Characterization Of Bm134, Bm51 From Bombyx Mori Nucleopolyhedrovirus (BmNPV)

The family Baculoviridae is a highly selective pathogen in arthropods, mainly in insects of the order Lepidoptera.Recently,the baculovirus have been successfully developed as expression vector system,recombinant bio-insecticide,gene transfer vector and surface display vector,providing huge benefits to nature science development.So far,the genomes of 29 baculoviruses,including 22 nucleopolyhedroviruses and 7 granuloviruses, have been sequenced.From the known genes,some are involved in the compositon of BV or ODV,some are involved in the virus replication, transcription and structural assemblage,and several other genes whose function are unknown.In this study,two baculovirus genes,Bombyx mori nucleopolyhedrovirus(BmNPV) ORF134,ORF51 were characterized.The gene transcription,gene expression,sub-cellular localization,virus structural location and gene knock-out were adopted to characterize these two genes.The object of this study is to provide theoretical basis and enhance our understanding of baculovirus molecular biology.The results were as following:1.The Bm134 is located at 127342-127671nt in the genome of BmNPV T3 strain.It contains 330 bp and is predicted to encode a 109 amino acid peptide with a deduced molecular weight of 12.4 kDa.A baculovirus consensus late transcriptional start motif GTAAG is found at 43nt upstream of the start codon ATG;Multiple sequence alignment showed that the deduced amino acid sequence of Bm134 shares 93% identity to ORF5 of AcMNPV and shared high level identity to the other 12 baculoviruses.The Bm51 is located at 45935-46402nt in the genome of BmNPV T3 strain.It contains 468 bp and is predicted to encode a 155 amino acid peptide with a deduced molecular weight of 18.5 kDa.A baculovirus early consensus transcriptional start motif(CAGT) is 26 nt upstream of the start codon,and a typical polyadenylation signal (AATAAA)is 454 nt downstream of the translation stop codon TAA.2.Two primers were designed as the BmORF134 gene sequence and used for PCR amplification.PCR products were cloned into pET30a(+) vector and BmORF134 were expressed as fusion protein.The purified fusion proteins were injected into New Zealand white rabbits to harvest polyclonal antibodies.Western blot analysis of extracts from BmNPV-infected BraN cells revealed a specific polypeptide with an apparent size of 12.4 kDa when using the BmORF134 antibody.RT-PCR results showed that BmORF134 genes transcribed in the infected cells from 12 h post infection(p.i.) to 72 h p.i.Western blot assay of occlusion-derived virus(ODV) and budded virus(BV) preparations revealed that Bm134 encodes a 12.4-kDa structural protein that is associated with ODV,consistent with predicted molecular weight. Immunofluorescence analysis showed that the Bm134 product was first detected in the cytoplasm at 24 h p.i and also located in nucleus during later infection.Using co-immunoprecipitation and peptide mass fingerprinting with MALDI-TOF,it was found that BmORF134 interacted with host protein X(unidentified).3.Special primers was designed to amply the Bm51 by PCR and expressed in E.coli as a fusion protein with the pET30a(+) vector.The result showed that Bm51 could be efficiently expressed in E.coli with a 24.5kD which is the together with of 18.5 kDa of Bm51 and 6 kDa of His-tag.Western blotting proved that the recombination protein expressed in E.coli BL21(DE3) is fused protein of His- Bm51.The 6×His-tagged Bm51 protein was purified and used to raise polyclonal antibodies in rabbits.The transcription and protein product of early viral gene Bm51 can be detected at 6 h post-infection(p.i.) in resistant strain NB by QRT-PCR and Western blotting,and the expression of Bm51 in NB reached maximal levels at 36 h post-infection in NB,and then gradually decreased to undetectable in 72 h.In contrast,the Bm51 protein was undetectable until 12 h post-infection in susceptible strain 306,and the expression of Bm51 progressively increased during 72 h post-infection. The results indicate that some host factors in BmNPV resistant strain inhibited the expression of viral Bm51.4.Bm134 was rapidly disrupted in E.coli DH10B by Red recombinant system and a Cm gene was inserted into the same site.BmNPV which disrupted Bm134 was rescued by the BmNPV/Bac to Bac system and Red system,vBm134-Wt,vBm134-Ko and vBm134-Repair viruses were constructed respectively.The gfp and polh genes were transposed into the recombinant bacmid that was used to infect BmN cells.Green fluorescence could be observed in BmN cells transfected by recombinant bacmid and the cells with green fluorescence scattered equally in the vision,which indicated that knockout of Bm134 have a little effect on the replication of virus.