Identification Of BmNPV PE38and VLF-1Interactive Proteins Using Yeast Two-hybrid System

Bombyx mori nuclear polyhedrosis virus (BmNPV) is a kind of serious pathogens ofsilkworm which always cause lot of loss to sericulture. In this study, we constructed theexpression library of BmNPV infected silkworm with the yeast two-hybrid system, and thenscreened the library with the core gene of BmNPV. The results may contribute to theunderstanding of the relationship between baculovirus and its host.cDNA library from the BmNPV infected silkworm was constructed by CLONTECHSMART technology. Generally, silkworms on the third day of the fifth instar werechallenged with109cfu/ml BmNPV. Total RNA of midguts at12and96h p.i.was extracted respectively and the first chain of cDNA was transcripted with a modifiedCDSⅢ primer and its terminal was automatically extended with CCC. The second chainwas synthesized with the SMARTⅢ-modified Oligo primer by Long Distance-PCR. Theresulted PCR products were purified by CLONTECH CHROMA SPINTE-400column．Finally, the purified PCR products were co-transformed with the linearized plasmidpGADT7-Rec into the competent yeast Y187．The transformed yeasts grew in the SD/-Leuplates．All the growing clones were harvested and then constituted the cDNA1ibrary． Thetiter of the library was4.4×10~7cfu/ml．The inserted fragment size was ranged from0.75to2.0kb. The yeast two hybrid cDNA library was successfully constructed and could be usedfor screening proteins related to BmNPV infection.To screen proteins interacted with PE38and VLF-1proteins of BmNPV, the codingregion of pe38and vlf-1was cloned into yeast-expressing vector pGBKT7to construct thebait plasmid named as pGBKT7-pe38and pGBKT7-vlf-1.Then the plasmids weretransformed into yeast Y2Hgold as the bait yeast strain. fusion protein pGBKT7-pe38andpGBKT7-vlf-1expressing in as bait, screening cDNA library construction of BmNPVinfected silkworm, searching the proteins interacting with PE38and VLF-1protein,sequenceing and analyzing the positive clones by bioinformatic methods. Eight proteinsinteracting with PE38in cDNA library construction of BmNPV infected silkworm weresuccessfully screened which are Bombyx mori translation elongation factor2, Bombyx moriserine protease, Bombyx mori catalase, putative membrane protein precursor,chymotrypsin-like serine protease precursor, Bombyx mori actin muscle-type A1, Bombyxmori putative alcohol dehydrogenase, Bombyx mori vacular ATPase B subunit. But No interaction protein with VLF-1were found.