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A method for constructing a koi brain cell line and its application

A construction method, a technology of brain cells, applied in microorganism-based methods, animal cells, cells of the nervous system, etc., can solve the problems of ineffective, invisible, and epithelial cell damage of fibers and connective tissue

Active Publication Date: 2019-07-16
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
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Problems solved by technology

Digestion method is currently commonly used trypsin digestion, but trypsin will cause damage to some epithelial cells, and will not be effective on fibers and connective tissues
So far, the bibliographical reports of preparation of koi cell lines by enzymatic digestion have: Dong Chuanfu, Xue Shuqun and Xiao Yi all adopted the tissue block separation method to establish koi fin ray cell lines (Virus Research, 2011, 161: 140-149; Aquatic Products Science Journal, 2012, 25(4): 16-23; Chinese Journal of Cell Biology 2012, 34(8): 767-774), Wang Yingying established a Koi snout cell line by digestion method (Aquaculture, 2015, 435:310–317), but there are no reports of koi brain-derived cell lines

Method used

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  • A method for constructing a koi brain cell line and its application
  • A method for constructing a koi brain cell line and its application
  • A method for constructing a koi brain cell line and its application

Examples

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Embodiment 1

[0054] Example 1 Construction method of carp brain cell line

[0055] (1) Preparation of cell culture solution and tissue digestion solution

[0056] 1. Preparation of koi brain cell culture medium:

[0057] L-15 medium (GIBCO) was added with 15v / v% fetal bovine serum (GIBCO), 10ng / ml epidermal growth factor (EGF) (Sigma) and 2ng / ml basic fibroblast-like growth factor (bFGF) (Sigma), along with 100 IU / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B, 20 mmol / L HEPES, pH 7.0-7.4.

[0058] After the cells were passed to 10 passages, the addition of fetal bovine serum in the koi brain cell culture medium was reduced to 10%, and growth factors and antibiotics were not added.

[0059] 2. Preparation of Tissue Digestive Solution

[0060] Special tissue digestion solution A: Dissolve 100mg type IV collagenase powder and 80mg trypsin powder in 100ml D-Hanks solution, stir and dissolve for 2h, 4°C overnight, filter and sterilize at 0.22um, and store at -20°C in aliqu...

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Abstract

The invention discloses a method for constructing a koi brain cell line and its application. The koi brain tissue is first put into digestion solution A containing collagenase and trypsin for digestion, and the cells are collected, and then added into a digestion solution containing EDTA‑4Na. Digest in solution B, collect the digested cells for primary culture and subculture to obtain the koi brain cell line; the present invention aims at the characteristics of koi brain tissue cells, and uses collagenase and trypsin joint digestion method to separate brain tissue The cells were cultured in the first generation, and then the koi brain cell line was successfully constructed, which has been continuously passed on for more than 100 generations. It can provide a large number of koi brain-derived cells, and the cells can maintain a good growth state, which can be cryopreserved . The koi brain cell line of the present invention is sensitive to the koi herpes virus, and cytopathy occurs after inoculation, and multiplied virus particles can be found by electron microscope observation. The cell line of the present invention can be directly applied to the research of pathogenic characteristics and the development of vaccines.

Description

technical field [0001] The invention relates to a method for constructing a koi brain cell line and its application in virus isolation, and belongs to the application technology of freshwater biological cell culture. Background technique [0002] Koi ( Cyprinus carpio ), also known as "scarlet carp" and "Japanese koi", is one of the large ornamental fish, a variant of carp, belonging to the family Cyprinidae and the genus Carp. According to the distribution of colors, markings, etc., koi are divided into red and white, Taisho three colors , Showa three colors and other 13 strains. Because of its good size and bright colors, koi are being farmed more and more. As the breeding environment is getting worse and worse, various infectious diseases of koi, especially koi herpes virus disease, have begun to spread widely. Koi herpesvirus disease first broke out in Israel in 1998, but it was only confirmed in 1999 that the real causative agent was Koi herpesvirus (KHV). Since the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079C12N7/00C12R1/93
Inventor 王英英王庆曾伟伟李莹莹石存斌吴淑勤
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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