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

  • Sun Jing-Chen (a1) (a2), Dai Wei-Jun (a1), Tan Yu-Rong (a1), Yang Yi-Feng (a1), Zhang Qin-Fen (a1), Xu Xing-Yao (a2) and Zhang Jing-Qiang (a1)...

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.

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*Corresponding author: Email: lsszhjq@sysu.edu.cn or Ls28@zsu.edu.cn

References

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