禽流感病毒H5N1亚型RNA聚合酶蛋白表达纯化及晶体生长  

Expression of purification and crystallization of PA_C257 & PB1_N25 protein complex of influenza A virus H5N1

作　　者：马建源[1] 贺晓静[2] 浦铜良[1] 刘迎芳[2] 

机构地区：[1]兰州大学生命科学学院,兰州730000 [2]中国科学院生物物理研究所,北京100101

出　　处：《兰州大学学报（自然科学版）》2009年第5期119-124,共6页Journal of Lanzhou University(Natural Sciences)

基　　金：国家自然科学基金项目(30599432)

摘　　要：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1～1.5 mol/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。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 peptide 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 pH 7.9, 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.

关 键 词：禽流感 H5N1 蛋白表达 纯化 晶体生长 

分 类 号：Q71[生物学—分子生物学]