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Mandarin fish spinal cord tissue cell line and construction method and application thereof

A technology of tissue cells and construction methods, applied in the field of mandarin fish spinal cord tissue cell lines and their construction, can solve the problems of high lethality, strong infectivity, and threats to the healthy development of the industry, and achieve the effect of simple formula and fast disease time

Pending Publication Date: 2021-12-07
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All three viruses are highly contagious and have a high fatality rate, causing significant economic losses to the mandarin fish farming industry and seriously threatening the healthy development of the industry
However, there is no mandarin fish cell line that is simultaneously sensitive to mandarin fish infectious spleen-kidney necrosis virus, mandarin fish frog iridescent virus and mandarin fish rhabdovirus

Method used

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  • Mandarin fish spinal cord tissue cell line and construction method and application thereof
  • Mandarin fish spinal cord tissue cell line and construction method and application thereof
  • Mandarin fish spinal cord tissue cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A method for constructing a mandarin fish spinal cord tissue cell line, specifically comprising the steps of:

[0034] (1) Treatment of spinal cord tissue: take the mandarin fish spinal cord tissue, disinfect it, cut it into pieces, soak it for later use after treatment, specifically: take fresh mandarin fish, disinfect the mandarin fish with 75% alcohol, and dissect it under aseptic conditions Take the spinal cord tissue, cut into pieces, soak in the rinse solution for rinsing;

[0035] The rinsing solution is HBSS (1X) (Hank's Balanced Salt Solution) solution containing 400 IU / ml penicillin and 400 μg / ml streptomycin.

[0036] (2) Primary culture: the mandarin fish spinal cord tissue after the above treatment was digested with trypsin digestion solution and then primary cultured, specifically: the spinal cord tissue obtained in step (1) was added into trypsin digestion solution (0.5% Trypsin- EDTA (10X)), wherein, the amount of trypsin digestion solution added is 10 ...

Embodiment 2

[0046] Example 2 Verifies the recovery ability of the SSC cell line cells prepared in Example 1 after cryopreservation

[0047] 1) Take the cells in the logarithmic growth phase of Example 1, digest with trypsin to obtain a single cell suspension, centrifuge at 1600 g for 10 min, and discard the supernatant. Add an appropriate amount of cell cryopreservation solution (ZENOAQ, JPN) to the cell pellet, resuspend, transfer to a 1.8ml sterile cryopreservation tube; put the cryopreservation tube into a programmed cooling box, freeze overnight at -80°C, and store it the next day Long-term storage in liquid nitrogen;

[0048] 2) Resuscitate the cryopreserved cells above, take the cryopreservation tube out of the liquid nitrogen tank, put it in a 37°C water bath and shake quickly until it melts, add it to a cell bottle containing 6-8ml of culture medium, and incubate at 28°C Cultured in the box, changing the medium every other day.

[0049] The recovery rate of different generations...

Embodiment 3

[0050] Embodiment 3 is to the chromosome analysis of the SSC cell line that embodiment 1 obtains

[0051] Take the SSC cell line (30th generation) of the above-mentioned implementation 1, and grow the 30th generation cells firmly on the wall for 36-48 hours, add colchicine at a final concentration of 0.6-1.0 μg / ml for 4-8 hours, trypsinize and centrifuge at 1600g Recover the cells for 10 minutes; treat with 75mM KCl at 37°C for 30 minutes, then add 2ml of fixative solution (methanol: glacial acetic acid 3:1) for pre-fixation; centrifuge at 1600g for 10 minutes, discard the supernatant, add fixative solution and fix at room temperature for 30 minutes; repeat fixation steps 2-3 For the last fixation, leave an appropriate amount of fixative solution and gently blow it evenly; absorb the fixation suspension, drop it on a pre-cooled glass slide at -20°C at a height of 15cm, quickly blow off the droplet, and dry it at room temperature; liquid staining for 10 min, the cell chromosome...

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Abstract

The invention discloses a mandarin fish spinal cord tissue cell line and a construction method and application thereof, the mandarin fish spinal cord tissue cell line is preserved in China Center for Type Culture Collection on August 31, 2021, the preservation number is CCTCC NO: C2021226, and the name is mandarin fish spinal cord tissue cell line SSC. The mandarin fish spinal cord tissue cell line can be applied to separation, culture, detection and vaccine preparation of mandarin fish infectious spleen and kidney necrosis virus, mandarin fish frog iridovirus and mandarin fish rhabdovirus. The mandarin fish spinal cord tissue cell line provides an important research platform and an experimental material for separating and identifying fish viruses and preparing virus vaccines.

Description

technical field [0001] The invention belongs to the technical field of aquatic organism cells and aquaculture disease prevention and control, and specifically relates to a mandarin fish spinal cord tissue cell line and its construction method and application. Background technique [0002] Mandarin fish (mandarin fish, Siniperca chuatsi) is delicious and has high nutritional value. In 2019, the total output of mandarin fish farming in my country was nearly 320,000 tons, and the output value exceeded 20 billion yuan. It is mainly cultivated in Guangdong, Hubei, Jiangxi, Anhui and Jiangsu provinces, among which Guangdong is the province with the highest production of mandarin fish. With the rapid expansion of the scale and density of mandarin fish farming, a variety of pathogenic microorganisms lead to disease outbreaks in mandarin fish. Diseases affecting mandarin fish mainly include viral diseases, bacterial diseases, and parasitic diseases. Viral diseases are the most seri...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12Q1/02C12R1/91
CPCC12N5/0618G01N33/5005C12N2509/00Y02A40/81
Inventor 潘晓艺沈锦玉蔺凌云姚嘉赟尹文林巩金鹏刘忆瀚
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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