幽门螺杆菌尿素酶B亚单位基因的克隆及其高效表达

目的克隆幽门螺杆菌尿素酶B亚单位(UreB)基因,构建原核表达载体,并进行高效表达。方法以幽门螺杆菌基因组DNA为模板,PCR扩增UreB基因,双酶切后,与质粒pET-22b(+)连接,构建表达载体pET-22b(+)/UreB,分别转化E.coliBL21(DE3)、Origam(iDE3)和Rossetta(DE3),经IPTG诱导后,进行SDS-PAGE和Western blot分析。结果经酶切及测序,证明幽门螺杆菌UreB基因的原核表达载体构建正确。3种重组菌的诱导表达产物经SDS-PAGE分析,均可见相对分子质量为64000的目的蛋白条带,Rossetta(DE3)重组菌目的蛋白表达量最高,约占菌体蛋白的35%。Western blot结果表明,表达的目的蛋白具有良好的反应原性。结论已成功克隆了幽门螺杆菌UreB基因,并在大肠杆菌Rossetta(DE3)中获得了高效表达。

Cloning and High Expression of Helicobacter pylori Urease B Subunit Gene

Objective To clone the gene encoding Helicobacter pylori (Hp) urease B subunit (UreB) and highly express in prokaryotic cells. Methods UreB gene was amplified by PCR using the genomic DNA of Hp as template, digested with Nde Ⅰ and Xho Ⅰ and inserted into plasmid pET-22b (+). The constructed recombinant plasmid pET-22b (+) / UreB was transformed to E. coli BL21 (DE3), Origami (DE3) and Rossetta (DE3) respectively for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-22b (+) / UreB was constructed correctly. The expressed product in each of the 3 kinds of recombinant E. coli showed a protein band with relative molecular mass of 64 000 on SDS-PAGE profile. The expression level of UreB in E. coli Rossetta(DE3) reached 35% of total somatic protein, which was the highest in the 3 kinds of recombinant E. coli. The expressed product in E. coli Rossetta (DE3) showed good reactogenicity as proved by Western blot. Conclusion UreB gene was successfully cloned and highly expressed in E. coli Rossetta (DE3).