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Immortalized rabbit small intestine epithelium cell line and construction method thereof

A small intestinal epithelial cell and construction method technology, applied in the field of immortalized rabbit small intestinal epithelial cell line and its construction, can solve the problems of restricting the development of technology in the field and the lack of scientific tools

Pending Publication Date: 2017-09-29
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the current research, scientific tools to study these diseases are seriously lacking, which restricts the technological development in related fields

Method used

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  • Immortalized rabbit small intestine epithelium cell line and construction method thereof
  • Immortalized rabbit small intestine epithelium cell line and construction method thereof
  • Immortalized rabbit small intestine epithelium cell line and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Isolation and culture of rabbit intestinal epithelial crypt cell clusters

[0035] The small intestinal cells of newborn rabbits were aseptically collected in the ultra-clean bench, the small intestine was cut longitudinally with sterile scissors, and then the intestinal tissue was chopped into 2mm pieces with a sterile surgical blade 2 tissue fragments. The intestinal tissue was rinsed 2 to 3 times with DMEM medium. Intestinal tissue was digested at 37°C for 20 minutes with enzymatic digestion solution, and the digestion solution needed to be stirred continuously during the digestion process. After digestion, the digested product was filtered using a copper filter with a pore size of 0.5 mm. The filtrate was centrifuged at 500 g for 5 min, and the supernatant was discarded. Use a sorbitol solution with a mass ratio (m / m) of 2.5% to resuspend the precipitate, centrifuge the suspension at 300g for 2 minutes, collect the supernatant in a new centrifuge tube, and the...

Embodiment 2

[0045] 1. Identification of cell lines (transcriptome sequencing analysis)

[0046] When the rabbit small intestinal epithelial cell line cultured to passage 50 grows to 100% confluence, add 0.25% trypsin digestion solution containing EDTA 0.5mL, in 5% CO 2 Incubate in a cell culture incubator at 37°C for 5 min, then pipette to dislodge the cells, add medium to stop digestion, collect cells by centrifugation at 800 g for 5 min, and add 1 mL of TRIzol reagent (Ambion, USA, Cat. No. 139605). Extract total RNA from the sample using the standard method provided by the reagent manufacturer.

[0047] The concentration and purity of the extracted RNA were measured with a spectrophotometer. Then, according to the results of 1% MOPS gel electrophoresis (the loading amount of total RNA is about 1 μg), the total RNA extracted was evaluated for RNA integrity and whether there was genomic DNA contamination.

[0048] After using the ribo-zero kit to digest the ribosomal RNA; add the inter...

Embodiment 3

[0055] Embodiment 3 Eimeria major in vitro culture

[0056] Digest the full length of a 25cm 2 Cell culture flasks of rabbit intestinal epithelial cells, counted, and diluted cells. Add 10 to each well of a 6-well plate for cell culture 6 cells and cultured for 12 h. After the cells adhered to the wall, 10 6 Sporozoites were cultured for 6 hours. After the cells are confluent, gently wash the cell culture plate with 5 times double antibody to wash away the non-invading sporozoites, and then add 5% FBS and MEM199 medium. Culture exchange time was set to 48h, 94h. After the extracted old culture medium was examined under a microscope, it was discarded after ensuring that there were no escaped merozoites. If there were merozoites, centrifuged and re-added to new cells to continue culturing. After 96 h, merozoites were continued to be cultured using low serum MEM199 medium containing 2.5% FBS. If the cell culture plate is overgrown, trypsinization with EDTA is used to pass ...

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Abstract

The invention relates to the field of molecular biology and specifically discloses an immortalized rabbit small intestine epithelium cell line and a construction method thereof. The small intestines of a newly born rabbit are collected, the enzymatic digestion juice containing collagenase and neutral protease is utilized to digest, and after the digestion, a method for re-suspending centrifuging and differential adherence is adopted for purifying the pit cell cluster, so that high-purity primary pit epithelium cells can be obtained. The pit epithelium cells can participate in culture and passage. The obtained pit epithelium cells are used for performing liposome transfection expression on the SV40 big T antigenic plasmid, the passage is continued, and the immortalized rabbit small intestine epithelium cell line is finally screened. The immortalized rabbit small intestine epithelium cell line can simultaneously express intestine epithelium surface marker molecules and pit basic stem cell surface molecules. The intestine epithelium surface marker molecules include but not limited to keratin 8, E-calcitonin and ki67; and the pit basic stem cell surface molecules include but not limited to Lgr5 and sox9. The immortalized rabbit small intestine epithelium cell line can be used for establishing a rabbit small intestine epithelium in vitro model and can be used for researching a nutrition absorbing mechanism, cellular pathway excavation and pathogen in vitro culture.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an immortalized rabbit small intestinal epithelial cell line and a construction method thereof. Background technique [0002] Rabbit is an important source of meat and fur, and plays an important role in livestock production, catering, public health and other fields. At present, major epidemic diseases of rabbits such as rabbit plague, rabbit coccidiosis, pasteurellosis, etc. have been paid more and more attention. Most of these pathogens attack the digestive tract. However, in the current research, scientific tools to study these diseases are seriously lacking, which restricts the technological development in related fields. At present, there are few cell lines derived from rabbits, so obtaining more cell lines derived from rabbits will promote the research on the prevention and control of major rabbit diseases. [0003] Cell lines derived from the small intestine are reliabl...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0625C12N5/0679C12N2500/25C12N2501/11
Inventor 刘贤勇索勋杜孟泽索静霞陶鸽如胡丹丹
Owner CHINA AGRI UNIV
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