CN113549625B - 与腐霉利特异性结合的核酸适配体Pr-A06及其应用 - Google Patents
与腐霉利特异性结合的核酸适配体Pr-A06及其应用 Download PDFInfo
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Abstract
本发明公开了一种与腐霉利特异性结合的核酸适配体Pr‑A06,其核苷酸序列如SEQ ID NO:1所示;所述核酸适配体Pr‑A06是ssDNA,由82个核苷酸组成,其二级结构含有突出的环和茎,核酸适配体Pr‑A06吉布斯自由能DG=‑12.84,且该适配体具有G‑四联体结构,使该结构更稳定;基于胶体金分光光度法进行核酸适配体特异性、亲和力性质评估,实验结果显示核酸适配体Pr‑A06能特异性与腐霉利结合,该核酸适配体具有高特异性、高亲和力的特点,其可以应用在核酸适配体在制备识别腐霉利的试剂或在制备检测腐霉利试剂盒中,在降低腐霉利对人体的侵害方面具有重要意义。
Description
技术领域
本发明属于生物医学技术领域,尤其涉及一种与腐霉利特异性结合的核酸适配体Pr-A06及其应用。
背景技术
腐霉利(Procymidone, PCD)是一个低毒的杀菌剂,通过抑制菌丝顶端细胞壁合成杀死霉菌。主要用于防治黄瓜、番茄、辣椒、葡萄、草莓、火龙果等瓜果蔬菜和三七、人参等的灰霉病、菌核病、灰星病、花腐病、褐腐病、蔓枯病等是一种杀菌剂类农药。腐霉利抗真菌效果良好,高效低毒,因此在世界各国得到了广泛应用。近年来,腐霉利在果蔬上的广泛使用,使其成为农药残留检出率和超标率较高的农药品种之一。在韭菜中的最大残留限量值为0.2 mg/kg, 植物油为0.5 mg/kg, 番茄、黄瓜为2 mg/kg, 茄子、辣椒、葡萄、蘑菇类(鲜)、鲜食玉米为5 mg/kg, 草莓为10 mg/kg。而研究表明,腐霉利进入机体后最终代谢产物二氯苯胺与其他芳香胺类一样对机体有毒性。不同的动物对腐霉利的毒性表现也不相同,但主要危害动物生殖功能。腐霉利少量摄入体内对人的身体没有影响,但如果长期超标食用,会影响人体健康,轻则刺激眼部和皮肤,重则可能在人体内定量沉积,对人体的神经、血液系统等造成危害。鉴于腐霉利残留对消费者存在危害作用,许多国家均对农产品中腐霉利的最大残留作出限量要求。
核酸适配体是单链DNA或RNA在没有互补链存在的条件下,通过卷曲、折叠为特定三级结构而具备一定功能的分子,主要通过体外指数富集的配体系统进化技术SELEX从随机寡核苷酸文库中筛选高灵敏度、强特异性结合靶标的适配体。人工化学合成一个文库容量为1014-1015nt的随机寡核苷酸文库,其总长度一般为70-100nt,中间包含20-40nt的随机序列。文库与靶标物质孵育一定时间后形成核酸-靶标的复合物,利用一定的方法除去未与靶标结合的文库序列,复合物热解离获得与靶标结合的序列,并以此为模板进行 PCR 扩增,进而制备下一级文库。经过8-20轮不断筛选获得对靶标具有高特异性与高亲和力的寡核苷酸序列,即核酸适配体。适配体经克隆测序后获得相应核酸序列用于后续的研究。核酸适配体在分析化学、生物医学、分子诊断领域都有越来越广泛的应用。
发明内容
本发明提供了一种与腐霉利特异性结合的核酸适配体Pr-A06,其核苷酸序列如SEQ ID NO:1 所示;该核酸适配体Pr-A06的二级结构具有突出的环和茎,且具有G-四联体结构,吉布斯自由能DG=-12.84。
本发明另一目的是将与腐霉利特异性结合的核酸适配体Pr-A06应用在制备识别腐霉利的试剂或在制备检测腐霉利试剂盒中。
本发明目的通过以下技术方案实现:
步骤一、筛选
采用SELEX技术通过能够偶联羟基的柱子筛选出能够与腐霉利特异性结合的核酸适配体群体;
步骤二、克隆
设计引物进行PCR扩增、连接转化、挑取单克隆,利用PMD 19-T载体将PCR产物连接转化到感受态细胞中,将连接转化好的感受态细胞通过划线的方法在带有氨苄的培养基平板上,37℃过夜培养,得到单菌落;
核酸适配体扩增引物为:Aptamer Fw:GACATATTCAGTCTGACAGCG;Aptamer Rv:GCTAGACGATATTCGTCCATC;
步骤三、鉴定阳性克隆、测序
随机挑选固体培养基上的单菌落,在含有氨苄抗性的液体培养基中培养至浑浊,将菌液进行PCR扩增、然后将PCR产物使用琼脂糖凝胶电泳进行验证,选取与目的大小一致的片段进行测序,测序结果表明与腐霉利特异性结合的核酸适配体(Pr-A06)由82个核苷酸组成,其序列(5’端至3’端)为:
gacatattcagtctgacagcgagggctgacatctttgtgcgtggctcaagtagagacaattgctagacgatattcgtccatc;
步骤四、核酸适配体(Pr-A06)单链DNA二级结构表征
使用MFOLD软件对核酸适配体Pr-A06单链DNA分子进行二级结构预测,结果表明,其二级结构具有突出的环和茎,且存在G-四链体结构,吉布斯自由能DG=-12.84,其二级结构如下:
步骤五、核酸适配体(Pr-A06)的亲和力、特异性和对腐霉利的敏感性实验
利用胶体金分光光度法鉴定核酸适配体的亲和力、特异性和敏感性,结果显示,Pr-A06的亲和力常数为46.94±8.142nmol/L,Pr-A06具有很高的特异性,其能检测到腐霉利的最低浓度为100ng/mL。
本发明的有益效果是:与市面上现有的腐霉利检测技术相比,利用SELEX技术所筛选出的核酸适配体Pr-A06能够高亲和力、高特异性的识别并结合腐霉利,基于该核酸适配体的检测技术可以实现食品中腐霉利的直接检测;核酸适配体Pr-A06的特异性和亲和力鉴定,确保了对果蔬中残留的腐霉利的检测结果的准确性,本发明提供了一种新型检测腐霉利的方法。
附图说明
图1是本发明核酸适配体Rr-A06的二级结构示意图;
图2是本发明胶体金分光光度法对核酸适配体Pr-A06的亲和力分析结果;
图3是本发明胶体金分光光度法对核酸适配体Pr-A06的特异性分析结果;
图4是本发明胶体金分光光度法核酸适配体Pr-A06对腐霉利灵敏度的分析结果。
具体实施方式
下面结合附图和实施例来进一步说明本发明的实质性内容,实施例仅为了更好理解本发明但不局限与本发明范围,实施例中方法如无特殊说明均为常规方法,使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1:核酸适配体Pr-A06的筛选、克隆,分离和测序以及单链DNA二级结构的预测
一、腐霉利与免疫亲和柱的偶联
1、将腐霉利稀释至 50μg/mL,总体积为1.5mL;
2、取3mL的免疫亲和柱Sepharose 6B(用丙酮保存)溶液与稀释好的腐霉利溶液使用15mL离心管进行混合;
3、在37℃、20rpm/min 条件下孵育 48 h;
4、48h后加入5mL1mol/L的氨基乙醇封闭未结合腐霉利的位点,轻轻混匀后放置摇床(37℃、20 rpm/min)孵育24h;
5、以4000rpm/min 转速离心2min后弃去上清;
6、分别依次加入pH8.0缓冲液、pH4.0缓冲液、偶联缓冲液洗涤免疫亲和柱,每个洗涤液每次加5mL,以4000rpm/min 转速离心2min后弃去上清;
7、最后加入6mL偶联缓冲液,将整个混合溶液移取到层析柱中使其形成过滤柱(防止免疫亲和柱干燥);放置于4℃冰箱,封口膜封口,竖直放置。
二、核酸适配体文库(ssDNA)的PCR
采用TaKaRa公司合成的ssDNA适配体文库;
1、94℃预热PCR仪;
2、将5μLssDNA、28.6μL去离子水、5μL10×Buffer、3μLMgCl2、4μL dNTP混合物(各2.5μmol/L)、2μL正向扩增引物(浓度为10mol/L)、2 μL反向扩增引物(浓度为10mol/L)以及0.4μLTaq酶离心管进行反应;正向扩增引物序列为5’-GACATATTCAGTCTGACAGCG-3’,反向扩增引物序列为5’-GCTAGACGATATTCGTCCATC-3’。
3、在PCR仪中按以下程序扩增
(1) 预变性:94℃,5min;(2) 30个循环:94℃,45s;58℃,30s;72℃,30s;(3) 后扩增:72℃,7min。
三、配体库的纯化、上样及洗脱
1、配体库的纯化
(1)核酸适配体文库PCR产物直接与胶回收试剂盒里的溶液按体积比1:1比例混合,进行DNA片段回收;
(2)DNA片段回收后,95℃水浴10min,冰浴10min;经过此变性处理,使双链DNA变成单链DNA。
2、亲和层析
(1)将单链DNA加入到偶联制备好的层析柱中,用结合缓冲液(10mmol/LKCl)将整个溶液定容至5mL;放置摇床37℃孵育并40rpm/min轻柔转动45min;
(2)弃去免疫层析柱中的结合缓冲液,加入超纯水洗涤,洗涤3次,每次2mL;
(3)然后用3-4柱体积的洗脱缓冲液(0.1mol/L NaCl,0.1mol/L醋酸盐,pH 4.0)进行线性梯度洗脱,收集洗脱组分;
(4)在离心机中12000rmp/min离心2min,甩干收集管内的液体;
(5)往收集管内加入50μLTE缓冲液,在离心机中以12000rmp/min离心1min,将收集管里的液体转移到PCR管里。
四、PCR优化和大量扩增核酸适配体
以步骤三制得的液体作为模板,按如下步骤操作:
1、94℃预热PCR仪;
2、将5μL模板、28.6μL去离子水、5μL10×Buffer、3μLMgCl2、4μL dNTP混合物(各2.5μmol/L)、2μL正向扩增引物、2μL反向扩增引物、0.4μLTaq酶,于PCR离心管中进行反应。正向扩增引物序列为5’-GACATATTCAGTCTGACAGCG-3’,反向扩增引物序列为5’-GCTAGACGATATTCGTCCATC-3’;
3、在PCR仪中按以下程序扩增:
(1) 预变性:94℃,5min;(2) 30个循环:94℃,45s;58℃,30s;72℃,30s;(3) 后扩增:72℃,7min;
4、循环结束后,将PCR产物用TIANGEN 公司的DNA产物纯化试剂盒进行抽提回收,步骤如下:
(1)将PCR产物与等体积的膜结合缓冲颠倒混匀,然后将混和液转入亲和住,室温静置5min,使DNA充分与硅胶膜结合,12000rpm/min离心1min,倒掉收集管中的废液;
(2)加入700µL的漂洗液(含乙醇)于离心纯化柱中,12000rpm/min离心1min,倒掉收集管中的废液;
(3)重复步骤(2);
(4)12000rpm/min离心3min;
(5)将离心纯化柱置于新的离心管中;
(6)加入30µL超纯水,在室温下静置5min;
(7)12000rpm离心1min,管底溶液即为纯化过的核酸适配体的PCR产物。
五、核酸适配体的克隆,分离和测序以及单链DNA二级结构的预测
1、大肠杆菌DH5α感受态细胞的制备
(1)挑取单个DH5α菌落,接种于3mL不含氨苄青霉素的LB培养基中,37℃培养过夜,次日取上述菌液按比例1:100再接种于50mL 液体LB培养基中,37℃振荡2h;当OD600值达到0.35时,收获细菌培养物;
(2)将细菌培养物转移到一个50mL预冷的无菌聚丙烯管中,冰上放置10min,使培养物冷却;
(3)于4℃下4000rpm/min离心10min,弃去培养液,并将管倒置1min以使残留的培养液流尽;
(4)各加150μL冰预冷的0.1mmol/LCaCl2溶液,合并两管,冰浴10min;
(5)于4℃下4000rpm/min离心10min,弃去上清液,并将管倒置l min以使残留的液体流尽;
(6)先加入800μL冰预冷的0.1mol/LCaCl2溶液重悬细胞,再加入25μL预冷的75%的甘油,之后于-80℃贮存备用。
2、连接及连接产物的转化
(1)在微量离心管中加入1μL Takara pMD19-T simple载体、4μL核酸适配体PCR产物及5μL的连接酶缓冲混合物;
(2)16℃反应过夜孵育;
(3)将感受态细胞从-80℃冰箱取出后立即放置冰上15min;
(4)连接好载体的体系(10μL)加入至100μL DH5α感受态细胞中,冰中放置30min;
(5)42℃加热90s后,再在冰中放置5min;
(6)加入37℃温浴过的LB液体培养基890μL(无氨苄),37℃缓慢振荡培养60min;
(7)取200μL涂布于含有氨苄青霉素的LB固体培养基上,37℃培养16h以形成单菌落。
3、核酸适配体的克隆筛选和测序以及单链DNA二级结构预测
挑取上述单菌落于含氨苄青霉素的LB培养基中,37℃、150rpm/min条件下振荡培养至浑浊,将菌液进行PCR扩增;扩增引物及扩增条件同前述核酸适配体的扩增条件。将经PCR确证的阳性克隆进行质粒提取后,以美国Applied Biosystems3730A全自动核苷酸序列测定仪进行核苷酸序列的测定;测序结果表明与腐霉利特异性结合的核酸适配体(Pr-A06)由82个核苷酸组成,其序列(5’端至3’端)为:
gacatattcagtctgacagcgagggctgacatctttgtgcgtggctcaagtagagacaattgctagacgatattcgtccatc;
与腐霉利特异性结合的核酸适配体Pr-A06的序列长度:82个碱基,序列类型:核酸,链数:单链,拓扑学:直链状,序列种类:ssDNA。
通过MFOLD软件设置温度为26℃、Na+浓度为150mmol/L、Mg2+浓度为1mmol/L(http://mfold.rna.albany.edu/q=mfold/DNA-Folding-Form)和QGRS映射(http://bioinformatics.ramapo.edu/QGRS/analyze.php)对与腐霉利特异性结合的核酸适配体Pr-A06单链DNA分子进行二级结构预测;结果表明,适配体含有突出的环和茎,其吉布斯自由能DG=-12.84,该结构具有较高的稳定性(见图1)。
实施例2:核酸适配体Pr-A06的亲和力、特异性和灵敏度检测
1、核酸适配体Pr-A06亲和力检测
胶体金分光光度法以胶体金为标记物,利用胶体金在高盐环境中发生聚集从而引起胶体金波长红移、特征峰向右偏移的特点测定胶体金溶液特定波长下的吸光值来鉴定。
(1)25nm胶体金的制备
1)配制1%的柠檬酸钠溶液,称0.1g 柠檬酸钠加入10mL超纯水;
2)取25μL氯金酸溶液(含Au量49%)稀释45倍至1125μL(此步骤在避光条件下进行);
3)取99mL纯净水,加入干净的锥形瓶中,纯净水、柠檬酸钠溶液和氯金酸溶液均需过45μm的滤膜;
4)在锥形瓶中加入1mL氯金酸溶液混匀用电热套调至80℃加热至沸腾(此过程用转子进行磁力搅拌);
5)快沸腾时将温度调至100℃加热至完全沸腾后加入1.5mL的1%的柠檬酸钠溶液;
6)将温度调至0℃,溶液由无色-黑色-紫色-红色,等颜色稳定后,将温度调至70℃,加热10min;
7)冷却至室温后转移至4℃避光存放。
(2)用超纯水将腐霉利稀释到1μg/mL,每管50 μL;
(3)将核酸适配体Pr-A06送到北京擎科生物公司合成;使用时,先进行短暂的离心,使核酸适配体全部都聚集在试管底部。依据说明,用灭菌水将核酸适配体充分溶解成浓度为10-4 mol/L的存储溶液,为了避免反复的冻融,可将其分装为小份;再用1×PBS将适配体稀释到50nmol/L、100nmol/L、200nmol/L、400nmol/L、800nmol/L、1600nmol/L,并添加到步骤(2)腐霉利溶液中,每管加入50μL,在室温条件下孵育30min;同时设置不加核酸适配体,加50μL1×PBS的空白对照,每个样品重复做3个平行实验;
(4)取步骤(1)制备的胶体金浓缩4倍,将4mL胶体金在4℃,8000 rpm/min离心12min,弃去3mL上清,剩余1mL用移液枪吹打均匀备用;
(5)在步骤(3)每管中加入50μL的浓缩后的胶体金,室温避光孵育30min;
(6)在步骤(5)每管中加入终浓度40mmol/L的NaCl,混匀后,使用酶标仪在520nm下测量吸光度值,计算(A’ -A 0)/A 0,其中A’是不同浓度适配体对应的吸光度值,A 0是不加入适配体的吸光度值;以(A’ -A 0)/A 0为纵坐标、核酸适配体浓度为横坐标,使用GraphPadPrism8软件进行分析、计算亲和力常数,结果表明,核酸适配体Pr-A06的K d = 46.94±8.142nmol/L(见图2),该适配体具有亲和力,其中亲和力常数越小表明适配体的亲和力越高该适配体具有亲和力。
2、核酸适配体Pr-A06特异性检测
(1)用超纯水将腐霉利稀释到1μg/mL,每管加入50μL,同时设置与腐霉利浓度相同的非特异性靶标对照样品:烯酰吗啉、五氯硝基苯、草甘膦,设置不加入腐霉利而加入50μL超纯水的空白对照,每个靶标重复做3个平行实验;
(2)用1×PBS将适配体稀释到400nmol/L,在步骤(1)每管加入50μL,在室温条件下孵育30min;
(3)取步骤1制备的胶体金浓缩4倍,将4mL胶体金在4℃,8000 rpm/min离心12min,弃去3mL上清,剩余1mL用移液枪吹打均匀备用;
(4)在步骤(2)每管中加入50μL浓缩后的胶体金,室温避光孵育30min;
(5)在步骤(4)每管中加入终浓度40mmol/L的NaCl,使用酶标仪在520nm和620nm下测量吸光度值,计算A620nm/A520nm,计算(A’ -A ’0)/A ’0,其中A’为不同浓度适配体对应的620nm吸光度值与520nm吸光度值的比值,其中A ’0是不加入靶标时620nm吸光度值与520nm吸光度值的比值;结果见图3,结果显示,加入腐霉利的管中(A’ -A ’0)/A ’0的值远远大于加入其他非特异性靶标的的值,适配体Pr-A06能够与腐霉利特异性的结合。
3、核酸适配体Pr-A06灵敏度检测
(1)用超纯水将腐霉利稀释到不同的浓度梯度50ng/mL、100ng/mL、200ng/mL、400ng/mL、600ng/mL、800ng/mL,每管加入50μL;设置不加入腐霉利加入超纯水作为空白对照,每个浓度重复做3个平行实验;
(2)用1×PBS将适配体稀释到400nmol/L,按每管加入50μL的量添加到步骤(1)中,在室温条件下孵育30 min;
(3)取步骤1制备的胶体金浓缩4倍,将4mL胶体金在4℃,8000 rpm/min离心12min,弃去3mL上清,剩余1mL用移液枪吹打均匀备用;
(4)在步骤(2)每管中加入50μL的浓缩后的胶体金,室温避光孵育30min;
(5)在步骤(4)每管中加入终浓度为40mmol/L的NaCl,使用酶标仪在波长520nm和620nm处测量吸光度值,计算A=A620nm/A520nm;结果显示,当腐霉利浓度达到100ng/mL,A620nm/A520nm值显著高于空白对照,随着腐霉利浓度的增加,A620 nm/A520 nm值逐渐增大,适配体Pr-A06灵敏度较高(见图4)。
本发明适配体Pr-A06显现出较好的亲和力、特异性、灵敏度,该适配体能用于后续实际样品中腐霉利的检测。
序列表
<110> 昆明理工大学
<120> 与腐霉利特异性结合的核酸适配体Pr-A06及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213> 人工序列(Artificial)
<400> 1
gacatattca gtctgacagc gagggctgac atctttgtgc gtggctcaag tagagacaat 60
tgctagacga tattcgtcca tc 82
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 2
gacatattca gtctgacagc g 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 3
gctagacgat attcgtccat c 21
Claims (2)
1.一种与腐霉利特异性结合的核酸适配体Pr-A06,其核苷酸序列如SEQ ID NO:1 所示。
2.权利要求1 所述的核酸适配体Pr-A06在制备识别腐霉利的试剂或在制备检测腐霉利试剂盒中的应用。
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