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International Journal of Virology

Year: 2005  |  Volume: 1  |  Issue: 1  |  Page No.: 5 - 5

Molecular Cloning and Expression of Nucleoprotein gene (NPs) of tomato spotted wilt virus in E.coli

E. T. Abdelsalam, Hala M. Habib, Hayam S. Abdelkader and Maha M. Al-Khazindar

Abstract

Tomato spotted wilt virus (TSWV) has become an economically important virus infecting major crop and causing losses in Egypt and worldwide. The nucleocapsid (NPs) protein gene was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from total nucleic acid extracts from TSWV-infected Physalis peruviana, and Datura metel. A fragment of the NPs gene of naturally occurring tomato spotted wilt virus (TSWV) isolate was amplified by RT-PCR using one set of primer TSWV1 (5`ATGTCTAAGGTTAAGCTC-3` and TSWV2 (5`-TTAAGCAAGTTCTGTGAG-3`). A successful PCR amplification of the virus-specific DNA fragment (777 bp) amplicon was cloned, and expressed into the pBAD-C expression vector in E.coli. The NPs fusion protein was induced using 0.002 % of the L-arabinose with a time course of induction for 4 hours time period. DBIA (Dot Blot Immuno Assay) of purified 6xHis-tag fusion nucleoprotein of TSWV strongly reacted with NINTA-AP conjugate after 15 minutes blocking in TBS-PVA. A strong reaction was also obtained in western immunoblots with the polyclonal antiserum prepared against the expressed nucleoprotein (NPs) gene.

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