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Cloning, expression and location of the RNA-dependent RNA polymerase gene from Bombyx mori cypovirus 1

Published online by Cambridge University Press:  12 February 2007

Sun Jing-Chen ,
Dai Wei-Jun ,
Tan Yu-Rong ,
Yang Yi-Feng ,
Zhang Qin-Fen ,
Xu Xing-Yao  and
Zhang Jing-Qiang
Sun Jing-Chen
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China College of Animal Science, South China Agricultural University, Guangzhou 510642, China
Dai Wei-Jun
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Tan Yu-Rong
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Yang Yi-Feng
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Zhang Qin-Fen
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Xu Xing-Yao
Affiliation:
College of Animal Science, South China Agricultural University, Guangzhou 510642, China
Zhang Jing-Qiang*
Affiliation:
State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
*
*Corresponding author: Email: [email protected] or [email protected]
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Abstract

The RNA-dependent RNA polymerase (RdRp) gene of Bombyx mori cypovirus 1 (BmCPV-1) (China strain) was cloned in three fragments using step-by-step reverse transcriptase-polymerase chain reaction (RT-PCR). The whole RdRp gene of 3691 bp was sequenced (GenBank accession number: AY496445). Vector pET-28b(+) was used to construct the expression vector pET28b–RdRp which was expressed in Escherichia coli BL21 (DE3) induced with isopropyl β-d-thiogalactoside (IPTG). The molecular weight of the expression product was about 138 Da. The primary antibody employed was rabbit antibody against BmCPV–RdRp recombinant protein and the secondary antibody was 15 nm immunogold-labelled goat anti-rabbit IgG. Immunogold was mostly bound to the virions which were dispersed in a virus generation matrix and polyhedron in the columnar cells of the midgut of the silkworms, and the average marking ratio was about 35%. This result demonstrated that BmCPV–RdRp complexes are indeed located at the BmCPV capsid.

Keywords

Bombyx mori cypovirus 1expressionimmuno-electron microscopyRNA-dependent RNA polymerase gene
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 2 , Issue 3 , December 2005 , pp. 213 - 218
Copyright
Copyright © China Agricultural University and Cambridge University Press 2005

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