肺炎衣原体重组的RNase HIIa的克隆和性质鉴定

Cloning and Characterization of Recombinant RNase HIIa from Chlamydia Pneumoniae

目的对肺炎衣原体AR39的RNase HIIa（CpRNase HIIa）进行克隆、表达和性质鉴定。方法按已知的CpRNase HIIa基因设计引物、扩增。用pET表达系统（表达载体、大肠杆菌BL21和Ni-NTA树脂）构建重组质粒，表达和纯化CpRNase HIIa。5’-^32P标记底物RNA／DNA，鉴定酶学性质。结摹CpRNase HIIa能切割RNA／DNA杂合体的寡聚核糖核苷酸链，产生3’羟基和5’磷酸基团的末端。CpRNase HIIa对Mg^2＋和Mn^2＋表现出明显的离子偏好性，且反应最适pH值为9～10。CpRNase HIIa切割12个碱基对的RNA／DNA杂合体底物的多个位点，但主要切割位点位于c10-g11之间与u7-g8之间。结论CpRNase HIIa可以有效的降解RNA／DNA杂合体的RNA链，于肺炎衣原体内担负着一定的生物学功能。

Objective The RNase HIIa of Chlamydia pneumoniae AR39 （CpRNase HIIa） was cloned, expressed and characterized. Methods A gene of CpRNase HIIa was amplified with the designed primers. Then, the recombinant plasmid was constructed, and CpRNase HIIa was expressed and purified with pET expression system including expression vectors, E. coli strain BL21 and nickel-nitrilotriacetic acid His-Bind Resin. 5＇ -^32P-labeled substrate was prepared to identify characterization of CpRNase HIIa. Results Ribonuclease H activity of CpRNase HIIa could cleave oligoribonucleotides strand, generating break with 3＇-OH and 5＇-phosphate ends. RNase H activity of CpRNase HIIa exhibited ions preference to Mg^2＋ and Mn^2＋ , and an optimum pH from 9 to 10. CpRNase HIIa cleaved the RNA strand of the 12 bp RNA/DNA substrate at multiple sites, but preferably at the sites of c10-g11 and u7-g8. Conclusion CpRNase HIIa can efficiently degrade the RNA strand of an RNA/DNA hybrid. It may have definite functions in C. pneumoniae.