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Squaliobarbus curriculus fin cell line as well as establishing method and application thereof

A technique for establishing a method and a cell line, which is applied in the field of red eye trout fin ray cell lines, can solve problems such as affecting the production of grass carp culture, and achieve the effects of increasing the adherence rate, reducing the impact and prolonging the time.

Active Publication Date: 2016-09-07
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of grass carp breeding, grass carp hemorrhagic disease has been very prominent, which has seriously affected the farming output of grass carp.

Method used

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  • Squaliobarbus curriculus fin cell line as well as establishing method and application thereof
  • Squaliobarbus curriculus fin cell line as well as establishing method and application thereof
  • Squaliobarbus curriculus fin cell line as well as establishing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Establishment of Trichogramma fin ray cell line of the present invention

[0022] (1) Preparation of cell culture medium

[0023] Take Leibovitz ， Based on sl-15 medium, add epidermal growth factor, fibroblast growth factor and fetal bovine serum to configure cell culture medium. In this nutrient solution, fetal bovine serum accounts for 25% of the total volume of this cell culture solution, the final concentration of epidermal growth factor is 10ng / ml, the final concentration of fibroblast growth factor is 10ng / ml, the cell culture solution prepared in Store at 4°C.

[0024] (2) Primary culture

[0025] Take a healthy red-eyed trout, soak it in 20mg / ml potassium permanganate solution for 30min, then spray the fish body with 70% mass concentration alcohol and dry it. Incubate in a petri dish containing 0.25% trypsin for 15 minutes, scrape off excess tissue with sterilized scissors and tweezers, and cut the tissue pieces into 1.5mm pieces with scissors 3 T...

Embodiment 2

[0028] Example 2 Preservation and Recovery of Cells

[0029] Configuration of liquid A and liquid B:

[0030] Solution A: 40% fetal bovine serum, 20% dimethyl sulfoxide and 40% Leibovitz by volume ， sl-15 medium prepared. Take 1ml of solution A and put it into a freezing tube, and mark the freezing date, cell name, medium type, and serum concentration on the freezing tube.

[0031] Liquid B: regular Leibovitz ， sl-15 culture medium (without fetal bovine serum).

[0032] Preservation of cells: Take vigorously growing red-eyed trout fin ray cells, wash them once with phosphate buffer, digest with 1ml of 0.25% trypsin, and suck out the trypsin when the cells shrink into single cells. Neutralize with 2ml of special medium (fetal bovine serum in this special medium accounts for 20% of the total volume of the medium), and then suck out the special medium. Suspend the cells with 1ml of solution B and add to the bottom of the cryopreservation tube containing solution A above. Pu...

Embodiment 3

[0034] Example 3 Determination of Optimal Growth Conditions for Cells

[0035] Determination of the optimum serum concentration: the initial density was 20×10 6 The fin ray cells of red-eyed trout per ml were planted in Leibovitiz containing 5%, 10%, 15% and 20% FBS respectively. ， sl-15 medium (this medium is also added with epidermal growth factor and fibroblast growth factor, the final concentration of epidermal growth factor in this medium is 10ng / ml, and the final concentration of fibroblast growth factor is 10ng / ml ,) of 25cm 2in a culture bottle. On days 2, 4, 6, 8, 10, and 12, the cells were digested with 0.25% trypsin, counted on a hemocytometer, and the growth curve was drawn. See results image 3 , the cell growth rate increased with the increase of FBS concentration, in the Leibovitiz containing FBS ， In sl-15 medium, the cell growth rate was the fastest when the volume fraction of FBS was 20%. Show: the red-eyed trout fin ray cell that the present invention ...

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Abstract

The invention discloses a squaliobarbus curriculus fin cell line as well as an establishing method and an application thereof. The preservation number of the squaliobarbus curriculus fin cell line is CCTCC NO: C201642; the squaliobarbus curriculus fin cell line is obtained from the tail fin of squaliobarbus curriculus through primary culture and subculture; and an adopted culture medium is obtained by cultivating through Leibovitz' sl-15. The squaliobarbus curriculus fin cell line has the biological characteristics as follows: cells are fibroblast-like cells, and the cells are cultivated to an optimal state by the Leibovitz' sl-15 culture medium which contains 20% of fetal calf serum by volume in a constant-temperature incubator at 25 degrees. The cells, which are resuscitated under the growth conditions, are dyed with trypan blue, so that a cell survival rate reaches 82.5% or above; and the cells are fibroblast-like cells which are vigorous in growth, and the cells include 48 characteristic chromosomes. In addition, a plasmid pEGFP-N1 is transferred to the cell line for expressing and displaying fluorescence. The cell line disclosed by the invention is used for supporting growth and replication of grass carp hemorrhage viruses; and the cell line is not only applicable to expression of exogenous genes but also suitable for toxicology.

Description

technical field [0001] The invention relates to the technical field of animal cells, in particular to a red-eyed trout fin ray cell line, and to a method for establishing the cell line and an application thereof. Background technique [0002] Grass carp belongs to the genus Grass carp of Cyprinidae Cyprinidae and is the main freshwater aquaculture object in China. However, during the cultivation of grass carp, grass carp hemorrhagic disease has been very prominent, which has seriously affected the cultivation output of grass carp. Red-eyed trout ( Squaliobarbus curriculum ) in taxonomy belongs to Cypriniformes Cyprinidae Cyprinidae subfamily Trichonymus genus, commonly known as red-eyed fish, is a high-quality economic fish, which is widely distributed, almost all over the major water systems in China. Red-eyed trout is often used for research because of its less disease, delicious meat, and strong adaptability. The usual research is to conduct distant hybridization exper...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12R1/94
CPCC12N5/0654C12N7/00C12N2501/10C12N2501/11C12N2509/00C12N2720/12051
Inventor 肖调义邓亚林乔庆李伟赵鑫周智愚何美凤金生振
Owner HUNAN AGRICULTURAL UNIV
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