耐辐射球菌PprI蛋白质表达及其纯化的实验研究  

Expression and purification of PprI protein from D.radiodurans R1 in escherichia coli

作　　者：张永芹[1] 周辉[1] 陈洁[1] 杨占山[1] 

机构地区：[1]苏州大学医学部放射医学与公共卫生学院,苏州215123

出　　处：《辐射研究与辐射工艺学报》2011年第2期117-122,共6页Journal of Radiation Research and Radiation Processing

基　　金：国防基础科研项目(A3820060138);国家自然科学基金项目(30570549)资助

摘　　要：为了在大肠杆菌中高效表达耐辐射球菌PprI融合蛋白并进行纯化,本研究以PCMV-HA-pprI质粒为模板,PCR扩增pprI基因全序列,经Nco I和EcoR I双酶切后定向克隆到pET-28a表达载体中,构建重组原核表达质粒pET-28a-His-pprI,并转入E.coli BL21(DE3)RP。IPTG诱导融合蛋白表达,SDS-PAGE和Western blot鉴定表达产物。对蛋白表达优化诱导时菌液的A600值、IPTG浓度、诱导时间和温度表达条件。诱导表达的PprI融合蛋白采用Ni-NTA His-Bind树脂和分子筛两步法进行纯化。结果显示,Western blotting检测的表达产物确为6×His-PprI融合蛋白,相对分子质量约为37 KD。不同诱导浓度、温度和时间在相对分子量为37 KD处见到特异性的PprI蛋白条带具有一定的差异,目的蛋白在上清和沉淀中均有表达。BCA法测得纯化蛋白质浓度为0.15 mg/mL。结果表明,本研究成功构建了耐辐射球菌pprI基因的pET-28a-His-pprI重组原核表达载体,并获得纯化的毫克级耐辐射球菌PprI融合蛋白,为进一步研究PprI蛋白的抗放作用和免疫等性能奠定了基础。In order to express and purify PprI protein from D.radiodurans R1 in E.coli,the full length of pprI gene was gained by PCR amplification using pCMV-HA-pprI as a template.The gene segment was inserted into vec-tor pET-28a after digested by two restriction endonucleases Nco I and EcoR I.Then the recombinant vector pET-28a-His-pprI was transfected into E.coli BL21（DE3） RP.The PprI protein expression was induced by IPTG and the fusion protein was confirmed by SDS-PAGE and Western blotting.The expressive conditions of the protein such as E.coli＇ A600,concentration of IPTG,time and temperature of culture,were optimized.Finally the fusion protein was purified by Ni-NTA His Bind Resins and molecule boult.The experimental results show the fusion protein con-firmed by Western blotting is 6×His-PprI and its molecular weight is 37 kDa.The ladders of PprI protein at molecular weight 37 kDa were different due to difference of the PprI protein expression conditions in E.coli.The PprI protein exists both in supernatant and precipitation.The concentration of purified protein is about 0.15 mg/mL which was measured by BCA method.It is concluded that the recombinant plasmid pET-28a-His-pprI is constructed and the PprI fusion protein is expressed and purified.The results lay a solid foundation for studying the radio-resistance and im-munity of PprI protein.

关 键 词：耐辐射球菌 pprI基因 原核表达质粒 蛋白表达 纯化 

分 类 号：R811.5[医药卫生—放射医学] R818.053[医药卫生—临床医学]