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Epinephelus lanceolatus head kidney cell line and construction method and application thereof

A saddle grouper and a construction method technology are applied to the saddle grouper head kidney cell line and the field of its construction, which can solve the problems of grouper breeding economic loss, deterioration of the breeding environment and the like, and achieve the reduction of culture cost and cell The effect of stable proliferation and good growth status

Active Publication Date: 2021-06-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the expansion of the grouper breeding scale, the single species, the improvement of the intensive cultivation and the deterioration of the breeding environment, etc., the outbreaks of diseases caused by bacteria, viruses or parasites are becoming more and more frequent. Breeding causes major economic losses

Method used

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  • Epinephelus lanceolatus head kidney cell line and construction method and application thereof
  • Epinephelus lanceolatus head kidney cell line and construction method and application thereof
  • Epinephelus lanceolatus head kidney cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction method of saddle grouper head kidney cell line

[0032] (1) Selection of fish body: select juvenile saddle grouper, and the length of the experimental fish is 10 cm.

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

[0034] L15 basal medium: Leibovitz's-15 (L-15) medium is a product of Gibco. The medium was prepared according to the company’s product instructions, and 0.266% NaCl and 5mM HEPES were added to adjust the pH value to 7.2-7.4. After being filtered through a 0.22μm filter membrane, the culture medium was subpackaged and stored at 4°C for later use; fetal bovine serum or Double antibody (penicillin, streptomycin).

[0035] Rinse solution: Add double-antibody penicillin and streptomycin to L-15 basal medium, the final concentration is 400IU / mL penicillin and 400μg / mL streptomycin.

[0036] Primary cell culture medium: L-15 basal medium was added with serum and double antibody to a final concentration of 20% fetal ...

Embodiment 2

[0049] Example 2 Detects the mycoplasma contamination in the saddle grouper head kidney cells of passage

[0050] Passage the saddle grouper head kidney cells (ELHK cell line) to a 35mm culture dish (glass slide at the bottom) for 48 hours, the culture medium is L-15 medium containing 10% fetal bovine serum, and the culture temperature is 28°C , the cell density reaches 80%-90%. Cells were fixed with 4% paraformaldehyde at room temperature for 1 h, rinsed with PBS, stained with Hoechst 33258 staining solution (0.05 μg / ml) for 10 min, rinsed with PBS three times, and observed under a 100-fold fluorescent microscope.

[0051] Fluorescent microscope observation results showed that the blue fluorescence of mycoplasma was not observed in the gap or cells of the passaged saddle grouper head kidney cells, indicating that the cells had no mycoplasma pollution ( figure 2 ).

Embodiment 3

[0052] Example 3 Detecting the Effects of Different Culture Temperatures and FBS Concentrations on the Growth of Saddle Grouper Head Kidney Cells

[0053] 1. The effect of different culture temperature on cell growth.

[0054] Using the 45th generation of saddle grouper head kidney cells (ELHK cell line) in Example 1, the growth curves of the cells at different culture temperatures were detected. The specific operations were as follows: discard the culture medium in the culture bottle, and add 0.25% pancreatic Rinse once with enzyme, add 0.2ml of 0.25% trypsin to digest the cells until they fall off completely, add medium (L-15 medium containing 10% fetal bovine serum) and gently blow the cell suspension to make the cells single. Count the cell density with a hemocytometer.

[0055] 5×10 4 Cells (ELHK cell line) were inoculated in a 12-well plate, and the culture medium was L-15 medium containing 10% fetal bovine serum. After 2 hours of adherent growth in a 28°C constant tem...

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Abstract

The invention discloses an epinephelus lanceolatus head kidney cell line as well as a construction method and application thereof. The ELHK cell line is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on December 04, 2020, the address is the 5th floor of No.59 building, No.100, Middle Xianlie Road, Guangzhou City, Guangdong Province, the postal code is 510070, and the preservation number is GDMCC No: 61340. The Epinephelus lanceolatus head kidney cell line-ELHK cell line is obtained, the growth state is good, cell proliferation is stable, the cell morphology takes a fibroblast sample as the main morphology, continuous passage can be achieved (at present, the cells have already been transferred to more than 120 generations), ultralow-temperature cryopreservation and recovery can be achieved. The establishment of the cell line lays a foundation for related research of grouper germplasm resource preservation.

Description

Technical field: [0001] The invention belongs to the technical field of seawater fish cell culture, and in particular relates to a saddle grouper head kidney cell line and its construction method and application. Background technique: [0002] Grouper (Epinephelus sp.) belongs to Osteichthyes, Perciformes, Serranidae, and Epinephelinate. It is a warm-water bottom-dwelling fish. The main economically farmed marine fish in Southeast Asian countries. Saddle band grouper (Epinephelus lanceolatus) is a grouper cultured species introduced in my country's southern coastal areas such as Guangdong and Fujian in recent years. It has the advantages of delicious meat, high nutritional value, fast growth rate, and strong adaptability. Moreover, the pearl saddle banded grouper bred as the male parent and the tiger grouper has accounted for 70% of the total grouper breeding in Hainan. However, with the expansion of the grouper breeding scale, the single species, the improvement of the in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12R1/91
CPCC12N5/0686C12N2509/00
Inventor 黄友华黄晓红刘泽天秦启伟
Owner SOUTH CHINA AGRI UNIV
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