CAJ | 학술논문

通过使用原核表达载体大量表达H5N1病毒RNA聚合酶亚基PA-C257,PB1_N25,再经过GST亲和层析和Sephadex G-200层析柱纯化,获得了高纯度的蛋白复合体.采用悬滴气相扩散法筛选蛋白晶体,在1～1.5mol/L乙酸钠和pH7.9条件下获得了理想的晶体,为解析禽流感病毒RNA聚合酶三维结构并进一步认识其生物功能奠定了基础.
통과사용원핵표체재체대량표체H5N1병독RNA취합매아기PA-C257,PB1_N25,재경과GST친화층석화Sephadex G-200층석주순화,획득료고순도적단백복합체.채용현적기상확산법사선단백정체,재1～1.5mol/L을산납화pH7.9조건하획득료이상적정체,위해석금류감병독RNA취합매삼유결구병진일보인식기생물공능전정료기출.
A method was set up to over-express two peptides from avian influenza A virus subtype H5N1 (A/goose/Guangdong/1/96) in escherichia coli strain BL21, including a PB1 amino terminal short pep-tide covering residues 1～25(PB1_N25 ) and a protein fragment from PA subunit covering residues 257~716 (PA_C257). After intensive purification by glutathione-Sepharose column and Sephadex G-200, these two peptides were co-purified as a high concentration complex. This protein complex was further applied for protein crystallization by hanging drops vapour diffusion method. The best crystals were obtained by the vapour diffusion method with 1～1.5 mol/L sodium acetate as the precipitant at pH7.9, which laid a foundation for the analysis of the tri-dimensional structure of H5N1 RNA polymerase subunit complex as well as for further understanding its biological function.