| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 91 === Abstract
Baculovirus expression vector system (BEVS) has many advantages and part of its ability to perform mass production of foreign proteins lies in the efficient transcriptional machinery equipped with unique enhancer, homologous region (hr). Identification of other enhancers will thus benefit BEVS. In this study, a reporter gene, luciferase, driven by CMV minimal promoter was randomly inserted into BmNPV genome by nonhomologous recombination to probe other possible enhancers. After screening for luciferase activity, 12 recombinant baculoviruses exhibiting high luciferase activity were isolated and found to be inserted into the same locus after sequence analysis. The location of viral genome integration was determined using genome walking, and 5 kb upstream and downstream sequences were cloned. After transfecting plasmids containing upstream fragment, pBS8ucmL, into Bombyx mori BmN cells, following by infecting with BmNPV, up to 100-fold increase of luciferase activity was observed; whereas the other fragment containing downstream sequence led to a 20-fold enhancement. This result indicated the lateral fragments, particularly the upstream sequence, could serve as an strong enhancer. However, such enhancerment only occurs upon infection with virus, which, in turn, suggesting that virus provide a factor in trans for the activation of the promoter. Furthermore, in an attempt to stimulate the CMV minimal promoter for high level foreign proteins production in insect cells, ie1, a well-known transcriptional activator in baculovirus, was tested. When pBS8ucmL and ie1 were co-transfected into cells following by viral infection, a 1,600-fold activation was achieved. These results indicated novel ways for high level protein expression in BmNPV had been found for better use of baculovirus in the future.
|
|---|
