The Expression Of Bombyx Mori Densovirus (BmDNV-1) Structure Genes And The Analysis Of Its Promoter Activity In Cultured Insect Cells

Bombyx mori densovirus (BmDNV-1) belongs to the Iteravirus genus within the Densovirinae subfamily of Parvoviridae family. The BmDNV-1 isolated from silkworms with flacherie disease in Ina City of Japan in 1968 mainly infects the columnar cells of midgut epithelium of the silkworm, and the infected cells in the diseased silkworms showing flacherie symptom include hypertrophy of infected nuclei. Therefore, the BmDNV-1 poses a major threat to silk industry.BmDNV-1 is about 20nm in diameter, the particle comprises of icosahedral capsid containing a single-stranded linear DNA genome of 5076kb. The genome has two large overlapping genes, the 5'-portion encodes the non-structural protein NS1 (89.3KDa) and NS2(56KDa), whereas the 3'-portion of the genome has a single open reading frame that codes for four structural capsid proteins, VP1 (74.9KDa), VP2 (64.3 KDa), VP3 (54.9 KDa), VP4 (51.6 KDa). The N-terminal part of the largest capsid protein VP1 called VP1-unique part (VPlu) has been demonstrated to exhibit a PLA2 activity that is required for successful infection. Analysis of the genome organization indicated that there are two promoters (promoters of structural gene and nonstructural gene) in the BmDNV-1's genome which are located in the 5'end and the middle of the viral DNA coding strain, respectively. To date, the research of BmDNV-1 is mainly focused on organization and expression of their genomes, little is known about functions and regulations of viral proteins as well as the transcription and translation strategy of BmDNV-1 genome due to lack of sensitive cell lines. The transcription and translation strategy of BmDNV-1 has not been reported.In present study, the vp1-u was amplified by primers by using the sequence of BmDNV-1's infectious clone PIN919 (GeneBank accession number:AY033435) as a tempelate, and the prokaryotic expression recombinant plasmids pET-28a-vplu and pMal-c2X-vp1u were constructed by inserting the vp1u into the vectors pET-28a and pMal-c2X, respectively. Then the VP1u protein was expressed and purified, and antibody preparation is underway. At the same time, we have successfully expressed the structural protien VP1 by the Bac-to-Bac baculovirus expression system to study the relationship between VP1 with VP1-u. Our data showed that vp1 was not truncated to form VP1-u protein in eukaryotic expression system. In this study, we also analyzed the structural gene (VP) promoter's activity utilizing the Luciferase Assay System in which the VP promoter was fused with a luciferase reporter gene. The enzymatic activity assay showed that VP promoter exhibited high activity in Ld652, Sf9 cell lines, detectable in C636 cell lines and no activity in Bmn (Bombyx mori) cell line. And the promoter activity of the VP gene was negatively regulated by co-transfection of the pIZT/V5-His-NS1/pIZT/ V5-His-NS2/pIZT/V5-His-NS constructs. The further study is needed to define function of BmDNV-1-VP1 and the transcription and translation mechanisms of this virus.