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

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