The Expression Analysis And Subcellular Localization Of BmNPV-pif-1

Bombyx mori nucleopolyhedrovirus (BmNPV) genome is 128 kb and predictive coding for 136 ORF. BmNPV ORF97 homologous with AcMNPV ORF119(Ac119) which is an important per os infectivity factors, named as pif-1. Researches have shown that the failure of infectivity of AcMNPV with PIF-1 knock out, but the infectivity of budded virus (BV) is not affected. PIF-1 is one membrane protein of ODV (occlusion derived virus). The main body of the BmNPV per os infection is ODV. So it is important to study the function and structure of PIF-1 on prevention and control the BmNPV in sericulture. And also can know the way of virus to through the midgut epithelium. Established the foundation for virus get through the membrance.In order to understand PIF-1 comprehensively of, our research including antibody preparation,5’RACE, sequence analysis, real-time PCR and cellular localization of protein.1) pif-1 sequence analysis:with the 5 ’RACE technology, we had cloned the 5’ upstream sequence (100 bp), in these sequence, a conservative sequences ATAAG of late virus promoter was found. In addition, PIF-1 protein contained two RGD motif. RGD motif can be mediated the target protein and protein aVβ3 combined and prompt virus integrated with the target cells.2)PIF-1 antibody preparation:we use the prokaryotic expression techniques to separate high purity PIF-1 protein successfully, which was appliedto immune the New Zealand white rabbit, higher specific antibodies was achieved that is the basis of protein interactions and protein localization study.3) The analysis of PIF-1 expression profile:The expression profile of PIF-1 by the q-PCR technology from the samples collected form different time points post infection. Results indicated that pif-1 can be detected from 14 hrs post infection and last to the late stage of infection, which indicated that pif-1 was a late factor, from.4) Subcellular localization of PIF-1:PIF-1 was fused to eGFP N terminal and was transient expressed in BmN cells by transfection, the fluoresence was observed by laser confocal microscope. The fused protein was expressedin cytoplasmic, however, it entry into nucleus with the help of BmNPV infection, in where PIF-1 was assembled into ODV membrane.On the basis ofthis research, we suggested that the PIF-1 protein in the process of peros infection may be mediated the complex of per os infection interaction with the integrinaVβ3, to make the ODV combination of intestinal epithelial cells, thereby ODV fusion with epithelial cell membrane.