本文關鍵詞： 白血病 粒細胞 慢性 K562細胞 SELEX 適配子　出處：《蘭州大學》2009年碩士論文　論文類型：學位論文

【摘要】： 目的: 利用SELEX技術篩選、鑒定出慢性粒細胞白血病K562細胞的寡核苷酸適配子,測定出ssDNA文庫與K562細胞的結合率,并對克隆適配子進行一級結構同源性分析和二級結構預測,為白血病的基因診斷和靶向基因治療奠定實驗基礎。 方法: 體外合成長度為88個堿基的隨機單鏈DNA(ssDNA)文庫,采用生物素-鏈霉親和素磁珠法制備次級文庫,以正常人血液中提取的中性粒細胞為反篩細胞,利用SELEX技術篩選出與慢性粒細胞白血病K562細胞特異結合的適配子。將篩選得到的適配子回收純化后連接pGEM-T質粒載體,經(jīng)藍白篩選后,隨機挑選24個克隆子進行序列測定。采用熒光標記引物法檢測ssDNA文庫與K562細胞的親和力,并用DNAMAN軟件對適配子序列進行一級結構同源性分析和二級結構預測。 結果: SELEX篩選產(chǎn)物PCR擴增時最佳退火溫度為45℃;每輪篩選的模板含量和循環(huán)次數(shù)都各不相同;瓊脂糖凝膠電泳結果顯示,電泳條帶隨SELEX輪數(shù)的增加逐漸變細變致密。經(jīng)過13輪篩選,慢性粒細胞白血病K562細胞適配子的結合率不斷增高,適配子的吸光度A值從0.12上升到1.25,至第13輪A值無明顯增高。測序結果顯示,絕大部分適配子與預期片段長度相同,但有幾個適配子短于預期片段長度。一級結構分析發(fā)現(xiàn)無同源序列,但可分為6個家族(family),其中5個家族各自具有保守序列,家族6無保守序列。二級結構分析表明:適配子形成的莖環(huán)、凸環(huán)結構可能是與K562細胞特異性結合的結構基礎。24個克隆適配子中,第10號適配子與慢性粒細胞白血病K562細胞的結合率最高,為45.2%,而與正常人血液中提取的中性粒細胞的結合率僅為2.8%。 結論: 1、利用SELEX技術從體外構建的88 nt的ssDNA文庫中成功篩選出高親和力、高特異性的慢性粒細胞白血病K562細胞適配子; 2、成功測出ssDNA文庫與慢性粒細胞白血病K562細胞的結合率; 3、成功將第13輪篩選得到的適配子群進行克隆、測序; 4、成功測出克隆適配子與慢性粒細胞白血病K562細胞的結合率; 5、推測適配子形成的莖環(huán)、凸環(huán)結構可能是與慢性粒細胞白血病K562細胞特異性結合的結構基礎。
[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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