Cyprinus carpio var. jian cerebral line and establishment and application methods thereof

A technique for establishing methods and brain cells, which is applied in the field of cell biology, can solve problems such as being in the blank, and achieve high-sensitivity effects

Active Publication Date: 2019-07-05
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, cell lines from different tissues of carp and koi have been established. However, even the genetic structure, genetic diversity and genetic differences of the same species cannot be ignored. There are great differen

Method used

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  • Cyprinus carpio var. jian cerebral line and establishment and application methods thereof
  • Cyprinus carpio var. jian cerebral line and establishment and application methods thereof
  • Cyprinus carpio var. jian cerebral line and establishment and application methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 The separation of Jian carp brain cells and the establishment of brain cell lines

[0041]Under sterile conditions, take the brain tissue of 1 healthy Jian carp fry (10-15cm), wash it three times with PBS, cut it into pieces and add 0.05% trypsin containing 36.5mM EDTA (EDTA:trypsin=1:100)( 0.25% trypsin:PBS=1:4) 5mL, digest at 25°C for 30min, remove the solution 1mL, centrifuge at 300g at 25°C for 5min, discard the supernatant, and use L-15 ( Gibco) medium (double antibody: medium = 1:100) was suspended, inoculated into a 6-well plate, and cultured at 25°C. 50% of the culture medium in the well was replaced every 3 days, and after 2 weeks of culture, the Jian carp brain cells obtained by primary isolation began to grow, as shown in figure 1 shown. When the cells in the well are full, trypsinize and transfer to 25cm 2 For cell culture flasks, after the cells grow to >90% confluence, they are digested with 0.05% trypsin and subcultured at 1:2. After 1:2 pas...

Embodiment 2

[0051] Example 2 Chromosome Analysis and 18S Molecular Identification

[0052] Select passaged cells (generation 30) with >75% confluence and add colchicine at a final concentration of 1 μg / mL, trypsinize after induction for 16 hours; collect cells by centrifugation at 200 g at 25°C for 5 minutes, add fixative solution (glacial acetic acid:methanol=1: 3) After fixing for 5 minutes, centrifuge at 200g 25°C for 5 minutes, discard the supernatant; repeat the fixation twice, each time for 25 minutes; suspend the precipitate obtained by centrifugation with 300 μL of fixative solution, and drop the pieces by cold drop method; after drying at room temperature, Giemsa staining 20min, microscopic examination.

[0053] Among them, the number of chromosomes in 100 metaphase cells was counted, and the results showed that the number of chromosomes in CCB-J was between 68 and 140, and its mode was 100. The results are as follows: Image 6 shown.

[0054] Using Genome Extraction Kit (Tiang...

Embodiment 3

[0055] Example 3 Application of Jian carp CCB-J brain cell line in KHV virological research

[0056] The herpes virus KHV isolated in the laboratory was used to infect the passaged cells of Jian carp (virus: L-15 complete medium = 1:10), and the electron microscope samples were made after the cytopathic changes reached 90%. Collect the medium in the cell flask, add 500 μL of trypsin to digest the adherent cells and use the collected medium to terminate the digestion. Centrifuge the cell suspension at 300g at 25°C for 5min, discard the supernatant; re-suspend twice with 2mL PBS, discard the supernatant; fix the precipitate with 2.5% glutaraldehyde solution (pH7.4) at 4°C for 3h, then wash with the buffer repeatedly Sample 3 times, 15-30min each time; then fix with 1% osmic acid for 3h, wash the buffer repeatedly 3 times, 15-30min each time; dehydrate with alcohol gradient and replace the alcohol with propylene oxide; finally soak the sample Embedded in epoxy resin. The sample...

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Abstract

The invention provides a Cyprinus carpio var. jian cerebral line and application thereof. Through primary isolation on Cyprinus carpio var. jian cerebral tissues with pancreatin of an appropriate concentration and reasonable digestion conditions, Cyprinus carpio var. jian cerebral cells can be obtained, and through subculture, the Cyprinus carpio var. jian cerebral line CCB-J is established for the first time; the Cyprinus carpio var. jian cerebral line is mainly represented as epithelioid cells and can be passaged for more than 60 generations, thereby be high in research and application the fields such as virology, toxicology, physiology, molecular genetics and biology of evolution. The Cyprinus carpio var. jian cerebral line can be applied to virus identification and virus strain isolation, to preparation of Cyprinus carpio var. jian virus preventing or killing vaccines, as biological models of drug screening, preparation and evaluation and the like.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular to a carp brain cell line and its establishment method and application. Background technique [0002] Jian carp (Cyprinus carpio var.jian) belongs to Cypriniformes (Cypriniformes), Cyprinidae (Cyprinidae), Cyprinus (Cyprinus), and is based on specific purse red carp (C.carpio var.wuyuanensis) and Yuanjiang carp (C. .carpio yuankiang) as the parent, a fine variety bred through 6 generations of directional selection. Jian carp has good meat quality and flavor, strong adaptability and disease resistance, and high feed conversion rate. It is suitable for various farming methods all over the country. Its characteristics are obviously superior to existing domestic carp and foreign imported species. one. [0003] Fish cell lines are important basic materials for research in virology, toxicology, physiology, molecular genetics, and developmental biology. At present, cell lines from different ...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N7/00C12Q1/02
CPCC12N5/0618C12N7/00C12N2710/16051G01N33/5044G01N2500/10
Inventor 刘振兴李盈马艳平马江耀郝乐梁志凌柯浩
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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