本文關(guān)鍵詞： FHL基因 熒光素酶報(bào)告基因 轉(zhuǎn)錄活性　出處：《軍事醫(yī)學(xué)》2013年06期 　論文類型：期刊論文

【摘要】：目的構(gòu)建含有FHL1基因3'-UTR區(qū)的熒光素酶報(bào)告基因載體,用以檢測(cè)與其相互作用的microRNA(miRNA)。方法 PCR擴(kuò)增出FHL1基因3'-UTR區(qū)片段,插入到經(jīng)過(guò)改造的熒光素酶報(bào)告基因載體pcDNA 3.0中;利用Target Scan5.1軟件預(yù)測(cè)可能與FHL1基因3'UTR區(qū)相互作用的miRNA,將miRNA與熒光素酶報(bào)告重組子共轉(zhuǎn)染至293T細(xì)胞中,檢測(cè)其熒光活性;構(gòu)建針對(duì)熒光活性結(jié)果變化明顯的miRNA的種子區(qū)缺失突變體,檢測(cè)熒光活性。結(jié)果測(cè)序結(jié)果表明,含有FHL1基因3'-UTR區(qū)的熒光素酶報(bào)告基因載體構(gòu)建正確;熒光素酶活性實(shí)驗(yàn)表明,與對(duì)照組相比,miR-200c、miR-146a、miR-146b-5p可使熒光素酶報(bào)告重組子的熒光素酶活性降低40%左右,并構(gòu)建上述miRNA缺失突變體,miR-200c使熒光素酶活性回復(fù)。結(jié)論成功構(gòu)建了FHL1基因3'UTR區(qū)的熒光素酶報(bào)告基因載體,而miR-200c、miR-146a、miR-146b-5p可以抑制其熒光素酶活性,其中miR-200c可能直接作用于FHL1基因3'-UTR區(qū)。
[Abstract]:Objective to construct a luciferase reporter gene vector containing the FHL1 gene 3r-UTR region. MicroRNAs and miRNAs interacting with them were detected. Methods the FHL1 gene 3- UTR region was amplified by PCR. Inserted into the modified luciferase reporter gene vector pcDNA 3.0; The Target Scan5.1 software was used to predict the miRNA that might interact with the 3UTR region of the FHL1 gene. MiRNA and luciferase reporter recombinant were co-transfected into 293T cells to detect their fluorescence activity. In order to detect the fluorescence activity of miRNA, we constructed the mutant of miRNA with obvious changes in fluorescence activity. Results the sequencing results showed that. The luciferase reporter gene vector containing the FHL1 gene 3 + -UTR region was constructed correctly. The results of luciferase activity test showed that miR-200cn miR-146a was higher than that of control group. MiR-146b-5p could reduce the luciferase activity of luciferase report recombinant by about 40%, and construct the miRNA deletion mutants mentioned above. Conclusion the luciferase reporter gene vector of FHL1 gene 3UTR was constructed successfully, and miR-200cnmmiR-146a was successfully constructed. MiR-146b-5p can inhibit the luciferase activity of FHL1 gene, and miR-200c may directly act on the 3- UTR region of FHL1 gene.
【作者單位】： 廣西醫(yī)科大學(xué);軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所;軍事醫(yī)學(xué)科學(xué)院生物工程研究所;
【基金】：中國(guó)博士后科學(xué)基金面上資助項(xiàng)目(201150M1523)
【分類號(hào)】：R346
【正文快照】： FHL1屬于FHL(41/2驁駘LIM結(jié)構(gòu)域,four and ahalf LIM domain)家族,該家族共有5個(gè)成員,即FHL1、FHL2、FHL3、FHL4、FHL5/ACT(activator ofCREM intestis),其蛋白結(jié)構(gòu)中含有4駘驁LIM結(jié)構(gòu)域,屬于LIM超家族。LlM是3種轉(zhuǎn)錄因子Lin-11、Isl-1和Mec-3的縮寫,由約55個(gè)氨基酸殘基組成,，

本文編號(hào)：1443189