In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.

2014-08-01·Current Microbiology4区 · 生物学

Bombyx mori Nucleopolyhedrovirus ORF101 Encodes a Budded Virus Envelope Associated Protein

4区 · 生物学

Article

作者: Guoping Huang ; Yajing Zhou ; Weijun Mai ; Huiqing Chen ; Mei Li ; Keping Chen

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

2003-09-01·Toxicon3区 · 医学

Novel neurotoxins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization

3区 · 医学

Article

作者: Long-Sen Chang ; Jau-Cheng Liou ; Charling Chung ; Chia-Wei Chang ; Chen-Chung Yang

Two novel neurotoxins BM10-1 and BM10-2 were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combinations of chromatography on a SP-Sephadex C-25 column and a reverse phase HPLC column. BM10-1 contained 66 amino acid residues including 10 Cys residues, while BM10-2 consisted of 65 amino acid residues with 8 Cys residues. The secondary structure of both BM10-1 and BM10-2 was dominated with beta-sheet, but their gross conformation differed as evidenced by CD spectra and acrylamide quenching studies. BM10-1 inhibited carbachol-induced muscle contraction in a reversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. BM10-2 exhibited an irreversible but weak inhibition on carbachol-induced muscle contraction. Sequence alignment of neurotoxins with BM10-1 and BM10-2 suggested that the manner in the manifestation of their activity could be partly elucidated by the residues at toxin second loop. The genomic DNAs encoding BM10-1 and BM10-1-like protein (BM10-1L) were amplified by PCR. The two genes shared virtually identical structural organization and high degree of sequence identity with B. multicinctus neurotoxin genes. Compared to intron sequences of these genes, the protein-coding regions were highly variable. The difference between BM10-1 gene and BM10-1L gene notably arose from the third exon. These results suggest the evolution of B. multicinctus neurotoxins via the path of gene duplication.

100 项与 BM-101 相关的药物交易

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