CN108314711B - 一种具有免疫原性的cry1C重组蛋白、分离的核酸分子及其应用 - Google Patents
一种具有免疫原性的cry1C重组蛋白、分离的核酸分子及其应用 Download PDFInfo
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Abstract
本发明提供一种具有免疫原性的cry1C重组蛋白，涉及基因工程技术领域，其氨基酸序列如SEQ ID No.1所示。编码该cry1C重组蛋白的氨基酸序列长度短于全长cry1C蛋白的氨基酸序列，且该cry1C重组蛋白能够与cry1C抗体发生免疫反应，能够用于快速高效地制备cry1C抗体。本发明还提供一种编码上述具有免疫原性的cry1C重组蛋白的核酸、能够扩增或检测该核酸分子的引物对以及包括该核酸分子的载体和宿主细胞，此外，本发明还提供一种cry1C抗体及其制备方法，该制备方法能够快速高效地获得cry1C抗体。
Description
技术领域
本发明涉及基因工程技术领域，具体而言，涉及一种具有免疫原性的cry1C重组蛋白、分离的核酸分子及其应用。
背景技术
苏云金芽孢杆菌(Bacillus thuringiensis Berliner,简称Bt)能产生对多种昆虫有害的杀虫晶体蛋白(Insecticidal Crystal proteins,ICPs)。Bt杀虫晶体蛋白(ICP)可特异性毒杀不同的昆虫，包括鳞翅目、鞘翅目、双翅目、螨类等害虫，而对人畜无害，是目前农业生产转基因育种中主要使用的抗虫蛋白。
水稻是我国的重要粮食作物之一。水稻害虫是水稻丰产和稳产的重要限制因子之一，每年给我国水稻生产造成的损失量可占总产量的5％～10％，其中鳞翅目害虫是水稻生产危害最大的害虫。
cry1C基因编码的Bt杀虫蛋白可以抑制和杀死鳞翅目害虫。利用生物技术手段将cry1C基因导入到各种植物中，包括水稻、大豆、玉米以及油菜等，能够获得对抗鳞翅目害虫具有抗性的转基因植物。随着转基因技术的发展，国内外大量的转基因植物已进入生物安全评价阶段，部分产品已进入商业化阶段。现有的cry1C抗体制备时间长，且成本高，如何快速、精准地检测cry1C蛋白及其相关产品是如今面临的问题。
发明内容
本发明的第一目的在于提供一种具有免疫原性的cry1C重组蛋白，其氨基酸序列长度短于全长cry1C蛋白的氨基酸序列，且能够与cry1C抗体发生免疫反应，用于快速高效地制备cry1C抗体。
本发明的第二目的在于提供一种分离的核酸分子，其能够编码上述具有免疫原性的cry1C重组蛋白，该核酸分子能够在宿主细胞中能够高效表达该cry1C重组蛋白，且该cry1C重组蛋白的氨基酸序列长度短于全长的cry1C蛋白的氨基酸序列，该cry1C重组蛋白能够与cry1C抗体发生免疫反应且用于高效快速地制备cry1C抗体。
本发明的第三目的在于提供一种能够扩增或检测上述核酸分子的引物对。
本发明的第四目的在于提供一种载体，其包括有上述核酸分子。
本发明的第五目的在于提供一种宿主细胞，其包括有上述核酸分子和上述载体。
本发明的第六目的在于提供上述蛋白在制备cry1C抗体中的应用。
本发明的第七目的在于提供一种制备上述cry1C重组蛋白的方法。
本发明的第八目的在于提供一种cry1C抗体的制备方法，其能够快速、高效地制备出该抗体，具有成本低、耗时短等优点。
本发明的第九目的在于提供一种cry1C抗体，其由上述制备方法制得。
本发明是这样实现的：
一种具有免疫原性的cry1C重组蛋白，该蛋白的氨基酸序列如SEQ ID No.1所示。
一种分离的核酸分子，其编码上述蛋白，该核酸分子的核苷酸序列如SEQ ID No.2所示。
一种扩增或检测上述核酸分子的引物对。
一种载体，其包括有上述核酸分子。
一种宿主细胞，其包括上述核酸分子或上述载体。
上述蛋白在制备cry1C抗体中的应用。
一种制备上述蛋白的方法，其包括培养上述宿主细胞。
一种cry1C抗体的制备方法，其包括步骤：用上述cry1C重组蛋白免疫动物。
一种cry1C抗体，其是由上述制备方法制成。
本发明具有以下有益效果：
本发明提供一种具有免疫原性的cry1C重组蛋白，其氨基酸序列如SEQ ID No.1所示。编码该cry1C重组蛋白的氨基酸序列长度短于全长的cry1C蛋白的氨基酸序列，且该cry1C重组蛋白能够与cry1C抗体发生免疫反应，能够用于快速高效地制备cry1C抗体。本发明还提供一种编码上述具有免疫原性的cry1C重组蛋白的核酸分子、能够扩增或检测该核酸分子的引物对以及包括该核酸分子的载体和宿主细胞，该核酸分子在宿主细胞中能够高效表达上述具有免疫原性的cry1C重组蛋白。此外，本发明还提供一种cry1C抗体及其制备方法，该制备方法能够快速高效地获得cry1C抗体。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明第一实施例中的核酸分子的PCR产物的扩增结果；
图2为本发明第一实施例中的pET28b(+)-cry1C质粒用NdeI和XhoI双酶切后的电泳图；
图3为本发明第一实施例中的His6-cry1C重组蛋白的SDS-PAGE结果图；
图4为本发明第一实施例中的纯化后的His6-cry1C重组蛋白的SDS-PAGE结果图；
图5为本发明第一实施例中cry1C抗体效价结果图；
图6为本发明第一实施例中的cry1C转基因水稻叶片的Western Blot结果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本发明实施例的一种具有免疫原性的cry1C重组蛋白、分离的核酸分子及其应用进行具体说明。
本发明实施例提供一种具有免疫原性的cry1C重组蛋白。该cry1C重组蛋白的氨基酸序列如SEQ ID No.1所示。上述免疫原性在于，该cry1C重组蛋白能够与cry1C抗体发生免疫反应。且该具有免疫原性的cry1C重组蛋白的氨基酸序列要短于cry1C蛋白的氨基酸序列，且能够高效、快速地制备抗cry1C的抗体。
本发明实施例提供一种分离的核酸分子。该核酸分子编码上述具有免疫原性的cry1C重组蛋白，该核酸分子的核苷酸序列如SEQ ID No.2所示。该核酸分子在宿主细胞(如大肠杆菌)中，能够高效表达上述具有免疫原性的cry1C重组蛋白，克服了外源基因在宿主细胞(大肠杆菌)中表达率不高的技术障碍，使得上述具有免疫原性的cry1C重组蛋白能够高效地在宿主细胞中转录并表达。
本发明实施例还提供一种扩增或检测上述核酸分子的引物对。进一步地，该引物对包括如SEQ ID No.3所示的上游引物和/或如SEQ ID No.4所示的下游引物。
本发明实施例还提供一种载体，其包括有上述核酸分子。进一步地，该载体是可以指表达载体或克隆载体。在本发明实施例中，该载体为原核表达载体，可以为pET28a(+)、pET28b(+)、pET30a(+)、pET30b(+)以及pET30c(+)中的任意一种。
本发明实施例还提供一种宿主细胞，其包括有上述核酸分子或上述载体。进一步地，在本发明中，宿主细胞为大肠杆菌：
载体构建过程中使用的宿主细胞可以为大肠杆菌DH5α、DH10B和JM109中的任意一种；重组蛋白诱导表达过程中使用的宿主细胞为BL21(DE3)。
另一方面，本发明实施例提供上述蛋白在制备cry1C抗体中的应用。
另一方面，本发明还提供一种cry1C抗体的制备方法，其包括步骤：用上述的cry1C重组蛋白免疫动物。上述已知该cry1C重组蛋白的氨基酸序列短于全长的cry1C蛋白的氨基酸序列，但该cry1C重组蛋白依然能够与cry1C抗体发生免疫反应，利用该cry1C重组蛋白能够更针对性地制得cry1C抗体，且其具有用时更短，成本更低，实用性更强等优点。
另一方面，本发明还提供一种cry1C抗体，其是由上述制备方法制得。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
第一实施例
本实施例提供的一种cry1C抗体及其制备方法。
1表达载体的构建
本发明提供的一种分离的核酸分子，该核酸分子为优化后人工合成的cry1C片段，其编码一种具有免疫原性的cry1C重组蛋白，该核酸分子的核苷酸序列如SEQ ID No.2所示。将该核酸分子作为模板进行PCR扩增，在PCR扩增过程中，采用扩增该核酸分子的引物对，引物对包括如SEQ ID No.3所示的上游引物和/或如SEQ ID No.4所示的下游引物。
具体地，上游引物的序列为：5`-CATATGGTCGAAGCATTCAAGGA-3`(下划线序列为NdeI酶切位点)；
下游引物的序列为：5`-CTCGAGTTATTTCTGCGCGCGTT-3`(下划线序列为XhoI酶切位点)。
利用该引物对核酸分子进行扩增后，得到扩增后的PCR产物，然后用电泳实验确定PCR扩增效果，请参照附图1，附图1为核酸分子的PCR扩增产物的电泳图，图1中，M为DNA标准分子量，1为核酸分子的PCR扩增结果，可以看出，PCR扩增效果与核酸分子的大小(1578bp)相匹配。
将该核酸分子与原核表达载体pET28b(+)质粒用NdeI和XhoI进行双酶切，然后用T4DNA连接酶把核酸分子与表达载体连接得到连接产物。将该连接产物转化到大肠杆菌感受态菌株TOP10，筛选阳性克隆，提取得到重组质粒pET28b(+)-cry1C，做电泳实验确认重组质粒的片段大小，采用NdeI和XhoI对重组质粒进行双酶切，双酶切后的片段大小如图2所示。附图2中，M为DNA标准分子量，1为pET28b(+)-cry1C质粒用NdeI和XhoI双酶切后的电泳图，由附图2可以看出，双酶切后，得到了两个片段，分别为5.3kb和1.6kb左右，5.3kb为载体片段，1.6kb为cry1C片段(核酸分子)，与预期片段的大小一致，表明该cry1C片段已正确插入到pET28b(+)载体之中。
2.cry1C重组蛋白(具有免疫原性的cry1C重组蛋白)的诱导性表达与可溶性分析
将上述重组质粒pET28b(+)-cry1C转化到大肠杆菌感受态细胞BL21(DE3)中，分为3组，其中两组加入0.5mM的IPTG诱导剂进行诱导表达，分别在20℃过夜和37℃诱导表达4h，未加IPTG诱导剂的一组作为阴性对照。在5000rpm，4℃的条件下将3组大肠杆菌离心10min，将离心收集的菌体用PBS缓冲液悬浮，冰浴下超声破碎，在12000rpm、4℃条件下离心30min，分别收集上清和沉淀。沉淀均采用500μL包涵体溶解液(8M Urea，50mM Tris-HCl，150mMNaCl，pH 8.0)溶解。
上清液和溶解的包涵体分别与蛋白质上样悬浮液(protein loading buffer)混匀、沸水处理10min，通过SDS-PAGE电泳，考马斯亮蓝染色液染色，观察分析重组蛋白存在的部分。
SDS-PAGE结果请参照附图3(附图中，M:Protein Marker；1：诱导前总蛋白；2：20℃上清；3：20℃沉淀；4：37℃上清；5：37℃沉淀)，由附图3可知，在包涵体中含有60kD左右的cry1C重组蛋白，其表达量高，且蛋白大小与预期大小一致，可以确定该蛋白就是cry1C重组蛋白，说明该蛋白在大肠杆菌包涵体中成功表达。
3.cry1C重组蛋白的扩增、纯化及鉴定
将含有重组质粒pET28b(+)-cry1C的BL21(DE3)菌液进行扩增培养至4L，在该菌液中加入0.5mM的IPTG诱导剂进行诱导表达，37℃诱导表达重组质粒4h，然后离心(5000rpm，4℃，离心20min)收集表达后的菌体。将收集的菌体用破碎缓冲液(20mM PB，300mM氯化钠，0.1％Triton X-100，1mM二硫苏糖醇，pH 8.0)溶解，冰浴中超声破碎菌体，弃上清、取沉淀。将收集的沉淀用破碎缓冲液(8mM尿素，20mM PB，300mM氯化钠，0.1％TritonX-100，1mM二硫苏糖醇，pH 8.0)溶解，冰浴中超声破碎菌体，离心(12000rpm，4℃，离心20min)后取上清，上清液含有His6-cry1C重组蛋白(His6为pET28b(+)中含有的标签蛋白)，得到的His6-cry1C重组蛋白采用镍琼脂糖亲和层析方法进行纯化，该蛋白经过0.45μm滤膜之后上样于Ni-NTASpin Columns亲和层析柱(Qiagen)结合1h，重复过柱3次，用洗涤液(Wash buffer：20mM/50mM Imidazole，8mM Urea，20mM PB，300mM NaCl，0.1％TritonX-100，1mM DTT，pH 8.0)洗杂4次，经不同浓度的咪唑洗脱缓冲液(Imidazole Elution buffer：20/50/500mM咪唑基，8mM尿素，20mM PB，300mM氯化钠，pH 8.0)洗脱。将纯度较好的500mM咪唑基洗脱组分透析至10mM磷酸盐缓冲液，0.1％SKL，pH8.0中，浓缩，过滤除菌得到纯化后的cry1C重组蛋白，将其保存于零下80℃。
纯化后的cry1C重组蛋白用SDS-PAGE电泳分析，电泳结果如附图4所示，该重组蛋白在相应位置出现明显条带，得到了60kD的His6-cry1C重组蛋白，该结果与预期结果相匹配。
4.一种cry1C抗体
cry1C多克隆抗体的制备
将纯化后的cry1C重组蛋白与弗氏完全佐剂(Sigma)混合，分别对4只4月龄新西兰大白兔(标记为A；B；C；D)按常规免疫周期进行免疫注射。共注射4次，第一次免疫(一免)后的第21天、第35天和第49天分别为第二次免疫(二免)、第三次免疫(三免)、和第四次免疫(四免)时期。第一次免疫用0.3mg cry1C重组蛋白与等体积弗氏完全佐剂混合进行，第二次免疫、第三次免疫、和第四次免疫用0.15mg cry1C重组蛋白与等体积弗氏完全佐剂混合后进行加强免疫注射。第三次免疫和第四次免疫后7～10天从兔耳缘静脉取血1mL，采用抗体间接ELISA法测定抗体效价，以免疫前的兔血清作为阴性对照。
抗体间接ELISA法具体为：将上述cry1C重组蛋白用0.05mol/L的碳酸盐缓冲液(pH9.6)稀释成2ng/μL后，包被酶标板(ELISA PLATE)，4℃孵育过夜后用PBST溶液洗涤该酶标板。然后，在该酶标板上用5％脱脂奶粉封闭液，37℃封闭60min，用PBST(0.05％Tween-20)洗该酶标板。将cry1C抗体血清分别按照1:1K，1:2K，1:4K，1:8K，1:16K，1:32K，1：64K,1:128K,1:256K,1:512K(K值为1000)倍比稀释，每个梯度设置3次重复，37℃孵育酶标板60min，用PBST洗酶标板。然后在酶标板上加入HRP标记的羊抗兔二抗，37℃孵育45min，用PBST洗涤。再在酶标板上加入底物TMB显色液反应15min，加入终止液终止反应。
在450nm波长下测定OD值，计算平均值。计算cry1C抗体效价，将OD阳性/OD阴性≥2.1时所对应的抗体最高稀释倍数作为抗体效价。
抗体效价结果请参照附图5，4只免疫兔抗体效价均在1：512K以上，结果表明4只免疫兔中获得的4中抗cry1C蛋白的多抗效价都达到了要求。抗体效价达到要求后，采集免疫兔颈动脉全血，血样先沉淀提纯血清，血清进行抗原抗体亲和纯化得到cry1C抗体。
cry1C抗体的特异性
得到的cry1C抗体采用Western Blot进行特异性分析，检测目标蛋白的表达情况。
利用植物蛋白提取试剂盒(北京康为世纪)提取cry1C抗虫转基因水稻叶片总蛋白，测定浓度。将20μg总蛋白用10％SDS-PAGE进行电泳。用半干转膜仪(BIO-RAD，美国)转到硝酸纤维素膜(Hybond-NC)之后，加入5％脱脂乳在37℃封闭1h。在硝酸纤维素膜上加入以1:2000稀释的cry1C抗体在4℃孵育12h，再加入以1:5000稀释的HRP标记的羊抗兔二抗在37℃孵育1h，加入ECL(GE Amersham，美国)在暗室中压上X-光片，化学发光显色在X-光片上，结果如附图6所示(M，Protein marker；NT，阴性对照；1～9，cry1C转基因水稻的不同株系)，获得71kD左右的明显条带，与预期条带大小一致，说明制备的cry1C多克隆抗体具有目的蛋白特异性，能够准确检测出目的条带。
综上所述，本发明提供一种具有免疫原性的cry1C重组蛋白，其氨基酸序列如SEQID No.1所示。编码该蛋白的氨基酸序列短于cry1C蛋白的氨基酸序列，且该蛋白能够与cry1C抗体发生免疫反应，其能够用于快速高效地制备cry1C抗体。本发明还提供一种编码上述具有免疫原性的cry1C重组蛋白的核酸分子、能够扩增或检测该核酸分子的引物对、包括该核酸分子的载体和宿主细胞，该核酸分子在宿主细胞中能够高效表达上述具有免疫原性的cry1C重组蛋白。此外，本发明还提供一种cry1C抗体及其制备方法，该制备方法能够快速高效地获得cry1C抗体。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 吉林省农业科学院
<120> 一种具有免疫原性的cry1C重组蛋白、分离的核酸分子及其应用
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Claims (10)
1.一种具有免疫原性的cry1C重组蛋白，其特征在于，所述蛋白的氨基酸序列如SEQ IDNo.1所示。
2.一种分离的核酸分子，其特征在于，其编码如权利要求1所述的cry1C重组蛋白，所述核酸分子的核苷酸序列如SEQ ID No.2所示。
3.一种扩增或检测权利要求2所述的核酸分子的引物对。
4.根据权利要求3所述的引物对，其特征在于，其包括如SEQ ID No.3所示的上游引物和/或如SEQ ID No.4所示的下游引物。
5.一种载体，其特征在于，其包括有权利要求2所述的核酸分子。
6.一种宿主细胞，其特征在于，其包括权利要求2所述的核酸分子或权利要求5所述的载体。
7.如权利要求1所述的cry1C重组蛋白在制备cry1C抗体中的应用。
8.一种制备权利要求1所述的蛋白的方法，其特征在于，其包括培养权利要求6所述的宿主细胞。
9.一种cry1C抗体的制备方法，其特征在于，其包括步骤：用权利要求1所述的cry1C重组蛋白免疫动物。
10.一种cry1C抗体，其特征在于，其是由权利要求9所述的制备方法制得。
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| US5942664A (en) * | 1996-11-27 | 1999-08-24 | Ecogen, Inc. | Bacillus thuringiensis Cry1C compositions toxic to lepidopteran insects and methods for making Cry1C mutants |
| US8609936B2 (en) * | 2007-04-27 | 2013-12-17 | Monsanto Technology Llc | Hemipteran-and coleopteran active toxin proteins from Bacillus thuringiensis |
| CN102120771A (zh) * | 2010-12-23 | 2011-07-13 | 浙江大学 | 一种抗Cry1c晶体蛋白兔单克隆抗体的制备方法 |
| CN102816235B (zh) * | 2011-05-18 | 2013-12-18 | 北京华大蛋白质研发中心有限公司 | 一种抗常用苏云金芽孢杆菌CryI单克隆抗体杂交瘤的制备方法及其单克隆抗体的应用 |
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