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A kind of saddle grouper kidney tissue cell line and its construction method

A technology of saddle grouper and kidney tissue, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve virus infection, pathogenesis mechanism cannot be studied in depth, virus isolation and culture and vaccine development progress is slow and other problems, to achieve the effect of fast cell lesion time, simple formula and good culture effect

Active Publication Date: 2019-01-18
ZHAOQING DAHUANONG BIOLOGIC PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of sensitive cell lines, the isolation, cultivation and vaccine development of marine fish viruses are slow, and the infection and pathogenesis of viruses in the process of marine fish farming cannot be studied in depth.

Method used

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  • A kind of saddle grouper kidney tissue cell line and its construction method
  • A kind of saddle grouper kidney tissue cell line and its construction method
  • A kind of saddle grouper kidney tissue cell line and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The construction method of saddle grouper kidney tissue cell line specifically comprises the following steps:

[0056] (1) Treatment of kidney tissue: Put the fresh and live grouper in the penicillin and streptomycin high double-antibody seawater with a concentration of 1000IU / ml for 24 hours, take the fresh and live grouper, and use medical alcohol Soak for 1-5 minutes to carry out overall disinfection of the saddle grouper, and then sterilize again with 75% alcohol. After disinfection, dissect the kidney tissue in an ultra-clean workbench under aseptic conditions, cut the kidney tissue into pieces with a blade, and use The rinsing solution (M199 solution containing 400 IU / ml penicillin and 400 μg / ml streptomycin) was washed 4-6 times to remove blood cells.

[0057] (2) Primary culture: add the kidney tissue obtained in step (1) to trypsin digestion solution, digest at room temperature for 20-30min, collect the cell suspension after digestion; add 1-2ml of serum-contai...

Embodiment 2

[0062] The method for constructing the kidney tissue cell line of the saddle grouper follows the steps in Example 1, and the difference from Example 1 is that in this example,

[0063] The basal culture solution is MEM basal culture solution.

[0064] The value-proliferating culture medium is the MEM culture medium containing 20% ​​fetal bovine serum, 0.046mM NaCl, 400IU / ml penicillin, 400μg / ml streptomycin, 20ng / ml epidermal growth factor and 20ng / ml fibroblast growth factor (referred to as MEM medium);

[0065] The subculture medium contains 5-20% fetal bovine serum, 0.046mM NaCl, 100-400IU / ml penicillin, 100-400μg / ml streptomycin, 0-20ng / ml epidermal growth factor and 0-20ng / ml synthetic MEM medium of fibroblast growth factor.

Embodiment 3

[0067] The method for constructing the kidney tissue cell line of the saddle grouper follows the steps in Example 1, and the difference from Example 1 is that in this example,

[0068] Described basal culture fluid is L-15 basal culture fluid;

[0069] The value-proliferating culture medium is the L-15 culture medium containing 20% ​​fetal bovine serum, 0.046mM NaCl, 400IU / ml penicillin, 400μg / ml streptomycin, 20ng / ml epidermal growth factor and 20ng / ml fibroblast growth factor (referred to as L-15 culture medium);

[0070] The subculture medium contains 5-20% fetal bovine serum, 0.046mM NaCl, 100-400IU / ml penicillin, 100-400μg / ml streptomycin, 0-20ng / ml epidermal growth factor and 0-20ng / ml synthetic L-15 medium of fibroblast growth factor.

[0071] On the basis of the above-mentioned Examples 1-3, further, when passing to the second generation, the concentration of FGF-basic and EGF in the subculture medium is reduced to 8ng / ml; when passing to the 5th-10th generation, the ...

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Abstract

The invention discloses a grouper kidney tissue cell line and a construction method thereof. The construction method specifically includes the following steps: (1) treatment of the kidney tissue: taking the kidney tissue of the grouper grouper, disinfecting it, and cutting it into pieces , soaked after treatment for use; (2) primary culture: the above-mentioned grouper kidney tissue after the above treatment was digested with trypsin digestion solution and treated with sterilized double distilled water for primary culture; (3) subculture: The primary culture cells were subcultured when the confluence reached 60-90%, every 5-7 days. The kidney tissue cell line constructed by the invention is sensitive to the grouper iridescent virus, and has a fast cytopathic time. Therefore, the cell line can be well applied to the isolation and propagation of grouper iridescent virus and the research of grouper iridescent virus vaccine. At the same time, it also provides cell materials for the isolation and cultivation of other seawater fish viruses and the development of vaccines.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, and in particular relates to a saddle grouper kidney tissue cell line and a construction method thereof. Background technique [0002] After more than 20 years of development, my country's mariculture has made great achievements. The scale, variety and output of aquaculture are all in the forefront of the world. The proportion of mariculture production in marine fishery production has risen from 10% in the past to 40% at present. Saddle band grouper (Epinephelus Lanceolatus), also known as gentian grouper, commonly known as gentian, is a kind of important marine economic fish in my country. Because of its fresh and tender meat, rich nutrition, fast growth and strong adaptability, it has been It has become an important target of mariculture in southern my country. In recent years, while my country's mariculture industry has developed rapidly, it is also facing outstanding problems such a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 欧阳征亮陈瑞爱高任吉华松
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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