Cloning of ureI gene from Helicobacter pylori and its expression in E. coli / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To clone the urea membrane channel gene (ureI) from Helicobacter pylori (Hp) for its expression in E. coli, and evaluate the expression conditions and immunological features of the fusion protein.</p><p><b>METHODS</b>ureI gene cloned by PCR from Hp was inserted into the plasmid pET32a (+) to construct the recombinant plasmid pET32a/ureI, followed by identification by BglII and HindIII digestion and sequencing. E. coli BL-21+(DE3) was transformed with pET32a/ureI to obtain the engineered bacterium BL21+/UreI, which was cultured at different temperatures and induced with 1.0 mmol/L IPTG for expression of the recombinant protein. The expressed proteins were identified by SDS-PAGE and analyzed by Pro-gel analyzer 4.0. Western blotting was performed to evaluate the immunogenicity of the expressed protein.</p><p><b>RESULTS</b>The cloned gene fragment was about 650 bp in length, and BglII and HindIII digestion of pET32a/ureI yielded a 650-bp band. Sequence analysis revealed that the cloned ureI gene contained 646 bp without reading frame alterations. Comparison against GenBank indicated a homology of 100% of the cloned gene with ureI gene of the corresponding Hp strains, and also one no less than 98.5% with ureI gene from other strains. The engineered E. coli BL21+/UreI could express recombinant UreI (rUreI) with His tag, and the target protein accounted for 20.2% of the total bacterial proteins after 1.0 mmol/L IPTG induction of the bacterium at 37 degrees C for 14 h. SDS-PAGE and Western blotting showed that the recombinant UreI protein was produced mainly in the inclusion bodies and fused with his-tag (rUreI/his), which could react with human anti-Hp and mAb to his tag but not with mAb to Hp UreB.</p><p><b>CONCLUSIONS</b>We have successfully cloned ureI gene and constructed the prokaryotic expression plasmid for efficient rUreI expression, and the fusion protein rUreI/his expressed in the inclusion bodies can react specifically with both Hp antibody and his-tag antibody.</p>