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

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

Example Embodiment

[0031] Example 1: Construction method of sea bass larvae cell line

[0032] (1) Larvae selection: choose sea bass fry that hatch for 3-4 days, and the experimental fish are 5-7mm long.

[0033] (2) Prepare rinsing fluid and cell culture fluid:

[0034] Basic medium: Leibovitz's-15 (L15) medium, M199 medium, and Eagle's minimum essential medium (MEM) medium are all products of Gibco. Each medium was prepared according to the company's product instructions, plus 0.266% NaCl and 5mM HEPES, filtered through a 0.22μm membrane, aliquoted, 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: The basic medium L-15 contains 400IU / mL penicillin, 400μg / mL streptomycin and 100μg / mL nystatin, with a pH value of 7.2-7.4.

[0036] Primary cell culture medium: Basic medium L-15 contains 20% fetal bovine serum, 0.266% NaCl, 5mM HEPES, 400IU / ml penicillin, 400μg / ml streptomycin and 400μ...

Example Embodiment

[0048] Example 2 Detection of the effects of different culture media and FBS concentrations on the growth of sea bass larvae cells

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

[0050] Using the 40th generation seabass larvae cells of this Example 1, the growth curves of the cells in different culture media were detected. The specific operations are as follows: aspirate the culture medium in the culture flask, rinse with trypsin once, and add 2ml of 0.25% Trypsin digest the cells until they fall off completely, add the culture medium and gently pipette the cell suspension to make the cells single. Count the cell density with a hemocytometer.

[0051] Respectively add 0.4×10 5 The cells were inoculated in a 24-well plate with 10% fetal bovine serum L-15, MEM and M199 medium, cultured in a constant temperature incubator at 28°C. At 1, 3, 5, and 7 days after culture, the cells were counted with a hemocytometer to draw the growth curve of sea bass larvae cells c...

Example Embodiment

[0057] Example 3 Verification of the cryopreservation and recovery capabilities of the sea bass larvae cell line cells of the present invention

[0058] 1. Cell cryopreservation

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

[0060] 2. Recovery of cryopreserved cells

[0061] The cryopreserved cells were recovered after 15 days of freezing. Remove the cells from liquid nitrogen and quickly thaw them in a 37°C water bath. The thawed cell suspension was centrifuged at 1000 rpm to collect the cell pellet, and the pellet wa...

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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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