APP695-siRNA慢病毒载体构建及其对SH-SY5Y细胞中APP695基因表达的作用

发布时间：2018-01-17 01:30

本文关键词：APP695-siRNA慢病毒载体构建及其对SH-SY5Y细胞中APP695基因表达的作用　出处：《郑州大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：构建针对APP695的siRNA慢病毒载体,并检测其对人神经纤维母细胞瘤细胞株SH-SY5Y中APP695基因表达作用的研究,可能为阿尔茨海默病的基因治疗提供新的途径。方法：设计并合成针对筛选确定的APP695基因寡核苷酸序列特异性siRNA有效靶序列；同时合成1对阴性对照寡核苷酸序列。合成后经退火形成双链DNA。将pFU-GW-iRNA质粒经HpaⅠ、XhoⅠ双酶切后，，将退火形成的双链DNA连接到线性化pFU-GW-iRNA质粒,构建重组APP695-siRNA表达质粒。将重组质粒转化后挑取阳性克隆经PCR和测序鉴定。同时完成阴性对照重组质粒的构建。将它们分别和包装质粒混合物共转染包装细胞293T,产生具有感染能力的慢病毒颗粒,并进行滴度测定。然后感染SH-SY5Y细胞,分未经病毒感染(CON)组、阴性对照病毒感染(NC)组和慢病毒介导阳性干扰(APP695-RNAi)组；感染72h应用实时定量荧光PCR(FQ-RT PCR)检测各组细胞APP695 mRNA的表达水平。结果：①阳性克隆的重组质粒经PCR鉴定和DNA测序证实目的寡核苷酸片段已被准确克隆到pFU-GW-iRNA质粒上。②各质粒与包装质粒共转染包装细胞293T后收获慢病毒颗粒。③各组慢病毒载体感染SH-SY5Y细胞72h后,APP695-siRNA组APP695 mRNA较CON组、NC组明显降低,差异有统计学意义(P0.001),而CON组与NC组间无明显差异(p=0.112)。结论：针对APP695基因的siRNA被正确地克隆到pFU-GW-iRNA质粒上,并成功构建了针对APP695基因的siRNA慢病毒载体；该载体可有效沉默SH-SY5Y细胞中APP695 mRNA的表达。
[Abstract]:Objective: to construct siRNA lentiviral vector targeting APP695 and detect its effect on APP695 gene expression in human neurofibroblastoma cell line SH-SY5Y, which may provide a new way for gene therapy of Alzheimer's disease.
Methods: the design and synthesis of the APP695 gene sequence specific oligonucleotide siRNA screening to determine the effective target sequence; at the same time on the synthesis of 1 negative control oligonucleotides. After synthesis annealed to form double stranded DNA. pFU-GW-iRNA plasmid by Hpa I, Xho I double enzyme digestion, the double stranded DNA annealing form connected to linear pFU-GW-iRNA to construct a recombinant plasmid, APP695-siRNA plasmid. The recombinant plasmid was transformed after positive clones by PCR and DNA sequencing. The constructed recombinant plasmid. Negative control respectively and the packaging plasmid mixtures were transfected 293T cell to produce lentiviral particles with the infection ability, and titered. Then SH-SY5Y cells were infected, divided without the virus infection (CON) group, negative control virus infection (NC) group and lentivirus mediated positive interference group (APP695-RNAi); 72h infection by real-time quantitative PCR (FQ-RT PCR) test the expression level of APP695 mRNA in each group.
Results: the recombinant plasmid of positive clone was confirmed by PCR analysis and DNA sequencing to oligonucleotide fragments were cloned into pFU-GW-iRNA plasmid. The accuracy of the plasmids and packaging plasmids were transfected into 293T packaging cells harvested after lentivirus infected SH-SY5Y cells in all groups. The 72h lentiviral vector, APP695-siRNA APP695 mRNA group compared with CON group, NC group significantly decreased, the difference was statistically significant (P0.001), CON group and NC group had no significant difference (p=0.112).
Conclusion: siRNA targeting APP695 gene has been cloned into pFU-GW-iRNA plasmid correctly, and siRNA lentiviral vector targeting APP695 gene has been successfully constructed, which can effectively silence APP695 mRNA expression in SH-SY5Y cells.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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