Construction of prokaryotic expression vector and expression of human beta defensin 4 gene.
- Author:
Yuhong CAO
1
;
Guocheng ZHANG
;
Guangyun ZHANG
;
Yanming XU
;
Lifeng WANG
Author Information
1. Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, China.
- Publication Type:Journal Article
- MeSH:
Anti-Infective Agents;
Cloning, Molecular;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
beta-Defensins;
biosynthesis;
genetics
- From:
Journal of Biomedical Engineering
2008;25(5):1166-1169
- CountryChina
- Language:Chinese
-
Abstract:
Human beta defensin 4 is a small cationic peptide with a broad range of antimicrobial activity. It plays an important role in innate immunity of human body, especially in mucosal and epithelial defense. In this study, the full-length encoding gene of HBD4 was synthesized by overlap extension polymerase chain reaction and inserted into cloning vector pMD18-T. The gene encoding mature peptide of HBD4 was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-2. Then pGEX-4T-2/mHBD4 was transformed into E. coli DH5 alpha, which was induced by isopropy-beta-D-thiogalactoside (IPTG). The identification was made by means of endonuclease digestion, DNA sequencing, sodium dodecyl sulphate-polyacrylamine gel electrophoresis (SDS-PAGE). The results showed that the synthesized gene and cloned gene were identical to the HBD4 gene sequence registered in GenBank and were successfully cloned into cloning vector pMD18-T and prokaryotic expression vector pGEX-4T-2. After IPTG induction, the GST-HBD4 fusion protein was successfully expressed in E. coli.
