普氏立克次体重组表面蛋白制备和免疫印迹抗原特异性分析

Preparation and immunoblotting of recombinant surface protein antigens of Rickettsia prowazekii

目的克隆与表达普氏立克次体(Rickettesia prowazekii)主要蛋白抗原基因,分析所表达重组蛋白的抗原特异性。方法采用PCR法从普氏立克次体全基因组中扩增其主要抗原基因,将扩得的基因片段插入原核表达质粒;将基因重组表达质粒转化大肠杆菌,用IPTG诱导转化菌表达重组蛋白。将普氏立克次体、莫氏立克次体、立氏立克次体、恙虫病东方体、黑龙江立克次体、贝氏柯克斯体等实验感染小鼠血清与重组蛋白做免疫印迹。结果经过PCR扩增后获得9个目的基因片段,用其构建出9个原核表达质粒;9个原核表达质粒转化菌均高效表达出相应的重组蛋白;结果显示FtsZ、GroEL、Rp828、EF-Ts等4个蛋白特异性最好,仅与普氏立克次体感染小鼠血清反应;其次是Omp,它除与普氏立克次体感染小鼠血清反应外,仅与莫氏立克次体感染血清反应。结论 FtsZ等4个重组蛋白为普氏立克次体的特异性抗原,可以作为研制流行性斑疹伤寒血清学诊断试剂的候选抗原。

The objective of the present study is to prepare recombinant proteins of the major protein antigens of Rickette- sia prowazekii （Rp） and to analyze their specificity in serological diagnosis of the rickettsial infection. The genes encoding 8 surface proteins of Rp were amplified from its genomic DNA by polymerase chain reaction and the gene fragments were ex- pressed in E. coli cells by molecular clone approaches. The resulting recombinant proteins were analyzed with sera from mice experimentally infected with R. prowazekii, R. mooseri, R. rickettsii, R. heilongjiangensis, Oreintia tsutsugarnushii, or Coxiella burnetii in immunoblotting. As a result, four proteins, FtsZ, GroEL, Rp828, and EF Ts, reacted with only R. prowazekii--infected sera and Omp reacted with the sera from mice infected with R. mooseri besides R. prowazekii. The result suggests that the four surface proteins are highly specific for R. prowazekii--infected sera and candidateantigens for se- rological diagnosis of R. prowazekii infection.