逆转录病毒整合酶真核表达载体的构建及其与Brd2相互作用初探

发布时间：2018-07-03 15:57

本文选题：整合酶 + 溴结合域蛋白　；参考：《华中科技大学》2012年硕士论文

【摘要】：逆转录病毒基因组整合到宿主细胞染色体是逆转录病毒感染的重要环节，而整合的位点是随机的，其机制尚不明了，病毒整合酶与宿主细胞之间的相互作用可能影响整合的过程。溴结合域蛋白2（Brd2）是一种含有多个磷酸化功能位点和溴区结合域的转录核因子，是与转录调控和表观遗传学相关的蛋白质家族，国外有学者初步发现Brd2与MLV、HIV的整合酶之间可能存在相互作用，本课题构建了绿色荧光蛋白标记的MLV、HIV整合酶和红色荧光蛋白标记的Brd2的真核表达质粒，应用激光共聚焦共定位的方法初步探讨了Brd2与MLV、HIV的整合酶之间的相互作用，，为揭示Brd2对逆转录病毒整合位点的影响打下实验基础。目的：构建绿色荧光蛋白标记的MLV、HIV整合酶和红色荧光蛋白标记的Brd2的真核表达质粒，应用激光共聚焦共定位试验探讨Brd2与MLV、HIV整合酶之间的相互作用。方法：用分子克隆技术构建绿色荧光蛋白标记的HIV整合酶的真核表达质粒psectag2A-GFP-hIN、MLV整合酶的真核表达质粒psectag2A-GFP-mIN和红色荧光蛋白标记的Brd2的真核表达质粒pDsred2-N1-Brd2。分别将psectag2A-GFP-hIN、pDsred2-N1-Brd2和psectag2A-GFP-mIN、pDsred2-N1-Brd2共转染293T细胞，观察相应的荧光，检测转染质粒的表达，通过激光共聚焦共定位试验探讨Brd2与HIV、MLV整合酶之间的相互作用。结果：本课题构建了psectag2A-GFP-hIN、 psectag2A-GFP-mIN、pDsred2-N1-Brd2的真核表达质粒，经测序结果与预期相符，将构建好的上述质粒分别转染到293T细胞，荧光检测到了转染质粒的表达，并初步应用激光共聚焦共定位试验探讨了Brd2与HIV、MLV整合酶之间存在的相互作用。结论：成功构建了HIV、MLV整合酶的真核表达质粒，并对其进行了绿色荧光标记，成功构建了红色荧光蛋白标记的Brd2真核表达质粒，而Brd2的荧光表达强度需要进一步加强。应用激光共聚焦共定位实验有待完善。同时，我们也正在利用Western Blot实验来证实重组质粒的表达。下一阶段，我们课题组拟通过敲除宿主细胞内的Brd2基因及过表达Brd2后，用逆转录病毒载体感染细胞，从而比较Brd2对整合位点选择的影响。
[Abstract]:The integration of retrovirus genome into host cell chromosomes is an important link of retrovirus infection, and the site of integration is random, and its mechanism is not clear. The interaction between virus integrase and host cells may affect the process of integration. Bromine binding domain protein 2 (Brd2) is a family of proteins associated with transcriptional regulation and epigenetics, which contains multiple phosphorylated functional sites and bromine binding domains. Some foreign scholars have preliminarily found that there may be interaction between Brd2 and MLVG HIV integrase. In this study, eukaryotic expression plasmids of MLVG HIV integrase labeled with green fluorescent protein and Brd2 labeled with red fluorescent protein were constructed. The interaction between Brd2 and MLV HIV integrase was preliminarily studied by confocal laser confocal localization, which laid the experimental foundation for revealing the effect of Brd2 on the integration site of retrovirus. Aim: to construct the eukaryotic expression plasmids of green fluorescent protein labeled MLVG HIV integrase and red fluorescent protein labeled Brd2, and to investigate the interaction between Brd2 and MLVG HIV integrase by confocal localization test. Methods: the eukaryotic expression plasmid psectag2A-GFP-hINMLV integrase and the eukaryotic expression plasmid psectag2A-GFP-mIN and Brd2 labeled Brd2 were constructed by molecular cloning technique. Psectag2A-GFP-hINP pDsred2-N1-Brd2 and psectag2A-GFP-mINP pDsred2-N1-Brd2 were co-transfected into 293T cells respectively. The fluorescence and expression of the transfected plasmid were observed. The interaction between Brd2 and HIV MLV integrase was studied by confocal localization test. Results: the eukaryotic expression plasmids psectag2A-GFP-hINand psectag2A-GFP-mIN-pDsred2-N1-Brd2 were constructed in this study. The constructed plasmids were respectively transfected into 293T cells after sequencing. The expression of the transfected plasmids was detected by fluorescence. The interaction between Brd2 and HIV MLV integrase was preliminarily studied by confocal laser confocal localization test. Conclusion: the eukaryotic expression plasmid of HIV-1 MLV integrase was successfully constructed and labeled with green fluorescence, and the red fluorescent protein labeled Brd2 eukaryotic expression plasmid was successfully constructed, and the intensity of Brd2 fluorescence expression needed to be further strengthened. The experiment of laser confocal localization needs to be improved. At the same time, we are using Western blot to confirm the expression of recombinant plasmid. In the next stage, our team intends to compare the effect of Brd2 on the selection of integration sites by knockout and overexpression of Brd2 gene in host cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416

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