基于信号放大检测miRNA和BACE1酶切多肽的电化学研究

发布时间：2018-06-10 05:08

本文选题：电化学信号放大检测 + miRNA　；参考：《中南大学》2012年硕士论文

【摘要】：人体内,生物分子miRNA和BACE1与胶质瘤和阿尔茨海默病存在密切的关系。1niRNA-182的表达水平可作为临床上胶质瘤早期诊断的依据,抑制BACE1的活性可在一定程度上减少Ap的产生,因此,研究miRNA和BACE1对产生Aβ的前体蛋白的剪切具有重要的生物学意义。鉴于miRNA在体内的低含量和以及BACE1对产生Ap的前体蛋白的切割缺乏有效的检测手段,本论文中,我们建立了基于一种巯基二茂铁修饰的纳米金/抗生物素蛋白复合物电化学信号放大方法来检测miRNA和BACE1对多肽的切割。 miRNA(MicroRNA)是一种广泛存在于真核生物中的小RNA,成熟体的miRNA长度大约为17-25nt。机体内miRNA-182的表达水平在一定程度上可反映胶质瘤的产生及发展过程。本论文中,我们采用电化学信号放大的方法,检测了胶质瘤中miRNA的表达水平。首先在金电极的表面修饰DNA单分子层,然后利用生物素标记的miRNA和靶点miRNA与表面固定的DNA发生竞争反应,随后巯基二茂铁修饰的纳米金/抗生物素蛋白复合物衍生到电极表面。二茂铁的电化学信号与靶点miRNA浓度成反比例。该方法成功应用于胶质瘤患者血清中miRNA-182的测定。结果表明,癌症患者中miRNA-182的含量是健康人的3倍左右,该结果和实时荧光定量聚合酶链式反应(RT-qPCR)结果一致。此外,电化学方法操作简单、重现性好(RSD5%)、检测限低至10fM。 BACE1是一种与阿尔茨海默病相关的重要的蛋白水解酶。Ap是β-淀粉样蛋白前体(APP)的水解产物之一,APP依次由p-分泌酶(BACE1)、γ-分泌酶特异性酶切产生Ap。目前大量的学者认为Aβ的异常表达与阿尔茨海默病存在密切的关系,因此抑制BACE1的活性成为治疗AD的重要途径之一。本论文中模拟BACE1酶切APP的生物过程,设计一条含BACE1酶切位点的一段生物素标记的多肽序列并修饰于金电极表面,经过BACE1剪切,随后用巯基二茂铁修饰的纳米金/抗生物素蛋白复合物衍生到电极表面,通过BACE1酶切前后电化学信号的变化来检测BACE1切割多肽的过程。该方法可初步用于BACE1抑制剂的筛选。当BACE1抑制剂存在时,电化学信号不降低或者降低程度较小。
[Abstract]:In human body, the expression of biomolecular miRNA and BACE1 is closely related to glioma and Alzheimer's disease. The expression level of .1niRNA-182 may serve as a basis for early diagnosis of glioma, and inhibition of BACE1 activity can reduce the production of AP to some extent. The study of miRNA and BACE1 has important biological significance for the shearing of precursor protein producing A 尾. In view of the low content of miRNA in vivo and the lack of effective detection of BACE1 for the cleavage of precursor proteins that produce AP, in this paper, We have established a novel electrochemical signal amplification method based on thioglycol-ferrocene modified nano-gold / biotin protein complex to detect the cleavage of polypeptides by miRNA and BACE1. MiRNA-MicroRNAs are a kind of small RNAs widely present in eukaryotes and mature. The length of miRNA is about 17-25 NT. The expression level of miRNA-182 can reflect the formation and development of glioma to some extent. In this paper, we detected the expression of miRNA in gliomas by electrochemical signal amplification. First, DNA monolayers were modified on the surface of gold electrode, then biotin-labeled miRNA and target miRNA were used to compete with the surface immobilized DNA, and then thioglycol-ferrocene modified nano-gold / biotin protein complex was derived to the electrode surface. The electrochemical signal of ferrocene is inversely proportional to the concentration of miRNA. The method has been successfully applied to the determination of miRNA-182 in serum of glioma patients. The results showed that the content of miRNA-182 in cancer patients was about three times as high as that in healthy persons, which was consistent with the results of real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). In addition, the electrochemical method is simple to operate, The detection limit is as low as 10fM.BACE1 is an important proteolytic enzyme associated with Alzheimer's disease. AP is one of the hydrolysates of 尾 -amyloid protein precursor APP. At present, a large number of scholars believe that the abnormal expression of A 尾 is closely related to Alzheimer's disease, so inhibiting the activity of BACE1 is one of the important ways to treat AD. In this paper, we simulated the biological process of BACE1 digesting app, designed a biotin labeled polypeptide sequence containing BACE1 restriction site and modified it on the surface of gold electrode, and then cut it by BACE1. Then the nano-gold / biotin protein complex modified by thioglycol-ferrocene was derived to the electrode surface. The process of peptide cleavage of BACE1 was detected by the change of electrochemical signal before and after BACE1 enzyme digestion. The method can be used to screen BACE1 inhibitors. When BACE1 inhibitor exists, the electrochemical signal does not decrease or decrease to a lesser extent.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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