【摘要】 目的构建绿色荧光蛋白标记的莫洛尼鼠白血病病毒(MLV)和人类免疫缺陷病毒(HIV)整合酶及红色荧光蛋白标记的Brd2的真核表达质粒,为研究整合酶与Brd2相互作用打下基础。方法以Prr88-MLV、HIV质粒pNL4-3及含有Brd2的质粒为模板,采用PCR技术扩增MLV-IN、HIV-IN、Brd2片段;利用酶切反应体系构建psectag2A-GFP-mIN、psectag2A-GFP-hIN、pDsred2-N1-Brd2质粒,0.8%琼脂糖凝胶电泳后进行测序分析;用重组质粒转染293T细胞,荧光显微镜观察目的蛋白表达情况,并进行WB鉴定。结果构建psectag2A-GFP-hIN、psectag2AGFP-mIN、pDsred2-N1-Brd2的真核表达质粒,酶切以及测序结果与预期相符。将上述质粒分别转染293T细胞,WB及荧光显微镜均检测到目的蛋白的表达。结论成功构建了绿色荧光标记的HIV、MLV整合酶真核表达质粒和红色荧光蛋白标记的Brd2真核表达质粒,尽管Brd2真核表达质粒荧光相对较弱,但这对整合酶与Brd2相互作用研究奠定了基础。更多还原

【Abstract】 Objectives To construct Moloney murine leukemia virus(MLV)and human immunodeficiency virus(HIV)integrase and Brd2 eukaryotic expression plasmids and label them with a fluorescent protein to study the basis for interaction between Brd2 and MLV and HIV integrases using laser confocal microscopy. Methods Prr88-MLV,HIV plasmid pNL4-3,and a Brd2-containing plasmid were used to amplify MLV-IN,HIV-IN,and Brd2 fragments with PCR.An enzyme digestion reaction system was used to construct the plasmids psectag2 A-GFP-mIN,psectag2 A-GFPhIN,and pDsred2-N1-Brd2.All plasmids were recovered using 0.8%agarose gel electrophoresis and sent out for sequencing.The recombinant plasmids were transfected into 293 Tcells,and transgene expression was tested using Western blotting.Expression of the transfected plasmid was determined by observing the corresponding fluorescence. ResultsThe HIV and MLV integrase plasmids psectag2 A-GFP-hIN and psectag2 A-GFP-mIN and the Brd2 plasmid pDsred2-N1-Brd2 were constructed.Results of sequencing and enzyme digestion matched expectations according to a comparison of NCBI blast modules and agarose gel electrophoresis.Each plasmid was transfected into 293 Tcells,and the expression of the transfected plasmid was detected by observing fluorescence with laser confocal microscopy and corresponding proteins with Western blotting. Conclusion HIV and MLV integrase plasmids have been successfully constructed and labeled with green fluorescent protein.A Brd2 plasmid was also successfully constructed and labeled with red fluorescent protein,though the Brd2 plasmid expressed rather weak fluorescence.However,this work is crucial to future research on Brd2 interaction.更多还原