乙型肝炎病毒靶向核糖核酸酶及其突变体的原核表达和活性分析

目的：研究原核表达的乙型肝炎病毒（HBV）靶向核糖核酸酶（RNase）及其突变体（点突变失去RNase活性）的活性。方法：将构建的靶向核糖核酸酶及其突变体基因克隆入原核表达载体pET32a（＋），转化大肠杆菌BL21（DE3），以IPTG诱导融合蛋白（HBV核心蛋白与人嗜酸性粒细胞来源的神经毒素的融合蛋白）的表达；表达产物经包涵体纯化、SDS-PAGE和Western印迹鉴定，将纯化的蛋白用透析方法复性；以酵母tRNA为作用底物，应用复性的蛋白进行RNase活性分析。结果：纯化和复性了HBV靶向核糖核酸酶及其突变体；复性的HBV靶向核糖核酸酶可以降解酵母tRNA且具有剂量依赖性，而复性后的突变的靶向核糖核酸酶体不具有RNase活性。结论：原核表达的HBV靶向核糖核酸酶具有较强的RNase活性，为探索HBV靶向核糖核酸酶抑制乙肝病毒复制的机理奠定了基础。

Prokaryotic Expression and Activity Analysis of HBV Targeted Ribonuclease and its Mutant

Objective： To study RNase activity of HBV targeted ribonuclease（fusion protein of HBVc and hEDN） and point-mutated HBV targeted ribonuclease. Methods： HBV targeted ribonuclease（TR） and point-mutated targeted ribonuclease（TRm） genes were inserted into prokaryotic expression vector pET32a（＋）. The recombinant vectors were transformed into E.coli BL21（DE3）. Expression of TR and TRm were induced by IPTG. The two kinds of target protein were purified by inclusion body, while characterized by SDS-PAGE and Western blot. Purified TR and TRm were refolded by using dialysis method. RNase activity of TR and TRm were assayed using HepG2 cell total RNA and yeast tRNA as the substrate. Results： TR and TRm were purified and refolded successfully, and refolded TR degraded yeast tRNA and showed a dose-dependent effect, but refolded TRm had no same activity. Conclusion： Purified and refolded TR demonstrates potent RNase activity. This research lays a foundation for further study on the mechanism inhibiting HBV by the targeted ribonuclease.