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Rhabdovirus-sensitive monopterus albus kidney tissue cell line and application

A rhabdovirus, cell line technology, applied in urinary tract/kidney cells, viruses, animal cells, etc., can solve problems of great significance in port security, affecting import and export economy and trade, and achieve stable cytopathic effect and good biological effect. The effect of biological activity and genetic stability

Active Publication Date: 2020-12-25
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

At present, the above three genera can infect a variety of cultured fish, have strong pathogenicity, and the mortality rate can reach 100%, causing significant economic losses to the global sea and freshwater aquaculture industry, especially according to "Import and Export Live Aquatic Animal Inspection and Quarantine According to the agreement, all imported and exported farmed fish, such as perch, carp, rainbow trout, crucian carp, flounder and other fish species used for breeding and seedlings, must be tested for rhabdovirus (IHNV, SVC V, HIRRV) disease, port security significance Significant, affecting import and export economy and trade

Method used

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  • Rhabdovirus-sensitive monopterus albus kidney tissue cell line and application
  • Rhabdovirus-sensitive monopterus albus kidney tissue cell line and application
  • Rhabdovirus-sensitive monopterus albus kidney tissue cell line and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The establishment of rice field eel kidney tissue cell line, its steps are as follows:

[0040] (1) Treatment of kidney tissue: take out rice field eel kidney tissue under aseptic conditions, place it in a petri dish, rinse it with PBS three times, and cut it into 50-100 mm with sterilized ophthalmic scissors 3 organization block;

[0041] (2) Primary culture: Put the shredded tissue pieces into 0.5W / V% trypsin digestion solution for digestion at 28°C for 15 minutes, shake 3 times during the period, add an equal volume of eel kidney tissue cell special culture medium after digestion ( Hereinafter referred to as the culture medium, the formula of the culture medium is to contain 20V / V% fetal bovine serum, 10ng / ml human basic fibroblast growth factor, 10ng / ml human epidermal growth factor, 100U / ml penicillin, 100μg / ml streptomycin 0.25 μg / ml amphotericin B), pipette evenly and filter once with 100-mesh nylon gauze, collect the filtrate into a centrifuge tube, centrifuge ...

Embodiment 2

[0045] Biological characteristics of rice field eel kidney tissue cell line CrE-K:

[0046] (1) Morphology: The cell type was fibroblast-like cells.

[0047] (2) Growth characteristics: The subcultured CrE-K cells began to adhere to the wall after 30 minutes, and completely adhered to the wall after 8 hours; the population doubling time was 45.5 hours.

[0048] (3) Stability: The eel kidney tissue cell line CrE-K has been propagated to 58 generations before the application date, and the proliferation is stable.

[0049] (4) Cryopreservation and recovery:

[0050] After thawing, CrE-K cells adhered to the wall quickly, and their growth shape and condition were basically similar to those of cells that had not been frozen, with no significant difference. The resuscitated cells were stained with trypan blue, and about (80.38±5.10)% of the cells were not stained and had cell viability by counting the cells.

[0051] (5) Chromosome analysis

[0052] The 18th generation rice fiel...

Embodiment 3

[0054] The application of rhabdovirus-sensitive eel kidney tissue cell line is as follows:

[0055] (1) Collection and treatment of rhabdovirus-infected eel disease materials

[0056] Collect kidneys and spleens from diseased eel rhabdovirus-infected fish (CCTCC NO: V201819) that have just died, cut them into pieces, add an equal volume of PBS to homogenate, centrifuge at 5,000rpm at 4°C for 30min, and filter through a 0.22μm filter membrane to prepare a sterile tissue homogenate. Slurry, aliquoted and stored at -80°C for later use;

[0057] (2) Proliferation of eel rhabdovirus in CrE-K

[0058] After CrE-K was cultured to 80% of the cell monolayer, the medium was discarded and washed twice with PBS, and 1ml of the supernatant of the homogenate of the above-mentioned treated diseased tissue was inoculated on the CrE-K cell monolayer, and the final concentration was 10μg / μl polybrene (Polybrene), adsorb at 25°C for 2 hours, and shake the culture bottle slightly every 15-20 m...

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Abstract

The invention belongs to the field of aquatic organism cells and the technical field of aquaculture disease prevention and control, and discloses a rhabdovirus-sensitive monopterus albus kidney tissuecell line and application. The cell line in the invention is preserved in the China centre for type culture collection; and the preservation number is CCTCC NO:C2019286. The monopterus albus kidney tissue cell line (CrE-K) has a good growth state and is sensitive to CrERV newly discovered, separated and identified at present; after continuous passage of CrERV on CrE-K cells to the 16th generation, viral nucleic acid can still be detected; cytopathy is stable; the cells with the pathological change effect are subjected to ultrathin electron microscopy slicing; and a large number of CrERV mature virus particles and the replication process thereof can be observed in the CrE-K cells. The construction method of the monopterus albus kidney tissue cell line in the invention is high in repeatability, scientific and reasonable in condition and suitable for in-vitro culture of the rhabdovirus; and a technical platform is provided for separation, detection and culture of the rhabdovirus and complete biological characteristic research of the rhabdovirus.

Description

technical field [0001] The invention belongs to the field of aquatic biological cells and the technical field of aquaculture disease prevention and control, and specifically relates to a rhabdovirus-sensitive eel kidney tissue cell line and its application. Background technique [0002] Rhabdoviridae viruses are a class of negative-strand RNA viruses with a wide range of hosts, and their particles are bullet-shaped or rod-shaped. Among them, Rabies virus is the most well-known, which can cause rabies in humans or other warm-blooded animals, and the fatality rate is almost 100%. Therefore, it is of great significance to conduct in-depth research on rhabdoviruses. In addition, rhabdoviruses can also cause a variety of acute infectious diseases in livestock (such as bovine epidemic fever, ephemeral fever), as well as infectious diseases in fish and a variety of crops. The International Committee on Taxonomy of Viruses (ICTV) published the latest tenth report on virus classifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12Q1/02C12Q1/70C12Q1/686A61K39/205A61P31/14C12R1/91
CPCA61K39/12A61K2039/552A61P31/14C12N5/0686C12N7/00C12N2760/20034C12N2760/20051C12Q1/686C12Q1/701G01N33/5044C12Q2521/107
Inventor 刘文枝曾令兵范玉顶周勇江南李逸群孟彦薛明洋
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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