本文選題：整合酶 + 溴結(jié)合域蛋白�。� 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：逆轉(zhuǎn)錄病毒基因組整合到宿主細(xì)胞染色體是逆轉(zhuǎn)錄病毒感染的重要環(huán)節(jié)，而整合的位點(diǎn)是隨機(jī)的，其機(jī)制尚不明了，病毒整合酶與宿主細(xì)胞之間的相互作用可能影響整合的過程。溴結(jié)合域蛋白2（Brd2）是一種含有多個(gè)磷酸化功能位點(diǎn)和溴區(qū)結(jié)合域的轉(zhuǎn)錄核因子，是與轉(zhuǎn)錄調(diào)控和表觀遺傳學(xué)相關(guān)的蛋白質(zhì)家族，國外有學(xué)者初步發(fā)現(xiàn)Brd2與MLV、HIV的整合酶之間可能存在相互作用，本課題構(gòu)建了綠色熒光蛋白標(biāo)記的MLV、HIV整合酶和紅色熒光蛋白標(biāo)記的Brd2的真核表達(dá)質(zhì)粒，應(yīng)用激光共聚焦共定位的方法初步探討了Brd2與MLV、HIV的整合酶之間的相互作用，，為揭示Brd2對(duì)逆轉(zhuǎn)錄病毒整合位點(diǎn)的影響打下實(shí)驗(yàn)基礎(chǔ)。 目的：構(gòu)建綠色熒光蛋白標(biāo)記的MLV、HIV整合酶和紅色熒光蛋白標(biāo)記的Brd2的真核表達(dá)質(zhì)粒，應(yīng)用激光共聚焦共定位試驗(yàn)探討B(tài)rd2與MLV、HIV整合酶之間的相互作用。 方法：用分子克隆技術(shù)構(gòu)建綠色熒光蛋白標(biāo)記的HIV整合酶的真核表達(dá)質(zhì)粒psectag2A-GFP-hIN、MLV整合酶的真核表達(dá)質(zhì)粒psectag2A-GFP-mIN和紅色熒光蛋白標(biāo)記的Brd2的真核表達(dá)質(zhì)粒pDsred2-N1-Brd2。分別將psectag2A-GFP-hIN、pDsred2-N1-Brd2和psectag2A-GFP-mIN、pDsred2-N1-Brd2共轉(zhuǎn)染293T細(xì)胞，觀察相應(yīng)的熒光，檢測(cè)轉(zhuǎn)染質(zhì)粒的表達(dá)，通過激光共聚焦共定位試驗(yàn)探討B(tài)rd2與HIV、MLV整合酶之間的相互作用。 結(jié)果：本課題構(gòu)建了psectag2A-GFP-hIN、 psectag2A-GFP-mIN、pDsred2-N1-Brd2的真核表達(dá)質(zhì)粒，經(jīng)測(cè)序結(jié)果與預(yù)期相符，將構(gòu)建好的上述質(zhì)粒分別轉(zhuǎn)染到293T細(xì)胞，熒光檢測(cè)到了轉(zhuǎn)染質(zhì)粒的表達(dá)，并初步應(yīng)用激光共聚焦共定位試驗(yàn)探討了Brd2與HIV、MLV整合酶之間存在的相互作用。 結(jié)論：成功構(gòu)建了HIV、MLV整合酶的真核表達(dá)質(zhì)粒，并對(duì)其進(jìn)行了綠色熒光標(biāo)記，成功構(gòu)建了紅色熒光蛋白標(biāo)記的Brd2真核表達(dá)質(zhì)粒，而Brd2的熒光表達(dá)強(qiáng)度需要進(jìn)一步加強(qiáng)。應(yīng)用激光共聚焦共定位實(shí)驗(yàn)有待完善。同時(shí)，我們也正在利用Western Blot實(shí)驗(yàn)來證實(shí)重組質(zhì)粒的表達(dá)。下一階段，我們課題組擬通過敲除宿主細(xì)胞內(nèi)的Brd2基因及過表達(dá)Brd2后，用逆轉(zhuǎn)錄病毒載體感染細(xì)胞，從而比較Brd2對(duì)整合位點(diǎn)選擇的影響。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R3416

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本文編號(hào)：2094178