Cloning and expression of Chlamydia trachomatis heat shock protein 60 gene
10.3760/cma.j.issn.0412-4030.2009.05.008
- VernacularTitle:沙眼衣原体热休克蛋白60基因的克隆和表达
- Author:
Juanhua LIU
;
Mukai CHEN
;
Qiman LIAO
;
Haipian LI
;
Yuying TU
;
Jiande HAN
- Publication Type:Journal Article
- Keywords:
Chlamydia trachomatis;
Heat shock protein 60;
Recombinant fusion proteins;
Geneclone
- From:
Chinese Journal of Dermatology
2009;42(5):318-320
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.
