天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

RNA聚合酶Ⅱ重組hAFP和hTERT雙啟動子調(diào)控針對IGF-Ⅱ基因siRNA表達載體的構(gòu)建

發(fā)布時間:2018-02-20 02:14

  本文關(guān)鍵詞: RNA干擾 肝細胞癌 人胰島素樣生長因子II基因 RNA聚合酶II 重組hAFP/hTERT啟動子 出處:《暨南大學(xué)》2012年碩士論文 論文類型:學(xué)位論文


【摘要】:目的:設(shè)計并篩選高效沉默IGF-II基因的小干擾RNA(small interference RNA,siRNA)序列,并構(gòu)建由RNA聚合酶II重組hAFP(人甲胎蛋白)和hTERT(人端粒酶逆轉(zhuǎn)錄酶)雙啟動子調(diào)控針對IGF-II基因siRNA表達載體。 方法:①根據(jù)siRNA設(shè)計原則,參照IGF-II mRNA序列設(shè)計3對siRNA序列(siRNA1、siRNA2、siRNA3)及1對陰性對照序列,,分別以25nM、50nM、100nM濃度,通過脂質(zhì)體法轉(zhuǎn)染肝癌細胞Huh7,轉(zhuǎn)染24h后采用實時熒光定量PCR檢測Huh7細胞中IGF-IImRNA表達量變化,篩選出干擾效率最高的siRNA序列及其最佳干擾濃度。②采用PCR技術(shù)擴增出RNA聚合酶II依賴啟動子hAFP及hTERT的核心序列,應(yīng)用基因重組技術(shù)構(gòu)建RNA聚合酶II重組hAFP及hTERT雙啟動子調(diào)控的針對IGF-II基因的siRNA表達載體。 結(jié)果:①實時熒光定量PCR顯示:轉(zhuǎn)染siRNA1、siRNA2、siRNA3的Huh7細胞中IGF-II mRNA表達量均顯著下降,干擾效率為67.18%-94.82%,其中siRNA3在25nM濃度時干擾效率最高,為94.82%。②成功擴增hAFP及hTERT啟動子核心序列,片段長度分別為269bp、456bp;將hAFP及hTERT啟動子核心序列分別克隆入pGL3-Basic載體,構(gòu)建成重組pGL3-hAFP-hTERT載體;將siRNA3克隆至pGL3-hAFP-hTERT載體,構(gòu)建成重組pGL3-hAFP-hTERT-siRNA3載體。DNA測序結(jié)果顯示:重組質(zhì)粒中各插入片段方向及序列正確,未見突變、缺失。 結(jié)論:①篩選出1對高效沉默IGF-II基因的siRNA序列,即siRNA3,其最佳干擾濃度為25nM;②成功構(gòu)建RNA聚合酶II重組hAFP及hTERT雙啟動子調(diào)控針對IGF-II基因的siRNA表達載體,即pGL3-hAFP-hTERT-siRNA3。
[Abstract]:Aim: to design and screen the small interfering RNA(small interference siRNAs sequence of highly efficient silencing IGF-II gene, and construct the IGF-II gene siRNA expression vector regulated by RNA polymerase II recombinant hAFP (human alpha-fetoprotein) and hTERT (human telomerase reverse transcriptase) double promoter. Methods according to the principle of siRNA design, three pairs of siRNA siRNA1siRNA2siRNA2siRNA3 and a pair of negative control sequences were designed according to the IGF-II mRNA sequence. Hepatocellular carcinoma cell line Huh7 was transfected by liposome method. After 24 hours of transfection, the changes of IGF-IImRNA expression in Huh7 cells were detected by real-time fluorescence quantitative PCR. The siRNA sequence with the highest interference efficiency and its optimal interference concentration .2 the core sequences of RNA polymerase II dependent promoter hAFP and hTERT were amplified by PCR technique. The recombinant hAFP of RNA polymerase II and the siRNA expression vector of IGF-II gene regulated by double promoter of hTERT were constructed by gene recombination technique. Results the IGF-II mRNA expression in Huh7 cells transfected with siRNA1 siRNA2siRNA2 siRNA3 was significantly decreased, and the interference efficiency was 67.18- 94.82%. The interference efficiency of siRNA3 was the highest at 25nM, and the core sequence of hAFP and hTERT promoter was successfully amplified at 94.82.2. The core sequences of hAFP and hTERT promoter were cloned into pGL3-Basic vector to construct recombinant pGL3-hAFP-hTERT vector, and siRNA3 was cloned into pGL3-hAFP-hTERT vector. The results of construction of recombinant pGL3-hAFP-hTERT-siRNA3 vector. DNA sequencing showed that the direction and sequence of the inserted fragments were correct, no mutation and deletion were found in the recombinant plasmid. Conclusion one pair of highly efficient siRNA sequences of silencing IGF-II gene, siRNA3, was screened by 1: 1. The best interference concentration was 25nmmf2 to construct the siRNA expression vector pGL3-hAFP-hTERT-siRNA3, which was regulated by RNA polymerase II recombinant hAFP and hTERT double promoter.
【學(xué)位授予單位】:暨南大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2012
【分類號】:R341

【參考文獻】

相關(guān)期刊論文 前6條

1 陳建發(fā);李宇華;陳引香;謝奎龍;傅明;李立;;ApollonsiRNA提高肝癌細胞化療敏感性的實驗研究[J];南方醫(yī)科大學(xué)學(xué)報;2011年10期

2 陳青;潘秋衛(wèi);蔡榮;錢程;;RNA聚合酶Ⅱ啟動子調(diào)控RNA干擾在腫瘤治療中的應(yīng)用前景[J];生物化學(xué)與生物物理進展;2007年08期

3 楊冬華,張鳴青,杜江,徐重,梁巧明,毛積芳,覃漢榮,范子榮;人胰島素樣生長因子Ⅱ反義RNA對肝癌細胞惡性表型的抑制作用[J];中華肝臟病雜志;1999年01期

4 楊冬華,徐重,楊波,顧健人,錢連方,曲淑敏;不同肝病患者肝細胞中胰島素樣生長因子及其受體的表達[J];中華醫(yī)學(xué)雜志;1996年01期

5 寧曉燕,楊冬華,杜江,徐重,梁巧明,楊波;反義胰島素樣生長因子-Ⅱ?qū)θ烁伟┘毎飳W(xué)特性的影響[J];中華肝臟病雜志;2001年04期

6 邢小康;何繼亮;陳智;;siRNA抑制感染細胞模型中HCV復(fù)制的研究[J];浙江大學(xué)學(xué)報(醫(yī)學(xué)版);2011年06期



本文編號:1518522

資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/xiyixuelunwen/1518522.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權(quán)申明:資料由用戶b1547***提供,本站僅收錄摘要或目錄,作者需要刪除請E-mail郵箱bigeng88@qq.com
黑鬼糟蹋少妇资源在线观看| 大香蕉伊人一区二区三区| 在线精品首页中文字幕亚洲| 亚洲妇女作爱一区二区三区| 五月婷婷六月丁香狠狠| 国产精品福利一二三区| 精品欧美一区二区三久久| 中文字幕一区二区免费| 制服丝袜美腿美女一区二区| 美女激情免费在线观看| 免费午夜福利不卡片在线 视频| 欧美一区二区三区喷汁尤物| 国产在线成人免费高清观看av| 精品人妻一区二区三区在线看| 国产精品亚洲欧美一区麻豆| 日韩欧美综合在线播放| 国产精品一区二区三区日韩av| 国产精品一区二区成人在线| 欧洲日韩精品一区二区三区| 日韩一区二区三区免费av| 黄色在线免费高清观看| 国产色一区二区三区精品视频| 精品国产av一区二区三区不卡蜜 | 亚洲精品中文字幕无限乱码| 国产日韩欧美专区一区| 国产高清一区二区白浆| 又黄又硬又爽又色的视频| 欧美日韩亚洲国产精品| 内射精品欧美一区二区三区久久久| 欧美精品久久男人的天堂| 东京热一二三区在线免| 日韩欧美国产三级在线观看| 久久国产亚洲精品成人| 我的性感妹妹在线观看| 亚洲综合日韩精品欧美综合区| 色丁香之五月婷婷开心| 日韩欧美三级中文字幕| 日韩精品视频一二三区| 国产精品大秀视频日韩精品| 在线观看免费视频你懂的| 国产丝袜美女诱惑一区二区|