Hostname: page-component-cd9895bd7-dzt6s Total loading time: 0 Render date: 2024-12-28T07:00:24.425Z Has data issue: false hasContentIssue false
  • English
  • Français

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: lsszhjq@sysu.edu.cn or Ls28@zsu.edu.cn
Get access Rights & Permissions [Opens in a new window]

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ahlquist, P (2002) RNA-dependent RNA polymerases viruses and RNA silencing. Sciences 296: 1270–1273.CrossRefGoogle ScholarPubMed
Hagiwara, K and Matsumoto, T (2000) Nucleotide sequences of genome segments 6 and 7 of Bombyx mori cypovirus 1, encoding the viral structural proteins V4 and V5, respectively. Journal of General Virology 81(4): 1143–1147.Google Scholar
Hagiwara, K and Naitow, H (2003) Assembly into single-shelled virus-like particles by major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1. Journal of General Virology 84: 2439–2441.Google Scholar
Hagiwara, K, Tomita, M, Kobayashi, J, Miyajima, S and Yoshimura, T (1998a) Nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 8. Biochemical and Biophysical Research Communications 247(3): 549–553.Google Scholar
Hagiwara, K, Tomita, M, Nakai, K, Kobayashi, J, Miyajima, S and Yoshimura, T (1998b) Determination of the nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 9 and its expression in BmN4 cells. Journal of Virology 72(7): 5762–5768.Google Scholar
Hagiwara, K, Kobayashi, J, Tomita, M and Yoshimura, T (2001) Nucleotide sequence of genome segment 5 from Bombyx mori cypovirus 1. Archives of Virology 146(1): 181–187.CrossRefGoogle ScholarPubMed
Hagiwara, K, Rao, S, Scott, SW and Carner, GR (2002) Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively. Journal of General Virology 83: 1477–1482.Google Scholar
Lin, W, Xu, AL, Yang, WL, Sun, JC, Lu, XY and Zhang, JQ (2002) Sequencing of genomic dsRNA segment V of Bombyx mori cytoplasmic polyhedrosis virus. Chinese Journal of Viology 18(4): 375–377.Google Scholar
Lu, HS (1998) Molecular Biology of Insect Virus. Beijing: China Agricultural Scientech Press, pp. 284–289.Google Scholar
Mertens, PPC, Arella, M, Attoui, H, Belloncik, S, Bergoin, M, Boccardo, G et al. , (2000) Family Reoviridae. In: van Regenmortel, MHV, Fauquet, CM, Bishop, DHL et al. , (editors) Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press, pp. 395–480.Google Scholar
Pu, ZL (1994) Insect Pathology. Guangzhou: Guangdong Science & Technology Press, pp. 130–141.Google Scholar
Sambrook, J, Fritsch, EF and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.Google Scholar
Tan, PC, Xu, XY and Sun, JC (2001) General technique about inoculation and purification of silkworm pathogen. Guangdong Sericulture 35(2): 26–28.Google Scholar
Tan, YR, Sun, JC, Lu, XY, Su, DM and Zhang, JQ (2003) Entry of Bombyx mori cypovirus 1 into midgut cells in vivo. Journal of Electron Microscopy 52(5): 485–489.Google Scholar
Yu, XK, Lu, XY, Zhang, H et al. , (1999) Structure of cytoplasmic polyhedrosis virus from Bombyx mori. Acta Biochimica et Biophysica Sinica 31(5): 563–566.Google ScholarPubMed
Zhang, H, Zhang, JQ, Yu, XK et al. , (1999) Visualization of protein–RNA interactions in cytoplasmic polyhedrosis virus. Journal of Virology 73(2): 1624–1629.Google Scholar