A Preliminary Research Of Molecular Mechanisms On BmCPV And BmNPV

Bombyx mori Cytoplasmic polyhedrosis virus(Bm CPV) and Bombyx mori nuclearpolyhedrosis virus(Bm NPV) are two kinds of virus that can infect silkworm in seri culture industry. This research conducted further research on the molecular mecha nisms of cell invasion based on existing research of Bm CPV. Through bioinform atics analysis software Bm CPV 9 candidate proteins for functional analysis, 9 kin ds of candidate receptor proteins were initially screened: CRT, CLAT, ITGB, BM KT, IPOA, AP, RACK, Gcy, OPB. Insight study into the biological function and the distribution of candidate receptors, through function of protein biological rol e, study of viral infection in silkworm using possible invasive way. In order to f urther identify candidate receptor protein, prokaryotic expression recombinant vect or of CAT, IPOA, Gcy, OPB and BMKETTIN were, and the polyconal antibody were prepared using inducible expressed recombinant protein. Antibody blocking experiments conducted to explore suppressed expression of candidate receptor fun ction Bm CPV proliferation of polyclonal antibody preparation.Bioinformatics analysis showed, CRT has three functional domains, namely a signal peptide, calreticulin and coiled-coil region, Calreticulin family major role to play in conjunction with the ER. CLAT mainly composition by clathrin and clathrin heavy chain region homologous repeat regions, CLAT is a cytoplasmic protein clathrin,gathered in intracellular membranes to form a lattice polyhedron protein coated membrane channels. ITGB contains integrin β subunit major, RPT, transmembrane and integrin-b-cyt domain, which mediates the interaction between cells and the extracellular matrix, cells and between cells. BMKETTIN mainly have IGc-2 and IG domains. IPOA have eight ARM repeat domain, which mediates β-catenin interaction with its ligand. There are adapter proteins AP-A domain and α-C2 domain adapter proteins, which may interact with the cytoplasmic tail membrane protein. RACK constructed by seven WD40 domains, and protein binding associated. Gcy mainly byANF binding domain, a transmembrane domain, a tyrosine kinase domain and adenylyl/guanylyl cyclase catalytic domains, the main function of intracellular signal transduction. OPB has a signal peptide, RPT zone, zone type IG, IG and transmembrane domains, OPB associated with cell recognition.The results of antibody blocking experiments showed that compared to the control group, both 100 μg/m L and 200 μg/m L concentrations of anti-candidate receptor antibody could significantly reduced relative expression level of Bm CPV S1 gene in Bm N cells. These results further confirmed the possibility of the four candidate receptor as members of Bm CPV receptor complex.Based on a series of studies Bm CPV, interaction relationship between Bm NPV and protein in cell also cause us concern. Through literature review, we guess Bm NPV interacting proteins and cells may be closely associated with lipid rafts.A virus overlay protein blot assay was used to screen and identify the interacted proteins from the lipid rafts. 65 proteins were identified with mass spectrometry and six proteins were predicted as binding proteins for the Bm NPV cellular invasion, including actin, kinesin light chain-like isoform X2, E3 SUMO-protein ligase PIAS3-like isoform X3, SKI family transcriptional corepressor 2-like, annexin B13, heat shock protein 90,barrier-to-autointegration factor B-like. Interestingly, the depletion of cholesterol with methyl-β-cyclodextrin can block the Bm NPV entry into Bm N cells, but supply cholesterol can rescue the Bm NPV infection. The results from this study will provide the scientific basis for the lipid raft regarding on cellular invasion. Further, it also can provide the targets for improving the resistance of silkworm to Bm NPV and therapeutic drug research and development for polyhedrosis of silkworm.The results provide new clues to a deep understanding of Bm CPV and Bm NPV invasion molecular mechanisms.