| Background and Objective1. Tetherin is a human cellular lipid raft associated transmembrane protein with specific topological structure. In 2008, it was found that Tetherin can inhibit retrovirus infection by preventing the diffusion of virus particles after budding from infected cells, showing the potential inhibition to various enveloped viruses. Influenza viruses are single-stranded RNA viruses with envelope, quite similar to HIV, Ebola and other enveloped viruses. In this study, we cloned and expressed innate immune molecule—human Tetherin and examined its effect on influenza virus multiplication2. Without mitotic checkpoint key protein Zwint-1, mitosis won't stop but lead to early separation of chromosomes as well as aneuploid cancer. In previous study, new Zwint-1 variant was found. In this study, using Cell Cycle Synchronization Technology to induce HeLa cells into interphase and mitotic phase, we analyzed sub-localization and changes of Zwint-1 wild type and Zwint-1v along with cell circle.Material and methods1. Tetherin RNA was extracted from human peripheral blood monouclear cells, while using RT-PCR its gene were cloned and then constructed with eukaryotic expression vector.2. Using Liposome transfection method, Tetherin was transfected to MDCK cells and using indirect immunofluorescence, Tetherin protein was expressed and cellular located.3. Using Liposome transfection method and G418 selection, Tetherin protein and empty vector were stable expressed in MDCK cells. Using CCK-8 to detect the cellular proliferation curve during post infection hours, cells resistant to influenza virus were analyzed.4. Plaque assay, Hemagglutination test and Real time PCR were detected to disclose influenza virus PR8 multiplication both in cells with stable transfection Tetherin and empty vector.5. HeLa cells were synchronized using Thymidine double-block method. The sub-localization of Zwint-1 wild type and variant were observed by fluorescence microscope after liposome transfection, indirect immunofluorescence and nucleus dyeing. Result1. The cloned gene human Tetherin was shown consistent with the GenBank sequence by endonuclease digestion and DNA sequencing results. Immunofluorescence results showed that the recombinant protein was mainly observed in the cell membrane.2. According to indirect immunofluorescence and flow cytometry stable transfection with empty vector and Tetherin MDCK cell lines were successfully constructed.3. After PR8 infection, cell viability of stable transfection Tetherin was higher than stable empty vactor transfection. In statistical analysis, at 36h post infection, difference had significant.4. Hemagglutination plaque and experiment results show that during different post infection time points, there were no differences between stable transfection Tetherin cells and stable transfection vector cells.5. Real time PCR results showed that at 24h, 36h the virus in cells with Tetherin were higher than empty vector.6. Immunofluorescence results show that in interphase of HeLa cells, Zwint-1 wild-type were localized in the cytoplasm, while Zwint-1v was in nucleus. In mitotic phase, Zwint-1 wild-type and Zwint-1v positioned on the spindle and centromere. Zwint-1v more approached than the wild type localization to the nucleus.Conclusion1. Human Tetherin was successfully cloned and expressed, stable transfection Tetherin and empty vector cell lines were constructed. Recombinant Tetherin cells can be effective against damaging effects on cells by influenza virus, and inhibit the release of progeny virus, initially showing that Tetherin had certain inhibition effects against influenza virus.2. In HeLa cells, the sub-localizations of Zwint-1v and Zwint-1 wild-type were different, providing fundamental research on role of Zwint-1v during mitosis and cell biological functions. |
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