Improving Baculovirus-mediated Foreign Gene Expression Level

Baculovirus is a group of virus that only infects arthropod in nature. It is used mainly in three aspects: used as pesticide; to infect lavers or insect cells to express hetero-proteins; and to deliver genes into mammalian cells. The last two aspects have the common ground that the virus is used as a vector for foreign genes expression, either in insect's cells, or in mammalian cells. Improving the expression level of foreign genes will be important for both applications. In this work, two approaches were used to increase the expression level in insect cells and mammalian cells respectively.HS4, an insulator derived from chicken, has two functions: insulating the effects of the enhancer or other cis-regulate elements, and blocking the chromosome heterochromotization. In the current work, it was shown that HS4 could increase the expression of report gene by about 4 times when inserted downstream of the reporter gene expression cassette controlled by thebaculovirus polyhedrin promoter, comparing with that without the insulator. The transcription level of the reporter gene is also up-regulated.It is reported that the reason of low transduction efficiency of baculovirus in some mammalian cells is that the virus capsid failed to enter the nucleus, and will reside in the cytoplasm. An 11 aa-long peptide Tat, which was derived from HIV-1 transcript factor TAT, was displayed on the surface of virus capsid by fusing with viral capsid protein VP39, in the hope that Tat may facilitate the capsid to enter the nucleus and increase the expression level of foreign genes in mammalian cells. Meanwhile, to avoid the fake results that might associate with cell fixation process, enhanced green fluorescence protein (EGFP) was also fused with VP39 and displayed on the capsid, so as to trace the baculovirus in the living cells. It was found that the Tat-presenting virus had imporvedlevel of expression the reporter gene in transduced Vero cells and CHO cells by 60% and 37%, respectively. Meanwhile, the Tat-presenting virus also showed more cells with green fluorescence in the nucleus, which suggested that Tat may improve the efficiency of baculovirus-mediated gene delivery into mammalian cells.In general, two approaches were used to improve the level of foreign genes expression in insect cells and mammalian cells, respectively, by modified the baculovirus vector system. These two approaches may expand the application of the baculovirus system.