Genome-wide Scanning For Baculoviral Factor Of Cis-acting Element And Applications Of Baculovirus Expression System

A genomic library of BmNPV was constructed by "partial filling-in" method. The genomic DNA of wildtype BmNPV was partially digested by Sau3A I, and the plasmid vector pUC19 was fully digested by Sal I , and subsequently filled in by the Klenow fragment. After ligation and transformation, the BmNPV genomic library with the representation of more than 99.9% was obtained.The viral factor involved in transactivation of the early helicase promoter was identified by co-transfection using the generated BmNPV genomic library. All of the screened plasmids contained intact ie-\ coding region. The ie-\ gene was cloned by PCR to further confirm that only IE-1 but not other factors encoded by flanking sequences functioned as transactivator. Other major baculovirus early genes exhibited no transactivation to the helicase promoter. Transient expression also revealed that IE-1 could give strong transactivation to other promoters from various origins.A plasmid hr3 enhancer failed to stimulate the expression of another plasmid containing luc gene under control of a promoter, but strong stimulation occurred when cells were infected by BmNPV. Co-transfections of hr3 enhancer, report plasmid, which containing luciferase gene driven by BmNPV helicase promoter, and the BmNPV genomic library revealed that IE-1, the essential viral factor, contributed to the hr3 enhancer function in trans. Further investigations proved that IE-1 could mediate hr3 enhancer function in trans as a broad-spectrum protein bridge to stimulate promoters from various origins. The stimulating effects ranged from 40 to more than 100 folds. Functional dissections of the hr3 enhancer clarified that the 30-bp imperfect palindrome was essential for the enhancer function in trans.During the screening of BmNPV transactivator for the helicase promoter, a relatively small library DNA was found to transactivate the target promoter distinctly but more slightly than the screened viral factor IE-1. No coding region of any viral early factor but the 5' non-coding region until 19 bp upstream of ie-1 gene ATG was found by sequencing analyses. Further transient assays confirmed that the 378 bp ie-\ promoter can stimulate transcription of promoters 40-100 folds via co-transfection. Functional dissection analyses of four overlapping sub-fragments and site-directed mutagenesis revealed that the transactivation was not contributed by the potential viral factor. To further confirm the suppose that it is an enhancer acting in trans via a cellular factor, a 238 bp fragment was inserted into reporter plasmids to verify if this fragment could stimulate transcription from a linked promoter in a distance- and orientation-independent manner. After transfection, the fragment was proved to have prominent stimulation effects of more than 10,000 folds and meet all the classical definitions of an enhancer. This non-homologous region enhancer (NHE) was inserted into reporter plasmids (downstream of luc gene) containing baculovirus late promoter and mammalian virus originated CMV promoter and exhibited stimulation on both these two promoters. The NHE was subsequently proved to be function simultaneously with the homologous region enhancer hr3.In the transfer vector BacPAK8, using ie-\ promoter substituted for polh promoter, the opd gene was inserted into the transfer vector under the control of ie-\ promoter. The homologous region enhancer hr3 was cloned downstream of the opd gene to construct a new expression cassette for the expression via BEVS. After co-transfection with linearized parent baculovirus DNA the recombinant baculovirus was obtained and further plaque screened. The results revealed that this expression cassette had advantages in improving expression level.The PfuPol gene was cloned into the baculovirai transfer vector pVL1393 under the control of the polyhedrin promoter. After co-transfection with Bsu36 I linearized BmBacPAK6 genomic DNA, the recombinant baculovirus harboring PfuPol gene was obtained and plaque screening was further conducted. The recombinant virus was used to infect silkworm larvae. SDS-PAGE analysis revealed that the recombinant enzyme was approximately 90 kDa in size and could be purified in a single heat-treatment step. The heat-treated enzyme was sufficient and retained good performance for amplification in PCR directly. The Pfu DNA polymerase was overexpressed up to 2X105 U per ml hemolymph.