Grass goldfish pelvic fin cell line constructing method

A construction method, the technology of goldfish grass, applied in the field of cell biology, can solve the problems that the pelvic fin cell line of goldfish grass has not been reported, and achieve the effect of strong cell reproduction ability

Inactive Publication Date: 2015-07-08
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the constructed goldfish cell lines are as described ...

Method used

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  • Grass goldfish pelvic fin cell line constructing method
  • Grass goldfish pelvic fin cell line constructing method
  • Grass goldfish pelvic fin cell line constructing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Treatment of tissue blocks: cut off the pelvic fins of goldfish, put them in a penicillin-streptomycin double-antibody solution (distilled water configuration) with a concentration of 1000IU / ml, and place them for 30 minutes, then sterilize them in 75% alcohol for 2 minutes, and carry out the first Disinfection; then wash 3 times with D-PBS containing 2.5ug / ml amphotericin B for secondary disinfection; after rinsing, cut the pelvic fins to about 1mm with scissors under sterile conditions 3 The small pieces of tissue were soaked in 0.25% trypsin and 0.02% EDTA for 10 minutes, and then digested with 0.5% hyaluronidase for 1 hour;

[0031] (2) Primary culture: collect the digested tissue fragments and transplant them to the bottom of 25cm 2In the cell culture flask, keep the bottom temperature at 25 °C. After 15 hours, add 2ml grass gold carp pelvic fin cell special-purpose proliferating culture fluid (culture fluid formula is to contain 18v / v% fetal calf serum, 15ng / ...

Embodiment 2

[0071] Example 2 The cell line is applied to the detection of crucian carp herpesvirus II (CyHV-2)

[0072] 1. Experimental method

[0073] 1. Virus infection

[0074] The cultured golden carp pelvic fin cell line was transferred to a 6-well plate and grown adherently overnight. Take 500 μl of infected cells from the positive sample (containing crucian carp herpesvirus II, CyHV-2) and the negative sample (alkaline phosphate buffered saline, PBS) to be detected respectively, incubate for 1 hour, discard the supernatant, wash 3 times with PBS, Add 500 μl of complete medium and place it in an incubator for culture, and observe the cell growth every day.

[0075] 2. Extraction of viral DNA

[0076] (1) Add an equal volume of PBS buffer, mix well, centrifuge at 8000rpm for 5 minutes, and discard the supernatant;

[0077] (2) Add 20 μL of proteinase k. Incubate in a water bath at 56°C for 3 hours;

[0078] (3) Centrifuge at 8000rpm for 5 minutes, take 750μL of supernatant and p...

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Abstract

The invention relates to a grass goldfish pelvic fin cell line and a constructing method thereof. Tissue blocks are digested through trypsin, EDTA and hyaluronidase according to the features of grass goldfish pelvic fin tissue histocyte, the digested tissue blocks are transplanted to a cultivation bottle, special multiplication cultivation solution is added to the cultivation bottle, primary culture is conducted, and then the grass goldfish pelvic fin cell line is successfully constructed. By means of the method, a large number of cells can be separated three days after primary culture is conducted, and the cells have the continuous passage capability. After passage is stable, the cells are in a typical fiber form, it proves that the Chinese grass goldfish pelvic fin cell line is stable in character and uniform in component and is a good material for studying the aquatic animal virology and the immunology. At present, the grass goldfish pelvic fin cell line has 60 generations, and the cells can stably increase and have the continuous passage capability.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for constructing a pelvic fin cell line of golden carp. Background technique [0002] After years of development of fish cell lines, various freshwater and seawater fish cell lines have become important in vitro research systems in various disciplines. Many important experiments and new technologies require further research on the basis of fish cell lines. The scope of application of fish cell lines is becoming more and more extensive, and has expanded from the initial virus isolation, purification, identification, and in vitro propagation to the toxicity mechanism of environmental pollutants and its monitoring, fish immunity, germplasm improvement, resource protection and many other fields. Fish cell lines can not only be used as a good in vitro research system for basic research in various disciplines such as cell biology, fish virology, fish physiology, fish immunology, en...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/70C12Q1/02C12R1/93
Inventor 王浩吕利群许丹喻文娟喻飞孔善云黄美秀
Owner SHANGHAI OCEAN UNIV
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