干扰素-γ核酸适配体的筛选及其在胞内干扰素检测方面的应用研究

发布时间：2019-06-19 19:52

【摘要】：目的：干扰素-γ (IFN-γ)是在特定刺激作用下由细胞产生的一种具有高度生物活性的糖蛋白,在同种动物细胞上具有抗肿瘤和抗病毒活性及广泛的免疫调控作用。内源性的IFN-γ表达水平的高低在很大程度上能反映机体的细胞免疫状态,因此IFN-γ的检测技术在免疫学的基础研究及多种病原感染的诊断中得到了广泛的应用。传统的IFN-γ检测方法,通常是以特异性抗IFN-γ抗体为基础所建立的。虽然这种方法已相对成熟,但目前商品化的试剂盒在大规模检测时存在成本较高的问题,因而有必要研发更为经济有效的检测方法及新型的可用于IFN-γ检测的配体。单链DNA或RNA可以借由自身所形成的三维空间结构与多种靶标如蛋白质、多肽及其他众多的化合物结合,这种能与靶标以高亲和力特异性结合的寡核苷酸称为核酸适配体(aptamer)。适配体具有靶标范围广,亲和力高,特异性强,易于合成价格低廉等特点,其作用机制与单克隆抗体类似,通过氢键或范德华力与靶标特异性地结合,起到识别靶标分子或抑制其生物学活性的作用。鉴于以上特点,适配体在肿瘤的靶向治疗以及分子检测等领域被广泛应用,因此有可能利用核酸适配体开发出更为经济、高效的IFN-γ检测方法。在本研究中,我们筛选到一种全新的INF-γ核酸适配体,并建立了一种基于该适配体的IFN-γ检测方法,用于测量淋巴细胞内所产生的IFN-γ的分泌水平。方法：我们选取正常及多聚甲醛固定过的重组人IFN-γ蛋白作为靶标,用SELEX技术筛选出了一种与该蛋白具有较高结合力的aptamer (B1-4);以流式细胞仪检测B1-4与白蛋白、正常及固定过的IFN-γ蛋白的结合情况；抽取健康志愿者外周血,常规分离外周血单个核细胞(peripheral blood mononuclear cells, PBMC),以phorbol12-myristate13-acetate(PMA)和Ionomycin共刺激,brefeldin A抑制蛋白转运：将荧光标记的B1-4作为抗体的替代物,以流式细胞仪检测其在刺激后的淋巴细胞内与IFN-γ的结合情况；构建基于适配体的胞内IFN-γ检测曲线。结果：(1)我们所筛选到的IFN-γ的适配体B14,其长度为59个碱基。经测序确定,其一级序列为5'-CCGCCCAAATCCCTAAGAGAAGACTGTAATGACATCAAACCAGACACACTACACACGC A-3'。(2)该适配体与IFN-γ蛋白产生较强的结合,其亲和系数为155nM。(3)该核酸适配体能够识别未经修饰的IFN-γ蛋白以及经多聚甲醛固定的IFN-γ蛋白。(4)进一步检测,该适配体与白蛋白的交叉结合相对较弱,明显优于国际文献报道过的IFN-γ适配体。(5)该适配体能够进入固定后的淋巴细胞,并与经刺激后细胞内产生的IFN-γ特异性地结合。(6)随着产生IFN-γ细胞比例的上升,适配体B14与细胞结合的荧光强度也相应的上升,二者间呈正相关性。(7)由此我们构建了基于核酸适配体的胞内IFN-γ的检测曲线,用于检测群体淋巴细胞INF-γ的分泌水平。结论：本研究筛选出了一种全新的IFN-γ适配体B1-4,能够识别未修饰及经多聚甲醛固定的IFN-γ蛋白。该适配体能够进入经多聚甲醛固定的淋巴细胞,并与细胞内的IFN-γ选择性地结合。由此我们建立了一种新型的基于核酸适配体的胞内IFN-γ的检测方法。由于内源性的IFN-γ表达水平的高低在很大程度上能反映机体的细胞免疫状态,在生物医学研究中被广泛检测,而适配体亲和力高、特异性强、易于合成且价格低廉,因此我们所建立的基于核酸适配体的新型胞内IFN-γ检测方法在临床检验和基础研究方面都具有较大的应用潜能,为测量淋巴细胞内干扰素的分泌提供了全新的技术手段。
[Abstract]:Objective: Interferon-1 (IFN-1) is a kind of glycoprotein with high biological activity under specific stimulation, and has anti-tumor and anti-viral activity and extensive immune regulation in the same animal cell. The level of endogenous IFN-1 expression can reflect the cellular immune status of the body to a large extent, so the detection technology of IFN-1 has been widely used in the research of immunology and the diagnosis of multiple pathogenic infections. The conventional IFN-antigen detection method is generally established on the basis of specific anti-IFN-antigen antibodies. Although the method is relatively mature, the currently commercialized kit has a high cost in large-scale detection, and therefore it is necessary to develop a more economical and effective detection method and a novel ligand which can be used for IFN-antigen detection. The single-stranded DNA or RNA can be combined with a variety of targets such as proteins, polypeptides and other numerous compounds by a three-dimensional space structure formed by itself, which is called a nucleic acid aptamer capable of specifically binding to a target with high affinity. The aptamer has the characteristics of wide target range, high affinity, strong specificity, easy synthesis and low cost, and the like, and the action mechanism is similar to the monoclonal antibody, and can be specifically combined with the target through the hydrogen bond or the Van der Waals force, so as to play the role of identifying the target molecule or inhibiting the biological activity of the target molecule. In view of the above features, the aptamer is widely used in the fields of tumor targeting therapy and molecular detection, and therefore it is possible to use the nucleic acid aptamer to develop a more economical and efficient method for detecting the IFN-antigen. In this study, we screened a brand-new type of INF-1 nucleic acid aptamer and a method of IFN-mediated detection based on the aptamer was established to measure the level of IFN-mediated secretion in lymphocytes. Methods: We selected the recombinant human IFN-2 protein fixed by normal and paraformaldehyde as the target, and the aptamer (B1-4) with a high binding force with the protein was screened by the SELEX technique, and the B1-4 and the white were detected by flow cytometry. Binding of proteins, normal and fixed IFN-I proteins; extraction of peripheral blood of healthy volunteers, conventional isolation of peripheral blood mononuclear cells (PBMCs), phorbol12-myrite13-acetate (PMA) and Ionein co-stimulation, break in A inhibition of protein transport: fluorescent labeled B1-4 as an antibody Generation of a surrogate, in the presence of a flow cytometer to detect its binding to the IFN-antigen in the stimulated lymphocytes, and the construction of an in-cell IFN-antigen assay based on the aptamer Curve. Results: (1) We screened the aptamer B14 of the IFN-1, the length of which is 59 bases. (3) the nucleic acid aptamer is capable of identifying an unmodified IFN-binding protein as well as a polyformaldehyde-immobilized IFN-binding protein. (4) further detection of the cross-junction of the aptamer with the albumin (5) the aptamer can enter the immobilized lymphocyte and specifically bind to the IFN-antigen produced in the post-stimulated cell. (6) With the generation of the IFN-antigen cell, In proportion, the fluorescence intensity of the aptamer B14 combined with the cells is also increased correspondingly, and the positive correlation is between the two. (7) The detection curve of the intracellular IFN-antigen based on the nucleic acid aptamer is thus constructed, and is used for detecting the group lymphocytes INF-1. secretion Conclusion: In this study, a new type of IFN-based aptamer B1-4 is selected, which can identify the unmodified and paraformaldehyde-immobilized IFN-I protein. The aptamer can enter the polymethylene Aldehyde-immobilized lymphocytes are selectively bound to the IFN-1 in the cell. Thus, a novel method for the detection of an intracellular IFN-antigen based on a nucleic acid aptamer is established. Since the level of endogenous IFN-antigen expression can be reflected to a large extent, The cellular immune state of the body is widely detected in the biomedical research, and the aptamer has high affinity, strong specificity, easy synthesis and low cost, so the novel intracellular IFN-antigen detection method based on the nucleic acid aptamer has the advantages of clinical examination and basic research has a large application potential and is used for measuring the secretion of the interferon in the lymphocytes,
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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