CN110923238A - 与呕吐毒素特异性结合的核酸适配体、制备方法及应用 - Google Patents
与呕吐毒素特异性结合的核酸适配体、制备方法及应用 Download PDFInfo
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Abstract
本发明属于生物医学技术领域,公开了与呕吐毒素特异性结合的核酸适配体、制备方法及应用,该核酸适配体是ssDNA,由82个核苷酸组成,其核苷酸序列如SEQ ID NO:1所示;其二级结构含有突出的环和茎,其中DON A16吉布斯自由能DG=‑8.96。基于酶联寡核苷酸吸附测定法(ELONA)、利用氧化石墨烯荧光法进行核酸适配体特异性、亲和力和灵敏度等一系列性质评估,显示DON A16能特异性与呕吐毒素结合,因此该核酸适配体具有高特异性、高亲和力的特点,可用于后续对呕吐毒素的快速检测在试纸条的应用中。
Description
技术领域
本发明属于生物医学技术领域,尤其涉及与呕吐毒素特异性结合的核酸适配体、制备方法及应用。
背景技术
脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)又称呕吐毒素(vomitoxin),主要是由镰刀菌的某些种产生的化学结构和生物活性相似的有毒代谢产物——单端孢霉烯族化合物中的一种。DON的主要产生菌是禾谷镰刀菌(Fusariumgraminearum),据报道也有其它一些镰刀菌可产生。DON广泛存在于全球,主要污染小麦、大麦、玉米等谷类作物,也污染粮食制品,人和动物在误食被该毒素污染的粮谷类后可以产生广泛的毒性效应。另外,它还常与其它的霉卤毒素如黄曲霉毒素共同污染农作物,进入人体后可以相互影响。近年来发现DON可能与人类食管癌、IgA肾病有关,对人类及动物的健康构成威胁。DON属于剧毒或中等毒物,研究表明,DON在体内可能有一定的蓄积,但无特殊的靶器官,具有很强的细胞毒性。人畜摄入了被DON污染的食物/饲料后,会导致厌食、呕吐、腹泻、发烧、站立不稳、反应迟钝等急性中毒症状,严重时损害造血系统造成死亡,但不同的动物对DON的敏感程度不一,猪是最敏感的动物。研究表明,DON可能对免疫系统有影响,有明显胚胎毒性和一定致畸作用,可能有遗传毒性,但无致癌、致突变作用。由于DON的危害严重,引起了各国的普遍重视。谷物及饲料中DON的含量有严格的限量标准。我国谷物中DON的限量标1.0mg/kg。
核酸适配体(aptamer)是基于SELEX技术从随机寡核苷酸文库中筛选获得的对靶物质具有很高特异性与亲和力的寡核苷酸序列。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。自此以来,适配体已经被广泛应用于细胞成像、新药研发、疾病治疗与微生物检测等众多方面。
发明内容
目前国内外并没有直接检测呕吐毒素的方法,为解决这一问题,本发明是通过以下技术方案来实现的:
一种与呕吐毒素特异性结合的核酸适配体,所述与呕吐毒素特异性结合的核酸适配体核苷酸序列如SEQ ID NO:1所示。
进一步的,所述与呕吐毒素特异性结合的核酸适配体的二级结构具有突出的环和茎,吉布斯自由能DG=-8.96。
进一步的,该适配体对应引物包括AptamerFw和Aptamer Rv,其中AptamerFw的序列如SEQ ID NO:2和SEQ ID NO:3,Aptamer Rv的序列如SEQ ID NO:4和SEQ ID NO:5。
与呕吐毒素特异性结合的核酸适配体的制备方法,所述制备方法包括以下步骤:
步骤一、筛选,采用SELEX技术筛选出能够与呕吐毒素特异性结合的核酸适配体群体;
步骤二、挑取单克隆,设计引物进行PCR扩增、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划线在带有氨苄的培养基平板上,37℃过夜;
步骤三、分离,利用划线法将大量的核酸进行分离,经过摇菌得到我们所需要的单个核酸,得到适配体DON A16。
与呕吐毒素特异性结合的核酸适配体的应用,与呕吐毒素特异性结合的核酸适配体可用于直接检测呕吐毒素的试剂盒中。
本发明的有益效果是:与市面上现有的呕吐毒素检测技术相比,利用SELEX技术所筛选出的核酸适配体DON A16能够高亲和力、高特异性的识别并结合呕吐毒素,因此后续基于核酸适配体的检测技术可以实现食品中呕吐毒素的直接检测;核酸适配体DON A16的特异性、亲和力及灵敏度鉴定,确保了对食品中残留的呕吐毒素的检测结果的准确性。基于核酸适配体的检测方法无需繁琐的样品前处理步骤,降低样液中呕吐毒素的损耗,提高检测结果的可靠性。因此本发明填补了目前国内外直接检测呕吐毒素方法的空白。
附图说明
图1是本发明实施例提供的核酸适配体DON A16的二级结构示意图;
图2为是本发明实施例提供的ELONA法对核酸适配体DONA16的特异性分析;
图3是本发明实施例提供的ELONA法对核酸适配体DON A16的亲和力分析;
图4是本发明实施例提供的基于ELONA法核酸适配体DONA16对呕吐毒素灵敏度的分析;
图5是本发明实施氧化石墨烯(GO)荧光法对氧化石墨烯的量进行优化;
图6是本发明实施氧化石墨烯(GO)荧光法对核酸适配体DON A16对呕吐毒素灵敏度的分析;
图7是本发明实施氧化石墨烯(GO)荧光法显示出明显变化趋势;
图8是本发明实施荧光法对核酸适配体DON A16的特异性分析。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下面结合附图对本发明的应用原理作详细的描述。
实施例1:
一、核酸适配体的筛选、克隆、分离、测序、单链DNA二级结构的预测
筛选:采用SELEX技术筛选出能够与呕吐毒素特异性结合的核酸适配体群体。
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划线在带有氨苄的培养基平板上,37℃过夜。
分离:利用划线法将大量的核酸进行分离,经过摇菌得到我们所需要的单个核酸,得到适配体DON A16。
测序:将挑单克隆过后的菌液送去上海生工测序,为了得到筛选出单一的核酸片段。
单链DNA二级结构的预测:通过MFOLD软件设置温度为26℃、Na+浓度为150mM,Mg2+浓度为1mM对与呕吐毒素特异性结合的核酸适配体DONA16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,DONA16吉布斯自由能DG=-8.96;结构具有较高的稳定性(见图1)。
二、应用ELONA方法检测核酸适配体DONA16的特异性、亲和力及对呕吐毒素的灵敏性
1、核酸适配体DONA16特异性检测
在传统的ELISA方法的基础上加以改进,用筛选出来的适配体来代替抗体,采用生物素-亲和素放大系统用以检测待测样品。
(1)毒素的包被
0.05MpH 9.6碳酸盐缓冲液与呕吐毒素体积比为1:1混匀,呕吐毒素的终浓度为50μg/mL。每孔100μL加入到酶标板中,用不干胶密封,于37℃、100rpm振荡器上孵育2小时,孵育完后弃去孔内的液体,每孔加入200μLPBST洗涤液,于水平恒温摇床上振荡洗涤3次,每次洗涤2分钟后于干净的吸水纸上拍干。同时设立空白对照和阴性对照(空白对照:脱脂奶;阴性对照为非靶标物质:AFB1,AFG1,ZEN,OTA;阳性组:DON。浓度保持一致,方法同上)。
(2)封闭
在包被有呕吐毒素的酶标板中,每个孔加入100μL的5%脱脂奶,用不干胶密封,之后,于37℃、100rpm振荡器上孵育2小时,封闭结束后,弃去孔内液体,重复(1)中的洗涤步骤。
(3)加带有生物素标记的核酸适配体孵育
筛选得到能够与呕吐毒素相结合的核酸适配体DONA16送到上海生工生物公司合成,并用生物素(Biotin)标记DONA16。使用时,先进行短暂的离心,使标记过生物素的核酸适配体全部都聚集在试管底部。依据说明,用灭菌水将标记过生物素的核酸适配体充分溶解成浓度为10-4M的存储溶液,为了避免反复的冻融,可将其分装为小份。
将标记过生物素的核酸适配体DON A16用1×PBS稀释到400nM的工作浓度,然后,对应地每个孔加入100μL,用不干胶或封口膜密封后,于37℃、100rpm振荡器上孵育2小时,孵育之后,弃去孔内液体,重复(1)中的洗涤步骤。
(4)加酶结合物孵育
每个孔中加入100μL标有链霉亲和素的辣根过氧化物酶结合物,然后,用不干胶密封,之后,于37℃、100rpm振荡器上孵育1小时,孵育之后,弃去孔内液体,重复(1)中的洗涤步骤。
(5)显色
每孔中加入显色剂TMB溶液100μL,之后于37℃下避光显色20分钟。
(6)终止
最后,往每孔中加入50μL的终止液(2M硫酸),并且要在反应终止的10分钟之内,使用酶标仪检测各孔在450nm处的吸光度值,得OD450 nm。
结果显示,适配体DONA16能够与呕吐毒素特异性的结合(见图2)。
2、核酸适配体DONA16的亲和力Kd值计算
(1)0.05MpH 9.6碳酸盐缓冲液与呕吐毒素体积比为1:1混匀,呕吐毒素的终浓度为50μg/mL。每孔100μL加入到酶标板中,用不干胶密封,于37℃、150rpm振荡器上孵育2小时,孵育完后弃去孔内液体,每孔加洗涤液200μL,于水平摇床上振荡洗涤3次,每次2分钟,同样的每次都要在于净的吸水纸上拍干;
(2)在包被有呕吐毒素的酶标板中,每个孔加入100μL的5%脱脂奶,用不干胶密封,之后,于37℃、100rpm振荡器上孵育2小时,封闭结束后,弃去孔内液体,重复1中的洗涤步骤;
(3)将生物素标记的适配体用1×PBS稀释到1nM、5nM、10nM、20nM、50nM、80nM、160nM、320nM、640nM,1280nM每个孔加100μL,用不干胶或封口膜密封,于37℃、100rpm振荡器上孵育2小时,孵育完后弃去孔内液体,重复1中的洗涤步骤;
(4)每孔中加入100μL辣根过氧化物酶结合物,用不干胶密封,于37℃、150rpm振荡器上孵育1小时,弃去孔内液体,重复1中的洗涤步骤;
(5)每孔加入TMB显色剂100μL,于37℃避光显色20分钟;
(6)加50μL终止液(2M硫酸),并在反应终止10分钟以内用酶标仪测各孔450nm处吸光度值OD450 nm;
结果表明,核酸适配体DONA16的Kd=110.9±29.39nM(见图2)。
3、核酸适配体DONA16对呕吐毒素灵敏度的检测
(1)0.05MpH 9.6碳酸盐缓冲液与呕吐毒素体积比为1:1混匀,使呕吐毒素形成不同浓度梯度。每孔100μL加入到酶标板中,用不干胶密封,于37℃、150rpm振荡器上孵育2小时,孵育完后弃去孔内液体,每孔加洗涤液200μL,于水平摇床上振荡洗涤3次,每次2分钟,同样的每次都要在于净的吸水纸上拍干;
(2)在包被有呕吐毒素的酶标板中,每个孔加入100μL的5%脱脂奶,用不干胶密封,之后,于37℃、100rpm振荡器上孵育2小时,封闭结束后,弃去孔内液体,重复1中的洗涤步骤;
(3)将生物素标记的适配体用1×PBS稀释到400nM,每个孔加100μL,用不干胶或封口膜密封,于37℃、100rpm振荡器上孵育2小时,孵育完后弃去孔内液体,重复1中的洗涤步骤;
(4)每孔中加入100μL辣根过氧化物酶结合物,用不干胶密封,于37℃、150rpm振荡器上孵育1小时,弃去孔内液体,重复1中的洗涤步骤;
(5)每孔加入TMB显色剂100μL,于37℃避光显色20分钟;
(6)加50μL终止液(2M硫酸),并在反应终止10分钟以内用酶标仪测各孔450nm处吸光度值OD450 nm;
结果表明,核酸适配体DONA16检测到呕吐毒素的最低浓度别为31.25μg/mL(见图3)。
三、利用氧化石墨烯(GO)荧光法检测核酸适配体DONA16的特异性、亲和力及对呕吐毒素的灵敏性
1、氧化石墨烯(GO)浓度的优化
将带有FAM荧光标记的呕吐毒素适配体溶解在Tris-HCl缓冲液(PH 7.4)中,并将金属浴调至90℃加热5分钟。待溶液冷却至室温后,然后将1mg/ml的GO溶液稀释其终浓度分别为0μg/ml、10μg/ml、20μg/ml、40μg/ml、60μg/ml、80μg/ml、100μg/ml添加到该混合物中。在37℃下以300rpm的转速振荡孵育60min后,在激发波长为479nm处记录518nm(发射波长)荧光强度。结果表明当氧化石墨烯(GO)的终浓度达到60μg/ml时,整个荧光值达到饱和点,因此氧化石墨烯的最适浓度为60μg/ml。(见图4)
2、利用氧化石墨烯(GO)荧光法检测对呕吐毒素灵敏度
(1)将和带有FAM标记的核酸适配体溶解在Tris-HCl缓冲液(PH 7.4)中,将其混合溶液用金属浴调至90℃加热5分钟。
(2)冷却至室温后,将不同浓度的呕吐毒素标准溶液添加到含有FAM标记的适配体中(200nM)孵育2h,呕吐毒素终浓度分别为0μg/ml、0.1μg/ml、0.2μg/ml、0.5μg/ml、1μg/ml、2μg/ml、4μg/ml。
(3)然后加入终浓度为60μg/ml的氧化石墨烯(GO)缓冲液在37℃下以300rpm转速振荡孵育60min。反应后,在激发波长为479nm处记录518nm(发射波长)荧光强度。
结果表明呕吐毒素的最低检测限在0.2μg/ml,灵敏度较高。(见图5)
3、荧光适配体对呕吐毒素的传感特性
为了获得最佳的感测平台,进行了对比荧光实验。
(1)将和带有FAM标记的核酸适配体溶解在Tris-HCl缓冲液(PH 7.4)中,将其混合溶液用金属浴调至90℃加热5分钟。
(2)冷却至室温后,将终浓度为0.2μg/ml的呕吐毒素标准溶液添加到含有FAM标记的适配体中(200nM)孵育2h。
(3)然后加入终浓度为60μg/ml的氧化石墨烯(GO)缓冲液在37℃下以300rpm转速振荡孵育60min。反应后,在激发波长为479nm处记录518nm(发射波长)荧光强度。
结果表明:与没有呕吐毒素的体系相比,在浓度为4μg/ml呕吐毒素下,其荧光强度明显提高。在没有呕吐毒素的情况下,该溶液显示出低荧光值,FAM标记的适配体被吸附在GO表面而被猝灭。(见图6)
4、利用氧化石墨烯(GO)荧光法检测对呕吐毒素特异性
(1)将和带有FAM标记的核酸适配体溶解在Tris-HCl缓冲液(PH 7.4)中,将其混合溶液用金属浴调至90℃加热5分钟。
(2)冷却至室温后,将ZEN、AFB1,AFG1、OTA、ZEN+DON、AFB1+DON,AFG1+DON、OTA+DON、DON分别添加到含有FAM标记的适配体中(200nM)孵育2h。
(3)然后加入终浓度为60μg/ml的氧化石墨烯(GO)缓冲液在37℃下以300rpm转速振荡孵育60min。反应后,在激发波长为479nm处记录518nm(发射波长)荧光强度。
结果表明溶液中带有呕吐毒素的混合溶液中,荧光值都明显提高,适配体DONA16特异性结合呕吐毒素。(见图7)
结论:通过氧化石墨烯(GO)荧光法可以证明该适配体的可行性,同时我们发现该方法操作简单,全程只需要3h。可广泛操作,并且检测限较低,可实际检测样品中有无呕吐毒素(DON),是一种新型的生物传感器。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
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Claims (5)
1.与呕吐毒素特异性结合的核酸适配体,其特征在于,所述与呕吐毒素特异性结合的核酸适配体核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的与呕吐毒素特异性结合的核酸适配体,其特征在于,所述与呕吐毒素特异性结合的核酸适配体的二级结构具有突出的环和茎,吉布斯自由能DG=-8.96。
3.如权利要求1所述的与呕吐毒素特异性结合的核酸适配体,其特征在于,该适配体对应引物包括AptamerFw和Aptamer Rv,其中AptamerFw的序列如SEQ ID NO:2和SEQ ID NO:3,Aptamer Rv的序列如SEQ ID NO:4和SEQ ID NO:5。
4.与呕吐毒素特异性结合的核酸适配体的制备方法,其特征在于,所述制备方法包括以下步骤:
步骤一、筛选,采用SELEX技术筛选出能够与呕吐毒素特异性结合的核酸适配体群体;
步骤二、挑取单克隆,设计引物进行PCR扩增、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划线在带有氨苄的培养基平板上,37℃过夜;
步骤三、分离,利用划线法将大量的核酸进行分离,经过摇菌得到我们所需要的单个核酸,得到适配体DON A16。
5.与呕吐毒素特异性结合的核酸适配体的应用,其特征在于,与呕吐毒素特异性结合的核酸适配体可用于直接检测呕吐毒素的试剂盒中。
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