立氏立克次体蛋白抗原基因的表达和重组蛋白抗原的免疫印迹分析

Expression of the genes encoding protein antigens of Rickettesia rickettesii and the analysis of the recombinant protein antigens by immunoblotting

目的表达立氏立克次体(Rickettsia rickettsii)主要抗原基因,免疫印迹初步分析重组蛋白抗原的抗原性。方法根据立氏立克次体全基因组序列选出主要抗原相关基因,采用PCR从全基因组中扩增抗原相关基因,将扩得的基因片段插入原核表达质粒构建抗原基因重组表达质粒,将重组质粒转化大肠杆菌并诱导转化菌表达重组蛋白抗原。采用立氏立克次体感染的豚鼠血清和斑点热患者血清分别与纯化的重组蛋白抗原做免疫印迹分析。结果经PCR扩增后获得43个目的基因片段,其中36个基因片段在大肠杆菌中高效表达重组蛋白抗原,其中5个蛋白抗原在免疫印迹中与感染豚鼠血清和斑点热患者血清反应均为阳性。结论免疫印迹筛选出5种立氏立克次体重组蛋白抗原,结果提示它们是能够诱导机体产生特异性免疫应答的主要抗原,可作研制斑点热诊断试剂或亚单位疫苗的新候选蛋白抗原。

In the present study, the genes encoding protein antigens of Ricleettesia rickettsii were expressed and its recombinant protein antigens were analyzed by immunoblot assay, in which the main antigen-associated genes were screened according to the complete genomic DNA sequence of R. riekettesii and amplified by PCR. The amplified gene fragment was then inserted to the prokaryotic expression plasmid to construct the recombinant expression vector for antigen associated genes, and the recombinant plasmid was transformed to E. coli later on. The protein antigens were expressed from E. eoli with induction and analyzed by immunoblotting with sera from guinea pigs infected with R. rickettsii and patients suffering from spotted fever. It was found that 43 target gene fragments were obtained through PCR amplification, from which 36 gene fragments could be highly expressed in E. coli with production of the recombinant antigen proteins. In the analysis of the expressed antigen proteins with immunoblotting assay. It was demonstrated that 36 of the 43 positive clones transformed with antigen associated genes were efficiently expressed, 9 of the expressed 36 recombinant antigen proteins were recognized by sera of the infected guinea pigs and 5 of 9 recombinant antigen protein were also recognized by sera from patients. It is evident from these observation that 5 recombinant protein antigens of R. rickettsii are screened by means of immunoblotting, which may be the major antigens of R. rickettsii capable to induce specific immune responses and may be used as the new candidate to develop diagnostic reagents or to prepare the subunit vaccine for spotted fever.