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Gibel carp dorsal fin cell line

A technology of cell line and crucian carp, which is applied in the field of life sciences to achieve the effect of good material resources, good effect and saving manpower

Inactive Publication Date: 2017-12-08
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on the establishment of the dorsal fin cell line of gibel crucian carp

Method used

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  • Gibel carp dorsal fin cell line
  • Gibel carp dorsal fin cell line
  • Gibel carp dorsal fin cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The construction method of embodiment 1 gibel carp dorsal fin cell line

[0032] 1. Preparation of cell culture medium

[0033] Take the M199 medium of GIBCO company, add fetal bovine serum accounting for 10-20% of the total volume of the medium (the percentages mentioned below are all volume percentages unless otherwise specified), and the final concentration is 20ng / ml basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) with a final concentration of 1 ng / ml, stored at 4°C, and used for later use.

[0034] 2. Primary culture

[0035] Cut off the dorsal fin of gibel carp, then disinfect it with 75% alcohol, and then use 1000IU mL -1 Penicillin, 1000ug mL -1 Streptomycin was used to remove the mucus on the dorsal fin of gibel carp, followed by 2.5ug mL -1 Wash 3 times with amphotericin B in D-PBS. After rinsing, cut the dorsal fin to about 1 mm with scissors under sterile conditions 3 The small pieces were then soaked in 0.25% trypsin and 0.02% EDT...

Embodiment 2

[0039] Embodiment 2 Gibel carp dorsal fin cell line

[0040] After in vitro culture, the present invention has obtained a strain of gibel crucian carp dorsal fin cell line (SCC-DF cell line, Silver crucian carp dorsal fin cell line) with stable passage and uniform components, which has been passed down to date.

[0041] The silver crucian carp dorsal fin cell line that the present invention obtains has been preserved in the China Typical Culture Collection Center, the silver crucian carp dorsal fin cell line SCC-DF, its preservation number is CCTCC NO:C201525, and the classification is called silver crucian carp (CarassiusC.auratus), China The address of the Typical Culture Collection Center is Wuhan University, Wuhan City, Hubei Province, China, and the date of deposit is March 27, 2015.

[0042] Further, the gibel carp dorsal fin cell line has the following biological characteristics:

[0043] 1) The cells isolated from the primary cell near the tissue block exhibit the mor...

Embodiment 3

[0047] Embodiment 3 virus infection experiment

[0048] 1. Experimental method

[0049] 1. Virus infection

[0050] The cultured golden carp pelvic fin cell line was transferred to a 6-well plate and grown adherently overnight. Contain crucian carp herpesvirus II, CyHV-2) and alkaline phosphate buffer, PBS (negative control group) to take 500 μl of infected cells, incubate for 1 hour, discard the supernatant, wash 3 times with PBS, add 500 μl of complete medium Place them in an incubator and observe the growth of the cells every day.

[0051] 2. Drawing of virus replication curve in cell supernatant

[0052] Transfer the cells to a 24-well plate, infect the virus according to step 1, take the supernatant every 24 hours, and discard the precipitate by high-speed centrifugation, and extract DNA from the supernatant according to the following steps for fluorescent quantitative PCR detection.

[0053] 3. Extraction of viral DNA

[0054] (1) Add an equal volume of PBS buffer, ...

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Abstract

The invention relates to a dorsal fin cell line of gibel crucian carp. The dorsal fin cell line of gibel crucian carp is obtained through primary culture and subculture of the dorsal fin of gibel crucian carp. The used cell culture medium is prepared from M199 medium. The invention also provides a construction method and application of the gibel carp dorsal fin cell line. The advantages are as follows: the dorsal fin cell line of gibel crucian carp established by the invention can be passed down continuously, and a large number of dorsal fin cell lines of gibel crucian carp can be provided. The cell line can be directly applied to the study of the immune-related functional genes of the gibel carp, and the cell line can be applied to the virus infection experiment of the gibel carp, which saves a lot of manpower and material resources and has a good effect. The dorsal fin cell line of gibel carp can be repeatedly frozen and thawed, and can be used in important researches such as cell biology, virology, toxicology, molecular biology, genetics and immunology.

Description

technical field [0001] The invention relates to the technical field of life sciences, in particular to a gibel carp dorsal fin cell line. Background technique [0002] C. auratus gibelio belongs to Percoiformes, Sparidae, Carassius, subspecies of C. auratus. The crucian carp distributed in my country includes black crucian carp, crucian carp and gibel crucian carp. The number of chromosomes of crucian carp is 100, which is diploid, and the number of chromosomes of gibel crucian carp is 150±, which is triploid. Gibel crucian carp individuals have good economic traits such as fast growth and strong disease resistance, and are the main freshwater aquaculture species in my country. [0003] Cell culture technology is the main research method in the field of life sciences, cell engineering, genetic engineering and biomedical engineering. The construction of fish cell lines provides the basic biological materials for the pathogenic research of fish viral diseases and the researc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 王浩吕利群黄美秀许丹喻文娟孔善云喻飞夏思瑶
Owner SHANGHAI OCEAN UNIV
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