Construction of the eukaryotic expression plasmid containing human epidermal growth factor gene with signal peptide
- VernacularTitle:应用RT-PCR构建人表皮生长因子基因真核表达质粒
- Author:
Huiqing XIE
;
Jianda ZHOU
;
Chengqun LUO
;
Yong CHEN
;
Kun XIA
;
Daojin CHEN
- Publication Type:Journal Article
- Keywords:
Epidermal growth factor;
Reverse transcription polymerase chain reaction
- From:
Journal of Chinese Physician
2001;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression plasmid containing human epidermal growth factor(hEGF) gene with signal peptide(SP).Methods After two pairs of primers were designed and synthesized,the cDNA fragment of hEGF and SP genes were amplified from total RNAs. The amplified cDNA fragments were cloned into pGEM-T vector.The expression plasmids were verified by double endonuclease digestion and DNA sequence analysis. Results With RT-PCR using two pairs of primers,two bands(about 90bp and 180bp) were obtained and confirmed as signal peptide and EGF cDNA fragment with electrophoresis analysis and DNA sequencing after cloned into pGEM-T vector.The SP and EGF cDNA fragments were inserted into plasmid pcDNA3.1(+).The bands of 240bp and 5.4kb were obtained and identified as the full length of SP-EGF cDNA fragment by DNA sequence analysis.Conclusion The eukaryotic expression plasmids containing hEGF gene is successfully constructed.
