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1.
Journal of Southern Medical University ; (12): 24-27, 2007.
Artigo em Chinês | WPRIM | ID: wpr-298253

RESUMO

<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>


Assuntos
Humanos , Proteínas de Bactérias , Genética , Metabolismo , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Genética , Helicobacter pylori , Genética , Proteínas de Membrana Transportadoras , Genética , Metabolismo , Plasmídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Metabolismo
2.
Journal of Southern Medical University ; (12): 31-35, 2006.
Artigo em Chinês | WPRIM | ID: wpr-234202

RESUMO

<p><b>OBJECTIVE</b>To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH(3)T(3) cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5).</p><p><b>METHODS</b>HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plasmid pET32a(+) followed by digestion with BamH I and Hind III. The recombinant plasmid pET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BamH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E. coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH(3)T(3) cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR.</p><p><b>RESULTS</b>The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 degrees C, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfected NIH(3)T(3) cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment.</p><p><b>CONCLUSIONS</b>The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E .coli and NIH(3)T(3) cells with stable expression of HPV16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.</p>


Assuntos
Animais , Humanos , Camundongos , Células 3T3 , Escherichia coli , Genética , Metabolismo , Células Eucarióticas , Metabolismo , Vetores Genéticos , Proteínas Oncogênicas Virais , Genética , Papillomaviridae , Genética , Infecções por Papillomavirus , Virologia , Plasmídeos , Genética , Células Procarióticas , Metabolismo , Proteínas Recombinantes de Fusão , Genética
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