H5N1亚型禽流感病毒RNA聚合酶PB1_P亚基片段的表达、纯化和结晶

摘要

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 polymerase 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/PBl_P plasmids were transformed into E. coli BL2KDE3)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/PBl_ 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.%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.