立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

Cloning,Expression and Immunogenicity of Outer Membrane Protein H Gene Fragment of Rickettsia Rickettsii in E.coli

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。

Objective：To express the gene encoding outer membrane protein H（OmpH） of Rickettsia rickettsii（R.r） in E.coli and to investigate the immunogenicity.Methods：The gene fragment encoding the outer membrane protein H（OmpH） was amplified from the genomic DNA of R.r by PCR,and was transferred into the prokaryotic expression vector pET32a to construct the recombinant plasmid pET32a/omph.The recombinant plasmid were transformed into the E.coli cells and the recombinant gene were induced to express pro-tein by IPTG.The recombinant protein was analyzes by immunoblottingg assay.Results：A ompH gene fragment with length of 486 bp was cloned,The recombinant protein of approximately 27kDa could recognized by sera of the infected guinea pigs and sera from patients.Before being added to suspended VERO cells to assess their infectivity by fluorescent quantitative PCR assays,hyperimmune antirick-ettsial sera,prepared in mouse by injections of the recombinant protein and R.r in growth medium were incubated in serum at room tem-perature for 30 min（pretreatment）.It was concluded that immune serum may inhibit rickettsial infection.Conclusions：E coli cells trans-formed by plasmid pET32a/ompH can express a 29 kDa recombinant protein and this protein may be the major antigens of R.r capable to induce specific immune responses and may be used as the new candidate to develop diagnostic reagents or to prepare the subunit vac-cine for spotted fever.