tailieunhanh - Báo cáo y học: " Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus"

Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus | Chakrabarti et al. Virology Journal 2010 7 181 http content 7 1 181 VIROLOGY JOURNAL RESEARCH Open Access Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus Mrinmay Chakrabarti Suvankar Ghorai Saravana KK Mani Ananta K Ghosh Abstract Background Antheraea mylitta cytoplasmic polyhedrosis virus AmCPV a cypovirus of Reoviridae family infects Indian non-mulberry silkworm Antheraea mylitta and contains 11 segmented double stranded RNA S1-S11 in its genome. Some of its genome segments S2 and S6-S11 have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 S1 and 3 S3 of AmCPV were converted to cDNA cloned and sequenced. S1 consisted of 3852 nucleotides with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of 141 kDa. Similarly S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of 137 kDa. BLAST analysis showed 20-22 homology of S1 and S3 sequence with spike and capsid proteins respectively of other closely related cypoviruses like Bombyx mori CPV BmCPV Lymantria dispar CPV LdCPV and Dendrolimus punctatus CPV DpCPV . The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins respectively in Escherichia coli M15 cells via pQE-30 vector purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles VLPs by transmission electron microscopy. .

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