Expression Of Actin In Insect Cell And Function Of Actin In Replication And Transportation Of The Baculovirus

Actin is a high-conserved protein with a WB of 43KD,aniversally exists in eucaryotic cell. F-actin is polymerized by G-actin which participates the formation of the netlike protein system of the complex skeleton in the eucaryotic cell. F-actin is so important a structure that it not only provides the tension for the cell, determines the cell's shape ,but also supports the cell and arranges the space organization of organelles and some micromolecules. The study shows that F-actin skeleton system is responsible for the processes of infection, transportation, replication and assembling of the progeny virus.Based on the foundation research of the interaction of virus and host actin, we recombine GFP gene-a reporter gene-with 5c actin gene of Drosophila melanogaster , a GFP-actin fusion gene was obtained. Put this fusion gene under the control of the promoter of IE1 gene, and then an eucaryotic cell expression plasmid vector Pgem/IE1' -GFP-actin resulted. pGEM/IE1' -GFP-actin was transfected into Hz cells by lipofection. GFP expression was monitored using a fluorescence microscope. The result showed that the fusion gene was expressed at a low level. Hz cells which expressed GFP-actin fusion gene but were or were not infected by HaSNPV-Cl virus were checked respectively under the FACS. The test showed that there is no distinctive intense difference between them.IEl gene is a very early stage gene and it expresses at a low level itself. Which leads to the low expression of the GFP-actin fusion gene, and so the Hz cells exhibited a low intense of fluorescence.To solve this problem, we used bac-to-bac system to recombine GFP-actin fusion gene and GFP gene under the control of the promoter of polyhedrin gene respectively. Two recombinant virus: AcMNPV-GFP-actin which contains GFP-actin fusion gene and AcMNPV-GFP which contains GFP gene were obtained.The latter was set as a control. Sf9 cells was infected by these two recombinant virus. The expression of GFP-actin fusion gene and GFP gene were checked with the fluorescence microscope and through SDS-PAGE. The distribution of fluorescence in the progress of the infection was checked under the confocal scanning light microscope. Because actin and GFP co-localized, the distribution of fluorescence suggested that of actin. The results showed that 24hr after infection, most Sf9 cells exhibit relatively intensive fluorescence in the nuclear, but little was shown in the cytoplasm. In the control, the cells were full green fluorescent, and there is no intense difference between nuclear and cytoplasm. From 36hr to 72hr after infection, the fluorescence gathered gradually to the cytoplasmic membrane, and eventually there was no fluorescence remained in nuclear and cytoplasm, and the membrane accumulated high level of fluorescence, and then there was no change taken place from 72hr to 168hr.But from 36hr to 168hr ,there was no change took place in the control. This result suggests that actin may participate the transportation of the AcMNPV BV from nuclear to cytoplasm and getting out of the cell. Some more experiments are needed to do to support this result. From this experiment we get some realization about the function of acitn in the replication and transportation of the virus, and the contribution of actin in the cell that is infected by the virus. This will be beneficial to realize the mechanism of the interaction of virus and host actin in the infection.