靶向23S rRNA的多肽核酸体外抑制细菌蛋白翻译

Inhibition of bacterial translation by peplide nucleic acids targeted domain Ⅱ of 23S rRNA in cell-free translation system

目的 尝试研究反义多肽核酸（peptide nucleic acid，PNA）在体外翻译系统中抑制细菌蛋白的翻译。方法以增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）为报告分子，将重组载体pET-28a—EGFP和靶向23S rRNA domainⅡ区的PNA在体外翻译系统中共同孵育后，用荧光显微镜观察荧光强度减弱，放射自显影证明荧光强度减弱是因为EGFP减少。为了分析PNA体外抑制蛋白翻译的浓度，我们用蛋白沉淀方法分析PNA体外抑制[^35S]methionine渗入蛋白比例。最后，用浊度分析观察PNA（G1138）在MH肉汤培养基中的抑菌作用。结果在体外翻译系统中，靶向23S rRNA do-mainⅡ区的PNA（G1138）以剂量和序列依赖方式抑制蛋白合成，抑制浓度（IC50）为0．15μmol／L。在MH培养基中，PNA（G1138）抑菌效果不明显（MIC〉50μmol／L）。结论靶向23S rRNA domainⅡ区的PNA（G1138）在体外翻译系统中能有效抑制细菌蛋白合成。在MH肉汤培养基中，PNA（G1138）抑菌效果不明显。

Objective To study inhibition of bacterial wanslation in a cell-free wanslation system with antisense peptide nucleic acids（PNA） targeted 23S rRNA domain Ⅱ.Methods Recombinant plasmid pET-28a- EGFP and PNA targeted to 23S rRNA domain Ⅱ were incubated together in a cell-free translation systems, fluorescence intensity of EGFP was observed by fluorescence microscopy. And to quantify the decline of fluorescence intensity potentially caused by the reduction of EGFP expression. Autoradiography was applied to analyze the inhibiriot effects of PNA.Then we measured the relative incorporation of [^35S]-methionine by TCA protein precipitation assay to calculate the inhibitory concentrations （IC50） of PNA.Finally, we used the turbidity assay to study the bacteriostasis of PNA（G1138） in MH broth. Results PNA（G1138） targeted 23S rRNA domain Ⅱ inhibited bacterial protein translation in the cell-free wanslation system in a dose- and sequence-dependent mariner, the inhibitory concentration （IC50） is 0.15/μmol/L. Conclusion PNA（G1138） targeted 23S rRNA domain Ⅱ can efficiently inhibite bacterial translation in a cell-free wanslation system.In MH broth,the bacteriostasis of PNA（G1138） is not imoressive.