Purification and characterization of Nipah virus nucleocapsid protein produced in insect cells

Abstract

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.

FIG. 1.

Strategy for amplification and cloning of the NiV N gene. Extracted viral RNA (vRNA) (using TRIZOL LS reagent; Life Technologies) was used as a template for cDNA synthesis using the Superscript II RNaseH (−) reverse transcriptase (RT; Life Technologies), which was subsequently used in a PCR amplification using the Platinum high-fidelity Taq DNA polymerase (Life Technologies). rTEV, recombinant tobacco etchvirus protease cleavage site.

FIG. 2.

The NiV N protein expressed by the recombinant baculovirus in insect cells. SDS-PAGE (A) and Western blot (B) analysis of Sf9 cells infected with wild-type baculovirus (lanes 1), noninfected Sf9 cells (lanes 2), and Sf9 cells infected with recombinant baculovirus (lanes 3). Arrows indicate the expected bands. Lanes M, molecular mass markers.

FIG. 3.

Characterization of purified NiV N protein expressed in insect cells (lanes 1) by SDS-PAGE (A) and Western blot analysis (B). Lanes M, molecular mass markers.

FIG. 4.

Transmission electron micrographs showing formation of full and empty spherical structures (A) and ring- and herringbone-like structures (B) in purified NiV His-N protein. Bars, 100 nm (A) and 50 nm (B).

FIG. 5.

Comparative analysis of reactivity of NiV-specific IgG (A and C) and IgM (B and D) antibodies to recombinant N protein (A and B) and inactivated NiV (C and D) in an indirect ELISA. The error bars represent standard deviations from the means of the results from triplicate determinations.