人幽门螺杆菌尿素通道蛋白UreI基因穿梭质粒的构建及表达

Construction and Expression of Shuttle Plasmid for Helicobacter pylori Urea Channel Protein UreI Gene

目的构建人幽门螺杆菌尿素通道蛋白UreI基因的大肠杆菌-分枝杆菌穿梭表达质粒,并在耻垢分枝杆菌中进行表达。方法PCR扩增幽门螺杆菌尿素通道蛋白UreI编码基因全长片段,克隆入pET32a(+)质粒,鉴定正确后,再亚克隆入大肠杆菌-分枝杆菌穿梭质粒pJHSP70,构建分枝杆菌穿梭表达质粒pJHSP70-UreI,转化耻垢分枝杆菌,诱导表达后,进行SDS-PAGE和Western blot检测。结果从幽门螺杆菌基因组中扩增出585bp的UreI基因片段。重组质粒pJHSP70-UreI经酶切和PCR鉴定,与预期结果一致。目的蛋白表达量约占菌体总蛋白的18.5%,且具有良好的反应原性。结论已成功构建了幽门螺杆菌尿素通道蛋白UreI基因大肠杆菌-分枝杆菌穿梭表达质粒,并在耻垢分枝杆菌中获得表达。

Objective To construct the E. coli-Mycobacterium shuttle plasmid for Helicobacter pylori（Hp ） urea channel protein UreI gene and express in Mycobacterium smegmatis. Methods The full-length of Urel encoding gene was amplified by PCR from the genome of HI） and cloned into plasmid pET32a （＋）. The constructed recombinant plasmid pET32a （＋）-Urel was identified by restriction analysis and sequencing, then subcloned to E. coli-Mycob（wterium shuttle plasmid pJHSP70. The constructed Mycobacterium shuttle plasmid pJHSP70-Urel was transtormed to M. smegmatis mc2 155 for expression by thermal induction. The expressed product was identified by SDS-PAGE and Western blot. Results The UreI gene fragment at a length of 585 bp was amplified. The results of rcstrietion analysis and PCR identification were consistent with those expected. Sequencing resuh proved that Urel gene was inserted correctly. The expressed Urel contained about 18.5% of total somatic protein and showed good immunogenicity. Conclusion The E. coli-Mycobacterium shuttle plasmid for UreI gene was successfully constructed and expressed in Mycobacterium smegrnatis.