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Cell line of pterygiophore tissue of cryprinus carpiod and construction method

A technology for organizing cells and fin rays, applied in the field of aquaculture disease prevention and control, can solve problems such as unreported and difficult proliferation, and achieve the effect of stable cell monolayer and clear cell shape.

Inactive Publication Date: 2012-04-04
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

KHV is very special and can only proliferate in cells derived from the original host, that is, in koi cells, and it is difficult to proliferate in cell lines commonly used for fish virus isolation
At present, the koi fin ray cell line KF-1 and the common carp brain cell line CCB are commonly used abroad to culture the virus, but there is no report in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] A method for constructing a koi fin ray tissue cell line, the steps of which are:

[0017] 1. The preparation of koi fin ray tissue block: at first the koi is placed in the potassium permanganate solution with a concentration of 20 g / liter, and after 30 minutes of disinfection treatment, the fish body surface is wiped with 70% (V / V) alcohol, The dorsal and pelvic fin tissues were aseptically removed in a class II biological safety cabinet (ESCO, Singapore). Wash the fin ray tissue 3 times with phosphate buffer containing 500 units / ml penicillin, 500 μg / ml streptomycin, and 12.5 μg / ml amphotericin B, and cut the fin ray tissue to about 1 mm with sterilized ophthalmic scissors 3 The left and right fragments were evenly transplanted into 25 ml cell culture flasks, and dried upside down in a 23°C incubator for 1.5 hours.

[0018] 2. Configuration of special proliferation culture medium for koi fin ray tissue cells: take 6.8-7.2 ml of MEM medium with a pH value of 7.0-7.4, ...

Embodiment 2

[0022] A method for constructing a koi fin ray tissue cell line, the steps of which are:

[0023] 1. The preparation of koi fin ray tissue block: at first the koi is placed in the potassium permanganate solution with a concentration of 20 g / liter, and after 30 minutes of disinfection treatment, the fish body surface is wiped with 70% (V / V) alcohol, The dorsal and pelvic fin tissues were aseptically removed in a class II biological safety cabinet. Wash the fin ray tissue 3 times with phosphate buffer containing 500 units / ml penicillin, 500 μg / ml streptomycin, and 12.5 μg / ml amphotericin B, and cut the fin ray tissue to about 1 mm with sterilized ophthalmic scissors 3 The left and right fragments were evenly transplanted into 25 ml cell culture flasks, and placed upside down in a 24°C incubator for 2 hours.

[0024] 2. Configuration of special proliferation culture medium for koi fin ray tissue cells: take 7 ml of MEM medium with a pH value of 7.0 or 7.1 or 7.2 or 7.3 or 7.4, add...

Embodiment 3

[0028] A method for constructing a koi fin ray tissue cell line, the steps of which are:

[0029] 1. The preparation of koi fin ray tissue block: first koi is placed in the potassium permanganate solution with a concentration of 20 g / liter, after disinfection treatment for 40 minutes, wipe the fish body surface with 70% (V / V) alcohol, The dorsal and pelvic fin tissues were aseptically removed in a class II biological safety cabinet. Wash the fin ray tissue 3 to 4 times with phosphate buffer solution containing 500 units / ml penicillin, 500 μg / ml streptomycin, and 12.5 μg / ml amphotericin B, and cut the fin ray tissue into approx. 1mm 3 The left and right fragments were evenly transplanted into 25 ml cell culture flasks, and placed upside down in a 25°C incubator for 2.5 hours.

[0030] 2. Configuration of special proliferation culture medium for koi fin ray tissue cells: take 7 ml of MEM medium with a pH value of 7.0 or 7.1 or 7.2 or 7.3 or 7.4, add 1 ml of fetal bovine serum,...

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Abstract

The invention discloses a cell line of pterygiophore tissue of cryprinus carpiod and a construction method. The construction method comprises the following steps of: A, preparing pterygiophore tissue blocks of cryprinus carpiod, namely putting cryprinus carpiod into potassium permanganate solution, disinfecting, transferring pterygiophore tissue to a culture dish, cleaning with an antibiotic, and transplanting the pterygiophore tissue blocks in cell culture flasks; B, preparing proliferation culture solution special for cells of pterygiophore tissue of cryprinus carpiod, namely adding fetal bovine serum, adding alkaline fibroblast growth factors and epidermal growth factors into the culture solution; C, performing primary culture of the cells of pterygiophore tissue of cryprinus carpiod, namely adding the special proliferation culture solution into pterygiophore tissue which is subjected to dry sticking in each culture flask, culturing in the culture box, and adding the proliferation culture solution special for the cells of pterygiophore tissue of cryprinus carpiod; and D, performing the subculture of the cells of pterygiophore tissue of cryprinus carpiod, namely after the cells of pterygiophore tissue of cryprinus carpiod are grown to form a single layer, preparing cell suspension, and performing the subculture. The method is easy and convenient to operate and has a scientific and reasonable process, and the established cell line of pterygiophore tissue of cryprinus carpiod is subcultured for over 60 generations at present.

Description

technical field [0001] The invention belongs to the technical field of aquaculture disease prevention and control, specifically relates to a koi fin ray cell line, and also relates to a construction method of a koi fin ray cell line, which is suitable for the establishment of a freshwater fish cell line and the pathogen of carp herpes virus isolation, culture and diagnosis. Background technique [0002] Koi (Cryprinus carpiod) is famous all over the world for its gorgeous colors, gorgeous markings, vigorous body, and docile habits. It is called "water treasure" and "swimming artwork". One of the most popular large ornamental fish. In addition to its ornamental value, koi are also edible fish with rich nutrition and tender meat. [0003] In recent years, with the increasing environmental pollution and the excessive expansion of the breeding industry, the disease problem of koi has become more and more serious. The outbreak of various infectious diseases, especially viral di...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12R1/91
Inventor 曾令兵肖艺徐进范玉顶周勇张辉
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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