CN108101970B - 基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原及其应用 - Google Patents
基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原及其应用 Download PDFInfo
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Abstract
本发明属于基因工程抗体和食品生物技术领域，涉及基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原及其应用，该Cry1Ab毒素模拟抗原氨基酸序列如SEQ ID NO.1所示，编码该氨基酸的核苷酸序列如SEQ ID NO.9所述，本发明提供的Cry1Ab模拟抗原可以替代价格昂贵且具有毒性的Cry1Ab标准品，并作为竞争抗原应用于Cry1Ab的竞争免疫学检测，其具有与Cry1Ab分子相似的免疫反应特性，效果良好。
Description
技术领域
本发明属于基因工程抗体和食品生物技术领域，具体涉及一种基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原及其应用。
背景技术
Bt毒素是苏云金芽孢杆菌(Bacillus thuringiensis,Bt)在形成芽孢时产生的一种伴孢晶体蛋白，可分为晶体蛋白(crystal protein,Cry蛋白)和细胞外溶解性蛋白(cytolytic protein,Cyt蛋白)两大类，其对鳞翅目、鞘翅目等多种昆虫具有杀虫活性。其毒性作用机理是Bt原毒素在昆虫中肠碱性环境及蛋白酶的作用下，水解为60-70KD的激活毒素，然后与中肠上皮细胞膜上的特异受体结合，从而导致细胞膜穿孔，最终导致昆虫死亡。Bt毒素对目标昆虫具有高度特异性，对人类和动物无害，已经被广泛应用于棉花、玉米和水稻等农作物的害虫防治中。其中，Cry毒素为Bt毒素中应用较为广泛的一类毒素，而Cry1Ab毒素是转基因作物中最常见的Cry毒素之一。
随着转基因作物的大规模推广和应用，人们开始对转基因作物的安全性问题产生顾虑，许多国家已经对转基因产品实行标签制度。因此建立快速、有效的Bt毒素检测方法是摆在各国政府面前的一项亟待解决的主要任务。
目前，Bt毒素的检测方法主要包括PCR方法和ELISA方法。PCR方法具有高灵敏度和高准确性，但局限于Bt毒素基因水平的检测，且需要相对完备的实验室和专业的操作人员。ELISA方法针对于Bt毒素蛋白表达水平的检测，具有简便、低耗、高通量等优点，是应用最广的Bt毒素检测方法，尤其以双抗夹心ELISA最为常见。然而，双抗夹心ELISA方法的建立需要进行两种抗体的制备及配对，过程复杂，周期长，且ELISA反应步骤繁多。与之相比，竞争ELISA具有过程简单、操作简便的优点，但竞争ELISA的建立必不可免的需要一个合适的竞争抗原或抗原模拟物。
纳米抗体(Nanobody)来源于骆驼和羊驼体内，是目前已知最小的功能性抗体片段，只含有3个CDR，却具有与普通抗体相同的抗体功能。与普通抗体相比，纳米抗体的CDR3更长，可以形成凸环结构，能够伸入酶的凹槽、裂缝或抗体的凹穴等普通抗体难以达到的表位。此外，纳米抗体的FR2区中4个疏水残基突变为亲水残基，使纳米抗体的水溶性更好。分子内CDR1与CDR3能形成一对二硫键，使纳米抗体具有好的稳定性。因此，纳米抗体是一种天然的、理想的模拟抗原材料。
发明内容
针对目前转Bt Cry毒素农作物及其毒素制剂的广泛应用所产生的安全性隐患和监管需求，本发明筛选基于抗独特型纳米抗体的Bt Cry1Ab毒素模拟抗原，建立简便、快速的竞争免疫分析法并应用于实际样品的检测。
本发明首先提供了一种驼源的基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原，其氨基酸序列如SEQ ID NO.1所示。
本发明所提及的Cry1Ab模拟抗原(纳米抗体)包括四个框架区FR和三个互补决定区CDR。其中，框架区(FR1-FR4)的氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.4、SEQ IDNO.6和SEQ ID NO.8所示；互补决定区(CDR1-CDR3)的氨基酸序列分别如SEQ ID NO.3、SEQID NO.5和SEQ ID NO.7所示。
本发明同时提供了编码氨基酸序列如SEQ ID NO.1所示Cry1Ab毒素模拟抗原的核苷酸，其核苷酸序列如SEQ ID NO.9所示。
本发明还提供了含有序列如SEQ ID NO.9所示的核苷酸的重组表达载体及重组工程菌。
此外，本发明进一步提供了氨基酸序列如SEQ ID NO.1所示的Cry1Ab毒素模拟抗原在农作物Cry1Ab毒素检测中的应用，即将该Cry1Ab模拟抗原作为竞争抗原，应用于免疫学检测分析；以及，该Cry1Ab模拟抗原Cry1Ab在模拟物试剂中的应用。
本发明通过构建噬菌体展示驼源天然纳米抗体库，筛选得到一种具有Cry1Ab单抗结合活性的独特型纳米抗体，并作为模拟抗原应用于Bt Cry1Ab毒素的竞争免疫检测。与现有技术相比，具有以下有益效果：
1)应用本发明提供的模拟抗原方法可避免传统双抗夹心法建立所需的两种抗体制备和配对的复杂过程，省时省力。
2)利用本发明提供的模拟抗原可减少Cry1Ab毒素标品的使用，节约成本，减少毒素对生态环境的危害。
3)本发明制备Cry1Ab模拟抗原、建立竞争免疫分析法的策略具有普遍适用性，可用于其他Bt毒素和蛋白竞争免疫分析方法的建立，易于推广应用。
附图说明
图1为纳米抗体的DNA电泳图；
其中，图1(A)为巢式PCR第一轮扩增，PCR产物约为700bp；图1(B)为巢式PCR第二轮扩增，PCR产物约为400bp；图1(C)为菌落PCR验证纳米抗体基因，PCR产物约500bp。
图2为以Cry1Ab模拟抗原建立的竞争ELISA标准曲线；
其检测范围为10.49-307.1ng/mL，IC50为42.68ng/mL。
具体实施方式
以下通过具体实施例对本发明技术方案思想进一步说明。
实施例中所涉及的试剂/培养基：
TRIZOL、cDNA synthesis via SuperScript III First-Strand SynthesisSuperMix Kit、购自美国Invitrogen公司；
限制性内切酶sfi I、T4 ligase、辅助噬菌体M13K07、购自美国NEB公司；
HRP标记抗M13二抗购、表达载体pET-26b、购自美国GE healthcare公司；
Cry1Ab标准品购自上海佑隆生物科技有限公司；
whatman滤纸购自杭州沃华滤纸有限公司；
噬菌体载体pComb3x为申请人实验室保存(具体实施时，也可通过Biovector公司购买)；
E.coli TG1为申请人实验室自制，制备方法参见文献：“徐重新,张霄，张存政，等.鼠源噬菌体抗体展示库的构建及初步应用.江苏农业学报，2017,01(33)：210-217”；
Cry1Ab单克隆抗体为申请人实验室自制，制备方法参见文献：“Dong,S.；Zhang,X.；Liu,Y.；Zhang,C.；Xie,Y.；Zhong,J.；Xu,C.；Liu,X.Establishment of a sandwichenzyme-linked immunosorbent assay for specific detection of Bacillusthuringiensis(Bt)Cry1Ab toxin utilizing a monoclonal antibody produced with anovel hapten designed with molecular model.Anal.Bioanal.Chem.2017,409,1985-1994”；
SOC培养基(1L)：20g蛋白胨，5g酵母提取物，0.5g氯化钠，0.19g氯化钾，0.95g氯化镁，3.6g葡萄糖；
SOB-AG平板(1L)：20g蛋白胨，5g酵母提取物，0.5g氯化钠，0.19g氯化钾，0.95g氯化镁，15g琼脂粉，100μg/mL的氨苄青霉素和2％(质量比)的葡萄糖；
2×YT-AG培养基(1L)：16g蛋白胨，10g酵母提取物，5g氯化钠，100μg/mL的氨苄青霉素和2％(质量比)的葡萄糖；
2×YT-AK培养基(1L)：16g蛋白胨，10g酵母提取物，5g氯化钠，100μg/mL的氨苄青霉素和50μg/mL的卡那霉素；
LB-A平板(1L)：10g蛋白胨，5g酵母提取物，5g氯化钠，15g琼脂粉和100μg/mL的氨苄青霉素；
LB-AG培养基(1L)：10g蛋白胨，5g酵母提取物，5g氯化钠，100μg/mL的氨苄青霉素和2％(质量比)的葡萄糖。
实施例1：天然纳米抗体库的构建
(1)提取未经免疫的骆驼外周血，分离淋巴细胞并通过TRIZOL提取总RNA；
(2)利用cDNA synthesis via SuperScript III First-Strand SynthesisSuperMix Kit反转录合成cDNA，并利用巢式PCR扩增纳米抗体基因(PCR引物参见文献“Ebrahimizadeh,W.；Gargari,S.M.；Rajabibazl,M.；Ardekani,S.；Zare,H.；Bakherad,H.Isolation and characterization of protective anti-LPS nanobody againstV.cholerae O1 recognizing Inaba and Ogawa serotypes.Appl MicrobiolBiotechnol.2013,97,4457–4466”)。
第一轮PCR反应体系(50μL)为：1μL cDNA，1μL上游引物，1μL下游引物，0.5μL DNA聚合酶，以去离子水补足余量；
第一轮PCR反应条件为：94℃ 4min、98℃ 10s、55℃ 15s、72℃ 45s、30 cycles、72℃ 7min；
第一轮PCR反应结果如图1(A)所示，可见PCR产物约为700bp；
第二轮PCR反应体系(50μL)为：1μL第一轮PCR产物，1μL上游引物，1μL下游引物，0.5μL DNA聚合酶，以去离子水补足余量；
第二轮PCR反应条件为：94℃ 4min、98℃ 10s、50℃ 15s、72℃ 40s、5 cycles，98℃ 10s、68℃ 40s、30 cycles、72℃ 7min；
第二轮PCR反应结果如图1(B)所示，可见PCR产物约为400bp。
(3)利用限制性内切酶sfiI分别对10μg纳米抗体基因(步骤(2)反应产物)和20μg噬菌体载体pComb3x进行酶切，50℃酶切4h；
将纳米抗体基因和载体pComb3x分别割胶回收，然后按摩尔比5:1进行混合，于16℃在T4ligase的作用下连接12h；
(4)取5μL步骤(3)获得连接产物加至80μL感受态细胞E.coliTG1中，充分混匀，冰上放置1min；然后转入0.1cm的电击杯中电击转化(电压为1.8kV)，立即向电击杯中加入900μL的SOC培养基，37℃180rpm培养1h；再将菌液涂布于SOB-AG平板，37℃倒置培养过夜；次日，从平板上随机挑取20个转化子，进行菌落PCR验证，分析目的基因正确插入率；其中纳米抗体基因插入正确的重组工程菌菌落PCR验证结果如图1(C)所示，可见其PCR产物约500bp；
上述菌落PCR验证程序为本领域常规程序：50μL PCR反应体系包含1μL菌液，1μL上游引物，1μL下游引物，0.5μL DNA聚合酶，以去离子水补足余量；
反应条件：94℃ 4min、94℃ 30s、55℃ 30s、72℃ 30s、30 cycles、72℃ 7min；
(5)将平板上所有的菌落洗下，合并混匀，用于噬菌体救援扩增，收集扩增后的噬菌体即为驼源天然纳米抗体文库。
实施例2 Cry1Ab模拟抗原的淘选及鉴定
(1)Cry1Ab模拟抗原的淘选
用10mM PBS(pH 7.4)将抗Cry1Ab单克隆抗体稀释至100μg/mL，包被Costar酶标板，4℃孵育过夜；用PBST(pH 7.4PBS，加入体积比为0.1％的Tween-20)洗涤3次，加入300μL的3％BSA-PBS(3％OVA-PBS)，37℃封闭2h；用PBST洗涤6次，加入100μL实施例1中制备得到的驼源天然纳米抗体库(滴度约2.0×1011pfu)，37℃孵育1h；用PBST洗涤10次，加入100μL的Glycine-HCl(0.2M,pH 2.2)洗脱8min后，立即用15μL Tris-HCl(1M，pH 9.0)中和，取10μL洗脱噬菌体测滴度，其余的用于感染20mL生长至对数前期的E.coli TG1菌株进行扩增，纯化后用于下一轮的筛选；在随后的3轮淘选中，包被的抗Cry1Ab单抗的浓度分别减少为50μg/mL，25μg/mL和10μg/mL，洗脱时采用竞争洗脱，所用Cry1Ab标准品浓度分别为5μg/mL，1μg/mL和0.5μg/mL，其余步骤同上。
(2)阳性噬菌体克隆的鉴定
从步骤(1)第三轮和第四轮的噬菌体滴度平板上随机挑取96个克隆，进行培养和扩增，随后采用phage-ELISA进行阳性噬菌体克隆的鉴定。
具体方法为：将Cry1Ab单抗用10mM PBS稀释至10μg/mL，4℃包被过夜；用PBST洗涤3次后，加入300μL的5％脱脂奶粉，37℃封闭2h；加入50μL噬菌体和50μL Cry1Ab标准品(1μg/mL)，37℃孵育1h；用PBST洗涤4次后，加入1:5000稀释的HRP标记抗M13二抗100μL，37℃孵育1h；加入100μL TMB底物溶液，避光显色10min，读取OD450，能结合Cry1Ab单抗，且能被Cry1Ab标准品所阻断的噬菌体克隆，初步鉴定为Cry1Ab的模拟抗原。将阳性噬菌体克隆进行测序，获得核苷酸序列如SEQ ID No.9所示；根据核苷酸测序结果及密码子表可翻译获得Cry1Ab模拟抗原的氨基酸序列，利用在线软件IMGT(www.imgt.org/)可对氨基酸序列进行分区，获得不同结构域(4个FR区和3个CDR区)的氨基酸序列，其中，框架区(FR1-FR4)的氨基酸序列分别如SEQ ID NO.2，SEQ ID NO.4，SEQ ID NO.6和SEQ ID NO.8所示，互补决定区(CDR1-CDR3)的氨基酸序列分别如SEQ ID NO.3，SEQ ID NO.5和SEQ ID NO.7所示。
实施例3 Cry1Ab模拟抗原的大量制备
(1)以噬菌体扩增的方式进行制备
将实施例2获得的阳性噬菌体克隆细胞接种于50mL 2×YT-AG培养基的三角瓶中，37℃220rpm振荡培养至OD600＝0.5；加入辅助噬菌体M13K07，37℃静置15min后，37℃220rpm继续培养45min；将培养物10000rpm离心10min，收集菌体，用50mL 2×YT-AK培养基重悬菌体，30℃ 220rpm振荡培养过夜；将培养物于4℃ 10000rpm离心10min，收集上清，加入1/6体积的PEG/NaCl，混匀后4℃静置4h；4℃ 10000rpm离心10min，弃上清，将沉淀重悬于1mL PBS中，加入等体积甘油于-80℃保存备用。
(2)以蛋白表达的形式进行制备
将噬菌粒载体pComb3x上的纳米抗体基因亚克隆至表达载体pET-26b中，再将重组表达载体转化至感受态细胞E.coli Rosetta中，将细胞培养后涂布于LB-A平板，37℃培养过夜；从平板上挑取单菌落接种于5mL LB培养基中，37℃，220rpm振荡培养过夜，将过夜培养物按1％接种量(v/v)接种于50mL的LB-AG培养基中，37℃，220rpm振荡培养；当培养物菌体浓度OD600达到0.5时，向培养物中加入终浓度为0.1mM的IPTG，30℃，220rpm振荡培养过夜；将培养物于4℃，8000rpm离心20min收集菌体沉淀；重悬细胞于5mL预冷的PBS溶液，超声破碎10min后，8000rpm离心20min取上清，即得纳米抗体粗提液；将上清经镍柱进行纯化，得到纯度90％以上的蛋白。
实施例4 Cry1Ab模拟抗原作为竞争抗原在ELISA中的应用
(1)样品提取
称取1g粉碎样品(市售大米、小麦和玉米)，加入5mL PBS(pH 7.4)溶液，充分振荡30min；10000rpm离心10min后，将提取液用whatman滤纸进行过滤，取1mL滤液与1mL PBS混匀，即为样品提取液，待用。
(2)包被及封闭
用10mM PBS(pH 7.4)将抗Cry1Ab单克隆抗体稀释至10μg/mL，4℃包被过夜。第二天用PBST洗涤3次，加入300μL的5％脱脂奶粉，37℃封闭2h后，用PBST洗板6次待用。
(3)标准曲线的建立
取出经步骤(2)处理好的板条，每孔分别投入实施例3步骤(1)获得的50μL展示有Cry1Ab模拟抗原的噬菌体(1.0×1011pfu)/实施例3步骤(2)获得的纳米抗体蛋白表达物(10μg/mL)及一系列不同浓度的50μL Cry1Ab标准品，37℃孵育1h。
加入HRP标记的抗M13二抗/抗His标签二抗，37℃孵育1h。然后用化学发光底物显色，读取化学发光强度。以Cry1Ab浓度对数为横坐标，结合率(加入Cry1Ab的孔的信号值/未加入Cry1Ab的孔的信号值×100％)为纵坐标，建立间接竞争标准曲线。
结果如图2所示，显示标准曲线的相关性系数R2为0.998，检测范围为10.49-307.1ng/mL，IC50为42.68ng/mL。
(4)样品的检测
取出经步骤(2)处理好的板条，每孔分别投入实施例3步骤(1)获得的50μL展示有Cry1Ab模拟抗原的噬菌体(1.0×1011pfu)/实施例3步骤(2)获得的纳米抗体蛋白表达物(10μg/mL)及不同浓度(50μg/kg、100μg/kg、200μg/kg和500μg/kg)的待测样品提取液，37℃孵育1h。加入HRP标记的抗M13二抗/抗His标签二抗，37℃孵育1h。然后用化学发光底物显色，读取化学发光强度，计算结合率，并根据标准曲线，测算出样品中Cry1Ab的含量，实验结果如表1所示：
表1 Cry1Ab毒素在大米、小麦和玉米样品中的加标回收实验结果
由表1可见，样品中Cry1Ab毒素的加标回收率为77.4％-126.7％，变异系数(CV)为2.7％-8.8％，表明基于抗独特型纳米抗体的Cry1Ab竞争免疫分析方法具有较好的准确性和实用性。
序列表
<110> 江苏省农业科学院
<120> 基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原及其应用
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Claims (6)
1.一种基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原，其氨基酸序列如SEQ ID NO.1所示。
2.一种编码如权利要求1所述基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原的核酸分子，其核苷酸序列如SEQ ID NO.9所示。
3.含有如权利要求2所述核酸分子的重组表达载体或重组工程菌。
4.如权利要求1所述基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原在非疾病诊断目的的免疫学检测分析中的应用。
5.如权利要求1所述基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原在制备Cry1Ab模拟物试剂中的应用。
6.如权利要求4所述基于抗独特型纳米抗体的Cry1Ab毒素模拟抗原的应用，其特征在于，所述应用是指，将该Cry1Ab模拟抗原作为竞争抗原，应用于免疫学检测分析。
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