草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

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，GCRVS2片段长为3877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。

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

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.