本文選題：miR-　切入點(diǎn)：慢病毒　出處：《吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年04期 　論文類型：期刊論文

【摘要】：目的:構(gòu)建miR-137過表達(dá)慢病毒載體并進(jìn)一步包裝慢病毒,探討miR-137在HEK293T細(xì)胞中的感染效率和表達(dá)水平。方法:化學(xué)合成miR-137序列并克隆到慢病毒載體GV209,得到并鑒定含有目的片段的重組質(zhì)粒,采用Lipofectamine 2000共轉(zhuǎn)miR-137重組質(zhì)粒與慢病毒輔助包裝質(zhì)粒到HEK293T細(xì)胞中生產(chǎn)慢病毒。以感染復(fù)數(shù)(MOI)值為40感染HEK293T細(xì)胞48h后,熒光顯微鏡下觀察細(xì)胞中綠色熒光蛋白(GFP)的表達(dá),采用熒光定量PCR法檢測(cè)HEK293T細(xì)胞中miR-137的表達(dá)水平。結(jié)果:測(cè)序結(jié)果,目的基因序列與GenBank公布的miR-137基因序列完全一致。在感染的HEK293T細(xì)胞中觀察到GFP的表達(dá)。過表達(dá)慢病毒感染細(xì)胞中miR-137表達(dá)水平是對(duì)照細(xì)胞的12.74倍。結(jié)論:成功包裝miR-137過表達(dá)慢病毒,并可高效感染HEK293T細(xì)胞。
[Abstract]:Objective: to construct miR-137 overexpression lentivirus vector and further package lentivirus. Methods: the miR-137 sequence was chemically synthesized and cloned into lentivirus vector GV209, and the recombinant plasmid containing the target fragment was obtained and identified. Lentivirus was produced by co-transfer of Lipofectamine 2000 miR-137 recombinant plasmid and lentivirus assisted packaging plasmid into HEK293T cells. The expression of green fluorescent protein (GFP) in HEK293T cells was observed by fluorescence microscope after 48 h infection of HEK293T cells with a number of infected plural moi values of 40. Fluorescence quantitative PCR assay was used to detect the expression of miR-137 in HEK293T cells. Objective the gene sequence was completely consistent with the miR-137 gene sequence published by GenBank. The expression of GFP was observed in infected HEK293T cells. The expression level of miR-137 in lentivirus infected cells was 12.74 times higher than that in control cells. And it can infect HEK293T cells efficiently.
【作者單位】： 廣東醫(yī)科大學(xué)附屬醫(yī)院精神心理科;廣東醫(yī)科大學(xué)神經(jīng)病學(xué)研究所;
【基金】：國家自然科學(xué)基金資助課題(81670252) 廣東省科技廳自然科學(xué)基金資助課題(2015A030313523) 廣東省湛江市科技局科技計(jì)劃項(xiàng)目資助課題(2016A01008)
【分類號(hào)】：R749.3

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