【摘要】 根据GenBank中H5N1亚型禽流感病毒RNA聚合酶PB1基因序列设计一对特异性引物,利用RT-PCR方法扩增H5N1亚型禽流感病毒的PB1_P基因,克隆到原核表达载体pET-28a载体中,经PCR、酶切和测序分析后,鉴定出阳性重组子。将阳性质粒pET-28a/PB1_P转化大肠杆菌BL21(DE3),用0.5mol/L IPTG,在37℃诱导表达4h,经SDS-PAGE和Western-blot检测,获得重组蛋白PB1_P表达。并经Ni-NTA柱亲和层析与柱层析纯化表达蛋白,采用悬滴气相扩散法对纯化蛋白进行结晶。结果成功构建pET-28a/PB1_P重组质粒,SDS-PAGE结果显示重组蛋白在大肠杆菌中获得了高效表达,Western-blot证明表达的融合蛋白具有良好的免疫原性,经蛋白纯化、初筛结晶,得到PB1_P重组蛋白初筛晶体,为深入研究流感病毒聚合酶的生物学功能、开发新型H5N1亚型禽流感病毒检测试剂盒奠定了良好的基础。更多还原

【Abstract】 This research was conducted to clone and express the PB1_P protein of avian influenza virus H5N1 RNA polymerase and purify PB1_P recombinant protein with high concentration and its preliminary crystals,which could be useful for the further study of the function and structure of the influenza virus po-lymerase and the development of a new diagnostic kit.According to the sequence of polymerase basic protein 1 PB1 gene in GenBank,a pair of specific primers were designed.Polymerase domain(PB1_P) of avian influenza virus PB1 gene was amplified by RT-PCR,and inserted into pET-28a,then the constructed recombinant plasmid was analyzed.Then,the positive pET-28a/PB1_P plasmids were transformed into E.coli BL21(DE3)competent cell and induced for 4 h,at 37 ℃ by 0.5 mmol/L IPTG.The recombinant fusion protein PB1_P was successfully expressed and identified by SDS-PAGE and Western-blot and then purified by Ni-NTA His Bind Resins and column chromatography.After purification,the preliminary crystals of PB1_P recombinant protein was obtained by the hanging drop vapor diffusion method.The pET-28a/PB1_P recombinant plasmid was successfully reconstructed.Analysis of SDS-PAGE showed that the fusion protein PB1_P was successfully cloned and expressed.The result of Western-blot indicated that the recombinant had specially immunologic reactionogenicty with postive serum.The preliminary crystals of PB1_P recombinant protein was obtained after purification and crystallization screening.This study provides a sound basis for further study on biological function and of PB1 and the development of a new avian influenza virus H5N1 detection kit.更多还原