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Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells

A suspension culture, cell technology, applied in the field of medicine and biology

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

Both Marc145 cells and MA104 cells are relatively sensitive to PRRS virus. At present, the production of PRRS vaccine is mainly carried out through the process of spinner bottle wall-attached culture or bioreactor microcarrier suspension culture of Marc145 cells. Full suspension culture has great limitations in terms of production cost, production efficiency and meeting market demand

Method used

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  • Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells
  • Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells
  • Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 Utilizes genetic modification suspension culture Marc145 cell

[0065] 1) Stable transfection of Marc145 cells with an expression vector containing the siat7e gene;

[0066] (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).

[0067] (2) Day 1: transfer 3×10 5 Marc145 cells (provided by the China Veterinary Drug Administration) were incubated in a 6-well plate for 22 hours with a confluence of about 70%.

[0068] (3) On the second day, 5 μg of plasmid DNA was diluted in 250 μL Opti-MEM medium, and mixed; 15 μL liposome was diluted in 250 μL Opti-MEM medium, mixed gently, and allowed to stand for 10 minutes.

[0069] (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 Opti-M...

Embodiment 2-4

[0082] Except that the experimental conditions were changed according to Table 1, the same operations as in Example 1 were carried out.

[0083] Table 1

[0084]

[0085]

Embodiment 5

[0086] Embodiment 5 utilizes the method for producing PRRS vaccine by the Marc145 cell of large-scale culture genetic modification suspension culture

[0087]A part of the cells verified by suspension culture (Marc145 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 2×10 5 The cells / mL cell density were inoculated into a 120L bioreactor (Beijing Qingda Tianyi Technology Co., Ltd.), and the cell culture conditions were as follows:

[0088] Working volume: 60L

[0089] Cell culture temperature: 37°C

[0090] pH: 7.2

[0091] Dissolved oxygen: 50%;

[0092] Cell culture medium: Marc145 bioreactor high-density cell culture medium (Cat No.MD900, Beijing Qingda Tianyi Technology Co., Ltd.)

[0093] Cultured to a cell density of 2×10 6 C...

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Abstract

The invention provides a method for suspension culture of sensitive cells, which comprises the following steps that: (1) expression vectors containing gene sequences, capable of weakening the cell adhesion performance, are used for stably transfecting Marc145 cells or MA104 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 blue ear disease vaccine vaccine by using the Marc145 cells or MA104 cells obtained through the suspension culture.

Description

technical field [0001] The invention relates to the field of medical biology, in particular to a method for suspending culture of sensitive cells, the sensitive cells including Marc145 cells or MA104 cells; and further relates to a method for producing PRRS vaccine using the cells. 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) It is easy to carry out ...

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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