High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Lan-lan ZHANG; Jin-yu SHEN; Cheng-feng LEI; Xiao-ming LI; Qin FANG

doi:10.1007/s12250-008-2921-3

February 2008

Lan-lan ZHANG, Jin-yu SHEN, Cheng-feng LEI, Xiao-ming LI and Qin FANG. High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells[J]. Virologica Sinica, 2008, 23(1): 51-56. doi: 10.1007/s12250-008-2921-3

Citation: Lan-lan ZHANG, Jin-yu SHEN, Cheng-feng LEI, Xiao-ming LI, Qin FANG. High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells .VIROLOGICA SINICA, 2008, 23(1) : 51-56. http://dx.doi.org/10.1007/s12250-008-2921-3

草鱼呼肠孤病毒VP7蛋白在原核细胞中的高效表达

张岚岚 ^1,2 ,
沈锦玉 ³ ,
类承凤 ¹ ,
李小明 ⁴ ,
方勤 ^1,,

1.
中国科学院武汉病毒研究所, 病毒学国家重点实验室，湖北武汉 430071; 2中国科学院研究生院，北京 100039;3浙江省淡水水产研究所，浙江湖州 313001;4武汉大学基础医学院，湖北武汉 430071

通讯作者： 方勤, qfang@wh.iov.cn
收稿日期： 2007-11-07
录用日期： 2007-12-25

摘要

序列分析表明 GCRV S10 片段长为909核苷酸，编码一个分子量为34kDa 的外衣壳蛋白VP7。为获得体外表达的外衣壳蛋白，通过RT-PCR扩增，得到一条特异的、大小约0.9 kb的草鱼呼肠孤病毒外衣壳蛋白片段；将其扩增片段克隆于含T7启动子的高效原核表达系统pRSET载体质粒，转化BL21（DE3）lysS 感受态细胞，获得重组表达菌株（pR/GCRV－VP7）。酶切重组克隆子及序列分析实验证实，所得重组克隆为目的基因插入序列。pR/GCRV-VP7表达菌株经IPTG诱导培养，SDS-PAGE结果显示，表达产物的蛋白分子量约37kDa，为目的表达融合蛋白。高效表达的融合蛋白以包含体形式存在，目的蛋白的表达量占总菌体量的60%以上。Westerblot分析表明，该表达产物与鼠抗Histag与兔抗GCRV抗体呈阳性反应。该结果为GCRV－VP7结构与功能分析奠定了基础。
- 草鱼呼肠孤病毒
- , VP7蛋白
- , 原核表达

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

1.
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2.
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
3.
Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001
4.
Basic Medical School, Wuhan University, Wuhan 430072, China

Corresponding author: Qin FANG, qfang@wh.iov.cn
Received Date: 07 November 2007
Accepted Date: 25 December 2007

Fund Project: National Natural Science Foundation of China 30470074Innovation Project of the Chinese Academy of Sciences KSCX2-YW-N-021Science and technology foundation of Zhejiang Province 2007C22052National Natural Science Foundation of China 30671615

Abstract

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid，which was named as pR/GCRV-VP7，was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover，the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.
- Grass carp reovirus (GCRV)
- , VP7 protein
- , Prokaryotic expression

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