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目的 克隆、表达幽门螺杆菌中性粒细胞激活蛋白基因napA ,为研究幽门螺杆菌致病机理提供材料。方法 用PCR从幽门螺杆菌DNA中扩增出目的基因napA ,定向插入原核表达载体pQE30中 ,测序分析确认后 ,转化大肠杆菌DH5α ,IPTG诱导表达 ,表达蛋白以NI2 + NTA柱进行纯化。结果 PCR扩增出 4 35bp目的基因片段napA ,克隆入pQE30质粒。工程菌诱导后SDS -PAGE显示新生表达蛋白带 ,相对分子质量为 170 0 0 ,与预期一致 ,约占菌体总蛋白的 38% ,经Ni2 + NTA柱纯化后可获得纯度为 95 5 %重组蛋白。Westernblot显示重组蛋白具有良好的抗原性。结论 克隆napA基因成功 ,并在大肠杆菌DH5α中高效表达。
Objective To clone and express Helicobacter pylori neutrophil activating protein gene napA and provide material for studying the pathogenesis of Helicobacter pylori. Methods The target gene, napA, was amplified from Helicobacter pylori by PCR and inserted into prokaryotic expression vector pQE30. After sequenced and confirmed, it was transformed into E. coli DH5α and induced by IPTG. The expressed protein was purified by NI2 + NTA column. Results The 35 35 bp fragment of napA was amplified by PCR and cloned into plasmid pQE30. After induced by engineering bacteria, SDS-PAGE showed that the expressed protein band was new, with a relative molecular mass of 170 0 0, consistent with the expectation, accounting for about 38% of the total bacterial protein. Purification by Ni2 + NTA column yielded 95 95% protein. Westernblot showed that the recombinant protein has good antigenicity. Conclusion The cloned napA gene was successfully expressed in E. coli DH5α.