Spinal cord tissue cell lines of Carassius auratus gibelio and construction method and application thereof

A technology of heterotrophic silver crucian carp and tissue cells, applied in cell dissociation methods, artificial cell constructs, bone/connective tissue cells, etc. Lesion effect stable effect

Active Publication Date: 2019-02-01
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the heterogeneous gibel carp brain tissue cell line has been established and is sensitive to CyHV-2, because the inventor does not supply it to the outside world, there is still a lack of CyHV-2 sensitive cell lines, which limits the research on

Method used

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  • Spinal cord tissue cell lines of Carassius auratus gibelio and construction method and application thereof
  • Spinal cord tissue cell lines of Carassius auratus gibelio and construction method and application thereof
  • Spinal cord tissue cell lines of Carassius auratus gibelio and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The establishment of heterogeneous gibel carp spinal cord tissue cell line, its steps are as follows:

[0040] (1) Treatment of spinal cord tissue: take out the spinal cord tissue of heterogeneous gibel crucian carp under aseptic conditions, and the aseptic treatment is 30-60mm 3 Put the tissue block into the culture dish containing L15 culture medium;

[0041] (2) Primary culture: Put the tissue pieces in step (1) evenly into a T25 cell culture bottle, with the side of the tissue pieces facing up, add 3ml of culture medium to the culture bottle, overnight, and slowly dissolve the culture bottle Turn it sideways to make the tissue block soak in the culture medium, and then turn the side with the tissue block up, and operate it once from time to time until cells grow out of the edge of the tissue block, place the cell culture bottle upright for culture, and replace the culture medium every 2 to 3 days Once; the morphology of primary cells and passage cells are as follow...

Embodiment 2

[0047] The biological characteristics of CSC of the allogenic gibel carp spinal cord tissue cell line:

[0048] (1) Morphology: The cell type was fibroblast-like cells.

[0049] (2) Growth characteristics: The subcultured CSC cells began to adhere to the wall after 30 minutes, and completely adhered to the wall after 6 hours; the population doubling time was 48 hours.

[0050] (3) Stability: The heterogeneous gibel crucian carp spinal cord tissue cell line CSC has been propagated to 42 passages before the application date, and the proliferation is stable.

[0051] (4) Cryopreservation and recovery:

[0052] After thawing, CSC cells adhered to the wall quickly, and their growth morphology and conditions were basically similar to those of cells that had not been frozen, with no significant difference. The revived cells were stained with trypan blue, and about (78.56±6.10)% of the cells were not stained and had cell viability through statistical counting of the cells.

[0053]...

Embodiment 3

[0056] The application of heterogeneous gibel crucian carp spinal cord tissue cell line, its process is as follows:

[0057] (1) Collection and processing of infected carp herpes virus type Ⅱ disease materials:

[0058] Collect the kidney, spleen, brain and spinal cord tissues of freshly dead fish infected with carp herpesvirus type Ⅱ, cut them into pieces, add an equal volume of PBS to homogenate, centrifuge at 5000rpm at 4°C for 15min, and filter through a 0.22μm filter membrane to prepare sterile tissue homogenate , stored at -80°C after aliquoting;

[0059] (2) Proliferation of carp herpesvirus type Ⅱ in CSC:

[0060] After the CSC is cultured to about 80% of the cell monolayer, discard the medium and wash it twice with serum-free cell maintenance solution, inoculate 0.2ml of the supernatant of the above-mentioned disease tissue homogenate on the CSC cell monolayer, and keep at 24°C Adsorb for 1 hour, during which time the flask was shaken slightly every 15-20 minutes fo...

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Abstract

The invention discloses spinal cord tissue cell lines of Carassius auratus gibelio and a construction method and application thereof. The class name of the cell lines is Spinal cord tissue cell linesof Carassius auratus gibelio (CSC), and the collection number is CCTCC NO: C2018211. The above cell lines can be applied to separation, culture and detection of the Herpesvirus cyprini Type II and preparation of a Herpesvirus cyprini Type II vaccine. The invention provides a necessary technical platform for the separation and identification of CyHV-2 and its complete biological characteristics, and lays an important foundation for the prevention and control of the hematopoietic necrosis of the carp.

Description

technical field [0001] The invention belongs to the technical field of aquatic biological cells and aquaculture disease prevention and control, and in particular relates to a heterogeneous gibel crucian carp spinal cord tissue cell line and its construction method and application. Background technique [0002] Cyprinid herpesvirus II (CyHV-2) is also known as Herpesviral haematopoietic necrosis virus (HVHNV) or Goldfish haematopoietic necrosis virus (GFHNV). The other two herpesviruses CyHV-1 (Carp pox) and CyHV-3 (Koi herpesvirus, KHV) belong to the family Alloherpesviridae and the genus Cyprinivirus. CyHV-2 was first reported in 1995, and caused huge economic losses to goldfish cultured in western Japan from 1992 to 1993, and the mortality rate of sick goldfish was as high as 100%. Subsequently, other countries and regions have also reported outbreaks of the disease one after another. In the spring of 1997, a large number of goldfish juveniles raised in recirculating wate...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12Q1/70C12Q1/686A61K39/245A61P31/22
CPCA61K39/12A61P31/22C12N5/0669C12N7/00C12Q1/686C12Q1/705C12N2710/16051C12N2710/16034C12N2509/00
Inventor 沈锦玉曹铮潘晓艺夏焱春姚嘉赟蔺凌云尹文林刘忆瀚陆裕肖邢刚岳丰雄周涛黄杰
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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