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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 clone and express the ompH gene of outer membrane protein Rickettsia rickettsii and analyze its immunogenicity. Methods: The ompH gene fragment was amplified from Rickettsia rickettsii by PCR and ligated with prokaryotic expression vector pET32a to construct recombinant prokaryotic expression plasmid pET32a / ompH. The pET32a / ompH was transformed into E. coli cells , Induced by IPTG transformation of E. coli expression of the target gene. RESULTS: An ompH gene fragment of 327 bp in length was obtained. SDS-PAGE analysis revealed that pET32a / ompH transformed cells expressed a protein of about 27 kDa in size. Western blotting analysis was performed on the serum of immunized guinea pigs with rickettsia rickettsia and spot blotch fever Positive reaction, the recombinant protein immune serum neutralizing Rieseltsia rickettsi infection VERO activity decreased. CONCLUSION: The ompH gene fragment was expressed in pET32a / ompH transformed E. coli. The resulting recombinant protein has good immunoreactivity and protection.