RNA聚合酶Ⅱ重组hAFP和hTERT双启动子调控针对IGF-Ⅱ基因siRNA表达载体的构建

发布时间：2018-02-20 02:14

  本文关键词： RNA干扰 肝细胞癌 人胰岛素样生长因子II基因 RNA聚合酶II 重组hAFP/hTERT启动子　出处：《暨南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：设计并筛选高效沉默IGF-II基因的小干扰RNA（small interference RNA，siRNA）序列，并构建由RNA聚合酶II重组hAFP（人甲胎蛋白）和hTERT（人端粒酶逆转录酶）双启动子调控针对IGF-II基因siRNA表达载体。 方法：①根据siRNA设计原则，参照IGF-II mRNA序列设计3对siRNA序列（siRNA1、siRNA2、siRNA3）及1对阴性对照序列，，分别以25nM、50nM、100nM浓度，通过脂质体法转染肝癌细胞Huh7，转染24h后采用实时荧光定量PCR检测Huh7细胞中IGF-IImRNA表达量变化，筛选出干扰效率最高的siRNA序列及其最佳干扰浓度。②采用PCR技术扩增出RNA聚合酶II依赖启动子hAFP及hTERT的核心序列，应用基因重组技术构建RNA聚合酶II重组hAFP及hTERT双启动子调控的针对IGF-II基因的siRNA表达载体。 结果：①实时荧光定量PCR显示：转染siRNA1、siRNA2、siRNA3的Huh7细胞中IGF-II mRNA表达量均显著下降，干扰效率为67.18%-94.82%，其中siRNA3在25nM浓度时干扰效率最高，为94.82%。②成功扩增hAFP及hTERT启动子核心序列，片段长度分别为269bp、456bp；将hAFP及hTERT启动子核心序列分别克隆入pGL3-Basic载体，构建成重组pGL3-hAFP-hTERT载体；将siRNA3克隆至pGL3-hAFP-hTERT载体，构建成重组pGL3-hAFP-hTERT-siRNA3载体。DNA测序结果显示：重组质粒中各插入片段方向及序列正确，未见突变、缺失。 结论：①筛选出1对高效沉默IGF-II基因的siRNA序列，即siRNA3，其最佳干扰浓度为25nM；②成功构建RNA聚合酶II重组hAFP及hTERT双启动子调控针对IGF-II基因的siRNA表达载体，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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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