[Cloning of ureI gene from Helicobacter pylori and its expression in E. coli].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Bao-Ning
BN
Department of Microbiology, School of Preclinical and Forensic Medicine, Sichuan university, Chengdu 610041, China. wbn@labsky.com
Shi
Qiao-Fa
QF
Li
Hong
H
Li
Ming-Yuan
MY
Chen
Cui-Ping
CP
Zheng
Qing-Wen
QW
Jiang
Zhong-Hua
ZH
Xiao
Li-Ying
LY
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C114527:UreI protein, Helicobacter pylori