幽门螺杆菌中性粒细胞激活蛋白的基因克隆与表达

Cloning and Expression of Neutrophil Activating Protein(napA) Gene of Helicoba cter pylori

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 the neutrophil a ct ivating protein (napA) gene of Helicobacter pylori and provide a basis for the study on pathogenic mechanism of the microorganism.Methods Amplify napA gene from DNA of H.pylori by PCR a nd insert into prokaryotic expression vector pQE30. Identify the recombinant pla msid by sequencing then transform into E.coli DH5α for expression under ind uction of IPTG.Purify the expressed protein by Ni 2+-NTA column chromatogr aphy.Results The napA gene fragment at a length of 435 bp w as amplified and successfully cloned into plasmid pQE30. SDS-PAGE showed a prot ein band with a relative molecular weight of 17 000,which was consistent with th e expectation. The expressed product contained about 38% of total somatic pr otein,and reached a purity of 95.5% after Ni 2+-NTA column chromatography . Western blot showed good antigenicity of the recombinant protein.Conclusion napA gene was successfully cloned and highly ex pressed in E.coli DH5α.