Promoter Analysis Of Baculovirus Ubiquitin And Polyhedrin Gene As Well As The Application Of BmNPV-Silkworm Expression System

We studied the characters of ubi promoter, the effect of IE1 and hr3 on late ubi and very late polh promoter activity, viral factors involved in late ubi gene expression as well as the expression of thermostable β-glucosidase from Pyrococcus furiosus.This paper mainly includes following parts:(1) Structure and functional element of AcMNPV and BmNPV ubi gene promoterSequence analysis of cloned ubi promoter showed that it was 99.5% and 98.9% identity to AcMNPV C6 and BmNPV T3 strain, respectively, and 92.8% of each other. Both of the promoter region have baculovirus late transcriptional initiation site, TAAG motif. They also have promoter conservative motif, CAAT and TATA box, respectively. Northern blot analysis testified that ubi gene is a late expression gene, it start its expression at about 12 hours post infection (h.p.i.) or earlier, and has two or three transcriptional product.Deletion analysis of ubi gene promoter region showed that cis-acting elements were mainly present within -595 to -382 bp upstream ATG of Acubi gene promoter and -382 to -124 bp upstream ATG of Bmwbi gene promoters. Interestingly, the region between -383 to -187 bp upstream ATG, which containing the distal TAAG, CAAT motif and TATA box, also can drive the reporter gene expression.Site mutation of TAAG, CAAT motif and TATA box showed that in the present of virus factors, both of the promoters were reduced obviously by TATA box and TAAG mutations, and increased remarkably by CAAT mutations.(2) Function of IE-1, hr3 on the late and very late gene promoters of baculovirusAbout ubi promoter, in the presence of BmNPV, hr3 could significantly enhance the activity of ubi promoter activity about 62- and 1.4-fold in cis- or in trans-, respectively. Co-transfected ie-1 and reporter plasmid, hr3 could enhance ubi promoter transcription about 210- in cis- or 9-fold in trans-. Co-transfected ie-1 and a series of deletion plasmid of ubi promoter. Transient expression result showed that cis-acting elements to IE-1 were mainly present above -595 to -382 bp of ATG in the promoter region.Toward very late polh promoter, in BmNPV-infected cells, hr3 could increase the expression of report gene about 283- in cis- and 1.8-fold in trans-. When co-transfected ie-1 and reporter plasmid, hr3 could increase polh promoter transcription about 621 -fold in cis-, and 4.2-fold in trans-, respectively.Hr3 also could enhanced ubi or polh promoter transcription in the presence of ie-I or BmNPV when transfected Bombyx mori larvae.(3) Study of virus factors which involving in the expression of baculovirus late gene ubiIn this study, we developed a method to identify baculovirus genes required for late gene expression that is based on accession of clones from a BmNPV genome DNA library. The results showed that some early genes, ieO, ie2, and pe38, et al. didn't activate the ubi promoter. A low level of luciferase activity was observed in cells when pBmubi595luc co-transfected with pIE-I plasmid only, implied there are other viral factors also participate in the transcription of ubi with the ie-1. Using pBmubi595luc, pIE-1 and BmNPV genome library co-transfected Bm cell lines in transient expression assay, and detected the reporter gene expression. Based on the result, nucleotide sequence of these plasmids, which can /raws-activated the transcription of ubiquitin promoter, was determined, several repeated gene in these fragments were subcloned to pGEM-3Z, then co-transfected with pBm595ubiluc and pIE-1. Subsequent subcloning and luciferase assays indicated that, in the present of IE-1, late expression factors LEF-6, LEF-11, HE65, P35 and other gene fragments, gpl 18-gpl 19 and gplO4 to gplO7, were required for the activation of baculovirus late ubi gene promoter.(4) Expression and purification of thermostable p-glucosidase in silkworm using baculovirus expression vector systemIn this study, we cloned thermostable p-glucosidase from Pyrococcus furiosus, and obtained the recombinant virus containing celB gene. The recombinant thermostable P -glucosidase was purified to above 80% homogeneity in a single heat-treatment step. The optimal activity of the expressed 3 -glucosidase was obtained at pH 5.0 and about 105 °C, divalent cations and high ionic strength did not affect on the activity remarkably. The enzymatic characteristics of recombinant p-glucosidase were similar to the native counterpart. The expressed p-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached to 10 199.5 U per ml haemolymph and 19 797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg.