Stduy On The Polyhedrin Gene(polh) Promoter Binding Proteins Of Bombyx Mori Nucleopolyhedro Virus

Polyhedrin promoter is widely used in the baculovirus system, which can improve exogenous gene expression, but the high-level transcription mechanism is still unknown. In this study, yeast one hybrid system was used to screen the transcriptional factors which can interact with polyhedron promoter.In order to screen the transcriptional factors, a c DNA library was constructed using the silkworm which infected with Bm NPV. Finally we find 18 proteins which can bind with polyhedron promoter. Furthermore, lef5, orf61 and p6.9 genes were selected for further studies.In addition, electrophoretic mobility shift assay(EMSA) was used to verify the biological activities of these proteins in vitro. The results showed that only Orf61 can bind with the promoter sequence. In order to further verify the regulation function, constructed transient expression vectors were transfected into Bm N cells. Then the cells were infected with the recombinant virus of Bacmid-Luc, 24 hours post infection. The results showed that lef5, orf61 and p6.9 could up-regulate the transcription level of luciferase gene. Constructed lef5, orf61 or p6.9 gene deletion Bacmids were called lef5-ko-Bacmid, orf61-ko-Bacmidand and p6.9-ko-Bacmid. Repaired Bacmids, named lef5-re-Bacmid, orf61-re-Bacmid and p6.9-reBacmid. TCID50 detection results showed that the titers of lef5-ko, orf61-ko and p6.9-ko virus were zero; however, the TCID50 of lef5-repaired, orf61-repaired and p6.9-repaired showed no obviously difference when compare with wild type virus(p>0.05), these results indicate that cells transfected with lef5-ko-Bacmid, orf61-ko- Bacmid or p6.9-ko-Bacmid would lead to fail assembling infectious virus particles. Moreover the deletion of orf61 would affect virus genome replication obviously(p<0.05), there were no obvious effects of lef5 and p6.9 deletion(p>0.05),indicating that orf61 is necessary for virus genome replication. Then q-PCR was used to analyzed the transcriptional level of virus early gene, late gene, very late gene and replication level of virus genome DNA in Bm N cells transfected with all the Bacmids, respectively. The results demonstrated that the knock-out of lef5, orf61 or p6.9 would lead to the replication level clearly lower and the transcriptional level of early gene lef3, late gene vp39 and very late gene p10 were lower, too(p<0.05).In conclusion, by using the yeast one-hybrid system, we successfully screened 17 polyhedrin promoter binding proteins. Our preliminary studies of lef5, orf61 and p6.9 geneswill lay a foundation for further understanding the polh promoter transcriptional mechanism and the construction of new type baculovirus vector.