分子信标用于活细胞内源性mRNA检测的实验研究

Experimental Research on Molecular Beacons used of Detection of Endogenous mRNA in Living Cells

目的:观察分子信标(molecular beacons,MB)在活细胞内及在体水平对内源性m RNA检测的可行性。方法:合成增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,e GFP)的MB,模拟体内条件,分别从MB的稳定性、与目标基因结合的特异性和敏感性进行检测,并通过在活细胞和斑马鱼胚胎内的应用来观察其对内外源性m RNA的检测的可行性。结果:MB在37℃条件下在体外48小时内经琼脂糖凝胶电泳鉴定未见降解,熔解曲线结果显示荧光强度差异无统计学意义(P>0.05);并且只有在靶序列存在的条件下MB的信号强度显著增强,MB的信号强度与靶基因浓度呈正相关并能基本稳定存在;经e GFP和靶向e GFP的MB共转染的293T细胞能看到荧光表达,靶向e GFP的MB可特异性结合细胞内e GFP的m RNA;在单细胞期斑马鱼胚胎内共注射MB以及目标单链后能观察到荧光表达。结论:结果表明分子信标可用于活细胞内内源性m RNA的检测。

Objective： To expand the application of molecular beacons（MB） to detect endogenous m RNA in living cells. Methods：As we have the transgenic zebrafish line containing e GFP, we synthetized the e GFP MB. We first tested the stability and sensitivity of e GFP MB under the in vitro conditions simulated like in vivo. We then checked the specificity of this MB through binding with synthetized target sequence assay. We further explore its use in living cells and zebrafish embryos respectively by transfection and microinjection. Results： The e GFP MB was stable under the experimental conditions（P〉0.05）and could specifically bind to target gene. The fluorescence of MB could not be detected with low concentration of target gene. Living 293 T cells have fluorescence expression after transfection of e GFP and e GFP MB. The fluorescence of MB also could be observed in 1-cell stage zebrafish embryos injected with e GFP MB. Conclusion： The results showed that the molecular beacons can be used in the detection of genes within living cells, and its application in zebrafish embryos is promising.