草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化  

Expression of Grass carp reovirus RNA Polymerase Gene and Purification of its Product

作　　者：方勤[1] 丁清泉[1] 汪亚平[2] 朱作言[2] 

机构地区：[1]中国科学院武汉病毒研究所,湖北武汉430071 [2]淡水生态与生物技术国家重点实验室,中国科学院水生生物研究所,湖北武汉430072

出　　处：《中国病毒学》2003年第2期169-173,共5页Virologica Sinica

基　　金：国家自然科学基金资助项目(30170730);"淡水生态与生物技术国家重点实验室"开放课题(2002FB05)

摘　　要：草鱼呼肠孤病毒是引起草鱼出血病的主要病原,隶属于呼肠孤病毒科水生呼肠孤病毒属。序列分析表明,GCRV S2 片段长为3 877核苷酸,编码一个分子量为138kDa 的蛋白VP2,具有RNA聚合酶性质。为进一步了解该病毒 RNA聚合酶特性,本研究在对GCRV RNA聚合酶基因(GCRV-RdRp)保守区(约1.5kb)重组质粒pR/RRp高效表达的基础上,分别构建了编码GCRV RNA聚合酶保守区N端与C端部分基因的 pR/RRpN及pR/RRpC重组表达载体,并在原核细胞中获得成功表达。筛选的重组表达菌株经IPTG诱导培养,得到分子量分别为98kDa、103kDa的目的表达融合蛋白。Western blot分析表明,该表达产物与兔抗GCRV-VP2血清呈阳性反应。通过ProBond柱亲和层析,纯化了融合有6个组氨酸的重组表达产物,并获得约90%纯的目的蛋白。上述结果为GCRV RNA聚合酶特性分析提供了依据。Grass carp reovirus (GCRV) is a disaster agent to aquatic animals, which belongs to genus Aquareovirus, family Reoviridae. Sequences analysis revealed GCRV S2 was 3877 nucleotides long encoding a 138kDa protein VP2, which is deduced as virus RNA polymerase. To understand the properties of its RNA polymerase, here we constructed 2 expression recombinants as pR/RRpN and pR/RRpC, that covered the gene sequences of N terminal and C terminal region of RNA polymerase. The 2 recombinants were demonstrated in frame expression by SDS-PAGE, and their molecular weight are about 98kDa and 103kDa, which were interest fusion proteins. It showed the fusion proteins were able to bind to rabbit serum anti GCRV-VP2 by using Western Blot analysis. In addition, 6XHis-tagged GCRV RNA polymerase products were purified by affinity chromatography and got around 90% purification of the interest proteins. This data provided the evidence for further GCRV RNA polymerase characterization.

关 键 词：草鱼呼肠孤病毒 草鱼 出血病 RNA聚合酶 基因表达 纯化 

分 类 号：S943[农业科学—水产养殖]