Construction Of Retrovirus Integrase Eukaryotic Expression Vectors And Interaction Of Integrase With Brd2

It is a critical step that retroviral genome integrates into the host chromosome inprocess of retroviral infection. However, the site on which the viral genome integratesinto the host chromosome is uncertain, and the integrating mechanism is not yet clear.Study showed that interplay between the integrase and host cell factors may beassociated with process of integation. Bromodomain-containing protein2(Brd2) is akind of host cell nuclear transcription factor which contains multiple phosphorylationsites and bromine binding domain, a family of proteins which are associated withtranscriptional regulation and epigenetics. Some researchers found that Brd2maybeinteracts with MLV and HIV integrase by preliminary studies. In this study weconstructed MLV, HIV integrase and Brd2eukaryotic expression plasmids whichwere labeled by fluorescent protein, and we try to confirm the interaction between theBrd2and HIV, MLV integrases by using of laser scanning confocal microscope inorder to clarify the impact of Brd2to the choice of retrovirus integrating site.Objective: To construct MLV, HIV integrase and Brd2eukaryotic expressionplasmids which are labeled by fluorescent protein, to confirm whether there is aninteraction between Brd2and MLV, HIV integrases by laser confocal microscopy.Methods: Constructed HIV and MLV integrase eukaryotic expression plasmid:psectag2A-GFP-hIN and psectag2A-GFP-mIN, then labeled them with greenfluorescent protein. We constructed Brd2eukaryotic expression plasmidpDsred2-N1-Brd2which was labeled by red fluorescent protein. Then one of the integrase plasmids and Brd2plasmid were cotransfected to293T cells. Thefluorescence was respectively detected after transfected plasmids by microscope.Finally, we tried to confirm the interaction between the Brd2and HIV, MLVintegrases by using of laser confocal test.Results: We constructed HIV, MLV integrase plasmids psectag2A-GFP-hIN,psectag2A-GFP-mIN and Brd2plasmid pDsred2-N1-Brd2. Sequencing resultsshowed that the sequences matched the expected. Then three plasmids wererespectively transfected to293T cells, and we detected the expression of thetransfected plasmids by observing fluorescence of laser confocal microscopy. Wehave preliminarily explored the interaction between HIV, MLV integrases by laserconfocal test.Conclusion: HIV, MLV integrase plasmids have been successfully constructedand labeled with green fluorescent protein. We have also constructed Brd2plasmidwhich is labeled by red fluorescent protein. The plasmids we have constructed can beexpressed in eukaryotic cells, though Brd2plasmids expressed weak fluorescence.The fluorescence in strength is needed so we will change another vector to improvethe visible fluorescence. Meanwhile, we are conducting the Western Blot experimentto confirm the expression of the plasmid which we have constructed. Next step, weare going to knock down the Brd2gene in host cells or overexpress the Brd2first, andthen infect the host cells with the retroviral vectors to find the effect of Brd2tointegrasing sites of the retrovirus.