Constructing method of stable-expression FGF-1 protein cell line

A technology of FGF-1 and stable expression, applied in the field of construction of cell lines stably expressing FGF-1 protein, can solve the problem of low mitogenic ability of recombinant FGF1 protein, and achieve the effect of convenient detection

Inactive Publication Date: 2019-05-10
GUIZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the structurally optimized recombinant FGF1 protein is less mitogenic

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0017] Embodiment of the present invention: construction method of cell line stably expressing FGF-1 protein

[0018] (1) FGF-1 gene amplification

[0019] 1. FGF-1 gene PCR amplification

[0020] According to the FGF-1 gene sequence (Gene ID: 2246) in NCBI, use the software LSPrimer (http: / / ccsipb.lnu.edu.cn / primer / ) to design the specific primers FGF-1-F and FGF of the FGF-1 gene -1-R, add the enzyme cutting site Not I and Avr II, the introduced enzyme cutting site Not I sequence is detailed in the sequence table SEQ ID 5, the introduced enzyme cutting site AvrII sequence is detailed in the sequence table SEQ ID 6, the For the sequence of the specific primer FGF-1-F, see SEQ ID 1 in the sequence listing, and for the sequence of FGF-1-R, see SEQ ID 2 in the sequence listing.

[0021] Use the pEmcherry-N1-FGF-1 plasmid as a template for PCR amplification. The PCR reaction system is:

[0022] PCR reaction program: Denaturation at 94°C for 4 minutes, renaturation at 56°C for ...

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Abstract

The invention discloses a constructing method of a stable-expression FGF-1 protein cell line. FGF-1 genes are inserted in Not I and Avr II polyclone sites of a pLenti-CMV-Gag-Mcherry-Puro carrier, co-transfection is conducted on 293T cells through the constructed pLenti-CMV-FGF-1-Emcherry-Puro recombinant plasmids and virus packaging plasmids, and culturing, virus extraction, virus supernate collecting and filtering are conducted to obtain recombinant slow virus liquid. The 293T cells are infected with packaged recombinant slow viruses, and through screening and identifying of puromycin, the stable-expression FGF-2 protein cell line is obtained. The cell line which is mixed with tag proteins and can stably express the FGF-1 proteins is prepared through a recombinant slow virus system, andthe cell line provides a quite good tool for the research of the biological function of the FGF-1 proteins.

Description

technical field [0001] The invention relates to the technical field of medical molecular biology, in particular to a method for constructing a cell line stably expressing FGF-1 protein. Background technique [0002] At present, more than 400 million adults worldwide suffer from diabetes, and it is estimated that the number will increase to more than 600 million in the next 10 years. Diabetes mellitus is a chronic metabolic disease caused by the comprehensive influence of heredity, environment and living habits. It is mainly divided into insulin-dependent diabetes mellitus (T1DM), non-insulin-dependent diabetes mellitus (T2DM) and pregnancy diabetes. Its clinical manifestation is that the human body itself cannot produce insulin or cannot use insulin, resulting in blood sugar higher than the normal blood sugar range, thus causing some concurrent diseases. Diabetes and its complications reduce the life expectancy and quality of life of patients, and the mortality, morbidity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/12
Inventor 郭建军田莹檀军赵帅郑玉林陈緖美尹志勇
Owner GUIZHOU UNIV
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