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Construction method of sea bass fry cell line

A technique for constructing method and cell line

Inactive Publication Date: 2020-07-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] To date, suitable cell lines for the isolation and propagation of marine fish iridoviruses, especially cytomegaloviruses, are very limited

Method used

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  • Construction method of sea bass fry cell line
  • Construction method of sea bass fry cell line
  • Construction method of sea bass fry cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the construction method of sea bass larvae cell line

[0032] (1) Selection of larvae: select sea bass fry hatched for 3-4 days, and the body length of the experimental fish is 5-7mm.

[0033] (2) Preparation of rinse solution and cell culture solution:

[0034] Basal medium: Leibovitz's-15 (L15) medium, M199 medium, Eagle's minimalessential medium (MEM) medium are all products of Gibco. Each medium was prepared according to the company's product instructions, and 0.266% NaCl and 5mM HEPES were added, filtered through a 0.22μm filter membrane, subpackaged, and stored at 4°C for later use; when used, add 10-20% fetal bovine serum, 1-4 times PSN Antibody mixture (penicillin, streptomycin, nystatin).

[0035] Rinsing solution: basal medium L-15 containing 400 IU / mL penicillin, 400 μg / mL streptomycin and 100 μg / mL nystatin, pH 7.2-7.4.

[0036] Primary cell culture medium: basal medium L-15 contains 20% fetal bovine serum, 0.266% NaCl, 5mM HEPES, 400IU / ml p...

Embodiment 2

[0048] Embodiment 2 detects the influence of different culture medium and FBS concentration on sea bass larvae cell growth

[0049] 1. The effect of different culture media on cell growth.

[0050] Using the 40th generation seabass larvae cells in Example 1, the growth curves of the cells in different culture media were detected, and the specific operations were as follows: suck out the culture medium in the culture bottle, rinse once with trypsin, and add 2ml of 0.25% Trypsinize the cells until they are completely detached, add medium and gently pipette the cell suspension to separate the cells. Count the cell density with a hemocytometer.

[0051] 0.4×10 respectively 5 Each cell was seeded in a 24-well plate, and the medium was L-15, MEM and M199 medium with 10% fetal bovine serum, respectively, and cultured in a constant temperature incubator at 28°C. The cells were counted with a hemocytometer at 1, 3, 5, and 7 days after culture, and the growth curves of sea bass larva...

Embodiment 3

[0057] Example 3 verifies the cryopreservation and resuscitative ability of sea bass larvae cell line cells of the present invention

[0058] 1. Cell cryopreservation

[0059] Take the adherent monolayer cells of passage 35 in Example 1, digest with trypsin to obtain a single cell suspension, centrifuge at 1000 rpm for 10 min, and discard the supernatant. The cell pellet was resuspended with 4°C pre-cooled freezing solution (L-15 medium containing 20% ​​fetal calf serum and 10% DMSO), and 1ml of each tube was transferred to a 2ml cryopreservation tube. The cryopreservation procedure is: 4°C, 30min; -20°C, 2h; -80°C overnight, and transfer to liquid nitrogen for long-term storage the next day.

[0060] 2. Recovery of cryopreserved cells

[0061] After 15 days of cryopreservation, the above-mentioned frozen cells were resuscitated. Remove the cells from liquid nitrogen and quickly thaw in a 37°C water bath. The thawed cell suspension was centrifuged at 1000 rpm to collect th...

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Abstract

The invention provides a construction method of a sea bass fry cell line. The construction method comprises the following steps: (1) primary culture: carrying out pancreatin digestion on sea bass frytissues to obtain a cell suspension, and adding a primary cell culture solution to culture primary cells; (2) subculture: when passage is carried out for 1-10 generations, the concentration of fetal calf serum in a passage cell culture solution is 20%; when passage is carried out for 11-20 generations, the concentration of fetal calf serum in the passage cell culture solution is 15%; after passageis carried out for 20 generations, the concentration of fetal calf serum in the passage cell culture solution is 10%; and (3) collecting passage cells to obtain the sea bass fry cell line. The sea bass fry cell line obtained by the construction method provided by the invention has a good growth state and stable cell proliferation, can be continuously passed, can be frozen and recovered at ultralow temperature and can be used for researches of exogenous gene expression, virus infection separation and the like.

Description

technical field [0001] The invention belongs to the technical field of seawater fish cell culture, and in particular relates to a method for constructing sea bass larvae cell lines by using sea bass larvae. Background technique [0002] Sea bass belongs to the order Perciformes and the genus Sea Bass, and is a carnivorous fish. Sea bass is a wide-salt and wide-temperature fish, usually living in estuary areas, and also directly enters freshwater lakes. At present, the annual output of sea bass in my country is about 250,000 tons, mainly concentrated in the Doumen area of ​​the Pearl River Delta in Guangzhou, and has become a pillar industry for mariculture in Doumen District, Zhuhai City, Guangdong Province. However, with the expansion of the scale of sea bass farming, the improvement of the degree of intensification, and the deterioration of the breeding environment, diseases caused by bacterial, viral or parasitic pathogens have become more and more frequent, and disease ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12R1/91
CPCC12N5/0602
Inventor 黄友华黄晓红秦启伟魏京广周胜
Owner SOUTH CHINA AGRI UNIV
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