CN104561010A - 同型半胱氨酸核酸适体HCy5及其制备方法 - Google Patents
同型半胱氨酸核酸适体HCy5及其制备方法 Download PDFInfo
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Abstract
本发明公开具有高特异性和高亲和力的同型半胱氨酸核酸适体及其制备方法。同型半胱氨酸核酸适体的核苷酸序列为：gggctaatgc？attgccatta？atgcctacct？ttgcctgggt？ggtctaaccc，命名为HCy5。同型半胱氨酸核酸适体制备方法包括：构建随机序列文库，筛选靶物质-核酸复合物，分离目的寡核苷酸适体，PCR扩增目的寡核苷酸适体，循环筛选出高度特异性适体，同型半胱氨酸核酸适体的修饰。制备获得的核酸适体无毒性，分子量小，易于合成与标记，只特异性识别同型半胱氨酸，对其他异型半胱氨酸或其类似物不识别结合，可成为检测同型半胱氨酸含量的分子信标。
Description
技术领域
本发明属于氨基酸检测技术领域，涉及一种核酸，特别是涉及高特异性和高亲和力的同型半胱氨酸核酸适体及其制备方法。
背景技术
同型半胱氨酸(Homocysteine,HCys)，或称为高半胱氨酸或同半胱氨酸，是氨基酸半胱氨酸的异种，在旁链部份硫醇基(-SH)前包含一个额外的亚甲基(-CH2-)。同型半胱氨酸是多种疾病的危险因子,目前认为同型半胱氨酸增高是动脉粥样硬化所致心脑血管疾病最广泛、最强独立的致病因素。与传统的血脂三项胆固醇、高密度蛋白、甘油三脂比较，同型半胱氨酸可以作为判断心脑血管疾病危险性的独立指标，由于同型半胱氨酸升高是渐进性的没有阈值，同时可增强传统危险因素的影响。测定同型半胱氨酸水平是心脑血管疾病死亡率最独特、最有效的预测指标。同型半胱氨酸检测与传统的指标相比，具有更高的应用价值。同型半胱氨酸其结构如下式所示：
目前常用的同型半胱胺酸的检测方法有三大类：色谱法、免疫学法、循环酶法。色谱法灵敏度高、特异性好，但样品处理、分离条件、色谱柱制备诸多变异，使其难以标准化；而且HPLC设备价格昂贵、技术条件要求高，需专门的维护人员，使其推广困难。免疫学法需要还原游离HCY形式，抗体荧光分析法和比浊法不能直接检测游离型同型半胱胺酸，只能检测血浆总同型半胱胺酸，用还原剂在37℃半小时卵育对血样进行还原处理。免疫学法需一小时以上才能出结果，操作步骤繁琐，因为需要进行还原处理可受一些不确定的因素影响。循环酶法过程繁琐，且检测限低、产生误差较大，价格昂贵，故得不到推广。
核酸适配体是近年发展起来的一类新型识别分子，近年来受到科学家的广泛关注，大量针对重要生理活性分子的核酸适配体被筛选出来；各种基于核酸适配体的分析方法和技术已被报道；核酸适配体药物“Macugen”也已于2005年被FDA正式批准上市。SELEX技术筛选得到的寡核苷酸序列被称为适配子,国内其翻译为核酸适体、核酸适配子、核酸识体或核酸适配体等。SELEX技术是指应用化学法合成大容量的随机寡核苷酸(由两端的固定序列和中间的随机序列组成)文库,通过施加选择压力(结合靶目标，淘选与靶目标高度特异结合片段的过程),并结合体外扩增技术,经过多轮的循环选择富集,获得与靶物质高度特异结合的寡核 苷酸分子,可以是RNA也可以是DNA,长度一般为25～60个核苷酸。
由上可知，核酸适体与靶物质结合所呈现的高敏感性和高特异性,使其在疾病诊断中具有良好的应用前景,尽管目前成熟的临床应用报道较少,但应用适体检测氨基酸的研究却不断增多,基于适体的检测新技术也不断出现。但是目前基于核酸适体的针对于同型半胱氨酸的高效特异性识别研究还很缺乏，且针对于同型半胱氨酸的核酸适体及其筛选制备方法尚未见有报道。
发明内容
本发明要解决的技术问题是克服现有的同型半胱氨酸检测技术的不足，填补还未见同型半胱氨酸的核酸适体及其筛选制备方法空白，提供一种同型半胱氨酸核酸适体及其制备方法，本发明所提供的核酸适体命名HCy5。
本发明的方案是通过这样实现的：一种同型半胱氨酸核酸适体，其特征在于，所述核酸适体的核苷酸序列其序列为：gggctaatgc attgccatta atgcctacct ttgcctgggt ggtctaaccc。
以上所述同型半胱氨酸核酸适体的衍生物，所述衍生物包括以下四种中的任意一种：
(1)将权利要求1中所述核酸适体任意位置上的碱基A、T、C或G置换成稀有碱基甲基化嘌呤、二氢尿嘧啶或次黄嘌呤后，得到的核酸适体衍生物；
(2)权利要求1中所述核酸适体的核苷酸序列的骨架衍生出的硫代磷酸酯骨架；
(3)权利要求1中所述核酸适体改造成的肽核酸；
(4)权利要求1中所述核酸适体改造成的锁核酸。
一种以上所述同型半胱氨酸核酸适体或其衍生物在识别、检测同型半胱氨酸，或者制备检测同型半胱氨酸的试剂盒方面的应用。
为了使本发明公开充分，本发明同型半胱氨酸核酸适体的制备方法步骤如下：
同型半胱氨酸核酸适体的制备方法包括以下步骤：
1)合成单链DNA随机序列寡核苷酸库：所述单链DNA随机寡核苷酸库的两端为固定序列，作为PCR扩增引物结合区，中间为60个碱基的随机序列，库容量1015以上。所述PCR引物为：
引物1：5’-ATACCAGCTTATTCAATT-3’
引物2：5’-Biotin-AGATTGCACTTACTATCT-3’
所述PCR引物2带有5’端生物素标记。
2)制备连接同型半胱氨酸的固相基质：以微磁珠为基质，通过化学方法把同型半胱氨酸通过其羧基共价连接到微磁珠上。
3)初次筛选目的寡核苷酸序列：将DNA随机寡核苷酸库与同型半胱氨酸混合，筛选除去DNA随机寡核苷酸库中不结合和非特异结合同型半胱氨酸的寡核苷酸序列，回收特异结合同型半胱氨酸的核酸序列。
4)制备次级单链DNA寡核苷酸库：将步骤3中所得与同型半胱氨酸特异结合的寡核苷酸序列进行PCR扩增，PCR扩增产物以链霉亲和素微磁珠为基质进行分离，经过碱变性解链，过滤，纯化，获得次级DNA寡核苷酸库，用于下一轮筛选。
5)筛选并鉴定同型半胱氨酸核酸适体：将步骤4所得的次级单链DNA寡核苷酸库进行下一轮筛选，经过15轮筛选后获得目标寡核酸序列。克隆并测序所述目标寡核酸序列，通过酶联核酸适体吸附测定法鉴定其与同型半胱氨酸结合的特异性和亲和力。
6)所述同型半胱氨酸核酸适体可作为检测试剂用于检测同型半胱氨酸。
本发明的有益效果如下：(1)所筛选到的核酸适体分子量小，无毒性，有利于分子探针的设计，易于合成与标记；(2)核酸适体只特异的识别同型半胱氨酸，不结合非同型半胱氨酸和其它氨基酸分子，结合同型半胱氨酸的能力是结合非同型半胱氨酸Hb的能力的20～80倍，可以成为鉴别同型半胱氨酸的试剂，提高检测方法的特异性和灵敏度，简化检测方法，降低成本。
附图说明
图1为本发明核酸适体与同型半胱氨酸的结合能力，图1中横坐标为核酸适体DNA浓度，纵坐标为解离常数(Kd)相对值，图中HCy曲线表示核酸适体HCy5与同型半胱氨酸的结合曲线，Cy曲线表示核酸适体HCy4与非同型半胱氨酸(半胱氨酸Cy)的结合曲线。
具体实施方式
实施1同型半胱氨酸特异结合的核酸适体HCy5的制备
构建随机序列寡核苷酸库：人工合成单链DNA序列，构建库容量为1×105的单链DNA随机序列寡核苷酸库，单链DNA随机寡核苷酸库包括的DNA序列为：
5’-GGATCCACCAGCGTCATCAGCA-N25～40-AGATAGTAAGTGCAATCTGGC-3’
所述单链DNA随机寡核苷酸库的DNA序列包括中间随机序列N25～60和两端固定序列，所述中间随机序列N25～40为30～40个碱基的随机序列，所述两端固定序列为：5’-GGATCCACCAGCGTCATCAGCA，3’-CGGTCTAACGTGAATGATAGA，所述两端固定序列为PCR扩增引物结合区。
1)将0.5ml(1x109微粒)Invitrogen公司的带有活化氨基的微磁珠与1mol/L同型半胱氨酸在偶联缓冲液(20mM磷酸钾盐缓冲液，0.15M NaCl,1mM DTT,pH 5.5)中混合，加 入200ul偶联剂溶液[57％1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)]，将上述反应置于25℃条件下轻混24小时。将藕连同型半胱氨酸的磁珠用磁珠分离装置和清洗液(PBS，1mM DTT，批H7.3)清洗后，重新悬浮于0.5ml PBS中。
2)将5nmol单链DNA文库溶于结合缓冲液(100mM NaCl,20mM Tris-HCl pH 7.6,2mM MgCl2,5mM KCl,1mM CaCl2,0.02％Tween 20,1mM DTT)，进行加热处理：90℃加热10min，置于冰上10min，然后温度放置5min。
3)将处理好的单链DNA文库与结合有半胱氨酸的微磁珠孵育后，收集不结合磁珠的液体。
4)将步骤3)收集的液体与25ul步骤1)得到的同型半胱氨酸-磁珠一起在结合缓冲液中37℃温育30min。
5)用结合缓冲液洗涤温育后的磁珠，然后加入200ul洗脱缓冲液(20mM Tris-HCl pH7.6,200mM NaCl,10mM EDTA)，92℃孵育5min后，回收带有单链寡核苷酸序列的洗脱缓冲液，进行PCR反应。
6)PCR反应程序为：94℃预变性5min；94℃ 30s，47℃ 1min，72℃ 1min,扩增20个循环；最终延伸反应为72℃ 10min。
7)PCR产物为5’端带有生物素标记的双链DNA，将产物与链霉亲和素磁珠混合，25℃孵育30min后，用0.15mol/L NaOH使双链DNA变性为单链DNA，通过脱盐柱纯化即得到下一轮筛选的单链DNA文库。
8)使用200pmol步骤7)得到的新单链DNA文库，重复进行步骤2)至步骤8)的筛选程序，共进行15轮筛选。最后克隆并测序第15轮单链DNA文库，得到HCy5核酸序列：gggctaatgc attgccatta atgcctacct ttgcctgggt ggtctaaccc。
实施例2以流式分析法检测核酸适体HCy5与同型半胱氨酸的结合能力
1)合成5’端带荧光基团FAM标记的同型半胱氨酸核酸适体。
2)使用0nml/L,5nml/L,10nml/L,20nml/L,50nml/L,100nml/L,200nml/L浓度梯度的FAM标记的核酸适体连接同型半胱氨酸(HCy)的微磁珠来测定同型半胱氨酸核酸适体的解离常数(kd)。用200μL结合缓冲液稀释的上述各种浓度核酸适体，加入150nmol/L连接同型半胱氨酸的微磁珠,37℃温育30min。用结合缓冲液洗涤磁珠后，重新悬浮在250μL结合缓冲液中。设置随机序列的寡核苷酸片段和连接半胱氨酸(Cy)的微磁珠作为对照。3)使用BD公司的流式细胞仪对微珠进行荧光测定，然后用Sigma plot软件作图，计算出所筛选到的核酸适体与糖化血红蛋白相互作用的解离常数(kd)相对值，结果如图1所示。
核苷酸序列表
<110>陈燕婷 张莘蔓 李红梅
<120>同型半胱氨酸核酸适体HCy5及其制备方法
<130>2014
<160>1
<170>PatentIn version 3.3
<210>1
<211>50
<212>DNA
<213>人工序列
<222>（1）...（50）
<400>1
gggctaatgc attgccatta atgcctacct ttgcctgggt ggtctaaccc
Claims (3)
1.一种同型半胱氨酸核酸适体，其特征在于，所述核酸适体的核苷酸序列其序列为：
gggctaatgc attgccatta atgcctacct ttgcctgggt ggtctaaccc。
2.一种权利要求1中任一所述同型半胱氨酸核酸适体的衍生物，所述衍生物包括以下四种中的任意一种：
（1）将权利要求1中所述核酸适体任意位置上的碱基A、T、C或G置换成稀有碱基甲基化嘌呤、二氢尿嘧啶或次黄嘌呤后，得到的核酸适体衍生物；
（2）权利要求1中所述核酸适体的核苷酸序列的骨架衍生出的硫代磷酸酯骨架；
（3）权利要求1中所述核酸适体改造成的肽核酸；
（4）权利要求1中所述核酸适体改造成的锁核酸。
3.一种权利要求1或2中所述同型半胱氨酸核酸适体或其衍生物在识别、检测同型半胱氨酸，或者制备检测同型半胱氨酸的试剂盒方面的应用。
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