發(fā)布時(shí)間：2018-06-01 21:04

  本文選題：CD2相關(guān)蛋白 + 轉(zhuǎn)錄調(diào)控��； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:克隆人CD2相關(guān)蛋白剪接異構(gòu)體(CD2AP002)啟動(dòng)子區(qū),探討microRNA-548k(miR-548k)能否通過靶向結(jié)合該剪接異構(gòu)體啟動(dòng)子序列參與其轉(zhuǎn)錄調(diào)控。方法:采用PCR方法獲取CD2AP002基因5'側(cè)翼序列及一系列5'端缺失體,定向插入到pGL3-Basic載體中,雙螢光素酶報(bào)告基因檢測(cè)系統(tǒng)鑒定其啟動(dòng)子活性;RegRNA2.0在線軟件預(yù)測(cè)CD2AP002啟動(dòng)子區(qū)域潛在的miR-548k的結(jié)合位點(diǎn);熒光原位雜交實(shí)驗(yàn)(fluorescence in situ hybridization,FISH)檢測(cè) miR-548k 在HEK293細(xì)胞內(nèi)的定位,細(xì)胞免疫熒光實(shí)驗(yàn)(immunofluorescence staining,IF)觀察RNA干擾蛋白(AG02)在HEK293細(xì)胞內(nèi)的分布;利用點(diǎn)突變/缺失突變技術(shù)、miR-548k 模擬劑(miR-548k mimic)/miR-548k 抑制劑(miR-548k inhibitor)的轉(zhuǎn)染、實(shí)時(shí)定量PCR(Real time quantitative PCR)技術(shù),鑒定miR-548k是否能通過靶向CD2AP002啟動(dòng)子的潛在結(jié)合位點(diǎn)調(diào)控CD2AP002的轉(zhuǎn)錄;轉(zhuǎn)染miR-548k 模擬劑(miR-548k mimic)后,染色質(zhì)免疫沉淀(chromatin immunoprecitation,ChIP)檢測(cè)RNA聚合酶Ⅱ在人CD2AP002啟動(dòng)子區(qū)域的富集情況。結(jié)果:成功構(gòu)建有活性的人CD2AP002質(zhì)粒;通過生物信息學(xué)預(yù)測(cè),在CD2AP002啟動(dòng)子區(qū)域含有兩個(gè)miR-548k靶向結(jié)合位點(diǎn);FISH證實(shí)miR-548k可見于HEK293細(xì)胞核、IF證實(shí)AG02蛋白在HEK293細(xì)胞的胞核里有分布;miR-548k通過結(jié)合CD2AP002啟動(dòng)子上兩個(gè)結(jié)合位點(diǎn)可在啟動(dòng)子和mRNA水平上正向調(diào)控CD2AP002;ChIP實(shí)驗(yàn)發(fā)現(xiàn)RNA聚合酶Ⅱ在CD2AP002啟動(dòng)子區(qū)的富集。結(jié)論:成功構(gòu)建出有活性的人CD2AP002質(zhì)粒;miR-548k通過靶向結(jié)合CD2AP002啟動(dòng)子上調(diào)CD2AP002的表達(dá),該過程中有RNA聚合酶Ⅱ的富集。
[Abstract]:Aim: to clone the promoter region of human CD2 related protein splicing isomer CD2AP002, and to investigate whether microRNA-548k- miR-548k) can participate in its transcriptional regulation by targeting the splicing isomer promoter sequence. Methods: the 5'flanking sequence of CD2AP002 gene and a series of 5'terminal deletions were obtained by PCR and inserted into pGL3-Basic vector. The double luciferase reporter gene detection system identified its promoter activity, RegRNA2.0 online software for predicting potential miR-548k binding sites in the CD2AP002 promoter region, fluorescence in situ hybridization assay (fish) for the localization of miR-548k in HEK293 cells, and fluorescence in situ hybridization assay (fish) for detecting the localization of miR-548k in HEK293 cells. The distribution of RNA interference protein (AG02) in HEK293 cells was observed by immunofluorescence assay, the transfection of miR-548k mimic agent miR-548k by point mutation / deletion mutation technique, and the real-time quantification of PCR(Real time quantitative PCR) were performed. To determine whether miR-548k can regulate the transcription of CD2AP002 by targeting the potential binding site of CD2AP002 promoter, and to detect the enrichment of RNA polymerase 鈪，

本文編號(hào)：1965626