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Molecular characterization of recombinant nucleocapsid protein of Peanut bud necrosis virus (To-K) isolate

Bhat, Amruta S and Bhushan, L and Narayanan, Srisathya D and Rathi, S and Savithri, HS (2008) Molecular characterization of recombinant nucleocapsid protein of Peanut bud necrosis virus (To-K) isolate. In: Indian Journal Of Virology, 19 (1). pp. 107-108.

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Peanut bud necrosis virus (PBNV) belonging to the genus Tospovirus, infects several economically important crops all over India. The NP gene of PBNV-tomato isolate from Karnataka was over expressed in E.coli and was purified by Ni-NTA chromatography. Different N- and C-terminal deletion clones, DN20NP, DN40NP and DC15NP, and double mutants -DN20DC15NP and DN40DC15NP were generated. Except DN40DC15NP, all the proteins were soluble and could be purified. The Far UV CD spectrum of NP was typical of globular proteins. The secondary structure predictions showed that it is indeed predominantly an alpha helical protein. Deletion of C-terminal 15 amino acids did not affect the secondary structure of the protein.Other mutant proteins were slightly different from wild type. Tm studies (melting temperature) have shown that mutant DC15NP was as stable as the wild type. Whereas, DN20NP, DN40NP were not as stable. The proteins were present as nucleoprotein complexes and the bound nucleic acid was shown to be RNA. Slot blot analysis indicated that NP specifically binds to ssRNA but not ssDNA and the binding is protein concentration dependent. The western blot analysis was carried out using monoclonal antibodies (MAbs) specific to NP. Among the 9 MAbs tested, 5 reacted with all the mutants. Mab H7A3 and A4F12 could recognize only the wild type. Epitope for F7C8 lies within the C-terminal 15 amino acids, and that for B8D12 is between N-terminal residues 20 to 40. Further epitope analysis of the MAbs is in progress.

Item Type: Journal Article
Publication: Indian Journal Of Virology
Publisher: Indian Virological Society
Additional Information: Copyright of this article belongs to Indian Virological Society.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 04 Nov 2009 08:23
Last Modified: 04 Nov 2009 08:23
URI: http://eprints.iisc.ac.in/id/eprint/17839

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