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

FANG Qin; DING Qing—quan; WAN G Ya-ping; ZHU Zuo—yan

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April 2003

FANG Qin, DING Qing—quan, WAN G Ya-ping and ZHU Zuo—yan. Expression of Grass carp reovirus RNA Polymerase Gene and Purification of its Product[J]. Virologica Sinica, 2003, 18(2): 169-173.

Citation: FANG Qin, DING Qing—quan, WAN G Ya-ping, ZHU Zuo—yan. Expression of Grass carp reovirus RNA Polymerase Gene and Purification of its Product .VIROLOGICA SINICA, 2003, 18(2) : 169-173.

草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

1.
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

摘要

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。Westernblot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。
- 草鱼呼肠孤病毒
- , 双链RNA病毒
- , RNA聚合酶
- , 基因表达与纯化

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

1.
Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071，China
2.
State Key Laboratory of Freshwater Ecology and Biotechnology,Institute ofHydrobiology,Chinese Academy ofSciences,Wuhan 430072，China

Abstract

Abstract：Grass carp reovirus(GCRV)is a disaster agent to aquatic animals，which belongs to genus Aquareovirus．family Reoviridae．Sequences an alysis revealed GCRV S2 was 3877 nucleotides long encoding a 1 38kDa protein VP2，which is deduced as virus RNA polymerase．To understan d the properties of its RN A polym erase，here we constructed 2 expression recombinan ts as pR／RRpN an d pR／RRpC，that covered the gene sequences of N terminal an d C terminal region of RN A polymerase． The 2 recombinan ts were demonstrated in frame expression by SDS—PAGE，an d their molecular weight are about 98kDa an d 103kDa，which were interest fusion proteins．It showed the fusion proteins were able to bind to rabbit serum an ti GCRV—VP2 by using W estern Blot an alysis．In addition，6XHis—tagged GCRV RN A polymerase products were purified by afinity chromatography an d got around 90％ purification of the interest proteins．Th is data provided the evidence for further GCRV RN A polymerase characterization．
- Key Wards：Grass carp reovirus(GCRV)
- , dsRNA
- , RNA polymerase
- , Gene expression and purification

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

1. Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071，China
2. State Key Laboratory of Freshwater Ecology and Biotechnology,Institute ofHydrobiology,Chinese Academy ofSciences,Wuhan 430072，China

Abstract: Abstract：Grass carp reovirus(GCRV)is a disaster agent to aquatic animals，which belongs to genus Aquareovirus．family Reoviridae．Sequences an alysis revealed GCRV S2 was 3877 nucleotides long encoding a 1 38kDa protein VP2，which is deduced as virus RNA polymerase．To understan d the properties of its RN A polym erase，here we constructed 2 expression recombinan ts as pR／RRpN an d pR／RRpC，that covered the gene sequences of N terminal an d C terminal region of RN A polymerase． The 2 recombinan ts were demonstrated in frame expression by SDS—PAGE，an d their molecular weight are about 98kDa an d 103kDa，which were interest fusion proteins．It showed the fusion proteins were able to bind to rabbit serum an ti GCRV—VP2 by using W estern Blot an alysis．In addition，6XHis—tagged GCRV RN A polymerase products were purified by afinity chromatography an d got around 90％ purification of the interest proteins．Th is data provided the evidence for further GCRV RN A polymerase characterization．