The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis.
- Author:
Shan CHEN
1
;
Feng-Tian HE
;
Yan-Lin DONG
;
Rong-Fen LI
;
Hui-Guang GAO
;
Min CHEN
;
Jia-He PENG
Author Information
1. Department of Biochemistry and Molecular Biology, the Third Military Medical University, Chongqing 400038, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
Plasmids;
Recombinant Fusion Proteins;
biosynthesis;
isolation & purification;
pharmacology;
Staphylococcus aureus;
drug effects;
Tetrahydrofolate Dehydrogenase;
genetics;
beta-Defensins;
biosynthesis;
genetics;
pharmacology
- From:
Chinese Journal of Biotechnology
2004;20(4):490-495
- CountryChina
- Language:Chinese
-
Abstract:
In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.
