Expression of truncated 56 kDa antigen gene of Orientia tsutsugamushi in different vectors.
- Author:
Yan-qin DENG
1
;
Yan-sheng YAN
;
Si HE
;
Yu-wei WENG
;
Liang CHEN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Bacterial; biosynthesis; Antigens, Bacterial; biosynthesis; genetics; immunology; Bacterial Proteins; biosynthesis; genetics; immunology; Cloning, Molecular; Escherichia coli; genetics; metabolism; Genetic Vectors; Humans; Membrane Proteins; biosynthesis; genetics; immunology; Orientia tsutsugamushi; immunology; Recombinant Fusion Proteins; biosynthesis; genetics; immunology; Scrub Typhus; immunology; prevention & control
- From: Chinese Journal of Epidemiology 2004;25(11):973-977
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct recombinant plasmids containing the truncated gene of the major surface antigen sta56 of Orientia tsutsugamushi (Ot.) Karp strain for expression antigen in E. coli so as to compare the expression efficiency in different systems.
METHODSFrom the recombinant plasmid TOPO-sta56 containing sta56 of Orientia tsutsugamushi Karp strain, several truncated genes of sta56 with different length were amplified and subcloned into the expression vectors pPROEX HTb and pET30a. These genes were expressed in E. coli DH5alpha and BL21(DE3) respectively when induced by IPTG. The expressed recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.
RESULTSSix recombinant plasmids containing truncated sta56 genes of different length were constructed as follow: pHTbOt957, pHTbOt498, pHTbOt342 and pETOt957, pETOt498, pETOt342. The recombinant sta56 proteins were highly expressed as 6 x His fusion proteins in E. coli DH5alpha and BL21(DE3) respectively. The fusion proteins showed as different bands of different molecular weight respectively when analyzed with SDS-PAGE. Western blot demonstrated that the recombinant proteins were recognized by the positive serum of Ot. patients.
CONCLUSIONThe sta56 gene of Orientia tsutsugamushi Karp strain could be highly expressed in E. coli and its expression showed better efficiency in pET30a than in pPROEX HTb. The recombinant sta56 antigen with immunoreactivity could be used as diagnostic reagent for Ot. infection.
