conditions (temperature, pH, oxygen, etc.). partial pressure and CO 2 etc.); (3) The culture conditions are stable and tend to be uniform, which is convenient for quantitative research; (4) I...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/68G01N33/53A61K39/12A61P31/14
Inventor 王建超陈文庆张韧
Owner BEIJING SKYWING TECH CO LTD
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