Purification and characterization of Nipah virus nucleocapsid protein produced in insect cells.
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 A(260)/A(280) 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.
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM 43400, Serdang, Selangor, Malaysia.
Majid
M
Eshaghi
Wen Siang
WS
Tan
Swee Tin
ST
Ong
Khatijah
K
Yusoff
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D045464:Henipavirus Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D008854:Microscopy, Electron; D045405:Nipah Virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000821:virology; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology
D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins