人RPP蛋白与HCV RNA体外相互作用的测定

摘要

目的检测人RPP蛋白与HCV RNA体外的相互作用.方法将人RPP各基因分别克隆至pET28a载体,在大肠杆菌中(BL21)表达的目标蛋白经蛋白纯化仪纯化获得人RPP蛋白;通过体外转录制备32P标记的HCV RNA片段作为探针,利用紫外交联试验研究各种人RPP 蛋白与HCV RNA的体外相互作用.结果 成功表达纯化了8种人RPP蛋白,紫外交联试验结果显示:Rpp20、Rpp21、Rpp29和Rpp304种蛋白可分别与HCV RNA片段结合,Rpp21和Rpp29结合力较强,Rpp20和Rpp30结合力则相对较弱,而其余几种RPP蛋白(包括Rpp14、Rpp25、Rpp40和Pop5)不能与HCV RNA发生结合.结论 人RPP蛋白可与HCV RNA片段在体外直接结合,推测这些RPP蛋白在人RNase P切割HCV RNA的过程中起重要作用.%Objective To identify the interaction between human RPP proteins and HCV RNA in vitro. Methods Each gene of human RPP was cloned into pET28a carrier,and the target proteins (Rpps) were separately expressed in Escherichia coli (BL21) and purified by instrument. The HCV RNA fragment labeled with 32P-UTP was prepared by in vitro transcription as the probe,and the interaction between each RPP protein and HCV RNA in vitro was studied by using the UV-cross linking method.Results Eight kinds of human RPP proteins were successful expressed and purified.UV-cross linking test showed that four RPP proteins including Rpp20,Rpp21,Rpp29 and Rpp30 may combine with HCV RNA. The binding force of Rpp21 and Rpp29 were strong,and the binding force of Rpp20 and Rpp30 were relatively weak. However, the rest of four RPP proteins including Rpp14,Rpp25,Rpp40 and Pop5 did not combine with HCV RNA. Conclusion This study confirms for the first time that some human RPP proteins can directly combine with HCV RNA fragment in vitro,which may play an important role in the process of RNase P cutting HCV RNA.