CN117247439A - 一种广谱识别Cry3类毒素的多肽及应用 - Google Patents
一种广谱识别Cry3类毒素的多肽及应用 Download PDFInfo
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- CN117247439A CN117247439A CN202311396662.0A CN202311396662A CN117247439A CN 117247439 A CN117247439 A CN 117247439A CN 202311396662 A CN202311396662 A CN 202311396662A CN 117247439 A CN117247439 A CN 117247439A
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Abstract
本发明涉及一种广谱识别Cry3类毒素的多肽及应用,该多肽的氨基酸序列如SEQ ID NO.4所示,其为可以同时识别Cry3Aa和Cry3Bb原毒素的玉米根萤叶甲钙粘蛋白片段,可作为包被原应用于Cry3类毒素的ELISA检测中,以至少同时检测Cry3Aa和Cry3Bb两种毒素,灵敏度和准确度高,可用于农作物组织中Cry3类毒素的广谱性检测。
Description
技术领域
本发明涉及免疫学技术领域,特别涉及一种用于Bt毒素中Cry3类毒素(Cry3Aa和Cry3Bb毒素)的检测方法。
背景技术
目前,农业转基因产业化应用最多的是转基因作物。全球已经大规模推广种植的转基因作物,主要目标性状涉及抗虫、耐除草剂、抗菌、抗病毒、抗干旱等已广泛种植的抗虫作物包括转基因大豆、转基因棉花、转基因玉米和转基因水稻,它们的抗虫基因均主要源自苏云金芽孢杆菌(Bacillus thuringiensis,Bt)的Bt杀虫蛋白基因(Bt基因)。
Cry3类毒素是Bt Cry毒素家族中的一类对鞘翅目昆虫有杀虫活性的晶体蛋白,其中Cry3Aa和Cry3Bb蛋白由于对马铃薯甲虫具有很好的防治效果而被应用于转基因作物或被加工为生物杀虫剂。先正达公司开发的MZIR098玉米所含有的mCry3A蛋白用于防治玉米根虫(Diabrotica spp.)。Cry3Bb存在于商业产品Raven Oil Flowable生物杀虫剂中,该生物杀虫剂自1995年起已在美国销售用于防治鞘翅目害虫。拜耳公司利用cry3Bb基因开发的抗虫玉米MON863最初于2003年在美国获准进行商业化种植;2005年,转Cry3Bb基因的抗虫耐除草剂玉米MON 88017先后在美国和加拿大被批准商业化种植。这两种抗虫玉米随后在澳大利亚/新西兰、巴西、阿根廷、哥伦比亚、欧盟、日本、韩国、马来西亚、墨西哥、菲律宾、俄罗斯、南非、新加坡等地区获得了进口批准。
转基因成分检测是进行转基因生物研究和安全管理的重要技术保障。对转基因作物中Bt蛋白的快速检测方法主要包括酶联免疫吸附试剂盒(ELISA)和胶体金试纸条两种方式,但二者均需以特异性的抗体作为基础材料。抗体制备过程需经过动物免疫,制备周期长、成本高,因此开发不依赖于动物免疫的Bt蛋白高效检测方法已成为目前研发的热点。
利用受体-配体结合反应这一特性替代原有检测技术中应用的抗原-抗体免疫反应原理可有效减少产品的生产周期和生产成本,研究显示来源于鞘翅目昆虫玉米根萤叶甲的钙粘蛋白能够与Cry3Aa和Cry3Bb活化毒素结合,但此类蛋白在Cry3类毒素的检测研究或产品还未见报道。此外,目前市场上应用的Cry3类毒素的单克隆抗体通常只针对一种蛋白(毒素),同时检测Cry3Aa和Cry3Bb的单克隆抗体尚未见报道。
发明内容
针对上述问题,本申请提供一种可用于同时检测Cry3Aa和Cry3Bb毒素的玉米根萤叶甲钙粘蛋白片段、抗体以及基于该钙粘蛋白片段和单克隆抗体建立的Cry3类毒素广谱检测方法,实现同时检测Cry3Aa和Cry3Bb两类毒素的目的。
为实现上述发明目的,本申请提供了以下技术方案:
首先,本申请提供一种可与Cry3类毒素广谱结合的玉米根萤叶甲钙粘蛋白片段,该多肽的氨基酸序列如SEQ ID NO.4所示,该片段位于玉米根萤叶甲钙粘蛋白全长序列(GI:156144975所示)的第1182-1441位;申请人将该多肽片段自命名为DvvCAD-F-2。上述Cry3类毒素至少包括Cry3Aa和Cry3Bb毒素。
其次,本申请提供了上述玉米根萤叶甲钙粘蛋白片段DvvCAD-F-2作为包被原(包被蛋白)在Cry3类毒素Elisa检测中的应用。
第三,本申请提供一种广谱检测Cry3类毒素检测方法,其具体检测步骤如下:
1)将氨基酸序列如SEQ ID NO.4所示的玉米根萤叶甲的钙粘蛋白片段DvvCAD-F-2置于96孔板中,4℃环境下包被12-16h,包被缓冲液为pH9.6的CBS,包被浓度为10μg/ml,包被体积为100μl;
2)包被结束后,加入封闭液(含有终浓度为1%BSA的PBS缓冲液)200μl于37℃下封闭2h;
3)封闭结束后,用PBST洗涤3-5次,加入待测样品100μl,37℃下孵育1h;上述待测样品制备方法为本领域常规方法,如专利CN115128281A中所采用的样品处理方法。
4)PBST洗涤3-5次,加入100μl经PBST稀释后的5μg/mL的单克隆抗体4C7,37℃下孵育1h;
5)PBST洗涤3-5次,加入100μl经PBST稀释后的0.5μg/mL商品化HRP标记羊抗鼠抗体,37℃下孵育1h;
6)PBST洗涤3-5次,加入100μl显色底物,37℃下孵育15min;
7)加入100μl终止液,使用酶标仪在450nm处读取吸光值;
8)将测定样品的OD450值(曲线方程中Y值)带入标准曲线方程计算样品中
Bt毒素(曲线方程中x值)的含量。
其中,Cry3Aa毒素的曲线方程为:
Y=(2.735-0.236)/[1+(x/209.6)^(-1.185)]+0.236;
Cry3Bb毒素的曲线方程为:
Y=(3.045-0.475)/[1+(x/250.8)^(-1.51)]+0.475;
上述单克隆抗体4C7制备方法如下:该抗体由Cry3Aa毒素单独免疫动物,细胞融合筛选后分别用Cry3Aa和Cry3Bb两类毒素交替筛选获得。该交叉筛选方法为本领域常规筛选方法,如文献“Dong S,Zhang C,Zhang X,Liu Y,Zhong J,Xie Y,Xu C,Ding Y,Zhang L,Liu X.2016.Production and Characterization of Monoclonal Antibody BroadlyRecognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten.AnalyticalChemistry,88,7023-7032.”中所公开的方法。
第四,本申请还提供了一株保藏号为CCTCC NO:C2023318的Cry3毒素鼠杂交瘤细胞株4C7(Hybridoma cell line 4C7)。
第五,本申请还提供了一种单克隆抗体4C7,所述单克隆抗体4C7是由保藏号为CCTCC NO:C2023318的杂交瘤细胞株4C7所产生的。即通过向小鼠腹腔内注射保藏号为CCTCC NO:C2023318的杂交瘤细胞株4C7,取腹水后纯化(如利用饱和硫酸铵和protein-G柱进行纯化),即获得所述单克隆抗体4C7。
本申请利用大肠杆菌原核表达***制备了能够同时与Cry3Aa和Cry3Bb毒素结合的玉米根萤叶甲钙粘蛋白片段DvvCAD-F-2。同时利用单克隆抗体制备技术,采用交替筛选的策略获得了一株能够同时识别Cry3Aa和Cry3Bb毒素的单克隆抗体4C7。之后选择玉米根萤叶甲钙粘蛋白片段作为捕获受体,单克隆抗体作为检测抗体开发了基于受体-抗体夹心的Cry3类毒素的广谱检测方法,可同时检测Cry3Aa和Cry3Bb毒素,检测限分别为32.85ng/ml和58.51ng/ml,回收率均在84.3%-92.8%之间。适宜对农作物或农产品中的Cry3类毒素进行定性和定量检测。
附图说明
图1为实施例1中基于广谱识别的Cry3类毒素检测方法的原理示意图。
图2为广谱识别Cry3Aa和Cry3Bb毒素的阳性杂交瘤细胞株的筛选结果。
图3为单克隆抗体4C7腹水纯化抗体的SDS-PAGE检测。
图4为来自棉铃虫(Helicoverpaarmigera)、烟芽夜蛾(Heliothisvirescens)、棉红铃虫(Pectinophora gossypiella)、小菜蛾(Plutella xylostella)、稻纵卷叶螟(Chaphalocrocis medina)、粉纹夜蛾(Trichoplusis ni)和玉米根萤叶甲(Diabroticavirgifera virgifera)的钙粘蛋白与Cry毒素结合的保守结构域的序列比对图。用于分析的序列来源于NCBI数据库,上述序列的登录号分别为JN836544、AAK85198.1、ASX13586.1、NM001305494、QNS31153.1、XP_026727179.1、EF531715.1。
图5为玉米根萤叶甲钙粘蛋白片段结构示意图。
图6为玉米根萤叶甲钙粘蛋白片段原核表达的Western检测。
图7为玉米根萤叶甲钙粘蛋白片段经镍柱纯化后的SDS-PAGE检测。
图8为玉米根萤叶甲钙粘蛋白片段与Cry3Aa和Cry3Bb毒素的配基结合检测。
图9为四参数拟合下广谱识别的Cry3类毒素检测方法针对Cry3Aa和Cry3Bb毒素的标准曲线。
具体实施方式
为方便理解本发明所述技术方案,以下结合附图和具体实施例做进一步阐述,下列实施例仅用于说明而非是对本发明权利要求范围的限制;下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和生物材料,如无特殊说明,均从商业途径获得。
实施例1Cry3Aa和Cry3Bb毒素广谱单克隆抗体的制备
1.1动物免疫
选用6-8周龄健康Balb/c小鼠(SPF级)5只(购自扬州大学比较医学中心),尾静脉采血,制备阴性血清,之后选用商品化Cry3Aa毒素标准品(购自美延(北京)农业科技有限责任公司)免疫5只小鼠。
采用注射器互推法乳化抗原,制备血清时,将采集的小鼠新鲜血液在室温下放置1-2h后,置于4℃过夜,使血清分离。次日,将其在4℃,10000g下离心10min,收集上清液即为所需要的血清。免疫及采血方案如表1所示。
表1免疫采血方案
之后对血清效价进行测定,测定取小鼠眼眶血,通过间接ELISA法进行血清效价测定。具体步骤为:
(1)包被抗原:用包被液(0.01M PBS)稀释抗原,按照2ug/ml,100ul/孔,4℃过夜包被;洗涤液(Tris-HCl+0.01%Tween-20%)洗3次,300ul/孔,拍干。
(2)封闭:200ul/孔加入封闭液(0.01M PBS+1%酪蛋白+0.1%Tween-20),37℃孵育2h;洗涤液洗3次,300ul/孔,拍干。
(3)以封闭液1:1000倍稀释小鼠血清,然后按照二倍稀释,进行加样,100ul/孔,37℃孵育1h;洗涤液洗3次,300ul/孔,拍干。
(4)将商品化二抗(HRP山羊抗小鼠IgG,购自博奥龙BIODRAGON)按照1:10000比例用封闭液稀释,100ul/孔,37℃孵育1h;洗涤液洗3次,300ul/孔,拍干。
(5)显色:100ul/孔显色液(TMB单组份显色液),室温孵育10min。
(6)读数:100ul/孔终止液(0.5M H2SO4),酶标仪中于450nm处读取吸光值。
检测小鼠第二次加强免疫7天后的效价如表2所示,5只小鼠均产生了针对Cry3Aa毒素的抗体,但1号小鼠的血清效价最高,因此选择1号小鼠进行腹腔冲击免疫。
表2检测小鼠第二次加强免疫7天后的效价表
1.2细胞融合
取1号小鼠脾脏与Sp2/0细胞(购自博奥龙BIODRAGON),采用PEG法进行融合;融合完的细胞在含有HAT的半固体培养基(购自博奥龙BIODRAGON,货号:BTYA0610-90ml)中进行筛选培养。具体步骤为:
(1)将状态良好的Sp2/0细胞轻柔地从培养瓶上吹打下来,放入50ml离心管中。
(2)小鼠摘眼球取血,然后拉颈处死,放入75%的酒精中浸泡5min。
(3)在平皿中倒入少量的无血清IMDM培养基(购自Gibco,货号12440061),将细胞筛及注射器内芯放入平皿中,用剪刀和镊子取下小鼠的脾脏,放到细胞筛上。用注射器的内芯轻轻地将脾脏充分碾碎,将研磨好的细胞吸入装有Sp2/0的离心管中,离心1500rpm,10min。
(4)用剪刀和镊子取下小鼠的胸腺,碾碎。将碾好的细胞放入15ml离心管中,再加入2mlHAT、1mlHT,放到培养箱中备用。
(5)将离心好的脾脏细胞,倒掉上清。用无血清的IMDM将离心好的细胞小心轻柔地吹匀与Sp2/0细胞混合,Sp2/0:脾脏细胞=1:3~1:10;离心1500rpm,10min。
(6)将离心好的细胞上清尽量倒掉。拍打离心管底充分混合细胞,将离心管放入37℃温水中,在1分钟内缓慢加入1mlPEG,加完后,在温水中静置1min。然后2min内缓慢加入8ml的无血清的IMDM,离心1000rpm,10min。
(7)倒掉上清,加入10ml的血清,小心地将细胞吹匀,再倒入5ml的HybridomaFeeder添加因子(购自博奥龙BIODRAGON),之前准备好的胸腺细胞混合物(2ml胸腺+2mlHAT+1mlHT),再加入25ml的含有HAT的半固体培养基,充分混匀。然后均匀倒入30个细胞培养皿中。将细胞培养皿放入湿盒中,放入培养箱中培养11~14天,待到克隆长到肉眼可见的时候,使用体视镜将克隆挑出。
1.3单克隆细胞筛选
使用20%新生牛血清+HT+10%Hybridoma Feeder添加因子进行铺板,150ul/孔,挑1100个克隆用于杂交瘤细胞第一次筛选。以Cry3Aa毒素为包被原,用间接ELISA检测细胞培养上清液,以融合之前所取的小鼠血清作阳性对照,以未融合细胞及饲养细胞的培养上清液作为阴性对照。
间接ELISA检测方法操作步骤与1.2中血清效价检测部分基本相同,只是在加样步骤中,本实验所加样为细胞培养上清。
将间接ELISA验证中OD450值高且细胞生长状态好的细胞孔内的细胞采用有限稀释法进行亚克隆,亚克隆2-3次之后,对所有孔均为阳性,此时得到14株阳性杂交瘤细胞株。
对得到的14株阳性杂交瘤细胞株扩大培养后进行第二次筛选,以Cry3Bb毒素为包被原,其余步骤与第一次筛选相同,共得到8株阳性杂交瘤细胞株。
1.4抗体亚型测定
对筛选获得的8株具有Cry3Aa和Cry3Bb广谱识别能力的阳性杂交瘤细胞株进行亚类鉴定,并分别自命名,鉴定结果如表3所示:
表3阳性杂交瘤细胞株的抗体亚型测定表
结果显示8株阳性细胞株的抗体亚型均为IgG亚型,有κ轻链。其中细胞株4C7对两种毒素的识别能力均较高,故选用该细胞株进行后续腹水的制备和抗体纯化。图2为8种阳性杂交瘤细胞株的筛选结果。申请人于2023/10/21将该Cry3毒素鼠杂交瘤细胞株4C7(Hybridoma cell line 4C7)保存于中国典型培养物保藏中心(CCTCC),其地址为:湖北省武汉市武昌区八一路299号,邮编:430072,保藏号为CCTCC NO:C2023318。
1.5腹水制备和抗体纯化
选择2只小鼠,分别向小鼠腹腔内注射阳性杂交瘤细胞株4C7,7天后检测,每只小鼠产生约5mL腹水,将腹水收集后利用饱和硫酸铵和protein-G柱进行纯化,SDS-PAGE结果(如图3所示)显示,纯化后的单克隆抗体纯度较高,测定浓度约为2mg/ml,可用于候选检测方法的建立。
上述利用饱和硫酸铵和protein-G柱纯化步骤参考文献“Dong S,Zhang C,ZhangX,Liu Y,Zhong J,Xie Y,Xu C,Ding Y,Zhang L,Liu X.2016.Production andCharacterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins byUse of Designed Polypeptide as Hapten.Analytical Chemistry,88,7023-7032.”中所公开的方法。本实施例中饱和硫酸铵溶液配置方法将500g硫酸铵加入500mL蒸馏水中,加热使其完全溶解;室温放置过夜,析出的结晶留在瓶内,用前取所需量,用NaOH调节pH至7.8。纯化试验所使用的protein-G柱购自Cytiva商品化的预装柱。
实施例2两种玉米根萤叶甲钙粘蛋白片段的原核表达
文献报道Park等对玉米根萤叶甲钙粘蛋白片段(CR8-10)(对应961-1329位氨基酸残基)进行了表达修饰,并测定修饰片段与胰蛋白酶活化的Cry3Aa和Cry3Bb毒素均具有一定的结合能力(Park Y,Abdullan MAF,Taylor MD,Rahman K,Adang MJ.2009.Enhancementof Bacillus thuringiensisCry3Aa and Cry3Bb Toxicities to Coleopteran Larvaeby a Toxin-Binding Fragment of an Insect Cadherin.Applied and EnvironmentalMicrobiology,(10):3086-3092.),但未涉及Cry3Aa和Cry3Bb原毒素的亲和性。
申请人将该片段命名为DvvCAD-F-1,其核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
昆虫钙粘蛋白上约19个氨基酸残基组成的保守结构域与Cry毒素的结合相关,通过同源序列比对发现DvvCAD-F-1片段中不含有该结构域(对应1396-1422位氨基酸残基)(附图4-5),因此,同样采用PET-26b(+)原核表达***在大肠杆菌中表达了包含该结构域的玉米根萤叶甲钙粘蛋白片段(对应1182-1441位氨基酸残基),申请人将该片段命名为DvvCAD-F-2,其对应核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ ID NO.4所示。
DvvCAD-F-1和DvvCAD-F-2这两种钙粘蛋白片段基因序列委托安徽通用生物公司合成并构建到pET-26b(+)载体上,并将重组质粒转入菌株E.coli BL21(DE3)中。
将测序验证正确的单菌落接种于10mL具有卡那霉素抗性的2XTY液体培养基(每1L培养基配方为:胰蛋白胨16g,酵母提取物10g,NaCl 5g,双蒸水溶解后定容至1L,分装后121℃高压灭菌)中,37℃过夜振荡培养,得到种子液。次日,按1:100的比例将种子液接种到200mL含卡那霉素的2XTY液体培养基中,37℃,250rpm震荡培养至OD600为0.6-1.0,加入IPTG至终浓度1mM,37℃250rpm,培养16h,诱导目的蛋白的表达。将诱导表达的菌液,4℃10000rpm离心15min,收集菌体加入20mL重悬液重悬(25mMTris-HCl,150mM NaCl,15mM咪唑),混匀后于冰浴中超声波破碎,每隔2s超声3s,60w,30min,使菌体充分破碎。破碎后的菌体沉淀用10mL溶解液进行重悬(20mM Tris,0.5M NaCl,8M尿素,20mM咪唑),混匀后于4℃放置过夜,之后4℃10000rpm离心30min,离心后取上清于-20℃保存(即为实施例3未纯化的上清蛋白),备用。
由于上述两种表达产物C末端均带有His标签,利用anti-His抗体进行Westernblot检测,结果显示两种钙粘蛋白片段均成功表达,其中DvvCAD-F-1片段分子量大小约为57kd,DvvCAD-F-1片段分子量大小约为37kd(附图6)。保存的上清解冻后通过His-Trap亲和柱(购自Cytiva品牌的HisTrapTMHP的5ML预装柱,依照产品说明书步骤进行纯化),纯化结果如图7所示,图7中,A、B分别为DvvCAD-F-1、DvvCAD-F-2的纯化结果。纯化获得的蛋白分别通过GE Desalting脱盐柱(Cytiva品牌的HiTrapTMDesalting的5ML预装柱,处理步骤参照说明书)脱盐处理。采用考马斯亮蓝法对蛋白浓度进行测定,每升培养物大约可获得2-3mg纯化蛋白。
实施例3两种玉米根萤叶甲钙粘蛋白片段与Cry3Aa和Cry3Bb毒素的结合活性鉴定
本实施例选用配基结合来分析两种玉米根萤叶甲钙粘蛋白片段与Cry3Aa和Cry3Bb毒素的结合特性。
在配基结合实验中,取10μg实施例2获得的未纯化的上清蛋白与对应体积的4×上样缓冲液混合,95℃加热5min进行变性,之后通过10%的SDS-PAGE胶进行分离;利用“三明治”结构将胶上的蛋白条带转移至PVDF膜,4℃条件下60V转移2h;将转好的PVDF膜用2.5%的BSA封闭2h,之后加入20nM Cry3Aa和Cry3Bb毒素37℃孵育1h,PBST洗涤3次后,加入经PBST稀释后的1μg/mL实施例1中的单克隆抗体4C7于37℃孵育1h,PBST洗涤3次后,加入经PBST稀释后的0.5μg/mL商品化HRP标记羊抗鼠抗体37℃孵育1h,将洗干净的膜置于显色工作液中5min后,用Versa DOC化学发光凝胶成像***拍摄。
结果如图8所示,图8中,A、B分别为蛋白与Cry3Aa、Cry3Bb毒素的配基结合检测结果。可见DvvCAD-F-2与Cry3Aa和Cry3Bb均具有结合活性,但DvvCAD-F-1与两种毒素均不结合。
实施例4基于广谱识别的Cry3Aa和Cry3Bb毒素检测方法的建立
1.1建立Cry3Aa和Cry3Bb毒素的检测标准曲线
采用夹心法检测Cry3Aa和Cry3Bb毒素,具体步骤如下:
(1)包被:玉米根萤叶甲的钙粘蛋白片段DvvCAD-F-2置于96孔板中,在4℃环境下包被12-16h,包被缓冲液为pH9.6的CBS,包被浓度为10μg/ml,包被体积为100μl;
(2)封闭:用PBST洗板3-5次,每次10s,300μl/孔,拍干,之后加入封闭液(含有1%BSA的PBS),200μl/孔,37℃孵育2h。
(3)加样:用PBST洗3-5次,每次10s,300μl/孔,拍干,之后加入梯度稀释的毒素样品样品100μl,37℃下孵育1h;
Cry3Aa和Cry3Bb两种毒素的浓度依次为2500、1250、625、312.5、156.25、78.13、39.06、19.53、9.76、4.88、2.44、0ng/ml,每个浓度设置3个重复;
(4)一抗孵育:用PBST洗3-5次,每次10s,300μl/孔,拍干,之后加入100μl经PBST稀释后的5μg/mL的单克隆抗体4C7,37℃下孵育1h;
(5)二抗孵育:用PBST洗3-5次,每次10s,300μl/孔,拍干,加入100μl经PBST稀释后的0.5μg/mL商品化HRP标记羊抗鼠抗体(购自博奥龙BIODRAGON),37℃下孵育1h;
(6)显色:用PBST洗3-5次,每次10s,300μl/孔,拍干,之后加入TMB单组分显色液(购自索莱宝,Solarbio),100μl/孔,37℃孵育10-15min。
(7)读数:加入100μl终止液(购自索莱宝,Solarbio),使用酶标仪在450nm处读取吸光值;
(8)数据分析:Cry3Aa和Cry3Bb毒素与对应的吸光值具有良好的剂量效应关系。利用四参数法进行标准曲线方程的拟合,所获的的标准曲线如图9所示,图9中,A、B分别为Cry3Aa和Cry3Bb毒素的标准曲线。
Cry3Aa毒素的标准曲线方程为:
Y=(2.735-0.236)/[1+(x/209.6)^(-1.185)]+0.236,R2=0.988,SC50为209.6ng/ml,检测限(LOD,SC10)为32.85ng/ml,线性区间(SC10-SC90)为32.85-1339ng/ml,方程中Y为OD450值,x为Cry3Aa毒素浓度。
Cry3Bb毒素的标准曲线方程为:Y=(3.045-0.475)/[1+(x/250.8)^(-1.51)]+0.475,R2=0.993,SC50为250.8ng/ml,检测限(LOD,SC10)为58.51ng/ml,线性区间(SC10-SC90)为58.51-1075ng/ml,方程中Y为OD450值,x为Cry3Bb毒素浓度。
以上检测原理示意图如图1所示。
以上标准曲线建立方法为本领域常规方法,如文献“Dong S,Gao M,Guan L,ZhangH,Wang Y,Liu B,Li P,Qiao K,Liu X,Zhang C.2020.Construction,Expression,andIdentification of Double Light Chain(VL-VL)Antibody from a Unique Bt Cry1-Specific Monoclonal Antibody.Food Analytical Methods,13:1570-1582.”、“侯亚莉等,人血清中Hib荚膜多糖抗体定量的ELISA方法建立及初步验证.微生物学免疫学进展,40(3):34-38.2012”所公开。
1.2添加回收试验
将玉米叶片剪成1mm2大小的碎片,然后利用冷冻干燥机器进行对待检样品进行24h的冷冻(-75℃)干燥处理,收集冷冻干燥处理后的样品。将不同浓度的毒素样品添加到叶片中,称取5mg的样品于1.5ml的离心管中,加入1ml样品提取液,颠倒混匀后,置于旋转仪上,40rpm摇匀40min后,10000rpm离心20min,取上清液用于样品测定(该样品处理方法为本领域常规方法,如中国专利202210751409.1所公开)。结果如表4所示:
表4Cry3Aa和Cry3Bb毒素在玉米叶片样品中的添加回收率
表4检测结果显示,两种毒素在玉米叶片样品中的添加回收率在84.3%-92.8%之间,变异系数在4.6-9.1之间。表明,该检测方法具有较高的准确性,可以用于农作物或农产品样品中Cry3Aa和Cry3Bb毒素的定量检测。
Claims (8)
1.一种广谱识别Cry3类毒素的多肽,其特征在于,所述多肽的氨基酸序列如SEQ IDNO.4所示。
2.如权利要求1所述多肽的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQID NO.3所示。
3.如权利要求1所述多肽作为包被蛋白在检测Cry3类毒素中的应用。
4.如权利要求3所述的应用,其特征在于,所述Cry3类毒素至少包括Cry3Aa和Cry3Bb毒素。
5.一种广谱检测Cry3类毒素检测方法,其特征在于,具体检测步骤如下:
1)将氨基酸序列如SEQ ID NO.4所示的多肽在4℃环境下包被12-16h;
2)包被结束后,加入封闭液,37℃下封闭2h;
3)封闭结束后,用PBST洗板3-5次,加入待测样品100µl,37℃下孵育1h;
4)BST洗板3-5次,加入100µl 经PBST稀释后的5 μg/mL的单克隆抗体,37℃下孵育1h;所述单克隆抗体由Cry3Aa毒素单独免疫动物,细胞融合筛选后分别用Cry3Aa和Cry3Bb两类毒素交替筛选获得;
5)PBST洗涤3-5次,加入100µl 经PBST稀释后的0.5 μg/mL商品化HRP标记羊抗鼠抗体,37℃下孵育1h;
6)PBST洗涤3-5次,加入100µl显色底物,37℃下孵育15min;
7)加入100µl终止液,使用酶标仪在450nm处读取吸光值;
8)将测定样品的OD450值带入标准曲线方程计算样品中Bt毒素的含量;
其中,Cry3Aa毒素的曲线方程为:
Y =(2.735-0.236)/[1+(x/209.6)^(-1.185)]+0.236;
Cry3Bb毒素的曲线方程为:Y=(3.045-0.475)/[1+(x/250.8)^(-1.51)]+0.475。
6.如权利要求5所述的方法,其特征在于,步骤4)所述的单克隆抗体是由保藏号为CCTCC NO:C2023318的杂交瘤细胞株4C7所产生的。
7.一株保藏号为CCTCC NO:C2023318的Cry3毒素鼠杂交瘤细胞株4C7(Hybridoma cellline 4C7)。
8.一种单克隆抗体,所述单克隆抗体是由保藏号为CCTCC NO:C2023318的杂交瘤细胞株4C7所产生的。
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