本文關(guān)鍵詞：APP695-siRNA慢病毒載體構(gòu)建及其對SH-SY5Y細(xì)胞中APP695基因表達(dá)的作用　出處：《鄭州大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： siRNA 慢病毒載體 APP695基因 實時定量熒光PCR

【摘要】：目的：構(gòu)建針對APP695的siRNA慢病毒載體,并檢測其對人神經(jīng)纖維母細(xì)胞瘤細(xì)胞株SH-SY5Y中APP695基因表達(dá)作用的研究,可能為阿爾茨海默病的基因治療提供新的途徑。 方法：設(shè)計并合成針對篩選確定的APP695基因寡核苷酸序列特異性siRNA有效靶序列；同時合成1對陰性對照寡核苷酸序列。合成后經(jīng)退火形成雙鏈DNA。將pFU-GW-iRNA質(zhì)粒經(jīng)HpaⅠ、XhoⅠ雙酶切后，，將退火形成的雙鏈DNA連接到線性化pFU-GW-iRNA質(zhì)粒,構(gòu)建重組APP695-siRNA表達(dá)質(zhì)粒。將重組質(zhì)粒轉(zhuǎn)化后挑取陽性克隆經(jīng)PCR和測序鑒定。同時完成陰性對照重組質(zhì)粒的構(gòu)建。將它們分別和包裝質(zhì)�；旌衔锕厕D(zhuǎn)染包裝細(xì)胞293T,產(chǎn)生具有感染能力的慢病毒顆粒,并進(jìn)行滴度測定。然后感染SH-SY5Y細(xì)胞,分未經(jīng)病毒感染(CON)組、陰性對照病毒感染(NC)組和慢病毒介導(dǎo)陽性干擾(APP695-RNAi)組；感染72h應(yīng)用實時定量熒光PCR(FQ-RT PCR)檢測各組細(xì)胞APP695 mRNA的表達(dá)水平。 結(jié)果：①陽性克隆的重組質(zhì)粒經(jīng)PCR鑒定和DNA測序證實目的寡核苷酸片段已被準(zhǔn)確克隆到pFU-GW-iRNA質(zhì)粒上。②各質(zhì)粒與包裝質(zhì)粒共轉(zhuǎn)染包裝細(xì)胞293T后收獲慢病毒顆粒。③各組慢病毒載體感染SH-SY5Y細(xì)胞72h后,APP695-siRNA組APP695 mRNA較CON組、NC組明顯降低,差異有統(tǒng)計學(xué)意義(P0.001),而CON組與NC組間無明顯差異(p=0.112)。 結(jié)論：針對APP695基因的siRNA被正確地克隆到pFU-GW-iRNA質(zhì)粒上,并成功構(gòu)建了針對APP695基因的siRNA慢病毒載體；該載體可有效沉默SH-SY5Y細(xì)胞中APP695 mRNA的表達(dá)。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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