Cloning Hap gene from non-typeable Haemophilus influenzae and expression of Hap protein in prokaryotic cell.
- Author:
Wanyi LI
1
;
Yu KUANG
;
Feng YAO
;
Yuan YANG
;
Changchun CHEN
;
Zhonghua JIANG
;
Mingyuan LI
Author Information
1. Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Bacterial Outer Membrane Proteins;
biosynthesis;
genetics;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Haemophilus influenzae;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Serine Endopeptidases;
biosynthesis;
genetics
- From:
Journal of Biomedical Engineering
2009;26(5):1072-1076
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
