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Cell line stably expressing chicken interleukin 10 protein and its construction method and application

A technology for stably expressing interleukin, which is applied in the field of preparation of recombinant chicken interleukin-10 protein, can solve the problems of low expression efficiency and unstable expression, and achieve the effect of wide application prospects

Inactive Publication Date: 2015-09-02
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing genetic engineering methods for preparing recombinant chicken interleukin-10 protein have defects such as low expression efficiency and unstable expression to varying degrees, which need to be improved

Method used

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  • Cell line stably expressing chicken interleukin 10 protein and its construction method and application
  • Cell line stably expressing chicken interleukin 10 protein and its construction method and application
  • Cell line stably expressing chicken interleukin 10 protein and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of a cell line stably expressing chicken interleukin-10 protein

[0035] 1. Experimental method

[0036] 1.1 Isolation, culture and total RNA extraction of chicken peripheral blood lymphocytes

[0037] Collect 10 mL of sodium citrate anticoagulant blood from an adult SPF chicken through sterile wing vein, and separate peripheral blood lymphocytes with chicken lymphocyte separation medium according to the product instructions. Adjust the cell density to 1×10 with complete RPMI1640 medium containing 10% fetal bovine serum 6 / mL, added to 6-well cell plate culture, and added to which the final concentration was respectively 10ug / mL, 20ug / mL LPS and the final concentration was respectively 10ug / mL, 25ug / mL, 30ug / mL ConA as a stimulator, At 37°C, 5% CO 2 The cells were cultured in an incubator, shaken every 4 h, and the cells were collected at different times. Total cellular RNA was extracted with Trizol Reagents total RNA extraction kit, and the s...

Embodiment 2

[0058] Example 2 Identification of cell lines stably expressing chicken interleukin-10 protein

[0059] 1. Experimental method

[0060] 1.1 RT-PCR detection

[0061] Trizol Reagents extraction kit was used to extract the total RNA of CHO-chIL-10F and CHO-chIL-10M cells constructed in Example 1, and the specific method was carried out according to the instructions of the kit. The reverse transcription product cDNA was taken, and the chIL-10 gene was identified by PCR amplification. The reaction program was: 95°C for 5 minutes; 30 cycles of 94°C for 30s, 64°C for 30s, and 72°C for 40s; 72°C for 10 minutes.

[0062] 1.2 IFA detection

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Abstract

The invention discloses a cell line for stable expression of a chicken interleukin 10 protein, as well as a construction method and application thereof. The construction method disclosed by the invention comprises the steps of cloning a chicken interleukin 10 encoding gene after removal of a signal peptide into a eukaryotic expression vector containing a neomycin resistance gene in a fixed direction to obtain a recombinant plasmid, transfecting cells of a Chinese hamster ovary cell line CHO-K1, and performing G418 pressurized screening and purification to obtain a cell line CHO-chIL-10M for stable and high-efficient expression of a chIL-10 protein, wherein the microorganism collection number is CGMCC NO. 7659. The cell line CHO-chIL-10M constructed by the construction method disclosed by the invention has extensive application prospects in preparation of recombinant chicken interleukin 10 proteins and chicken interleukin 10 monoclonal antibodies and other aspects.

Description

technical field [0001] The present invention relates to a cell line expressing recombinant chicken interleukin, in particular to a cell line stably expressing chicken interleukin-10 fusion protein. The present invention further relates to the construction method and application of the cell line, belonging to the preparation of recombinant chicken interleukin-10 protein field. Background technique [0002] Interleukin, referred to as interleukin, is a cytokine produced by white blood cells that mediates intercellular interactions and plays a regulatory role in cell activation, proliferation and differentiation. With the development of molecular biology, research on chicken interleukin genes and functions has gradually become a hot spot. So far, a variety of interleukins have been isolated from chickens, such as: IL-1, IL-2, IL-6, IL- 10. IL-12, IL-16, IL-18, etc. These interleukins can be used in immunotherapeutic agents, immune enhancers, and construction of new genetic eng...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12P21/02
Inventor 韩凌霞廉传江文辉强司昌德林欢陈洪岩
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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