绿脓杆菌RNA降解蛋白PNPase与RNaseE表达纯化及相互作用  

作　　者：方葆 沈国森 郭珍 

机构地区：[1]浙江省开化县临床检验检测中心,324300 [2]浙江大学医学院附属第二医院,310014

出　　处：《浙江临床医学》2017年第12期2319-2321,共3页Zhejiang Clinical Medical Journal

摘　　要：目的RNA降解复合体是RNA在生物体内更新的主要工具。PNPase通过3’到5’核酸外切酶降解RNA，RNaseE参与其调控。通过对绿脓杆菌RNA降解蛋PNPase与RNaseE进行克隆表达纯化，并获取复合物蛋白，进而揭示PNPase与RNaseE对绿脓杆菌RNA的降解机制。方法对PNPase与RNaseE基因序列进行PCR扩增，构建重组质粒，分别转入E．coliB L21中表达，应用镍离子亲和层析，离子亲和层析和分子筛纯化目的蛋PNPase与RNaseE；通过Pull-down方法验证PNPase和RNaseE的相互作用。结果成功克隆、表达、纯化绿脓杆菌PNPase与RNaseE蛋白，并通过Pull-down获得大量PNPase-RNaseE复合物。结论PNPase与RNaseE蛋白能相互作用并形成PNPase-RNaseE复合物。Objective To analyze the molecular mechanism on Pseudomonas Aeruginosa, the protein PNPase and RNase from P. Aeruginosa were cloned, expressed, purified and crystallized. Methods SdrE gene was amplified and cloned into the vector. The recombinant plasmid, pW28- SdrE, was transformed into E. coli B834 to express SdrE protein. The SdrE protein was purified by Ni2＋-NTA affinity chromatography column and DEAE anion-exchange chromatography column, the state of aggregation in solution was analyzed by Hiload Superdex 200 gel flittration colunm. The SdrE crystal was screened with Hampton kit and optimized crystal with chessboard method. Results （ 1 ） Recombinant plasmid pW28-SdrE was constructed and expressed in soluble form in E.coli B834 strain. （ 2 ） The puffed SdrE exhibited as monomer in solution. （ 3 ） The single crystals of SdrE were obtained. Conclusions The SdrE crystals are grown by classic methods of protein purification and crystal cultivation. This date provides a key base for structural and functional study of SdrE in future, which is necessary for developing new antibacterial drugs.

关 键 词：PNPASE RNASE E 蛋白表达纯化 相互作用 

分 类 号：R392-33[医药卫生—免疫学]