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Journal of Clinical Microbiology, July 2005, p. 3172-3177, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3172-3177.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells

Majid Eshaghi,1 Wen Siang Tan,1,2 Swee Tin Ong,1 and Khatijah Yusoff1,2*

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,1 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia2

Received 1 October 2004/ Returned for modification 5 November 2004/ Accepted 7 February 2005

The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A260/A280 ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM 43400, Serdang, Selangor, Malaysia. Phone: 603 89466701. Fax: 603 89430913. E-mail: kyusoff{at}putra.upm.edu.my.

Journal of Clinical Microbiology, July 2005, p. 3172-3177, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3172-3177.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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