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利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧光标记,进而在活细胞(HeLa)内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3′UTR和24×MS2,构建重组质粒pSG5/AGTR1-3′UTR/24×MS2,并将该质粒与重组质粒pERFP/MS2和pEGFP/C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3′UTR-24×MS2 mRNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3′UTR-24×MS2 mRNA片段可在胞浆中形成与应激蛋白G3BP-GFP共定位的颗粒。该结果表明,针对AGTR1-3′UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3′UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3′UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。
Using the high affinity between the bacteriophage capsid protein MS2 and the RNA with a sequence-specific stem-loop structure containing the MS2 protein binding site, the angiotensin II receptor type 1 (AGTR1 ) 3’untranslated region (3’UTR) fragment was labeled with red fluorescent dye, and then the stress biological behavior of the mRNA fragment was studied in living cells (HeLa). The recombinant plasmid pSG5 / AGTR1-3’UTR / 24 × MS2 was constructed by inserting two double stranded DNA target fragments AGTR1-3’UTR and 24 × MS2 on the empty plasmid vector pSG5, and the recombinant plasmid pERFP / MS2 and pEGFP / C1-G3BP were co-transfected into Hela cells. Fluorescence microscopy results showed that the AGTR1-3’UTR-24 × MS2 mRNA fragment could carry MS2-RFP fusion protein with nuclear entry signal and leave the nucleus to enter the cytoplasm, and under arsenite stimulation, the red fluorescence labeled The AGTR1-3’UTR-24 × MS2 mRNA fragment can form particles in the cytoplasm that co-localize with the stress protein G3BP-GFP. This result indicates that the MS2-RFP fluorescent labeling system targeting AGTR1-3’UTR fragment has been successfully constructed, and the fluorescent labeling system can effectively avoid the false positive fluorescent signal. AGTR1-3’UTR is recruited to the stress particle structure in the cytoplasm when the cells are oxidatively stressed, suggesting that the AGTR1-3’UTR region plays an important role in the regulation of the AGTR1-3 mRNA stress localization in the cell.