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慢性粒细胞白血病K562细胞适配子的筛

发布时间:2018-02-24 18:28

  本文关键词: 白血病 粒细胞 慢性 K562细胞 SELEX 适配子 出处:《兰州大学》2009年硕士论文 论文类型:学位论文


【摘要】: 目的: 利用SELEX技术筛选、鉴定出慢性粒细胞白血病K562细胞的寡核苷酸适配子,测定出ssDNA文库与K562细胞的结合率,并对克隆适配子进行一级结构同源性分析和二级结构预测,为白血病的基因诊断和靶向基因治疗奠定实验基础。 方法: 体外合成长度为88个碱基的随机单链DNA(ssDNA)文库,采用生物素-链霉亲和素磁珠法制备次级文库,以正常人血液中提取的中性粒细胞为反筛细胞,利用SELEX技术筛选出与慢性粒细胞白血病K562细胞特异结合的适配子。将筛选得到的适配子回收纯化后连接pGEM-T质粒载体,经蓝白筛选后,随机挑选24个克隆子进行序列测定。采用荧光标记引物法检测ssDNA文库与K562细胞的亲和力,并用DNAMAN软件对适配子序列进行一级结构同源性分析和二级结构预测。 结果: SELEX筛选产物PCR扩增时最佳退火温度为45℃;每轮筛选的模板含量和循环次数都各不相同;琼脂糖凝胶电泳结果显示,电泳条带随SELEX轮数的增加逐渐变细变致密。经过13轮筛选,慢性粒细胞白血病K562细胞适配子的结合率不断增高,适配子的吸光度A值从0.12上升到1.25,至第13轮A值无明显增高。测序结果显示,绝大部分适配子与预期片段长度相同,但有几个适配子短于预期片段长度。一级结构分析发现无同源序列,但可分为6个家族(family),其中5个家族各自具有保守序列,家族6无保守序列。二级结构分析表明:适配子形成的茎环、凸环结构可能是与K562细胞特异性结合的结构基础。24个克隆适配子中,第10号适配子与慢性粒细胞白血病K562细胞的结合率最高,为45.2%,而与正常人血液中提取的中性粒细胞的结合率仅为2.8%。 结论: 1、利用SELEX技术从体外构建的88 nt的ssDNA文库中成功筛选出高亲和力、高特异性的慢性粒细胞白血病K562细胞适配子; 2、成功测出ssDNA文库与慢性粒细胞白血病K562细胞的结合率; 3、成功将第13轮筛选得到的适配子群进行克隆、测序; 4、成功测出克隆适配子与慢性粒细胞白血病K562细胞的结合率; 5、推测适配子形成的茎环、凸环结构可能是与慢性粒细胞白血病K562细胞特异性结合的结构基础。
[Abstract]:Objective:. The oligonucleotide aptamers of chronic myeloid leukemia K562 cells were identified by SELEX technique, the binding rate of ssDNA library to K562 cells was determined, and the homology analysis and secondary structure prediction of cloned aptamers were carried out. To lay an experimental foundation for gene diagnosis and targeted gene therapy of leukemia. Methods:. A random single strand DNA ssDNA library with 88 bases was synthesized in vitro. The secondary library was prepared by biotin-streptavidin magnetic bead method. The neutrophils extracted from normal human blood were used as anti-sieve cells. The aptamers specifically binding to K562 cells of chronic myeloid leukemia were screened by SELEX technique. The selected aptamers were purified and ligated into pGEM-T plasmid vector. Twenty-four clones were randomly selected for sequencing. The affinity of ssDNA library to K562 cells was detected by fluorescence labeling primer method. The primary structural homology analysis and secondary structure prediction of aptamer sequence were carried out by DNAMAN software. Results:. The optimum annealing temperature for PCR amplification of SELEX screening product was 45 鈩,

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