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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) hp1188 gene and lay a foundation for studying the mechanism of Helicobacter pylori adhesion. Methods The hp1188 gene was amplified by PCR from the genomic DNA of NCTC11637 of H.pylori and cloned into prokaryotic expression vector pQE-30. The recombinant plasmid pQE30-hp1188 was constructed and transformed into E.coli DH5α for expression under IPTG. SDS-PAGE analysis of expression and expression levels. The expressed protein was purified by Ni2 + -NTA resin and identified by Western blot. Results The constructed recombinant plasmid pQE30-hp1188 had a complete and inserted sequence of 810bp, which shared 98% homology with hp1188 in GenBank. The expressed recombinant protein had a molecular weight of about 30,600 and was induced by 1.0mmol / L IPTG for 4h. The highest level of expression was detected in both the supernatant and the precipitate, and the soluble protein accounted for 47% of total bacterial total protein. Purified recombinant protein purity of up to 90%, and can be H.pylori serum identification. Conclusion The H.pylori hp1188 gene has been successfully cloned and highly expressed in E. coli DH5α.