| Acting during DNA replication and gene expression regulation, DNA helicases convert the duplex DNA to single strands and thereby activate the DNA for nascent strand synthesis. Helicases also play essential roles during DNA repair and recombination During infection baculovirus genes are expressed in a coordinately regulated cascade fashion in which each successive phase is dependent on the expression of genes during the previous phase. In this study, we examined the structure and function of the helicase gene promoter of BmNPV, the function of homologous region 3 (hr3) enhancer in BmNPV, and viral factors transactivating the helicase gene promoter. We also investigated the mechanism of host range determination between AcMNPV and BmNPV, and discussed the importance of baculovirus helicase involved in DNA replication, stimulating late gene transcription, closing host protein synthesis, and changing baculovirus host range.On the basis of studying the regulation of baculovirus early gene expression, we then successfully co-expressed chicken anaemia virus proteins VP1 and VP2 in silkworm using BmNPV expressing vector. This will provide an efficient method for development of CAV genetic engineering subunit vaccine. This paper mainly includes following parts:(1) Establishment of in vitro and in vivo transient expression system in insect cellsIn gene expression and regulation studies, Cationic liposomes have been commonly used to delivery functional genes into cells. Several cationic liposomes generally prepared by evaporation and sonication, are now commercially available;however, their large-scale use is confined for the great expense. So we prepared a cationic liposome reagent, composed of dirnethyldioctadecylammonium bromide (DDAB) and dioleoyl phosphatidylethanolamine (DOPE) (1:2 mol ratio). Then we investigated and determined the optimum conditions of this liposome mediated a reporter plasmid expressing luciferase into insect cell lines (Sf-21 and Bm-N) and silkworm larvae. Together the data demonstrated that Bomhyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA (128 kb) was successfully transfected into Bm-5 cells using this liposome These results suggest that DDAB/DOPH liposome will be useful as delivery agents for gene transfer to insect cells both in vitro and in vivo. It is significant to understand the function of the helicase gene promoter deeply.(2) Structure and function of BmNPV helicase gene promoterTo examine the expression and regulation of DNA helicase gene of BmNPV, the promoter of the helicase gene (BmNPV ZJ strain), including 510 bp upstream of ATG, was amplified by PCR. Sequence analysis showed that it was 99% and 95% identity, respectively, with the homologues in BmNPV T3 strain and AcMNPV This promoter has both early (TGTGC) and late (GTAAA) RNA initiation sites. It has a minicistron at about -70 bp upstream of ATG. It also has the TATA box at the upstream of transcriptional initiation sites. To detect the activity of the helicase gene promoter, the initiation codon ATG was deleted by using point mutation, and luciferase gene, as a reporter gene, was fused with the promoter region to construct the plasmid pBmhe15101uc. When pBmhe15101uc was transfected into Bm-N, Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and had the characteristics of early promoters. Deletion analysis of the helicase gene promoter by PCR showed that cis-acting elements were mainly present within 5' region of the promoter(3) Functional analysis of BmNPV homologous region 3 enhancerBaculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to augment the expression of a small number of baculovirus genes in vitro. In this study, BmNPV hr3 was cloned into upstream or downstream of he!510-reporter gene construct, to study the effect of this enhancer on hel510 promoter activity. The results showed that hr3 could significantly enhance the activity of he!510 promoter. The promoter activity was increa...
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