幽门螺杆菌hp1188基因的克隆及高效表达

Cloning and High Expression of Helicobacter pylori hp1188 Gene

目的克隆并表达幽门螺杆菌(Helicobacter pylori,H.pylori)hp1188基因,为研究幽门螺杆菌的黏附机制奠定基础。方法用PCR法从H.pylori标准株NCTC11637基因组DNA中扩增hp1188基因,克隆入原核表达载体pQE-30,构建重组原核表达质粒pQE30-hp1188,转化大肠杆菌DH5α,IPTG诱导表达。SDS-PAGE分析表达形式和表达量。表达蛋白经Ni2+-NTA树脂纯化,Western blot鉴定其反应原性。结果所构建的重组表达质粒pQE30-hp1188序列完整,插入的基因片段全长810bp,与GenBank中的hp1188基因同源性达98%。表达的重组蛋白相对分子质量约为30600,以1.0mmol/L IPTG诱导4h,表达量最高,破菌上清和沉淀中均有表达,可溶性蛋白表达量占全菌总蛋白的47%。重组蛋白纯化后纯度可达90%,并可被H.pylori患者血清识别。结论已成功克隆了H.pylori hp1188基因,并在大肠杆菌DH5α中获得高效表达。

Objective To clone and express Helicobacter pylori hp1188 gene and lay a foundation of study on the adhesion mechanism of H. pylori. Methods hp1188 gene was amplified from the genome of H. pylori standard strain NCTC 11637 by PCR and cloned into prokaryotic expression vector pQE-30. The constructed recombinant plasmid pQE30-hp1188 was transformed to E. coli DH5α for expression under induction of IPTG. The expressed protein was purified by Ni2^＋-NTA chromatography, then analyzed for form and expression level by SDS-PAGE and for reactogenicity by Western blot. Results The constructed recombinant plasmid carried the target gene fragment, at a full-length of 810 bp, which showed a homology of 98% to that reported in GenBank. The relative molecular mass of expressed protein was about 30 600. The expression level reached a peak value after induction with 1.0 mmol/L IPTG for 4 h. The expressed product was found in both supernatant and precipitate of recombinant E. coli, while that in supernatant contained 47% of total somatic protein. The recombinant protein reached a purity of more than 90% after purification and was recognized by the sera of patients with H. pylori infection. Conclusion hpl188 gene was successfully cloned and highly expressed in E. coli DH5α.