鼠疫耶尔森菌Pla毒力蛋白的表达、纯化及其活性研究  

Expression and purification of virulence protein Pla of Yersinia pestis and its activity

作　　者：范艳晓 周亚洲[2] 冯娜[1,2] 汪琼[2] 毕玉晶[2] 韩延平[2] 杨瑞馥[2] 王效义[1,2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2015年第9期677-681,687,共6页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31430006,81171529);国家重点基础研究发展计划资助项目(2014CB744405)

摘　　要：目的制备鼠疫耶尔森菌(Yersinia pestis)重组纤溶酶原激活剂(Pla)蛋白,为研究鼠疫菌蛋白之间相互作用和鼠疫的免疫学诊断奠定基础。方法应用PCR从鼠疫菌基因组中扩增pla基因,将其克隆到p ET28a表达载体中;转化大肠杆菌BL21中,IPTG诱导表达;SDS-PAGE电泳检测表达结果;应用8 mol/L尿素将包涵体变性,梯度稀释透析法将其复性,SDS-PAGE电泳和Western印迹检测目的蛋白;奶粉平板法检测纤溶酶原激活剂活性。结果与结论琼脂糖凝胶电泳和测序结果证明表达质粒构建成功;SDS-PAGE电泳结果表明Pla蛋白以包涵体形式表达,表达产物主要为目的蛋白,且目的蛋白的表达量较高;包涵体经变性、复性得到了电泳纯Pla蛋白,并具有纤溶酶原激活剂活性。该研究提供了一种简单、快速、高效制备具有活性的Pla蛋白的方法。Objective To prepare recombinant plasminogen activator（Pla） protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside （ IPTG） was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.

关 键 词：鼠疫耶尔森菌 纤维蛋白酶原激活剂 表达 纯化 

分 类 号：R378[医药卫生—病原生物学]