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Micropterus salmoides brain cell line and application thereof

A technology of largemouth bass and brain cells, applied in animal cells, cell dissociation methods, nervous system cells, etc., can solve the problems of poor drug effect and large economic losses, achieve small errors, reduce dosage, and avoid environmental pollution Effect

Active Publication Date: 2021-06-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, California perch rhabdovirus disease has broken out in large numbers during the cultivation of California perch seedlings. When it is severe, 80% of the ponds are infected, and the mortality rate can be as high as 90% in a short period of time. The drug effect is not good, and the economic loss is huge.

Method used

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  • Micropterus salmoides brain cell line and application thereof
  • Micropterus salmoides brain cell line and application thereof
  • Micropterus salmoides brain cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Separation and culture of large mouthfuls

[0037] 1, experimental reagents and materials

[0038] (1) Experimental animals: Repeatedly verify the healthy large mouth of the unwaffected aquatic animals virus by molecular methods, about 10 cm, from the large mouth of the large mouth of the branch of Zhijiang, Hubei Province, and cultivation in the base of the University of Huazhong Agricultural University. Culture bucket.

[0039] (2) Experimental equipment: Anatomy, ophthalmic scissors, ophthalmology tweezers, 70 μm cell network sieve, syringe, alcohol cotton and gauze, etc.

[0040] (3) Experimental reagents: AIM culture solution, liquid medium M199, trypsin-Edta, penicillin / streptomycin 10000u / ml, fetal bovine serum (FBS) purchased from Gibco company; braithycin B, gentamicin Autumn Shui Xian Su was purchased from Biosharp, Diamel (DMSO) purchased from Sigma, fibroblast growth factor (FGF), epidermal cell growth factor (EGF) purchased from Peprotech.

[0041] ...

Embodiment 2

[0060] Example 2 Optimistic culture environment, growth curve and chromosome analysis of large mouthfuls

[0061] 1. Optimal culture temperature of MSBR in large mouth black bass cell line

[0062] Select 16, 22, 28, 37 ° C, four different culture temperatures, use the M199 culture solution to add 10% FBS, adjust the cell density of 1 × 10 5 A / ml, the cell suspension was inoculated with a 12-well plate and placed in 4 different culturing temperature incubator in an amount of 500 μl / well. Each 1D removes 3-well cells from each experimental group, collecting cells with trypsin-EDTA digestion, count, co-cultivate 5D, continuous count 5, plotted its growth curve, cell growth curve at different culture temperatures figure 2 Indicated. The results showed that the large mouthfuls of black beam cell lines were grown at four temperatures, of which 28 ° C is the optimal culture temperature of MSBR cells.

[0063] 2, large mouth black bass cell line MSBR's most appropriate bovine blood c...

Embodiment 3

[0069] Example 3 Sensitivity Test for Msbr of Dagged Black Brain Cell Line for Different Viruses

[0070] 1. Sensitivity experiment of MSBR on different aquatic viruses in large mouth black bass cell lines

[0071] The cell density is 1 × 10 after digestion with Trypsin-EDTA. 5The / ml is inoculated on a 6-well plate at an amount of 1 ml / well. When the cells have just long, PBS buffer is cleaned twice, 1 mOI different viruses (large mouth black props) MSRV, squid virus blood disorder virus SVCV, grass fish, solitary, solitary virus GCRV, frog, color RGV , Non-point grouper necrosis virus RGNNV, infectious hematoprockecepter necrosis virus IHNV, infectious spleen and necrosis virus ISKNV) Inoculated in the large mouth of the present invention, the amount of black tattocyte cell line MSBr was 200 μl. After inoculation, the cells were observed to produce CPE, and the cells without virus were taken as a control (NC), and the results are as Figure 6 Disted; and qualitative detection ...

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Abstract

The invention discloses a micropterus salmoides brain cell line and application thereof, the micropterus salmoides brain cell line has been preserved in the China Center for Type Culture Collection on March 25, 2021, and the preservation number is CCTCC NO: C202166. The micropterus salmoides brain cell line MSBr is stable in passage, has been successfully passed for more than 65 generations at present, is good in growth state, is wide in application range, not only can be used for virus isolation and identification, pathogen function research, environmental pollutant detection, gene screening and function analysis, fish tumor research and the like, but also can be used for preparing the micropterus salmoides brain cell line MSBr. Meanwhile, the method can also be used for monitoring and diagnosing viral diseases of aquatic animals, investigating and researching epidemiology, developing diagnostic kits for related viral diseases, developing vaccines and the like, and has relatively high application value and social and economic value.

Description

Technical field [0001] The present invention belongs to the technical field of prevention and control of aquatic bioecary and aquaculture disease. Background technique [0002] Aquatic (mainly fish) cell culture studies are late in mammals, starting in the 1960s. In 1962, WOFT and Quimby have established the world's first fish cell line, namely the randamati genital cell line. Since then, fish cell culture studies have been very rapid, and 275 fish cells have been reported in the world. my country's fish cell culture begins in the late 1970s, and the grassfish kiss end tissue diploid cells established by Zhang Nianci et al. It is the first fish cell line in my country. The currently established fish cell lines have about 70 strains, mostly from sea waterfish. The cell line is used as a biologically active in vitro material, and there is an advantage that the living fish cannot match: the culture and maintenance of the cells do not require large breeding equipment, the cost is low...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12Q1/02C12R1/91
CPCC12N5/0618G01N33/5058C12N2509/00C12N2509/10C12N2503/02G01N2500/10
Inventor 袁军法戴彩姣余利李莉娟
Owner HUAZHONG AGRI UNIV
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