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Rhabdovirus-sensitive eel kidney tissue cell line and its application

A rhabdovirus and cell line technology, applied in urinary tract/kidney cells, viruses, antiviral agents, etc., can solve the problems of great significance in port security, affecting import and export economy and trade, and achieve the effect of good biological activity

Active Publication Date: 2021-09-07
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 eel kidney tissue cell line and its application
  • Rhabdovirus-sensitive eel kidney tissue cell line and its application
  • Rhabdovirus-sensitive eel kidney tissue cell line and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Eel kidney cell line established, the following steps:

[0040] (1) treated kidney tissues: kidney eel taken under sterile conditions placed in tissue culture dishes, rinsed three times with PBS, cut by an ophthalmic scissors 50 ~ 100mm sterilized 3 Tissue blocks;

[0041] (2) Primary culture: The tissue was cut into pieces 0.5W / V% of renal cell eel specific medium tryptic digestion digestive fluids 28 ℃ 15min, three times during the shock, after incubation with an equal volume of ( hereinafter referred to as culture solution, culture medium formulations containing 20V / V% fetal bovine serum, 10ng / ml human basic fibroblast growth factor, 10ng / ml human epidermal growth factor, 100U / ml penicillin, 100μg / ml streptavidin Su, M199 medium 0.25μg / ml amphotericin B), evenly pipetting 100 mesh nylon gauze with a filtration, the filtrate was collected in a centrifuge tube, centrifuged at 1500rpm 5min cell collection digested filtration treatment, aspirated on clear, medi...

Embodiment 2

[0045] Eel kidney cell line biological characteristics of CrE-K:

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

[0047] (2) Growth characteristics: passaged CrE-K cells began to adhere after 30min, after completely adherent 8h; population doubling time of 45.5h.

[0048] (3) Stability: eel kidney cell line CrE-K has spread to 58 generations before the filing date, stable proliferation.

[0049] (4) Cryopreservation and Recovery:

[0050] After recovery CrE-K adherent cells fast, growth morphology, and no conditions substantially similar frozen cells, no obvious differences. Recovery of cells stained with trypan blue, the cells counted statistics, about (80.38 ± 5.10)% of the cells did not stain with cell viability.

[0051] (5) chromosome analysis

[0052] Kidney eel passage 18 CrE-K cell line in the logarithmic growth phase, a final concentration of 20μg / ml of colchicine, after 4h of incubation cells were harvested 28 ℃, treated with 0.075mol / L KCl solution hypoto...

Embodiment 3

[0054] Rhabdovirus sensitive applications eel kidney cell line, which process is as follows:

[0055] Collection and Processing (1) eel rhabdovirus infection virus material

[0056] Death collected just rhabdovirus infection eel (CCTCC NO: V201819) diseased fish kidney and spleen, cut into pieces and homogenized with an equal volume PBS, centrifuged at 5000rpm 4 ℃ after 30min was prepared as a sterile 0.22μm membrane filter tissue homogenates the slurry, aliquots were stored to -80 deg.] C;

[0057] Proliferation (2) eel rhabdoviruses of the CrE-K

[0058] CrE-K after cell monolayers grown to 80%, the medium was discarded and after washing with PBS 2 times, the thus-treated diseased tissue homogenate supernatant 1ml inoculated cell monolayer CrE-K, the final concentration of 10μg / μl polybrene (polybrene), 25 ℃ adsorption 2h, every 15 ~ 20min a slight shake flask to evenly during adsorption. After completion of the adsorption, the serum concentration of 2% change of M199 culture ...

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Abstract

The invention belongs to the field of aquatic biological cells and the technical field of aquaculture disease prevention and control, and discloses a rhabdovirus-sensitive eel kidney tissue cell line and its application. The cell line of the present invention is preserved in the China Center for Typical Culture Collection, and the preservation number is: CCTCC NO: C2019286. The rice field eel kidney tissue cell line (CrE-K) was in good growth state and was sensitive to the newly discovered and isolated CrERV; CrERV could still be detected in the 16th passage on CrE-K cells and the cytopathic changes were stable; The ultra-thin electron microscope sections of the cells with pathological effects can be observed in CrE-K cells in a large number of CrERV mature virions and their replication process. The construction method of the eel kidney tissue cell line constructed by the present invention has strong repeatability, scientific and reasonable conditions, is suitable for in vitro culture of rhabdoviruses, and provides technology for the isolation, detection, cultivation and complete biological characteristics of rhabdoviruses. platform.

Description

Technical field [0001] The present invention is in the field of cell and disease control in aquaculture BACKGROUND aquatic organisms, particularly to sensitive rhabdovirus eel kidney cell line and application. Background technique [0002] Rhabdoviridae family (Rhabdoviridae) virus is a negative strand RNA virus type having a broad host, which exhibit a bullet-like or rod-like particles. Which rabies virus (Rabies virus) is best known, can cause rabies in humans or other warm-blooded animals, the mortality is almost 100%, attracted worldwide attention, causing social panic safety of pet breeding. Therefore, rhabdovirus in-depth study has important significance. In addition, livestock rhabdovirus can cause a variety of acute infectious diseases (such as bovine ephemeral fever, ephemeral fever), as well as infectious diseases of fish and a variety of crops. International Committee on Taxonomy of Viruses (International Committee on Taxonomy of Viruses, ICTV) published in 2017, the l...

Claims

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Application Information

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Patent Type & Authority Patents(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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