幽门螺杆菌尿素通道蛋白基因UreI载体的构建、融合表达及纯化

Construction urea channel protein(UreI) of human Helicobacter pylori and expression in E.coli

目的构建幽门螺杆菌(H.pylori,Hp)尿素通道蛋白基因UreI的融合表达载体,并在E.coliBL21中表达,为进一步研究UreI的功能奠定基础。方法利用分子克隆技术以Hp DNA染色体为模板,扩增Hp UreI基因片段,将目的基因UreI与载体pET32a(+)分别经kpnⅠ和HindⅢ双酶切,纯化,连接。筛选阳性重组载体,转化大肠杆菌BL21,以IPTG诱导表达pET32a(+)/UreI融合蛋白。表达产物经Ni-NTA琼脂糖树脂纯化后,用SDS-PAGE及Western blot分析。结果经酶切、测序分析表明,插入的基因片段为Hp UreI编码基因,由585 bp组成,与GenBank相应菌株HP AM417 609序列完全一致,无碱基突变。经SDS-PAGE分析显示重组质粒pET32a(+)/UreI在原核细胞中得到了高效融合表达,其重组融合蛋白分子量为130 ku,经Ni-NTA琼脂糖树脂纯化后,其纯度达95%以上,Western blot分析显示,该重组融合蛋白可被6-His鼠抗单克隆抗体所识别,具有良好的反应原性。结论成功地克隆和构建了原核表达载体pET32a(+)/UreI,并在原核细胞中得到了高效融合表达。

Objective To construct the prokaryotic expression vector of the urea channel protein gene（UreI） of Helicobacter pylori and make it expressed in E.coli BL21. Methods The target gene encoding UreI was amplified from Hp DNA by PCR,digested by restricted endonuclease enzyme of Hin dⅢ,Kpn Ⅰ simultaneously,and inserted into prokaryotic expression vector pET32a（＋） digested by corresponding restricted endonuclease enzyme.The recombinant plasmid was used to select and identify by restricted endonuclease enzyme digestion and sequence analysis and transform,meanwhile induce it to be expressed in E.coli BL21 by IPTG.The recombination fusion protein was purified by 6-His marked Ni-TED TM kit and analysed by Western blot. Results Restriction endonuclease analysis and sequencing showed that the target gene was found to be 585 base pairs and had been inserted into recombinant vector.As compared with gene HP AM417 609 reported by GenBank,cloned UreI gene sequence was completely matched and composed of 585 bp.SDS-PAGE analysis showed that the recombinant plasmid pET32a（＋）/UreI could be expressed in E.coli BL21,its relative molecule mass of expressed product was 130 ku.After purified with Ni-NTA agarose resin,the purity of recombinant fusion protein was about 95%.Western blot results showed that the recombinant fusion protein could be identified by anti-6-His monocloned antibody,suggesting that this fusion protein has good reactionogenicity. Conclusion The gene coding for UreI is cloned and expressed successfully.