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Baculovirus has been widely used for production of recombinant proteins in insect cells. Previous studies found when a reporter gene under the control of a mammalian promoter was cloned into the baculovirus genome, it was expressed in hepatoma cells (Huh7 and HepG2), primary rat hepatocytes, epithelioid cell lines and rabbit intervertebral disc cells (Hela and Cos7) in vitro (2, 7, 14). Since these findings demonstrated that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded (23). Interestingly, although baculovirus can transduce mammalian cells very efficiently it only replicates in insect cells (10). Therefore, it has been suggested that baculovirus could be used as a gene therapy vector (11, 12).
Baculovirus p35 gene is from the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and is known to inhibit cell apoptosis in different organisms as diverse as Caenorhabditts elegans, Drosophila melanogaster and mammals, including humans (1, 4). Expression of p35 prevents apoptosis induced by various stimuli in carcinoma cells, neurons, oligdendrocytes, vascular smooth muscle cell and pancreatic beta-cell (8, 17, 19). Compared with cardiomyocytos from wild-type mice, cardiomyocytes from p35 expressing transgenic mice had longer lifetime under hypoxia conditions, and markedly reduced cytochrome C release, and caspase 3 acti-vation. Additionally, purified P35 has been found to inhibit all the mammalian caspases except caspases 5 and 9 (20). Based on previous studies, the molecular mechanism of inhibiting apoptosis is most likely the direct inactivation of caspase by P35 protein. Because of these characteristics, P35 is also commonly referred to as a pan-caspase inhibitor (21). In this study, we investigated whether the expression of p35 gene mediated by baculovirus vector can protect human embryo kidney 293 cells against apoptosis. The results demonstrated that baculovirus-mediated expression of p35 effectively protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV and serum-free media, and it could be widely used in molecular research and even gene therapy.
Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells*
- Received Date: 18 June 2007
- Accepted Date: 29 August 2007
Abstract: Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35 expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.