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Tilapia brain cell line and application thereof

A tilapia and brain cell technology, applied in the field of biological cytology, can solve the problems of amplification and purification, lack of Luohu virus, inability to identify TiLV infection, etc., and achieves the effect of a simple and convenient preparation method

Inactive Publication Date: 2018-11-23
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The etiology of many tilapia diseases has not been thoroughly investigated, and the infection situation of TiLV in my country cannot be clarified. One of the major obstacles is that there are currently no sensitive cell lines derived from tilapia to Luohu virus in China, and the virus cannot be expanded. Purification cannot provide a material basis for the isolation, identification, diagnosis and vaccine development of Luohu virus

Method used

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  • Tilapia brain cell line and application thereof
  • Tilapia brain cell line and application thereof
  • Tilapia brain cell line and application thereof

Examples

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Effect test

Embodiment 1

[0026] Embodiment 1 A kind of construction method of tilapia brain cell line

[0027] The above-mentioned method of the present invention is described in detail as follows according to detailed steps:

[0028] 1. Preparation of tilapia brain tissue:

[0029] Soak fresh tilapia in 75% alcohol for 1-2min, put it in an ultra-clean bench, remove the brain tissue aseptically, rinse it twice with PBS, and put it in a 5ml sterile penicillin vial (containing 5% fetal bovine serum). M199 culture medium), cut it into about 1mm with surgical scissors 3 For fragments, add 5ml PBS to collect them into a 15ml centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant after centrifuging to collect tissue fragments, and repeat once. Add collagenase type Ⅰ about 5 times the volume of the tissue block, digest at 37°C for 30 minutes, remove the digestion solution, wash twice with PBS, then add 0.25% trypsin, digest at room temperature for 6-7min, add culture medium to stop d...

Embodiment 2

[0039] The culture condition research of embodiment 2 tilapia brain cells

[0040] 1 Determination of the best culture medium for tilapia brain cells

[0041] Four cell culture media, DMEM, M199, MEM, and L-15, were selected, and FBS with a final concentration of 10% was added to prepare the cell culture medium. Adjust the cell density to 4 x 10 5 mL -1 , each of the four mediums was inoculated in a 6-well plate at an amount of 2.5 mL / well, and cultured in an incubator at 27°C. Cells in 3 wells were taken out from each experimental group every 1 day, collected and counted by Trypsin-EDTA digestion method, co-cultured for 7 days, counted 7 times continuously, and the growth curve was drawn. Determine its optimum medium as M199 or L-15 medium ( figure 2 A).

[0042] 2 Determination of optimal serum concentration of tilapia brain cells

[0043] Prepare culture solutions with FBS concentrations of 5%, 10%, 15%, and 20% respectively, and adjust the cell density to 4×10 5 mL...

Embodiment 3

[0046] Example 3 Chromosome determination of tilapia brain cells

[0047] The 5th and 60th generation tilapia brain cells were added with colchicine at a final concentration of 20 μg / mL in the logarithmic growth phase, and the cells were collected after treatment at 27°C for 4 hours, treated with 0.075mol / L KCl for 25 minutes, and added 1 mL Pre-cooled Carnot's fixative, centrifuged at 1000r / min for 5min to remove the supernatant, then fixed with pre-cooled Carnot's fixative for 3 times, 15min each time. The slices were dropped by cold drop method, and after drying, stained with 5% Giemsa for 25min. For microscopic examination, 100 split phases were selected for karyotype analysis and statistics. The results show( Figure 4 ), 60% of the chromosomes of the tilapia brain cells of the 5th generation were 2n=44; 43% of the chromosomes of the tilapia brain cells of the 60th generation were 2n=50. This indicates that the cells are a permanent cell line and can proliferate indefi...

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Abstract

The invention discloses a construction method of a tilapia brain cell line. The method comprises the following steps: disinfecting fresh and live tilapia, taking brain tissue, cutting the tilapia intominced blocks, treating the minced blocks with collagenase, and adding trypsin; performing full suspension with a culture solution, and performing primary culture; when the bottom of a bottle is fullof cells, performing digestion with pancreatin-EDTA, and performing inoculation for secondary culture; and after 60 generations of secondary culture, adding colchicine in the logarithmic growth phasefor treatment, and performing karyotype analysis. The preparation method of the tilapia brain cell line, disclosed by the invention, is simple and convenient; and the tilapia brain cell line preparedby the method can perform secondary culture for a long time, is very sensitive to hemorrhagic disease viruses of tilapia, and can be widely applied to experiments.

Description

technical field [0001] The invention relates to the field of biological cytology, in particular to a tilapia brain cell line and its application. Background technique [0002] Tilapia is the second largest aquaculture species in the world. It is omnivorous and highly tolerant to high-density farming methods. This low-cost, high-quality fish has become an important source of food for people in developing countries. According to data from the Food and Agriculture Organization of the United Nations (FAO), in 2015, the global tilapia farmed and captured production reached 6.4 million tons, worth about US$9.8 billion, and the global trade volume reached US$1.8 billion. Currently, China, Indonesia and Egypt are the three major producers of tilapia. Among them, China is a big tilapia farming country, and its farming output accounts for about 50% of the world's total output, mainly concentrated in Guangdong, Guangxi, Hainan and Fujian. [0003] Compared with other fish species, t...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00
CPCC12N5/0602C12N7/00C12N2501/11C12N2501/115C12N2509/00C12N2760/16051
Inventor 王英英曾伟伟王庆任燕李莹莹石存斌朱新平
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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