幽门螺杆菌外膜蛋白18的克隆表达及纯化研究

目的克隆表达幽门螺杆菌（HP）外膜蛋白，为HP的疫苗开发及诊断试剂盒的研究奠定基础。方法用PCR方法从HP临床分离株9806的染色体DNA中扩增出OMP18基因片段，将目的基因插入表达载体pQE30中，重组载体pQE30-OMP18经DNA测序鉴定后，将重组质粒转化大肠杆菌E．coli．M15，IPTG诱导表达。采用镍离子亲和层析纯化蛋白，并用SDS-PAGE和Western blot分析鉴定其表达。结果PCR扩增出长度为537bp的OMP18基因，片段测序分析其与Gen—Bank公布的核苷酸序列相似性达99％；SDS—PAGE显示，重组工程菌经IPTG诱导表达了分子量为20．0kDa的目的蛋白，占总蛋白的20％，经纯化后，目的蛋白纯度达85％以上；Western blot结果表明，该蛋白可与兔抗HP的多克隆抗血清发生特异的抗原抗体结合反应。结论成功构建了OMP18表达载体pQE30-OMP18，并在大肠杆菌中获得高效表达，为HP疫苗有效抗原筛选及诊断试剂的开发奠定基础。

Cloning,expression and purification of outer membrane protein 18 of helicobacter pylori

Objective To explore the cloning ,expression and purification of an outer membrane protein of helicobacter pylori(HP) in order to lay a foundation for the development of HP vaccine and diagnostic reagent kit for HP infection. Methods The OMP18 gene of chromosomal DNA in HP clinical isolate 9806 was amplified by PCR,then inserted into the expression vector pQE30. After the recombinant vector pQE30-OMP18 was identified by DNA sequencing,recombinant plasmid were transformed to E. coli M15 and induced to express by IPTG. Nickel chelating chromatography was used to purify protein,SDS-PAGE and Western blotting to identify its expression. Results OMP18 gene with a length of 537bp was amplified by PCR,and determined by DNA sequencing to have a similarity of 99% with nucleotide sequence records in GenBank. It was shown by SDS-PAGE that recombinant engineering bacteria was induced by IPTG to express the protein with a molecular weight of 20.0 kDa,accounting for 20% of total somatic protein,and the protein purity reached more than 85%. As shown by Western blotting,the protein could produce specific antigen-antibody reaction with rabbit antiserum. Conclusion The expression vector pQE30-OMP18 has been successfully constructed and OMP18 has been effectively expressed in E.coli ,which can provide the foundation for antigen screening of HP vaccine and diagnostic reagent for HP infection.