CN112533625A - 用于蛋白质检测的组合物以及方法 - Google Patents
用于蛋白质检测的组合物以及方法 Download PDFInfo
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Classifications
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- C07K7/04—Linear peptides containing only normal peptide links
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明总体上涉及具有特定电离特性的肽生物标记物,以通过液相色谱与串联质谱联用型多反应监测(MRM)直接定量包括转基因植物样品的生物样品中的一种或多种转基因靶蛋白。所述肽生物标记物与基于MRM的方法的组合可单独或组合使用选择的多种肽生物标记物用于定量堆叠的转基因作物诸如玉米中的单一转基因靶蛋白或多种转基因靶蛋白。本披露允许在包括植物基质的不同生物基质中进行广泛、可靠的定量。本发明的肽生物标记物可以进一步用作性状生物标记物以支持特定转基因事件的鉴定和/或选择。还提供了可用于执行本发明的方法的不同肽生物标记物组合。
Description
对电子提交的序列表的引用
该序列表的官方副本是通过EFS-Web以ASCII格式的序列表以2018年8月7日生成的名为“81319-US-L-ORG-NAT-1_SeqList_ST25.txt”的文件进行电子提交的,并且该序列表的大小为94千字节并且与本说明书同时提交。包含在该ASCII格式文件中的序列表是本说明书的一部分,并且通过引用以其全文并入本文。
技术领域
本发明总体上涉及质谱用于选择性检测、定量和表征复合生物样品中的靶转基因蛋白的用途。
背景技术
转基因作物由越来越复杂的遗传修饰(包括赋予不同性状的多个转基因,也称为“基因堆叠”或“性状堆叠”)组成。例如,目前市场上的许多转基因玉米产品在同一植物内含有用于控制广谱昆虫有害生物的多种杀昆虫蛋白、赋予植物对广谱化学除草剂的耐受性的多种蛋白和被用作在植物转化过程中的可选择标记物的多种蛋白。许多用于控制昆虫有害生物的转基因蛋白(例如来自苏云金芽孢杆菌的晶体内毒素(称为Cry蛋白))可以彼此结构上密切相关并且具有相似的总体氨基酸序列同一性或含有具有显著同一性的基序或结构域。许多Cry蛋白对鳞翅目或鞘翅目昆虫有害生物具有活性。鳞翅目活性Cry蛋白的实例包括Cry1A、Cry1B、Cry1C、Cry1D、Cry1E、Cry1F和Cry9。鞘翅目活性Cry蛋白的实例包括Cry3A、Cry3B、Cry3C、Cry8、二元Cry23-Cry37以及二元Cry34-Cry35。在给定的昆虫目内,大多数个体Cry蛋白具有针对窄谱昆虫物种的生物活性。
在文献中已经披露了许多成功的创造具有增强的活性谱的杂合Cry蛋白的尝试。例如,将来自Cry1Aa蛋白的蚕蛾(家蚕(Bombyx mori))特异性结构域移动至Cry1Ac蛋白,由此将一种新的杀昆虫活性赋予生成的Cry1Aa-Cry1Ac嵌合蛋白(Ge等人1989,PNAS[美国科学院院刊]86:4037 4041)。Thompson等人,1996和1997(美国专利5,527,883和5,593,881)用Cry1Ab蛋白的原毒素尾区替换野生型Cry1F蛋白和Cry1C蛋白的原毒素尾区以制造Cry1F-Cry1Ab杂合Cry蛋白和Cry1C-Cry1Ab杂合Cry蛋白,它们在某些表达宿主细胞中都具有改进的表达。Bosch等人,1998(美国专利5,736,131)通过如下创造了新的鳞翅目活性蛋白:用Cry1Ca蛋白的结构域III取代Cry1Ea蛋白和Cry1Ab蛋白的结构域III,从而产生称为G27的Cry1E-Cry1C杂合Cry蛋白和称为H04的Cry1Ab-Cry1C杂合Cry蛋白,它们都具有比野生型Cry蛋白亲本分子更宽的鳞翅目活性谱。Malvar等人,2001(美国专利6,242,241)将Cry1Ac蛋白的结构域I与Cry1F蛋白的结构域II和III以及原毒素尾部组合以产生比亲本野生型Cry蛋白具有更广杀昆虫活性的Cry1Ac-Cry1F杂合Cry蛋白。Bogdanova等人,2011(美国专利8,034,997)将Cry1Ab蛋白的结构域I和II与Cry1Fa蛋白的结构域III组合,并添加Cry1Ac蛋白原毒素尾部以产生称为Cry1A.105的新的鳞翅目活性杂合Cry蛋白。而且,Hart等人,2012(美国专利8,309,516)将Cry3A蛋白和修饰的Cry3A蛋白的结构域I和II与Cry1Ab蛋白的结构域III组合,并添加Cry1Ab蛋白原毒素尾部的一部分以产生称为FR8a的鞘翅目活性杂合Cry蛋白(也称为eCry3.1Ab)。迄今为止报道的大多数杂合Cry蛋白已经使用了全部或部分相同类型的野生型Cry蛋白,如Cry1Aa、Cry1Ab、Cry1Ac、Cry1C、Cry1F和Cry3A。
若干野生型Cry蛋白(例如Cry1Ab、Cry1Ac、Cry1C、Cry1F、Cry2A、Cry2Ba、Cry3A、Cry3B、Cry9C和Cry34-Cry35)以及营养期杀昆虫蛋白(例如Vip3A(参见美国专利5,877,012))已经在转基因作物植物(包括玉米、棉花、水稻和大豆)中表达,早自1996年以来,其中一些已经在商业上被用于控制某些鳞翅目和鞘翅目昆虫有害生物。最近,已经在商业上引入了转基因作物产品,例如玉米,该转基因作物产品含有其中一个或多个氨基酸被取代、缺失或插入的工程化的Cry蛋白(例如,修饰的Cry3A(mCry3A;US专利7,230,167))和杂合Cry蛋白(例如上述eCry3.1Ab和Cry1A.105)。
重组DNA技术越来越多地用于产生商业和工业用途的转基因植物,这需要开发分析转基因植物系的诊断方法。需要此类方法来通过连续世代的育种维持转基因植物品种,监测环境中或衍生自转基因植物的生物样品中的转基因植物或植物部分的存在,以及协助快速产生并且开发具有期望的或最佳的表型的新转基因植物。此外,目前许多国家的监管机构关于转基因植物的安全性评估指导原则需要在DNA和蛋白水平的表征,从而获得并且维持监管批准。堆叠到如上所述的转基因植物中的基因和蛋白越来越复杂,使得在复合混合物内检测和定量任何一种靶蛋白的特异性变得困难,特别是在堆叠的转基因蛋白彼此相似、或与环境中的野生型非转基因蛋白相似、或与相对于转基因植物而言内源的非转基因蛋白相似的情况下。
免疫测定(例如酶联免疫吸附测定(ELISA))是目前农业产业中用于检测和定量通过植物遗传修饰而引入的蛋白的优选方法。免疫测定的关键部分是对靶蛋白(抗原)具有特异性的抗体。免疫测定可以是高度特异性的,并且样品在被分析前通常只需要一个简单的准备。此外,免疫测定可以在很宽的浓度范围内定性或定量地使用。典型地,免疫测定需要针对每种目的蛋白进行单独的测试。抗体可以是在动物中产生的多克隆的或者是由细胞培养物产生的单克隆的抗体。就其性质而言,多克隆抗体的混合物将具有多个识别表位,该识别表位可以增加灵敏度,但是也可能降低特异性,因为序列和结构与其他蛋白具有同源性的可能性随着不同抗体互补位的存在数量而增加。单克隆抗体比多克隆抗体具有一些优点,因为它们对单个表位或抗原决定簇表达均匀的亲和力和特异性,并且可以大量生产。然而,所有抗体中存在固有特性限制其在更高要求的应用(例如相似的转基因蛋白或内源蛋白的复合混合物中的单个转基因蛋白的选择性检测和定量)中的使用。此外,多克隆抗体和单克隆抗体都可能需要进一步纯化步骤来增强灵敏度并降低测定中的背景。此外,ELISA系统可能无法检测到靶蛋白的细微变化,而这些变化可能对其物理和生物学特性产生巨大影响。例如,该抗体可能无法识别已被翻译后修饰(诸如磷酸化或糖基化)改变或构象模糊或被部分降解修饰的蛋白或肽的特定形式。鉴定此类修饰至关重要,因为这些蛋白的物理和生物学特性的变化可能在其酶促、临床或其他生物学活性中起重要作用。此类变化可能会限制基于ELISA的定量方法的可靠性和实用性。
目前,对含有转基因蛋白的转基因植物产品进行有效的鉴定或定量商业作物产品中的转基因蛋白取决于免疫测定的准确性。成功的免疫测定的发展取决于用于开发抗体所使用的抗原的某些特征,即抗原的大小、疏水性和三级结构以及抗体的品质和准确性。必须仔细检查抗体的特异性以阐明与类似物质的任何交叉反应性,该类似物质可能导致假阳性结果。目前行业存在的问题在于,可商购的测试试剂盒中的许多抗体不能区分各种产物中的相似转基因蛋白、或区分转基因蛋白与野生型蛋白,这使得差异化的产物鉴定和定量变得困难或不可能。例如,在使用一种或多种相同的野生型Cry蛋白(例如Cry1Ab、Cry1Ac、Cry1F和Cry3)的许多目前商业的转基因作物产品的情况下,并且在引入表达杂合Cry蛋白(该杂合Cry蛋白由全部或部分的已经在转基因作物产品中的相同野生型Cry蛋白组成)的作物的情况下,仍然需要开发新的和改进的诊断方法,以能够当一起处于复合生物样品(例如来自转基因植物、转基因植物部分或转基因微生物的样品)中时将野生型Cry蛋白彼此区分,以及将野生型Cry蛋白与含有全部或部分的相同野生型Cry蛋白的杂合Cry蛋白进行区分。
质谱(MS)提供了一个替代平台,该平台克服了ELISA用于蛋白分析的许多限制。基于MS的分析的领域已得到靶向蛋白分析的重要进展,诸如通过电喷雾液相色谱与串联质谱联用(LC-MS/MS)进行多反应监测(MRM)。基本概念是蛋白可以通过蛋白水解消化后测量其特定的组成肽(替代肽)来定量。仅针对所选的肽的数据采集允许以更高的精度、灵敏度和通量进行测量。通过对替代肽的基于MRM的测量进行蛋白定量是MS在蛋白分析中发展最快的应用。与基于免疫的测定相比,基于MRM的蛋白测定具有两个引人注目的优点,第一个优点是无需使用抗体即可针对基本上任何蛋白系统地配置特异性测定的能力。第二个是靶向MS测定在单个分析中对许多肽进行多重分析的能力。此外,MRM是直接分析,而基于免疫的测定是间接的。基于免疫的测定依赖于结合测定,该结合测定包括可固定在固相上的连接试剂以及将特异性结合并使用酶产生可正确定量的信号的检测试剂。
商业转基因作物产品包含杀昆虫蛋白、除草剂耐受蛋白和可选择标记蛋白的堆叠。在有许多使用一种或多种相同的野生型杀昆虫Cry蛋白(例如Cry1Ab、Cry1F和Cry3)的商业转基因作物产品的情况下,以及在引入了表达由转基因作物产品中已经存在的相同野生型Cry蛋白的全部或部分组成的杂合Cry杀昆虫蛋白(例如mCry3A,eCry3.1Ab和Cry1A.105)的农作物的情况下,基于MRM的测定必须能够区分这些紧密相关的转基因靶杀昆虫蛋白以及除草剂耐受蛋白和可选择标记蛋白。因此,持续需要鉴定具有在基于MRM的测定中起作用的所有生化特性并且具有附加特性的替代肽,使得这些替代肽对靶向可能具有大部分的重叠氨基酸序列的转基因蛋白有绝对特异性,即替代肽的一个或多个过渡态能够清楚地、无干扰地在多个复合基质中区分两个紧密相关的靶蛋白。此类选择性替代肽及其过渡态应能够区分彼此相似、或与环境中的野生型非转基因蛋白相似、或与转基因植物内源的非转基因蛋白相似的靶转基因蛋白。
发明内容
本发明提供了标记的替代肽及其各自的过渡离子,其可用于使用质谱选择性地检测或定量复合生物基质中的靶转基因蛋白。本发明进一步提供了使用标记的替代肽和过渡离子在复合生物基质中选择性检测或定量靶转基因蛋白的方法和系统。
在本发明的一方面,通过经验分析和计算机模拟消化分析来设计内标肽标记物;并在稳定的同位素标记的一种或多种氨基酸或代谢中间体存在下,通过用重氨基酸残基化学合成或通过表达合成基因遗传合成。在某些实施例中,可以通过质谱(MS)分析,包括串联质谱分析(MS/MS),更具体地,液相色谱与串联质谱分析联用(LC-MS/MS),对内标进行单独表征。表征后,可以收集肽的预选参数,诸如每种肽的单同位素质量、其替代电荷状态、替代m/z值、m/z过渡离子以及每种过渡离子的离子类型。其他考虑因素包括优化肽大小,避免翻译后修饰,避免过程诱导的修饰以及避免高比率的蛋白酶切割遗漏。
本文提供了独特的稳定同位素标记的(SIL)替代肽的示例性清单,其包括包含SEQID NO:1-98中的任一个或其组合的肽,以选择性检测或定量选自由以下组成的组的转基因蛋白:杀昆虫蛋白Cry1Ab、eCry3.1Ab、mCry3A和Vip3,除草剂耐受蛋白dmEPSPS和PAT,以及可能包含在具有单个转基因事件、多个事件的育种堆叠或多种靶转基因蛋白的分子堆叠的植物中的植物转化可选择标记蛋白PMI。衍生自七种蛋白的每种肽的每个替代肽序列和过渡离子都可用于基于质谱的多反应监测(MRM)测定。
在本发明的另一方面,该标记的替代肽选择性地检测或定量Cry1Ab蛋白,并包含SEQ ID NO:1-26中任一个的氨基酸序列。在另一方面,该标记的替代肽选择性地检测或定量Cry1Ab蛋白,并产生具有选自SEQ ID NO:99-141或肽PIR、TY、VW、HR、YR或PPR中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQ ID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量eCry3.1Ab蛋白,并包含SEQ ID NO:27-32中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量eCry3.1Ab蛋白,并产生具有选自SEQ ID NO:142-150或肽DGR、IEF或LER中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量mCry3A蛋白,并包含SEQ ID NO:33-35中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量mCry3A蛋白,并产生具有选自SEQ ID NO:151-155或肽IDK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列LQSGASVVAGPR(SEQ ID NO:252),并产生由氨基酸序列SGASVVAGPR(SEQ ID NO:253)或SVVAGPR(SEQ ID NO:254)组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量Vip3蛋白,并包含SEQID NO:36-73中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量Vip3蛋白,并产生具有选自SEQ ID NO:156-212或肽TCK、FEK、DVS、FTK、HK、VNI、MIV、EAK、HLK、NK、DNF、LLC、NAY、YE、SDK、NEK、DK或VDK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列DGGISQFIGDK(SEQ ID NO:36),并产生由氨基酸序列SQFIGDK(SEQ ID NO:156)或氨基酸序列GDK组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量dmEPSPS蛋白,并包含SEQ ID NO:74-77中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量dmEPSPS蛋白,并产生具有选自SEQ ID NO:213-219或肽PIK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SLTAAVTAAGGNATYVLDGVPR(SEQ ID NO:257),并产生由氨基酸序列GVPR(SEQ ID NO:258)或氨基酸序列PR组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量PAT蛋白,并包含SEQID NO:78-86中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量PAT蛋白,并产生具有选自SEQ ID NO:220-231或肽DFE、DF、PER、SHR、GYK或NFR中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列LGLGSTLYTHLLK(SEQ ID NO:79),并产生由氨基酸序列YTHLLK(SEQ ID NO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。
在本发明的另一方面,该标记的替代肽选择性地检测或定量PMI蛋白,并包含SEQID NO:87-98中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量PMI蛋白,并产生具有选自SEQ ID NO:232-251或肽LK、PVK、HN或PNK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SALDSQQGEPWQTIR(SEQ ID NO:89),并产生由氨基酸序列PWQTIR(SEQ ID NO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。
在本发明的其他方面,本发明的标记的替代肽及其产生的过渡离子选择性地检测或定量包含SEQ ID NO:259的Cry1Ab蛋白、或包含SEQ ID NO:260的eCry3.1Ab蛋白、或包含SEQ ID NO:261的mCry3A蛋白、或包含SEQ ID NO:262的Vip3蛋白、或包含SEQ ID NO:263的dmEPSPS蛋白、或包含SEQ ID NO:264的PAT蛋白、或包含SEQ ID NO:265或SEQ ID NO:266的PMI蛋白。
在其他方面,当靶蛋白在来自转基因植物的生物样品中时,本发明的标记的替代肽选择性地检测或定量本发明的靶蛋白。在该方面的一些实施例中,该生物样品来自该转基因植物的叶组织、种子、籽粒、花粉或根组织。
在本发明的其他方面,本发明的标记的替代肽及其产生的过渡离子选择性地检测或定量来自包含转基因事件Bt11的玉米植物的Cry1Ab蛋白,或来自包含转基因事件5307的玉米植物的eCry3.1Ab蛋白,或来自包含转基因事件MIR604的玉米植物的mCry3A蛋白,或来自包含转基因事件MIR162的玉米植物或来自包含转基因事件COT102的棉花植物的Vip3蛋白,或来自包含转基因事件GA21的玉米植物的dmEPSPS蛋白,或来自包含转基因事件Bt11、DAS-59122、TC1507、DP4114或T25的玉米植物的PAT蛋白,或来自包含转基因事件MIR162、MIR604、5307或3272的玉米植物的PMI蛋白。
替代肽的许多不同组合可以通过MRM测定用一种或多种来自Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI蛋白的特定替代肽同时进行监测和定量,并因此提供通过质谱测量从生物样品获得的给定蛋白制品中那些蛋白质中每种的总量的方法。这些肽与基于MRM的测定的结合具有许多应用,包括定量肽/蛋白分析以用于确定转基因植物不同生长阶段的表达水平,确定不同转基因植物组织和器官(包括但不限于叶组织、种子和籽粒、花粉和根组织)中的表达水平,确定潜在的暴露水平以进行监管风险评估,确定食品加工、比较性和世代研究中蛋白的不同水平。从广义上讲,这七种蛋白质的独特替代肽可与MRM测定组合使用以用于多种应用,包括农业应用,生物等效性测试,所有类型的生物和非生物基质中生物标记物、诊断性、发现性、食品性、环境性、治疗性监测。在本发明的一些方面,提供了一种测定盒,其包含一种或多种本发明的标记的替代肽、允许同时和选择性地检测或定量本发明的任一种或多种靶蛋白,这些替代肽包含SEQ ID NO:1-98中的任一个。
本发明还提供了用于选择性地检测或定量复合生物基质内的转基因靶蛋白的方法,该复合生物基质诸如来自表达这些转基因靶蛋白的转基因植物的生物样品。这种方法包括从该转基因植物中获得样品,例如从叶、种子或籽粒、花粉或根中获得样品;从该植物样品中提取蛋白;通过减少提取物中的非转基因不溶性蛋白的量来浓缩靶蛋白库;用选择的酶(例如胰蛋白酶)消化提取物中的可溶性蛋白,得到包含肽碎片的提取物,其中这些肽碎片包含至少一种对每种靶转基因蛋白具有特异性的替代肽;添加特异性检测靶蛋白的SIL肽的测定盒,其中每种标记的替代肽都具有与该靶转基因蛋白的每种替代肽相同的氨基酸序列,并且其中添加的标记的替代肽的数目等于混合物中靶转基因蛋白的数目;通过减少混合物中非替代肽的量来浓缩这些替代肽和标记的替代肽;使用液相色谱使该肽碎片混合物分解;使用质谱分析该肽碎片混合物,其中检测到标记的替代肽的过渡离子碎片指示存在衍生该替代肽的靶转基因蛋白;以及,任选地,通过比较由该过渡离子碎片产生的质谱信号与由标记的替代肽的过渡离子产生的质谱信号,计算该生物样品中靶转基因蛋白的量。衍生自本发明的转基因蛋白的SIL替代肽各自在基于质谱的多反应监测(MRM)测定中具有独特的过渡离子。这样,这些肽将由于碰撞能量的微小变化而产生选择性MS离子,从而导致不同程度的电离。例如,三重四极杆MS可用于产生具有肽特异性的高m/z离子。结果,相对于使用本领域中已知的较低m/z强离子标记物,本发明的方法可以提供选择性优势,从而减少内源性背景。
在本发明的一些方面,在本发明的方法中选择性检测或定量的靶蛋白是Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白或磷酸甘露糖异构酶(PMI)蛋白。
在本发明的其他方面,可用于本发明的方法以检测或定量Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白或磷酸甘露糖异构酶(PMI)蛋白的标记的替代肽包含SEQ IDNO:1-98中的任一个。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量Cry1Ab并包含SEQ ID NO:1-26中任一个的氨基酸序列。在另一方面,该标记的替代肽选择性地检测或定量Cry1Ab,并产生具有选自SEQ ID NO:99-141或肽PIR、TY、VW、HR、YR或PPR中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQ ID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。在另一方面,通过比较由氨基酸序列PLTK(SEQID NO:132)组成的过渡离子碎片产生的质谱信号,对该生物样品中的Cry1Ab靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量eCry3.1Ab蛋白,并包含SEQ ID NO:27-32中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量eCry3.1Ab蛋白,并产生具有选自SEQ ID NO:142-150或肽DGR、IEF或LER中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。在另一方面,通过比较由氨基酸序列TDYHIDQV(SEQ ID NO:142)组成的过渡离子碎片产生的质谱信号,对该生物样品中的eCry3.1Ab靶蛋白进行定量。在该方面的另一实施例中,该标记的替代肽包含氨基酸序列AVFNELFTSSNQIGLK(SEQ ID NO:28),并产生由氨基酸序列TSSNQIGLK(SEQ ID NO:144)或SSNQIGLK(SEQ ID NO:145)组成的过渡离子。在另一方面,通过比较由氨基酸序列TSSNQIGLK(SEQ ID NO:144)组成的过渡离子碎片产生的质谱信号,对该生物样品中的eCry3.1Ab靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量mCry3A蛋白,并包含SEQ ID NO:33-35中任一个的氨基酸序列。在该方法的另一方面,该标记的替代肽选择性地检测或定量mCry3A蛋白,并产生具有选自SEQ ID NO:151-155或肽IDK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列LQSGASVVAGPR(SEQ ID NO:252),并产生由氨基酸序列SGASVVAGPR(SEQ ID NO:253)或SVVAGPR(SEQ ID NO:254)组成的过渡离子。在另一方面,通过比较由氨基酸序列SGASVVAGPR(SEQ ID NO:253)组成的过渡离子碎片产生的质谱信号,对该生物样品中的mCry3A靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量Vip3蛋白,并包含SEQ ID NO:36-73中任一个的氨基酸序列。在该方法的另一方面,该标记的替代肽选择性地检测或定量Vip3蛋白,并产生具有选自SEQ ID NO:156-212或肽TCK、FEK、DVS、FTK、HK、VNI、MIV、EAK、HLK、NK、DNF、LLC、NAY、YE、SDK、NEK、DK或VDK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列DGGISQFIGDK(SEQ IDNO:36),并产生由氨基酸序列SQFIGDK(SEQ ID NO:156)或氨基酸序列GDK组成的过渡离子。在另一方面,通过比较由氨基酸序列SQFIGDK(SEQ ID NO:156)组成的过渡离子碎片产生的质谱信号,对该生物样品中的Vip3靶蛋白进行定量。在该方面的另一实施例中,该标记的替代肽包含氨基酸序列FTTGTDLK(SEQ ID NO:255),并产生由氨基酸序列TGTDLK(SEQ ID NO:256)或氨基酸序列LK组成的过渡离子。在另一方面,通过比较由氨基酸序列TGTDLK(SEQ IDNO:256)组成的过渡离子碎片产生的质谱信号,对该生物样品中的Vip3靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量dmEPSPS蛋白,并包含SEQ ID NO:74-77中任一个的氨基酸序列。在该方法的另一方面,该标记的替代肽选择性地检测或定量dmEPSPS蛋白,并产生具有选自SEQ ID NO:213-219中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SLTAAVTAAGGNATYVLDGVPR(SEQ ID NO:257),并产生由氨基酸序列GVPR(SEQ ID NO:258)或氨基酸序列PR组成的过渡离子。在另一方面,通过比较由氨基酸序列PR组成的过渡离子碎片产生的质谱信号,对该生物样品中的dmEPSPS靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量PAT蛋白,并包含SEQ ID NO:78-86中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量PAT蛋白,并产生具有选自SEQ ID NO:220-231或肽DFE、DF、PER、SHR、GYK或NFR中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列LGLGSTLYTHLLK(SEQ ID NO:79),并产生由氨基酸序列YTHLLK(SEQ ID NO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。在另一方面,通过比较由氨基酸序列YTHLLK(SEQ ID NO:220)组成的过渡离子碎片产生的质谱信号,对该生物样品中的dmEPSPS靶蛋白进行定量。
在本发明的方法的另一方面,该标记的替代肽选择性地检测或定量PMI蛋白,并包含SEQ ID NO:87-98中任一个的氨基酸序列。在本发明的另一方面,该标记的替代肽选择性地检测或定量PMI蛋白,并产生具有选自SEQ ID NO:232-251或肽LK、PVK、HN或PNK中至少一个的氨基酸序列的过渡离子。在该方面的一个实施例中,该标记的替代肽包含氨基酸序列SALDSQQGEPWQTIR(SEQ ID NO:89),并产生由氨基酸序列PWQTIR(SEQ ID NO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。
本发明进一步提供了用于高通量检测或定量转基因靶蛋白的系统。这样的系统包含对转基因靶蛋白具有特异性的预先设计的标记的替代肽的盒;和一个或多个质谱仪。
本发明的各种目的、特征、方面和优点将根据以下对本发明的优选实施例的详细描述以及附图和序列表而变得更加清楚。
序列简要说明
SEQ ID NO:1-26是用于选择性检测和定量转基因Cry1Ab蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:27-32是用于选择性检测和定量转基因eCry3.1Ab蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:33-35是用于选择性检测和定量转基因mCry3A蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:36-73是用于选择性检测和定量转基因Vip3蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:74-77是用于选择性检测和定量转基因dmEPSPS蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:78-86是用于选择性检测和定量转基因PAT蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:87-98是用于选择性检测和定量转基因PMI蛋白的稳定的同位素标记的替代肽的氨基酸序列。
SEQ ID NO:99-141是具有SEQ ID NO:1-26的SIL替代肽的过渡离子的氨基酸序列。
SEQ ID NO:142-150是具有SEQ ID NO:27-32的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:151-155是具有SEQ ID NO:33-35的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:156-212是具有SEQ ID NO:36-72的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:213-219是具有SEQ ID NO:74-77的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:220-231是具有SEQ ID NO:79-86的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:232-251是具有SEQ ID NO:87-98的SIL替代肽的过渡产物的氨基酸序列。
SEQ ID NO:252-254是用于选择性检测和定量转基因mCry3A蛋白的SIL替代肽及其过渡产物的氨基酸序列。
SEQ ID NO:255-256是用于选择性检测和定量转基因Vip3A蛋白的SIL替代肽及过渡产物的氨基酸序列。
SEQ ID NO:257-258是用于选择性检测和定量转基因dmEPSPS蛋白的SIL替代肽和过渡产物的氨基酸序列。
SEQ ID NO:259-270是本发明的示例性靶转基因蛋白的氨基酸序列。
具体实施方式
本说明不旨在是可以实施本发明的所有不同方式,或可以添加到本发明中的所有特征的详细目录。例如,关于一个实施例所说明的特征可以并入其他实施例中,并且关于一个特定实施例所说明的特征可以从那个实施例删除。因此,本发明考虑了,在本发明的一些实施例中,可以排除或省略本文阐述的任何特征或特征的组合。此外,鉴于本披露,本文建议的不同实施例的众多变化以及附加对于本领域技术人员是显而易见的,这不脱离本发明。因此,以下说明旨在阐述本发明的一些特定实施例,并且并没有穷尽地叙述其所有排列、组合和变化。
除非另外定义,本文所使用的所有技术和科学术语均具有与本发明所属领域的普通技术人员通常所理解的相同的含义。在本文的发明的说明中使用的术语是仅出于描述特定实施例的目的,且并不旨在限制本发明。还应当理解的是在此使用的术语仅仅是出于描述具体实施例的目的并且不旨在限制本发明的范围。与本发明有关的一般参考文献包括:Alwine等人(1977)Proc.Nat.Acad.Sci.[美国国家科学院学报]74:5350-54;Baldwin(2004)Mol.Cell.Proteomics[分子和细胞的蛋白质组学]3(1):1-9;Can和Annan(1997)Overview of peptide and protein analysis by mass spectrometry[肽和蛋白质质谱分析综述].In:Current Protocols in Molecular Biology[摘自:最新分子生物学方法汇编],Ausubel等人编辑,纽约:威利,第10.21.1-10.21.27页;Chang等人(2000)PlantPhysiol.[植物生理学]122(2):295-317;Domon和Aebersold(2006)Science[科学]312(5771):212-17;Nain等人(2005)Plant Mol.Biol.Rep.[植物分子生物学导报]23:59-65;Patterson(1998)Protein identification and characterization by massspectrometry[蛋白质的质谱鉴定与表征].In:Current Protocols in MolecularBiology[摘自:最新分子生物学方法汇编],Ausubel等人编辑,纽约:威利,第10.22.1-10.22.24页;Paterson和Aebersold(1995)Electrophoresis[电泳]16:1791-1814;Rajagopal和Ahern(2001)Science[科学]294(5551):2571-73;Sesikeran和Vasanthi(2008)Asia Pac.J.Clin.Nutr.17[亚太临床营养杂志]增刊1:241-44;以及Toplak等人(2004)Plant Mol.Biol.Rep.[植物分子生物学导报]22:237-50。
定义
如本文和所附权利要求书中使用的,单数形式“一个/种(a/an)”和“该/所述(the)”可以表示一个或多于一个。因此,例如,提及“一种植物”可以指单种植物或多种植物。
如本文所使用的,“和/或”是指并且涵盖一个或多个相关的列出项的任何及全部可能组合,连同当以可替代性(“或”)解释时组合的缺少。
术语“约”在此用于意指大约、大致、约或在……左右。当术语“约”结合数值范围来使用时,它通过将边界延伸至高于以及低于所阐述的数值来限定这个范围。一般而言,术语“约”本文用于将数值限定至以20%的变化,优选地10%上下(更高或更低)地高于以及低于规定值。关于温度,术语“约”意指±1℃,优选±0.5℃。当术语“约”被用于本发明的上下文中(例如与温度或分子量值组合)时,确切值(即,无“约”)是优选的。
术语“包含(comprises和/或comprising)”当在本说明书中使用时,指明所列举特征、整体、步骤、操作、元件、和/或部件的存在,但是不排除一种或多种其他特征、整体、步骤、操作、元件、部件、和/或其组的存在或添加。
如本文使用的,过渡短语“基本上由……组成”(以及语法变体)意指,权利要求书的范围有待被解读为涵盖权利要求书中所列举的指定材料或步骤以及不实质上改变所要求的发明的一个或多个基本和新颖特征的那些。因此,当用于本发明的权利要求中时,术语“基本上由……组成”并不旨在被解释为等同于“包含(comprising)”。
如本文使用的,术语“Cry蛋白”是指在芽孢杆菌属物种(例如苏云金芽孢杆菌)生长周期的孢子形成阶段期间,在天然条件下以结晶形式积聚为原毒素的呈球状蛋白质分子的杀昆虫蛋白。术语“Cry毒素”以及“δ-内毒素”可以与术语“Cry蛋白”互换地使用。对于Cry蛋白和编码Cry蛋白的基因的当前命名法基于氨基酸序列同源性(Crickmore等人,(1998),Microbiol.Mol.Biol.Rev.[微生物分子生物学评论],62:807-813)。在本领域公认的分类中,每种Cry蛋白被指定唯一的名称,所述名称合并了初级等级(阿拉伯数字)、二级等级(大写字母)、三级等级(小写字母)、以及四级等级(另一个阿拉伯数字)。例如,根据Crickmoe等人,具有<45%同源性的两个Cry蛋白将被指定唯一的初级等级,例如Cry1和Cry2。具有>45%但<70%同源性的两个Cry蛋白将接受相同的初级等级,但是将被指定不同的二级等级,例如Cry1A和Cry1B。具有70%至95%同源性的两个Cry蛋白将被指定相同的初级等级和二级等级,但是将被指定不同的三级等级,例如Cry1Aa和Cry1Ab。并且具有>95%但<100%同源性的两个Cry蛋白将被指定相同的初级等级、二级等级和三级等级,但是将被指定不同的四级等级,例如Cry1Ab1和Cry1Ab2。
如本文使用的,“Cry1Ab蛋白”意指衍生自苏云金芽孢杆菌的杀昆虫晶体蛋白,其(无论是天然存在的还是合成的)包含与全型Cry1Ab氨基酸序列(根据Crickmore等人(同上))具有至少96%同一性的氨基酸序列并且在因特网站“lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt”上披露为登录号AAA22330)。Cry1Ab蛋白(具有登录号)的实例包括但不限于Cry1Ab1(AAA22330)、Cry1Ab2(AAA22613)、Cry1Ab3(AAA22561)、Cry1Ab4(BAA00071)、Cry1Ab5(CAA28405)、Cry1Ab6(AAA22420)、Cry1Ab7(CAA31620)、Cry1Ab8(AAA22551)、Cry1Ab9(CAA38701)、Cry1Ab10(A29125)、Cry1Ab11(I12419)、Cry1Ab12(AAC64003)、Cry1Ab13(AAN76494)、Cry1Ab14(AAG16877)、Cry1Ab15(AAO13302)、Cry1Ab16(AAK55546)、Cry1Ab17(AAT46415)、Cry1Ab18(AAQ88259)、Cry1Ab19(AAW31761)、Cry1Ab20(ABB72460)、Cry1Ab21(ABS18384)、Cry1Ab22(ABW87320)、Cry1Ab23(HQ439777)、Cry1Ab24(HQ439778)、Cry1Ab25(HQ685122)、Cry1Ab26(HQ847729)、Cry1Ab27(JN135249)、Cry1Ab28(JN135250)、Cry1Ab29(JN135251)、Cry1Ab30(JN135252)、Cry1Ab31(JN135253)、Cry1Ab32(JN135254)、Cry1Ab33(AAS93798)、Cry1Ab34(KC156668)、Cry1Ab35(KT692985)、和Cry1Ab36(KY440260)。本发明的Cry1Ab蛋白的示例性实例由SEQ ID NO:259表示。
如本文使用的,术语“Cry3”是指与分类为Cry3的先前描述的序列(根据Crickmore等人(同上))共享高度序列同一性或相似性的杀昆虫蛋白,其实例披露在因特网站“lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/”上并且包括(具有登录号)Cry3Aa1(AAA22336)、Cry3Aa2(AAA22541)、Cry3Aa3(Caa68482)、Cry3Aa4(AAA22542)、Cry3Aa5(AAA50255)、Cry3Aa6(AAC43266)、Cry3Aa7(CAB41411)、Cry3Aa8(AAS79487)、Cry3Aa9(AAW05659)、Cry3Aa10(AAU29411)、Cry3Aa11(AAW82872)、Cry3Aa12(ABY49136)、Cry3Ba1(CAA34983)、Cry3Ba2(CAA00645)、Cry3Ba3(JQ397327)、Cry3Bb1(AAA22334)、Cry3Bb2(AAA74198)、Cry3Bb3(I15475)、和Cry3Ca1(CAA42469)。已经通过插入、取代或缺失氨基酸而工程化的Cry3蛋白在本文称为“修饰的Cry3蛋白”或“mCry3蛋白”。与从其衍生出“修饰的Cry3蛋白”的野生型Cry3蛋白相比,此类“修饰的Cry3蛋白”典型地具有针对某些昆虫有害生物(例如玉米根虫(根萤叶甲属物种))的增强的活性。“修饰的Cry3蛋白”的实例是由SEQID NO:262的氨基酸序列表示的“mCry3A”。“修饰的Cry3”蛋白的其他实例包括但不限于披露于美国专利8,247,369中的“mCry3A蛋白”、披露于美国专利9,109,231中的“mCry3A蛋白”、和披露于美国专利6,060,594中的“mCry3B蛋白”。
术语“eCry3.1Ab”是指工程化的杂合杀昆虫蛋白,其在N-末端至C-末端方向包含融合至如描述于美国专利8,309,516中的Cry1Aa或Cry1Ab蛋白的C-末端区域的Cry3A的N-末端区域。“eCry3.1Ab蛋白”的实例由SEQ ID NO:260的氨基酸序列表示。
如本文使用的,术语转基因“事件”是指通过用异源DNA(例如,包含目的基因的表达盒)转化和再生单个植物细胞而产生的重组植物。术语“事件”是指包含异源DNA的原始转化体和/或所述转化体的子代。术语“事件”也是指通过该转化体和另一种玉米品系之间进行有性异型杂交(sexual outcross)而产生的子代。即使在重复回交至一个轮回亲本后,来自所述转化的亲本的插入DNA和侧翼DNA存在于在所述杂交子代的同样的染色体位置。通常,植物组织的转化产生多个事件,每个上述事件代表DNA构建体插入至植物细胞的基因组中的不同位置中。基于转基因或其他期望的特征的表达,选择特定的事件。本发明的此类转基因事件的非限制性实例包括“事件Bt11”,其包含cry1Ab和pat基因,并描述于US 6114608中(也称为“Bt11事件”或仅“Bt11”);“事件5307”,其包含eCry3.1Ab和PMI基因,并描述于US8466346中(也称为“5307事件”或仅称为“5307”);“事件MIR604”,其包含mCry3A和PMI基因,并描述于US 7361813中(也称为“MIR604事件”或仅称为“MIR604”);“事件MIR162”,其包含Vip3A和PMI基因,并在描述于US 8232456中(也称为“事件MIR162”或仅称为“MIR162”);“事件GA21”,其包含dmEPSPS基因,并描述于US 6566587中(也称为“GA21事件”或仅称为“GA21”);“事件3272”,其包含α-淀粉酶797E和PMI基因,并描述于US 7635799中(也称为“3272事件”或仅称为“3272”);“事件MON810”,其包含Cry1Ab,并描述于US 6713259中(也称为“MON810事件”或仅称为“MON810”);“事件MON89034”,其包含Cry1A.105和Cry2Ab基因,并描述于US 8062840中(也称为“MON89034事件”或仅称为“MON89034”);“事件TC1507”,其包含Cry1F和PAT基因,并描述于US 7288643(也称为“TC1507事件”或仅称为“TC1507”);“事件DAS59122”,其包含Cry34/Cry35和PAT基因,并描述于US 7323556中(也称为“DAS59122事件”或仅称为“DAS59122”)和“事件DP4114”,其包含Cry1F、Cry34/Cry35和PAT基因,并描述于US 9790561中(也称为“DP4114事件”或仅称为“DP4114”)。
如本文使用的,术语“杂合Cry蛋白”是天然不存在的工程化的杀昆虫蛋白,并且其至少一部分包含至少一个27%连续的Cry1Ab蛋白氨基酸序列。该27%限制是通过将杂合Cry蛋白中的连续的Cry1Ab氨基酸数量除以杂合Cry蛋白中的氨基酸总数来计算的。例如,该杂合Cry蛋白eCry3.1Ab(SEQ ID NO:261)具有174个Cry1Ab氨基酸(位置480-653)和总共653个氨基酸。因此,eCry3A.1Ab具有至少一个27%连续的Cry1Ab蛋白的氨基酸序列。根据本发明的杂合Cry蛋白Cry1A.105的另一个实例由SEQ ID NO:267表示。
“dmEPSPS”(5-烯醇丙酮酰莽草酸-3-磷酸合酶)是工程化的蛋白,赋予植物对草甘膦除草剂的耐受性,如PCT公开号WO 97/04103中所述。本发明的dmEPSPS的示例性实例由SEQ ID NO:263表示。
如本文使用的,“高度相关的杀昆虫蛋白”是指具有至少95%总体序列同一性的蛋白,或具有存在至少80%序列同一性的共同基序的蛋白。“高度相关的”杀昆虫蛋白的实例包括Cry1Ab(SEQ ID NO:259)和eCry3.1Ab(SEQ ID NO:260)(它们具有存在至少80%序列同一性的共同基序),以及eCry3.1Ab(SEQ ID NO:260)和mCry3A(SEQ ID NO:261)(它们具有存在至少80%序列同一性的共同基序)。
术语“分离的”核酸分子、多核苷酸或毒素是不再存在于其天然环境中的核酸分子、多核苷酸或毒性蛋白。本发明的分离的核酸分子、多核苷酸或毒素可以按照纯化的形式存在,或者可以存在于重组宿主中,例如转基因细菌细胞或转基因植物中。
如本文使用的,一般术语“质谱”是指任何合适的质谱方法、设备或配置,包括例如电喷雾电离(ESI)、基质辅助激光解吸/电离(MALDI)MS、MALDI-飞行时间(TOF)MS、大气压(AP)MALDI MS、真空MALDI MS、串联MS或其任何组合。质谱设备通过测量分子在一组磁场和电场中的飞行路径,来测量分子的分子量(作为分子的质荷比的函数)。质荷比是带电粒子的电动力学中广泛使用的物理量。特定肽的质荷比可由本领域技术人员事先计算。当经历相同的电场和磁场时,质荷比不同的两个粒子在真空中不会沿相同的路径移动。本发明尤其包括使用高效液相色谱(HPLC),随后对该肽进行串联MS分析。在“串联质谱”中,可以在MS仪器中过滤替代肽,并在随后将该替代肽碎裂以产生一个或多个“过渡离子”,在第二个MS程序中对这些过渡离子进行分析(检测和/或定量)。
质谱方法和设备的详细概述可在以下参考文献中找到,在此通过引用将其并入:Can和Annan(1997)Overview of peptide and protein analysis by mass spectrometry[肽和蛋白质质谱分析综述].In:Current Protocols in Molecular Biology[摘自:最新分子生物学方法汇编],Ausubel等人编辑,纽约:威利,第10.21.1-10.21.27页;Paterson和Aebersold(1995)Electrophoresis[电泳]16:1791-1814;Patterson(1998)Proteinidentification and characterization by mass spectrometry[蛋白质的质谱鉴定与表征].In:Current Protocols in Molecular Biology[摘自:最新分子生物学方法汇编],Ausubel等人编辑,纽约:威利,第10.22.1-10.22.24页;以及Domon和Aebersold(2006)Science[科学]312(5771):212-17。
肽是一种短聚合物,由α-氨基酸按定义顺序连接而成。肽也可以通过用蛋白酶消化多肽(例如蛋白质)来产生。
“植物”是在发育的任何阶段的任何植物,特别是种子植物。
“植物细胞”是植物的结构和生理学单位,包含原生质体和细胞壁。植物细胞可以处于分离的单个细胞或培养细胞的形式,或者是作为较高级的组织单位(诸如像植物组织、植物器官、或全株植物)的一部分。
“植物细胞培养物”意指植物单元(诸如像原生质体、细胞培养物细胞、植物组织中的细胞、花粉、花粉管、胚珠、胚囊、接合子以及处于不同发育阶段的胚)的培养物。
“植物材料”是指叶、茎、根、花或花的部分、果实、花粉、卵细胞、接合子、种子、切条、细胞或组织培养物、或植物的任何其他部分或产物。
“植物器官”是植物的独特而明显的已结构化并且分化的部分,如根、茎、叶、花蕾或胚。
如本文使用的“植物组织”意指组织化成结构和功能单元的一组植物细胞。包括植物中或培养物中的任何植物组织。这个术语包括但不限于全株植物、植物器官、植物种子、组织培养物以及被组织化成结构和/或功能单元的任何植物细胞群组。这个术语与如以上列出的或由该定义以其他方式涵盖的任何具体类型的植物组织的联合应用或单独应用并不旨在排除任何其他类型的植物组织。
如本文使用的,术语“替代肽”是指通过蛋白水解消化,例如胰蛋白酶消化,从靶转基因蛋白衍生的肽,该靶转基因蛋白在质谱测定中起作用以产生一个或多个过渡离子,当在复合生物基质(诸如来自转基因植物的样品)中该靶转基因蛋白伴随存在一种或多种其他转基因蛋白和/或非转基因蛋白时,该一个或多个过渡离子与该替代肽组合差异检测和/或定量该靶转基因蛋白,并且不检测和/或定量该生物基质中的该一种或多种其他转基因蛋白或该非转基因蛋白。“替代肽”也可以称为该靶转基因蛋白的“特征(signature)肽”。例如,本发明的Cry1Ab替代肽产生一个或多个过渡离子,当该Cry1Ab转基因蛋白伴随存在一种或多种非Cry1Ab转基因蛋白例如本发明的eCry3.1Ab杀昆虫蛋白或mCry3A杀昆虫蛋白和/或非转基因蛋白时,其与Cry1Ab-替代肽组合差异检测和/或定量复合生物基质中的靶Cry1Ab转基因杀昆虫蛋白。在另一个实例中,本发明的eCry3.1Ab替代肽与eCry3.1Ab-替代肽组合产生一个或多个过渡离子,当在复合生物基质中该eCry3.1Ab转基因蛋白伴随存在一种或多种非eCry3.1Ab转基因蛋白例如本发明的Cry1Ab或mCry3A和/或非转基因蛋白时,其在复合生物基质中差异检测和/或定量靶eCry3.1Ab转基因蛋白。根据本发明的实施例,本发明的两种或更多种标记的替代肽可以同时用于质谱测定中,以检测和/或定量复合生物基质中的两种或多种靶转基因蛋白。
“标记的替代肽”是一种非天然存在的替代肽,其被标记使得易于在质谱测定中检测该替代肽。例如,该标记可以是稳定的同位素标记的氨基酸(SIL),诸如赖氨酸、异亮氨酸、缬氨酸或精氨酸。因此,SIL标记的替代肽具有与未标记的替代肽相同的氨基酸序列,不同之处在于该替代肽的一个或多个氨基酸被重同位素标记。例如,如本文所述,将该替代肽SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21)用重赖氨酸(K)标记,并且可以被命名为SAEFNNIIPSSQITQIPLTK[C13N15-K];将该替代肽TDVTDYHIDQV(SEQ ID NO:27)用重缬氨酸(V)标记,并可以命名为TDVTDYHIDQV[C13N15-V];将该替代肽LQSGASVVAGPR(SEQ ID NO:252)用精氨酸(R)标记,并可以命名为LQSGASVVAGPR[C13N15-R];将该替代肽DGGISQFIGDK(SEQ ID NO:36)用重赖氨酸(K)标记,并可以命名为DGGISQFIGDK[C13N15-K];将该替代肽FTTGTDLK(SEQ ID NO:255)用重赖氨酸(K)标记,并可以命名为FTTGTDLK[C13N15-K];将该替代肽SLTAAVTAAGGNATYVLDDGVPR(SEQ ID NO:257)用重精氨酸(R)标记,并可以命名为SLTAAVTAAGGNATYVLDDGVPR[C13N15-R];将该替代肽LGLGSTLYTHLLK(SEQ ID NO:79)用重赖氨酸标记,并可以命名为LGLGSTLYTHLLK[C13N15-K];将该替代肽SALDSQQGEPWQTIR(SEQ IDNO:89)用重精氨酸(R)标记,并可以命名为SALDSQQGEPWQTIR[C13N15-R],依此类推。
如PCT公开号WO 87/05629中所述,“PAT”(草丁膦N-乙酰基转移酶)蛋白赋予植物对草铵膦除草剂的耐受性。本发明的PAT蛋白的示例性实例由SEQ ID NO:264表示。
“PMI”(甘露糖6-磷酸异构酶)蛋白赋予植物细胞利用甘露糖的能力,如US5767378中所述。本发明的PMI蛋白的示例性实例由SEQ ID NO:265和SEQ ID NO:266表示。
如本文使用的,术语“堆叠(stacked或stacking)”是指植物的基因组中存在掺入的多个异源多核苷酸或转基因蛋白或转基因事件。
如本文所用,“靶蛋白”是指如下蛋白,典型地是转基因蛋白,当该靶蛋白处于复合生物基质中时,旨在通过标记的替代肽进行选择性检测和/或定量。
如本文使用的,术语“转基因蛋白”意指在非天然形式、位置、生物等中产生的蛋白或肽。因此,“转基因蛋白”可以是与天然存在的蛋白具有相同的氨基酸序列的蛋白,或它可以是具有非天然存在的氨基酸序列的蛋白。例如,当在转基因植物或细菌中产生时,与来自苏云金芽孢杆菌(产生天然Cry1Ab的生物)的野生型Cry1Ab蛋白具有相同氨基酸序列的Cry1Ab蛋白是“转基因蛋白”。
核苷酸在本文中用以下标准缩写表示:腺嘌呤(A)、胞嘧啶(C)、胸腺嘧啶(T)、以及鸟嘌呤(G)。氨基酸也由以下标准缩写表示:丙氨酸(Ala;A)、精氨酸(Arg;R)、天冬酰胺(Asn;N)、天冬氨酸(Asp;D)、半胱氨酸(Cys;C)、谷氨酰胺(Gln;Q)、谷氨酸(Glu;E)、甘氨酸(Gly;G)、组氨酸(His;H)、异亮氨酸(Ile;1)、亮氨酸(Leu;L)、赖氨酸(Lys;K)、甲硫氨酸(Met;M)、苯丙氨酸(Phe;F)、脯氨酸(Pro;P)、丝氨酸(Ser;S)、苏氨酸(Thr;T)、色氨酸(Trp;W)、酪氨酸(Tyr;Y)、以及缬氨酸(Val;V)。
本发明包括可用于进行质谱的组合物、方法和系统,用于差异检测和/或定量衍生自转基因植物的复合生物样品中的包含转基因和非转基因蛋白的混合物中的一种或多种靶转基因蛋白,例如,来自一种或多种转基因植物的叶、茎、根、花粉和种子的生物样品,每种生物样品对质谱测定结果的影响可能不同。本发明的组合物、方法和系统也可用于测试如下非转基因植物,该非转基因植物具有例如通过异花授粉而被来自邻近植物的转基因污染的风险。通过这些实施例,可以监测和限制转基因的偶然存在。在其他实施例中,本文披露的方法可用于筛选植物转化程序的结果,以鉴定表现出转基因蛋白的所需表达特征的转化体。
熟练技术人员可自行选择针对待分析的特定靶蛋白的偏好。此类蛋白可以是但不限于来自植物、动物、细菌、酵母等的那些,并且可以是见于非转化的细胞或见于转化的细胞的蛋白。在转基因植物中表达的特别适合的蛋白是赋予对除草剂、昆虫、或病毒的耐受性的那些,以及提供改进的营养价值、增加的产量、耐旱性、氮利用、生产有用的工业化合物、加工植物特征、或生物除污的潜力的基因。此类蛋白质的实例包括杀昆虫晶体蛋白(即Cry蛋白)和营养期杀昆虫蛋白(即来自苏云金芽孢杆菌的Vip)或由其衍生的工程化的蛋白(用于赋予昆虫抗性)、除草剂耐受蛋白(诸如5'-烯醇丙酮酸-3'-磷酸莽草酸合酶(EPSPS)或草丁膦乙酰转移酶(PAT)或可选择标记蛋白诸如磷酸甘露糖异构酶(PMI)。如本领域技术人员容易理解的,赋予所希望的性状的任何蛋白可以使用重组DNA技术在植物细胞中表达,并且因此可以是根据本发明的靶转基因蛋白。
更具体地,本发明提供了组合物、诊断方法和用于进行该诊断方法的系统,其允许对来自转基因植物组织(诸如叶、根、茎、花粉、种子或籽粒)的样品中的复合生物基质中的Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和PMI转基因蛋白进行特异性差异检测和/或定量。本发明的组合物、诊断方法和系统特别适用于在包含该转基因杀昆虫蛋白的复合生物样品中差异检测和/或定量高度相似的转基因杀昆虫蛋白,例如Cry1Ab、mCry3A和eCry3.1Ab。当前的现有技术是这样的,可商购的基于抗体的免疫测定不能用于从使用大量的Cry1Ab蛋白的氨基酸序列工程化的杂合Cry蛋白(当这两种蛋白在同一生物样品中时)示差检测Cry1Ab蛋白,这是因为在这两种类型的蛋白之间存在抗体的高度交叉反应性。例如,针对用于Cry1Ab检测免疫测定中的野生型Cry1Ab而产生的抗体与具有衍生自野生型Cry1Ab蛋白的少至27%的其氨基酸的杂合Cry蛋白(当这两种蛋白在同一生物样品中时)交叉反应。因此,例如,在这种复合生物样品中对野生型Cry1Ab的定量可能由于存在一种或多种非靶野生型Cry蛋白或非靶杂合Cry蛋白而混淆。此外,由于抗体对转基因植物产品中的Cry1Ab蛋白和杂合Cry蛋白的交叉反应性,使用所表达的蛋白质的检测用于包含野生型Cry1Ab和一种或多种本发明的杂合Cry蛋白的商品化转基因植物产品的身份保存是困难的。本文披露的方法和组合物提供了针对这些问题的解决方案,并依赖于来自靶转基因蛋白的替代肽和衍生自替代肽的过渡离子来进行靶蛋白的差异检测和/或定量,即使当该靶蛋白处于其他非常紧密相关的转基因蛋白和非转基因蛋白的混合物中。
通过质谱多反应监测测定(MRM)进行靶蛋白定量的准确性完全取决于适当替代肽的选择以及该替代肽/过渡离子组合对该靶蛋白的区分能力。可以用一种或多种来自Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI蛋白的特定肽通过MRM测定同时监测和定量本发明替代肽的许多不同组合,并因此提供通过质谱鉴定和定量给定生物样品中每种靶蛋白的方法。该七种靶蛋白的替代肽可以组成盒以定量每种相应的靶蛋白,即Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI。组成该盒的可用替代肽可在单个MRM测定中单独或以任何组合进行分析,或在多个MRM测定中进行分析。
本发明的替代肽结合基于MRM的测定具有众多应用,包括定量肽/蛋白质分析,用于确定不同生长阶段的表达水平,确定潜在的暴露水平以进行环境风险评估,确定食品加工中靶蛋白的不同水平,确定比较研究中的表达水平,并比较世代研究中的表达水平。从广义上讲,这七种蛋白质的独特替代肽可与MRM测定组合使用,以监测或定量可能处于特定组织(即叶、根、谷粒、花粉)中的单个转基因事件中或多个转基因事件的育种堆叠中的可选择标记物、除草剂耐受性或杀昆虫性状。
该基于MRM的测定可以定量或测量来自如下蛋白质的特定替代肽的相对或绝对水平,这些蛋白质包括Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI。这些蛋白质的相对定量水平可以通过MRM测定,通过相互比较特征峰面积来确定。可以从不同样品或组织类型中定量个体Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI替代肽的相对水平。通常,通过将MRM测量中的肽丰度与稳定的同位素标记的(SIL)合成肽类似物作为每种靶替代肽的内标进行比较,来确定相对定量水平。与本领域典型地教导的相反,典型地,通过掺入[13C6 15N2]赖氨酸或[13C6 15N4]精氨酸来标记SIL肽,但还可以包括其他氨基酸,诸如异亮氨酸和缬氨酸。该SIL标准需要具有高纯度,并应通过氨基酸分析进行定量标准化。与本领域典型地教导的相反,本发明的SIL在蛋白质消化后立即加标到样品中,从而用于校正后续的分析步骤。该SIL在液相色谱分离中与未标记的替代肽共洗脱,并显示出相同的MS/MS碎裂图谱,但由于该同位素标记,其仅质量不同。标记的替代肽和产物离子的质量偏移结果使该质谱仪区分未标记的肽和标记的肽。由于复合肽消化物通常包含多组可能被误认为该靶肽的共洗脱过渡物,因此该同位素标记的标准品的共洗脱可鉴定正确的信号,并提供最佳的保护,以防止假阳性定量。由于将已知浓度的加标SIL标准品加标到每个样品中,因此可以确定Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和/或PMI的来自不同靶蛋白的每种相应替代肽的相对量。由于可以相对于样品内部或样品之间另一种肽或多种肽的量进行一种或多种个体肽的相对定量,因此可以通过确定峰面积是否在该生物样品内彼此相对来确定存在的肽的相对量。从各个特征峰面积得出的不同样品之间的相对定量数据通常会归一化为每个样品分析的蛋白质的量。可以在一个样品和/或多个样品中同时对来自多种蛋白质的多种肽进行相对定量,以进一步了解一种肽/蛋白相对于其他肽/蛋白的相对蛋白量。
可以通过基于MRM的测定,通过比较一个生物样品中来自相应蛋白的个体替代肽的特征峰面积与该样品中已知数量的一种或多种内标来确定Cry1Ab、eCry3.1Ab、mCry3A、Vip3、mEPSPS、PAT和/或PMI的绝对定量水平。这可以通过将已知浓度的这些蛋白加标到不含该靶蛋白的阴性对照基质中来实现。该多重反应监测(MRM)测定包括用七种蛋白质中每种的确切加标浓度称量该非转基因样品;在裂解缓冲液中提取和均化样品;离心样品以分离可溶性和不溶性蛋白,以丰富和减少提取的复杂性;用胰蛋白酶消化可溶性蛋白样品(该组织或生物样品可以用一种或多种蛋白酶处理一段时间以充分消化该样品,该一种或多种蛋白酶包括但不限于胰蛋白酶、胰凝乳蛋白酶、胃蛋白酶、蛋白内切酶Asp-N和Lys-C),离心样品,添加固定浓度的SIL肽(绝对定量时,该SIL用作指示剂);通过利用阳离子交换的固相提取进行脱盐,以最小化基质效应或干扰并减少离子抑制;并且通过液相色谱与串联质谱联用分析该样品。典型地,执行典型地离子阱质谱仪或能够执行全局分析的另一种形式的质谱仪,以从单个复合蛋白/肽裂解物中鉴定出尽可能多的肽,以进行分析。尽管可以在任何类型的质谱仪上开发和执行基于MRM的测定,但通常认为MRM测定最有利的仪器平台是三重四极杆仪器平台。通过LC-MS/MS测量了对于这七种蛋白而言独特的目的替代肽和SIL。确定每种目的肽的峰面积比(替代肽的峰面积/相应的SIL肽的峰面积)。使用该峰面积比从校准曲线中反算出七种目的蛋白的浓度。可以对许多肽进行绝对定量,这允许同时在单个样品中和/或在多个样品中定量测定多种蛋白质,从而深入了解单个生物样品或大样品集中的绝对蛋白量。
在一些实施例中,本发明包括在质谱测定例如多反应监测测定中起作用以选择性地检测或定量靶转基因蛋白的标记的替代肽,所述靶转基因蛋白选自由以下组成的组:来自一种或多种转基因植物的一个或多个生物样品中的转基因蛋白和非转基因蛋白的混合物中的Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白和磷酸甘露糖异构酶(PMI)蛋白,该替代肽包含标记和选自由以下组成的组的氨基酸序列:GSAQGIEGSIR(SEQ ID NO:1)、IVAQLGQGVYR(SEQ ID NO:2)、TLSSTLYR(SEQ ID NO:3)、DVSVFGQR(SEQ ID NO:4)、TYPIR(SEQ ID NO:5)、TVSQLTR(SEQ ID NO:6)、WYNTGLER(SEQ ID NO:7)、EWEADPTNPALR(SEQ IDNO:8)、VWGPDSR(SEQ ID NO:9)、APMFSWIHR(SEQ ID NO:10)、WGFDAATINSR(SEQ ID NO:11)、NQAISR(SEQ ID NO:12)、IEEFAR(SEQ ID NO:13)、SGFSNSSVSIIR(SEQ ID NO:14)、LSHVSMFR(SEQ ID NO:15)、EIYTNPVLENFDGSFR(SEQ ID NO:16)、LEGLSNLYQIYAESFR(SEQ ID NO:17)、YNQFR(SEQ ID NO:18)、YNDLTR(SEQ ID NO:19)、SPHLMDILNSITIYTDAHR(SEQ ID NO:20)、SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21)、QGFSHR(SEQ ID NO:22)、MDNNPNINECIPYNCLSNPEVEVLGGER(SEQ ID NO:23)、ELTLTVLDIVSLFPNYDSR(SEQ ID NO:24)、RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR(SEQ ID NO:25)、SGTVDSLDEIPPQNNNVPPR(SEQ IDNO:26)、TDVTDYHIDQV(SEQ ID NO:27)、AVNELFTSSNQIGLK(SEQ ID NO:28)、ITQLPLTK(SEQID NO:29)、GLDSSTTK(SEQ ID NO:30)、QCAGIRPYDGR(SEQ ID NO:31)、IEFVPAEVTFEAEYDLER(SEQ ID NO:32)、ITQLPLVK(SEQ ID NO:33)、MTADNNTEALDSSTTK(SEQ ID NO:34)、VYIDK(SEQ ID NO:35)、DGGISQFIGDK(SEQ ID NO:36)、LITLTCK(SEQ ID NO:37)、ELLLATDLSNK(SEQ ID NO:38)、FEELTFATETSSK(SEQ ID NO:39)、EVLFEK(SEQ ID NO:40)、TASELITK(SEQID NO:41)、DVSEMFTTK(SEQ ID NO:42)、LLGLADIDYTSIMNEHLNK(SEQ ID NO:43)、IDFTK(SEQID NO:44)、TDTGGDLTLDEILK(SEQ ID NO:45)、DIMNMIFK(SEQ ID NO:46)、ALYVHK(SEQ IDNO:47)、VNILPTLSNTFSNPNYAK(SEQ ID NO:48)、ITSMLSDVIK(SEQ ID NO:49)、QNLQLDSFSTYR(SEQ ID NO:50)、DSLSEVIYGDMDK(SEQ ID NO:51)、MIVEAKPGHALIGFEISNDSITVLK(SEQ IDNO:52)、VYFSVSGDANVR(SEQ ID NO:53)、NQQLLNDISGK(SEQ ID NO:54)、VESSEAEYR(SEQ IDNO:55)、YMSGAK(SEQ ID NO:56)、DGSPADILDELTELTELAK(SEQ ID NO:57)、VYEAK(SEQ IDNO:58)、LDAINTMLR(SEQ ID NO:59)、GKPSIHLK(SEQ ID NO:60)、DENTGYIHYEDTNNNLEDYQTINK(SEQ ID NO:61)、DNFYIELSQGNNLYGGPIVHFYDVSIK(SEQ ID NO:62)、LLCPDQSEQIYYTNNIVFPNEYVITK(SEQ ID NO:63)、SQNGDEAWGDNFIILEISPSEK(SEQ IDNO:64)、NAYVDHTGGVNGTK(SEQ ID NO:65)、LDGVNGSLNDLIAQGNLNTELSK(SEQ ID NO:66)、IANEQNQVLNDVNNK(SEQ ID NO:67)、YEVTANFYDSSTGEIDLNK(SEQ ID NO:68)、QNYALSLQIEYLSK(SEQ ID NO:69)、QLQEISDK(SEQ ID NO:70)、LLSPELINTNNWTSTGSTNISGNTLTLYQGGR(SEQ ID NO:71)、YVNEK(SEQ ID NO:72)、QNYQVDK(SEQ ID NO:73)、MAGAEEIVLQPIK(SEQ ID NO:74)、FPVEDAK(SEQ ID NO:75)、EISGTVK(SEQ ID NO:76)、ILLLAALSEGTTVVDNLLNSEDVHYMLGALR(SEQ ID NO:77)、DFELPAPPRPVRPVTQI(SEQ ID NO:78)、LGLGSTLYTHLLK(SEQ ID NO:79)、MSPER(SEQ ID NO:80)、HGGWHDVGFWQR(SEQ ID NO:81)、NAYDWTVESTVYVSHR(SEQ ID NO:82)、TEPQTPQEWIDDLER(SEQ ID NO:83)、AAGYK(SEQ IDNO:84)、YPWLVAEVEGVVAGIAYAGPWK(SEQ ID NO:85)、RPVEIRPATAADMAAVCDIVNHYIETSTVNFR(SEQ ID NO:86)、ENAAGIPMDAAER(SEQ ID NO:87)、ALAILK(SEQ ID NO:88)、SALDSQQGEPWQTIR(SEQ ID NO:89)、GSQQLQLKPGESAFIAANESPVTVK(SEQ ID NO:90)、FEAKPANQLLTQPVK(SEQ ID NO:91)、STLLGEAVAK(SEQ ID NO:92)、LINSVQNYAWGSK(SEQ IDNO:93)、HNSEIGFAK(SEQ ID NO:94)、VLCAAQPLSIQVHPNK(SEQ ID NO:95)、TALTELYGMENPSSQPMAELWMGAHPK(SEQ ID NO:96)、LSELFASLLNMQGEEK(SEQ ID NO:97)和QGAELDFPIPVDDFAFSLHDLSDK(SEQ ID NO:98)。在其他实施例中,通过掺入稳定的同位素标记的(SIL)氨基酸来标记该替代肽。在其他实施例中,通过掺入[13C6 15N2]赖氨酸、[13C6 15N2]异亮氨酸、[13C6 15N2]缬氨酸或[13C6 15N2]精氨酸来标记该SIL肽。
在一些实施例中,所述标记的替代肽选择性地检测或定量转基因和非转基因蛋白混合物中的Cry1Ab蛋白,并包含选自由以下组成的组的氨基酸序列:GSAQGIEGSIR(SEQ IDNO:1)、IVAQLGQGVYR(SEQ ID NO:2)、TLSSTLYR(SEQ ID NO:3)、DVSVFGQR(SEQ ID NO:4)、TYPIR(SEQ ID NO:5)、TVSQLTR(SEQ ID NO:6)、WYNTGLER(SEQ ID NO:7)、EWEADPTNPALR(SEQ ID NO:8)、VWGPDSR(SEQ ID NO:9)、APMFSWIHR(SEQ ID NO:10)、WGFDAATINSR(SEQ IDNO:11)、NQAISR(SEQ ID NO:12)、IEEFAR(SEQ ID NO:13)、SGFSNSSVSIIR(SEQ ID NO:14)、LSHVSMFR(SEQ ID NO:15)、EIYTNPVLENFDGSFR(SEQ ID NO:16)、LEGLSNLYQIYAESFR(SEQ IDNO:17)、YNQFR(SEQ ID NO:18)、YNDLTR(SEQ ID NO:19)、SPHLMDILNSITIYTDAHR(SEQ IDNO:20)、SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21)、QGFSHR(SEQ ID NO:22)、MDNNPNINECIPYNCLSNPEVEVLGGER(SEQ ID NO:23)、ELTLTVLDIVSLFPNYDSR(SEQ ID NO:24)、RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR(SEQ ID NO:25)和SGTVDSLDEIPPQNNNVPPR(SEQ IDNO:26)。
在其他实施方案中,本发明的Cry1Ab特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:GIEGSIR(SEQ ID NO:99)、EGSIR(SEQ ID NO:100)、AQLGQGVYR(SEQ ID NO:101)、GQGVYR(SEQ ID NO:102);SSTLYR(SEQ ID NO:103)、STLYR(SEQ ID NO:104)、SVFGQR(SEQ ID NO:105)、FGQR(SEQ ID NO:106)、PIR、TY、SQLTR(SEQ IDNO:107)、QLTR(SEQ ID NO:108)、NTGLER(SEQ ID NO:109)、YNTGLER(SEQ ID NO:110)、PTNPALR(SEQ ID NO:111)、DPTNPALR(SEQ ID NO:112)、GPDSR(SEQ ID NO:113)、VW、HR、SWIHR(SEQ ID NO:114)、ATINSR(SEQ ID NO:115)、DAATINSR(SEQ ID NO:116)、AISR(SEQID NO:117)、ISR、EFAR(SEQ ID NO:118)、EEFAR(SEQ ID NO:119)、SNSSVSIIR(SEQ ID NO:120)、SSVSIIR(SEQ ID NO:121)、SMFR(SEQ ID NO:122)、VSMFR(SEQ ID NO:123)、ENFDGSFR(SEQ ID NO:124)、GSFR(SEQ ID NO:125)、YAESFR(SEQ ID NO:126)、LEG、NQFR(SEQ ID NO:127)、QFR、DLTR(SEQ ID NO:128)、NDLTR(SEQ ID NO:129)、TIYTDAHR(SEQ ID NO:130)、YTDAHR(SEQ ID NO:131)、PLTK(SEQ ID NO:132)、SAEFNNII(SEQ ID NO:133)、FSHR(SEQ IDNO:134)、GFSHR(SEQ ID NO:135)、EVLGGER(SEQ ID NO:136)、GGER(SEQ ID NO:137)、FPNYDSR(SEQ ID NO:138)、PNYDSR(SEQ ID NO:139)、PSAVYR(SEQ ID NO:140)、YR、PPR和SGTVDSLDE(SEQ ID NO:141)。
在仍其他实施例中,本发明的Cry1Ab特异性标记的替代肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQ ID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。
在一些实施例中,本发明的标记的替代肽选择性地检测或定量eCry3.1Ab蛋白,并包含选自由以下组成的组的氨基酸序列:TDVTDYHIDQV(SEQ ID NO:27)、AVNELFTSSNQIGLK(SEQ ID NO:28)、ITQLPLTK(SEQ ID NO:29)、GLDSSTTK(SEQ ID NO:30)、QCAGIRPYDGR(SEQID NO:31)和IEFVPAEVTFEAEYDLER(SEQ ID NO:32)。
在其他实施例中,该eCry3.1Ab特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:TDYHIDQV(SEQ ID NO:142)、DYHIDQV(SEQ ID NO:143)、TSSNQIGLK(SEQ ID NO:144)、SSNQIGLK(SEQ ID NO:145)、QLPLTK(SEQ ID NO:146)、TQLPLTK(SEQ ID NO:147)、DSSTTK(SEQ ID NO:148)、SSTTK(SEQ ID NO:149)、PYDGR(SEQID NO:150)、DGR、IEF和LER。
在仍其他实施例中,本发明的eCry3.1Ab特异性标记的替代肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。
在一些实施例中,该标记的替代肽选择性地检测或定量mCry3A蛋白,并包含选自由以下组成的组的氨基酸序列:ITQLPLVK(SEQ ID NO:33)、MTADNNTEALDSSTTK(SEQ ID NO:34)、VYIDK(SEQ ID NO:35)和LQSGASVVAGPR(SEQ ID NO:252)。
在其他实施例中,该mCry3A特异性替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:QLPLVK(SEQ ID NO:151)、TQLPLVK(SEQ ID NO:152)、ALDSSTTK(SEQ IDNO:153)、EALDSSTTK(SEQ ID NO:154)、YIDK(SEQ ID NO:155)和IDK。
在仍其他实施例中,本发明的mCry3A特异性标记的替代肽包含氨基酸序列LQSGASVVAGPR(SEQ ID NO:252),并产生由氨基酸序列SGASVVAGPR(SEQ ID NO:253)或SVVAGPR(SEQ ID NO:254)组成的过渡离子。
在一些实施例中,本发明的标记的替代肽选择性地检测或定量Vip3A蛋白,并包含选自由以下组成的组的氨基酸序列:DGGISQFIGDK(SEQ ID NO:36)、LITLTCK(SEQ ID NO:37)、ELLLATDLSNK(SEQ ID NO:38)、FEELTFATETSSK(SEQ ID NO:39)、EVLFEK(SEQ ID NO:40)、TASELITK(SEQ ID NO:41)、DVSEMFTTK(SEQ ID NO:42)、LLGLADIDYTSIMNEHLNK(SEQ IDNO:43)、IDFTK(SEQ ID NO:44)、TDTGGDLTLDEILK(SEQ ID NO:45)、DIMNMIFK(SEQ ID NO:46)、ALYVHK(SEQ ID NO:47)、VNILPTLSNTFSNPNYAK(SEQ ID NO:48)、ITSMLSDVIK(SEQ IDNO:49)、QNLQLDSFSTYR(SEQ ID NO:50)、DSLSEVIYGDMDK(SEQ ID NO:51)、MIVEAKPGHALIGFEISNDSITVLK(SEQ ID NO:52)、VYFSVSGDANVR(SEQ ID NO:53)、NQQLLNDISGK(SEQ ID NO:54)、VESSEAEYR(SEQ ID NO:55)、YMSGAK(SEQ ID NO:56)、DGSPADILDELTELTELAK(SEQ ID NO:57)、VYEAK(SEQ ID NO:58)、LDAINTMLR(SEQ ID NO:59)、GKPSIHLK(SEQ ID NO:60)、DENTGYIHYEDTNNNLEDYQTINK(SEQ ID NO:61)、DNFYIELSQGNNLYGGPIVHFYDVSIK(SEQ ID NO:62)、LLCPDQSEQIYYTNNIVFPNEYVITK(SEQ IDNO:63)、SQNGDEAWGDNFIILEISPSEK(SEQ ID NO:64)、NAYVDHTGGVNGTK(SEQ ID NO:65)、LDGVNGSLNDLIAQGNLNTELSK(SEQ ID NO:66)、IANEQNQVLNDVNNK(SEQ ID NO:67)、YEVTANFYDSSTGEIDLNK(SEQ ID NO:68)、QNYALSLQIEYLSK(SEQ ID NO:69)、QLQEISDK(SEQID NO:70)、LLSPELINTNNWTSTGSTNISGNTLTLYQGGR(SEQ ID NO:71)、YVNEK(SEQ ID NO:72)、QNYQVDK(SEQ ID NO:73)和FTTGTDLK(SEQ ID NO:255)。
在其他实施例中,该Vip3A特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:SQFIGDK(SEQ ID NO:156)、GDK、TLTCK(SEQ ID NO:157)、TCK、ATDLSNK(SEQ ID NO:158)、LATDLSNK(SEQ ID NO:159)、TFATETSSK(SEQ ID NO:160)、FATETSSK(SEQ ID NO:161)、FEK、LFEK(SEQ ID NO:162)、SELITK(SEQ ID NO:163)、ASELITK(SEQ ID NO:164)、SEMFTTK(SEQ ID NO:165)、DVS、IMNEHLNK(SEQ ID NO:166)、MNEHLNK(SEQ ID NO:167)、DFTK(SEQ ID NO:168)、FTK、TLDEILK(SEQ ID NO:169)、LTLDEILK(SEQID NO:170)、MNMIFK(SEQ ID NO:171)、NMIFK(SEQ ID NO:172)、YVHK(SEQ ID NO:173)、HK、VNI、VNIL(SEQ ID NO:174)、SMLSDVIK(SEQ ID NO:175)、TSMLSDVIK(SEQ ID NO:176)、DSFSTYR(SEQ ID NO:177)、LDSFSTYR(SEQ ID NO:178)、IYGDMDK(SEQ ID NO:179)、VIYGDMDK(SEQ ID NO:180)、SNDSITVLK(SEQ ID NO:181)、MIV、SGDANVR(SEQ ID NO:182)、SVSGDANVR(SEQ ID NO:183)、LLNDISGK(SEQ ID NO:184)、LNDISGK(SEQ ID NO:185)、SSEAEYR(SEQ ID NO:186)、ESSEAEYR(SEQ ID NO:187)、SGAK(SEQ ID NO:188)、MSGAK(SEQID NO:189)、TELTELAK(SEQ ID NO:190)、DGSPADI(SEQ ID NO:191)、YEAK(SEQ ID NO:192)、EAK、NTMLR(SEQ ID NO:193)、AINTMLR(SEQ ID NO:194)、PSIHLK(SEQ ID NO:195)、HLK、DYQTINK(SEQ ID NO:196)、NK、DNF、DNFY(SEQ ID NO:197)、PNEYVITK(SEQ ID NO:198)、LLC、SPSEK(SEQ ID NO:199)、LEISPSEK(SEQ ID NO:200)、NAY、DHTGGVNGTK(SEQ IDNO:201)、GNLNTELSK(SEQ ID NO:202)、NTELSK(SEQ ID NO:203)、LNDVNNK(SEQ ID NO:204)、NDVNNK(SEQ ID NO:205)、YE、DLNK(SEQ ID NO:206)、QIEYLSK(SEQ ID NO:207)、LQIEYLSK(SEQ ID NO:208)、SDK、QEISDK(SEQ ID NO:209)、YQGGR(SEQ ID NO:210)、TLYQGGR(SEQ ID NO:211)、NEK、VNEK(SEQ ID NO:212)、DK和VDK。
在仍其他实施例中,本发明的Vip3A特异性标记的替代肽包含氨基酸序列FTTGTDLK(SEQ ID NO:255),并产生由氨基酸序列TGTDLK(SEQ ID NO:256)和LK组成的过渡离子。
在一些实施例中,本发明的标记的替代肽选择性地检测或定量dmEPSPS蛋白,并包含选自由以下组成的组的氨基酸序列:MAGAEEIVLQPIK(SEQ ID NO:74)、FPVEDAK(SEQ IDNO:75)、EISGTVK(SEQ ID NO:76)、ILLLAALSEGTTVVDNLLNSEDVHYMLGALR(SEQ ID NO:77)和SLTAAVTAAGGNATYVLDGVPR(SEQ ID NO:257)。
在其他实施例中,该EPSPS特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:PIK、EIVLQPIK(SEQ ID NO:213)、PVEDAK(SEQ ID NO:214)、VEDAK(SEQ ID NO:215)、SGTVK(SEQ ID NO:216)、GTVK(SEQ ID NO:217)、ILLLAA(SEQ ID NO:218)和HYMLGALR(SEQ ID NO:219)。
在仍其他实施例中,本发明的dmEPSPS特异性标记的替代肽包含氨基酸序列SLTAAVTAAGGNATYVLDGVPR(SEQ ID NO:257),并产生由氨基酸序列PR和GVPR(SEQ ID NO:258)组成的过渡离子。
在一些实施例中,本发明的标记的替代肽选择性地检测或定量PAT蛋白,并包含选自由以下组成的组的氨基酸序列:DFELPAPPRPVRPVTQI(SEQ ID NO:78)、LGLGSTLYTHLLK(SEQ ID NO:79)、MSPER(SEQ ID NO:80)、HGGWHDVGFWQR(SEQ ID NO:81)、NAYDWTVESTVYVSHR(SEQ ID NO:82)、TEPQTPQEWIDDLER(SEQ ID NO:83)、AAGYK(SEQ ID NO:84)、YPWLVAEVEGVVAGIAYAGPWK(SEQ ID NO:85)和RPVEIRPATAADMAAVCDIVNHYIETSTVNFR(SEQ ID NO:86)。
在其他实施例中,该PAT特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:DFE、DF、YTHLLK(SEQ ID NO:220)、THLLK(SEQ ID NO:221)、PER、SPER(SEQ ID NO:222)、GFWQR(SEQ ID NO:223)、VGFWQR(SEQ ID NO:224)、STVYVSHR(SEQ IDNO:225)、SHR、TEPQT(SEQ ID NO:226)、DLER(SEQ ID NO:227)、GYK、AGYK(SEQ ID NO:228)、GPWK(SEQ ID NO:229)、GIAYAGPWK(SEQ ID NO:230)、TSTVNFR(SEQ ID NO:231)和NFR。
在仍其他实施例中,该PAT特异性标记的替代肽包含氨基酸序列LGLGSTLYTHLLK(SEQ ID NO:79),并产生由氨基酸序列YTHLLK(SEQ ID NO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。
在一些实施例中,本发明的标记的替代肽选择性地检测或定量PMI蛋白,并包含选自由以下组成的组的氨基酸序列:ENAAGIPMDAAER(SEQ ID NO:87)、ALAILK(SEQ ID NO:88)、SALDSQQGEPWQTIR(SEQ ID NO:89)、GSQQLQLKPGESAFIAANESPVTVK(SEQ ID NO:90)、FEAKPANQLLTQPVK(SEQ ID NO:91)、STLLGEAVAK(SEQ ID NO:92)、LINSVQNYAWGSK(SEQ IDNO:93)、HNSEIGFAK(SEQ ID NO:94)、VLCAAQPLSIQVHPNK(SEQ ID NO:95)、TALTELYGMENPSSQPMAELWMGAHPK(SEQ ID NO:96)、LSELFASLLNMQGEEK(SEQ ID NO:97)和QGAELDFPIPVDDFAFSLHDLSDK(SEQ ID NO:98)。
在其他实施例中,该PMI特异性标记的替代肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:PMDAAER(SEQ ID NO:232)、GIPMDAAER(SEQ ID NO:233)、AILK(SEQID NO:234)、LK、PWQTIR(SEQ ID NO:235)、GEPWQTIR(SEQ ID NO:236)、ANESPVTVK(SEQ IDNO:237)、PVTVK(SEQ ID NO:238)、LTQPVK(SEQ ID NO:239)、PVK、GEAVAK(SEQ ID NO:240)、LGEAVAK(SEQ ID NO:241)、QNYAWGSK(SEQ ID NO:242)、NYAWGSK(SEQ ID NO:243)、NSEIGFAK(SEQ ID NO:244)、HN、VLCAAQ(SEQ ID NO:245)、PNK、WMGAHPK(SEQ ID NO:246)、TALTE(SEQ ID NO:247)、NMQGEEK(SEQ ID NO:248)、LNMQGEEK(SEQ ID NO:249)、SLHDLSDK(SEQ ID NO:250)和HDLSDK(SEQ ID NO:251)。
在仍其他实施例中,该PMI特异性替代肽包含氨基酸序列SALDSQQGEPWQTIR(SEQID NO:89),并产生由氨基酸序列PWQTIR(SEQ ID NO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。
根据一些实施例,本发明的Cry1Ab特异性标记的替代肽检测和/或定量包含SEQID NO:259的氨基酸序列的Cry1Ab蛋白。在其他实施例中,该Cry1Ab蛋白来自该转基因玉米事件Bt11。
在一些实施例中,本发明的eCry3.1Ab特异性标记的替代肽检测和/或定量包含SEQ ID NO:260的氨基酸序列的eCry3.1Ab蛋白。在其他实施例中,该eCry3.1Ab蛋白来自转基因玉米事件5307。
根据一些实施例,本发明的mCry3A特异性标记的替代肽检测和/或定量包含SEQID NO:261的氨基酸序列的mCry3A蛋白。在其他实施例中,该mCry3A蛋白来自该转基因玉米事件MIR604。
根据一些实施例,本发明的Vip3特异性标记的替代肽检测和/或定量包含SEQ IDNO:262的氨基酸序列的Vip3Aa蛋白。在其他实施例中,该Vip3Aa蛋白来自该转基因玉米事件MIR162。
根据一些实施例,本发明的dmEPSPS特异性标记的替代肽检测和/或定量包含SEQID NO:263的氨基酸序列的dmEPSPS蛋白。在其他实施例中,该dmEPSPS蛋白来自该转基因玉米事件GA21。
根据一些实施例,本发明的PAT特异性标记的替代肽检测和/或定量包含SEQ IDNO:264的氨基酸序列的PAT蛋白。在其他实施例中,该PAT蛋白来自该转基因玉米事件Bt11、59122、TC1507、DP4114或T25。
根据一些实施例,本发明的PMI特异性标记的替代肽检测和/或定量包含SEQ IDNO:265或SEQ ID NO:266的氨基酸序列的PMI蛋白。在其他实施例中,该PMI蛋白来自该转基因玉米事件MIR162、MIR604、5307或3272。
在一些实施例中,本发明的标记的替代肽特异性地检测或定量转基因蛋白的混合物中的Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、dmEPSPS蛋白、PAT蛋白或PMI蛋白,该转基因蛋白包含至少两种选自由以下组成的组的转基因蛋白:Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3A蛋白、dmEPSPS蛋白、PAT蛋白和PMI蛋白。在其他实施例中,转基因蛋白的混合物包含Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3A蛋白、dmEPSPS蛋白、PAT蛋白和PMI蛋白。在仍其他实施例中,转基因蛋白的混合物进一步包含选自由以下组成的组的至少一种转基因蛋白:Cry1A.105蛋白(SEQ ID NO:267)、Cry1F蛋白(SEQ ID NO:268)、Cry34蛋白(SEQ ID NO:269)和Cry35蛋白(SEQ ID NO:270)。
在一些实施例中,本发明的标记的替代肽特异性地检测或定量来自转基因植物的生物样品中的蛋白转基因混合物中的Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、dmEPSPS蛋白、PAT蛋白或PMI蛋白,其中该转基因植物是玉米植物、大豆植物、棉花植物、水稻植物、小麦植物或卡诺拉油菜植物。在其他实施例中,该转基因植物是玉米植物,其包含选自由以下组成的组的转基因事件:事件Bt11、事件5307、事件MIR604、事件MIR162、事件3272和事件GA21。在仍其他实施例中,该转基因玉米植物进一步包含事件MON89034、事件DP4114、事件TC1507、事件59122或事件T25。
在一些实施例中,本发明的标记的替代肽特异性地检测或定量来自转基因植物的叶组织、种子、籽粒、花粉或根组织的生物样品中的Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、dmEPSPS蛋白、PAT蛋白或PMI蛋白。在其他实施例中,该叶组织、种子、籽粒、花粉或根组织来自转基因玉米植物,其包含一种或多种该转基因玉米事件Bt11、5307、MIR604、MIR162、GA21、3272、59122、DP4114、TC1507和T25。
本领域中有许多参考文献提出了许多不同的方法来预测哪种替代肽对于任何给定的靶蛋白而言是最佳的,许多参考文献提出了使用质谱定量靶蛋白的捷径,例如Mead等人2009.Mol.Cell.Proteomics[分子和细胞的蛋白质组学]8:696-705和美国专利号8,227,252。但是,对此类预测方法和捷径的依赖会导致混淆结果,因为不可预测的因素会干扰基于质谱的测定,从而导致灵敏度损失和定量结果不准确。至少一个主要因素在于生物基质本身。例如,从与来自转基因植物的叶、根、花粉和种子的生物样品同样有效的替代肽中鉴定出单个过渡离子是非常不可预测且困难的。该基质的化学组成、pH或离子强度的差异可能会影响MS仪器中的蛋白水解、肽稳定性、聚集或电离。因此,必须鉴定和凭经验测试所有相关基质(特别是转基因植物的基质)的替代肽和特定替代肽/过渡离子组合,以克服此类测定的不可预测的性质。本发明在开发质谱测定中采用了两步途径,以特异性地检测和/或定量靶转基因蛋白,包括1)从蛋白水解切割的靶蛋白衍生的肽库中测试和选择替代肽,以及测试SIL替代肽和过渡离子肽的组合,并选择能在所有生物基质(例如来自转基因植物的叶、根、花粉或种子的生物样品)中特异性检测和定量该靶蛋白的组合;以及2)根据经验确定适合所有生物基质(包括转基因植物,特别是转基因玉米植物的叶、根、花粉和种子)中所有替代肽和替代肽/过渡离子组合的样品制备方法和质谱条件。
因此,在一些实施例中,本发明包括同时检测和/或定量来自转基因植物的包含靶转基因蛋白和非转基因蛋白的混合物的复合生物样品中的一种或多种靶转基因蛋白的方法,其中该方法包括以下步骤:a)从转基因植物中获得生物样品;b)从所述生物样品中提取蛋白,得到包含蛋白的混合物的提取物;c)减少步骤b的提取物中非转基因不溶性蛋白的量,得到浓缩的可溶性蛋白的提取物;d)消化步骤c的提取物中的可溶性蛋白,得到包含肽碎片的提取物,其中所述肽碎片包含至少一种对每种靶转基因蛋白具有特异性的未标记的替代肽;e)浓缩步骤d的提取物中的肽碎片;f)添加一种或多种本发明的标记的替代肽,其中每种标记的替代肽具有与衍生自该靶转基因蛋白的每种未标记的替代肽相同的氨基酸序列,并且其中添加的标记的替代肽的数目等于该混合物中靶转基因蛋白的数目;g)通过减少所述混合物中非替代肽的量来浓缩所述未标记的替代肽和所述标记的替代肽;h)通过液相色谱使步骤g的所述肽碎片混合物分解;i)通过质谱分析从步骤h得到的所述肽碎片混合物,其中检测到未标记的替代肽的过渡离子碎片指示存在衍生所述替代肽的靶转基因蛋白;以及,任选地,j)通过比较由步骤i的所述过渡离子碎片产生的质谱信号与由标记的替代肽的过渡离子产生的质谱信号,计算所述生物样品中靶转基因蛋白的量。
在一些实施例中,通过上述方法检测和/或定量的靶转基因蛋白是Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白或磷酸甘露糖异构酶(PMI)蛋白。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是Cry1Ab,并且该标记的替代肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQ ID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列PLTK(SEQ ID NO:132)组成的未标记的和标记的过渡离子产生的质谱信号,对该Cry1Ab靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是eCry3.1Ab,并且该标记的替代肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列TDYHIDQV(SEQ ID NO:142)组成的未标记的和标记的过渡离子产生的质谱信号,对该eCry3.1Ab靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是mCry3A,并且该标记的替代肽包含氨基酸序列LQSGASVVAGPR(SEQ ID NO:252),并产生由氨基酸序列SGASVVAGPR(SEQ ID NO:253)或SVVAGPR(SEQ ID NO:254)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列SGASVVAGPR(SEQ ID NO:253)组成的未标记的和标记的过渡离子产生的质谱信号,对该mCry3A靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是Vip3A,并且该标记的替代肽包含氨基酸序列FTTGTDLK(SEQ ID NO:255),并产生由氨基酸序列TGTDLK(SEQ ID NO:256)或LK组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列TGTDLK(SEQ ID NO:256)组成的未标记的和标记的过渡离子产生的质谱信号,对该Vip3A靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是dmEPSPS,并且该标记的替代肽包含氨基酸序列SLTAAVTAAGGNATYVLDGVPR(SEQ ID NO:257),并产生由氨基酸序列PR或GVPR(SEQ ID NO:258)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列PR组成的未标记的和标记的过渡离子产生的质谱信号,对该eCry3.1Ab靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是PAT,并且该标记的替代肽包含氨基酸序列LGLGSTLYTHLLK(SEQ ID NO:79),并产生由氨基酸序列YTHLLK(SEQ IDNO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列YTHLLK(SEQ ID NO:220)组成的未标记的和标记的过渡离子产生的质谱信号,对该PAT靶蛋白进行定量。
在本发明的方法涵盖的其他实施例中,该靶转基因蛋白是PMI,并且该标记的替代肽包含氨基酸序列SALDSQQGEPWQTIR(SEQ ID NO:89),并产生由氨基酸序列PWQTIR(SEQ IDNO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。在仍其他实施例中,通过比较由氨基酸序列PWQTIR(SEQ ID NO:235)组成的未标记的和标记的过渡离子产生的质谱信号,对该PMI靶蛋白进行定量。
在其他实施例中,本发明包括用于高通量检测或定量转基因靶蛋白的系统。这样的系统包含对该转基因靶蛋白具有特异性的预先设计的标记的替代肽的盒;和一个或多个质谱仪。在该实施例的一方面,该盒包含对选自由以下组成的组的靶蛋白具有特异性的标记的替代肽:Cry1Ab、eCry3.1Ab、mCry3A、Vip3、dmEPSPS、PAT和PMI。在该实施例的其他方面,该标记的替代肽包含SEQ ID NO:1-98中的任一个。在该实施例的其他方面,该标记的替代肽产生一个或多个过渡离子,其包含选自由SEQ ID NO:99-251、SEQ ID NO:254、255、256,肽PIR、TY、VW、HR、ISR、LEG、QFR、YR、PPR、DGR、IEF、LER、IDK、GDK、TCK、FEK、DVS、FTK、HK、VNI、MIV、EAK、HLK、NK、DNF、LLC、NAY、YE、SDK、NEK、DK、VDK、PIK、DFE、DF、PER、SHR、GYK、NFR、LK、PVK、HN、PNK和PR中至少一个组成的组的肽序列。
包括以下特定实例,以说明本发明的优选实施例。本领域技术人员应当理解,以下实例中披露的技术代表本发明人发现的在本发明的实践中良好地起作用的技术,因此可以认为构成本发明实践的优选模式。然而,根据本披露,本领域技术人员应当理解,可以在所披露的特定实施例中进行许多改变,并且在不脱离本发明的概念、精神和范围的情况下仍可获得相似或类似的结果。更具体地,将清楚的是,化学和生理学均相关的某些药剂可以用本文描述的药剂取代,同时实现相同或相似的结果。对于本领域技术人员而言清楚的所有此类类似取代和修改被认为在如所附权利要求书所限定的本发明的精神、范围和概念之内。
实例
实例1-替代肽选择
基于MRM的测定依赖于选择一组预定的肽,并取决于每种选择的替代肽的特定碎裂/过渡离子。选择合适的替代或特征肽需要几个标准。首先,必须选择构成该靶向蛋白盒的蛋白质。其次,对于每种靶蛋白,必须鉴定出表现出良好质谱响应并独特地鉴定该靶蛋白或其特异性修饰(即,翻译后修饰)的那些肽。第三,对于每种质谱合适的肽,必须鉴定出那些提供最佳信号强度并将该替代肽与该样品中存在的其他肽种类独特地区分开的过渡离子。这些标准对于执行基于MRM的测定至关重要。
鉴定并选择了来自七种转基因蛋白Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI的替代肽,用于基于MRM的测定。使用微生物产生的蛋白开发了该MRM测定,这些微生物产生的蛋白用胰蛋白酶消化。用水将微生物产生的蛋白分别重构。然后将三氟乙醇(TFE)添加到每种蛋白质的等分试样中,然后添加100mM碳酸氢铵和胰蛋白酶(1:10(w:w)酶:蛋白比例)。将该样品在约37℃下消化过夜,然后添加约0.05M的三(2-羧乙基)膦(TCEP)。等分每种蛋白质以形成终浓度为约200pmol/μL的库。将该肽混合物用于在QTRAP6500质谱仪(爱博才思公司(AB Sciex LLC),弗雷明汉,美国马萨诸塞州)上开发该MRM测定。使用选择的反应监测MS/MS确定每种肽的最佳的两个过渡(由质谱仪监测的肽替代物和碎片离子质荷比(m/z)的组合)。通过针对每种肽计算双电荷和三电荷肽离子以及第一和第二y碎片离子(其m/z大于[m/z(替代)+20Da])的特征质量来开发该MS/MS方法。如果在该MRM扫描过程中观察到这些计算的过渡,则该仪器将自动切换到MS/MS模式,并获得该替代肽离子的完整MS/MS谱。对于每种获得的肽,确定了该MS/MS谱中两个最强的碎片离子(仅b或y碎片离子)及其洗脱时间。然后针对每个选定的过渡优化碰撞能量(CE)。所开发的MRM测定用于校准曲线样品的分析。
该MRM测定靶向来自7种转基因蛋白的193种蛋白型肽(proteotypic peptide)。其中,111种肽是对于这7种蛋白而言独特的,并且与已知的玉米蛋白不重叠。表1列出了每种靶蛋白的替代肽和过渡离子的特征,包括氨基酸序列(包括包含至少四个氨基酸的肽的序列表标识符)、单同位素质量、特征电荷状态、特征m/z和产物过渡m/z。鉴定了所有七种蛋白的独特替代肽;Cry1Ab(26)、eCry3.1Ab(6)、mCry3A(4)、Vip3Aa20(39)、dmEPSPS(5)、PAT(9)和PMI(12)。这些来自Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI的替代肽经鉴定可用于确定Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI转基因蛋白的绝对或相对量。表1列出的这些肽或肽组合中的每一种均通过质谱在裂解物中检测到,并且是基于MRM的测定中用于定量Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI的潜在候选物。
表1.替代肽和过渡离子的特征。
鉴定了靶蛋白Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI的多种潜在替代肽后,根据提供最佳信号强度并具有将该靶替代肽与生物样品基质(例如,玉米叶、根、花粉或谷粒(种子))中存在的其他种类中区分开的能力的过渡离子进一步选择个体替代肽。这既包括基质干扰(即基质干扰是在目的肽处或附近检测到的该基质中的一种或多种特定成分)和潜在的遗留物(即遗留物是由于该样品分析系统的化学/物理特性或二者,先前注入的样品在后续分析中洗脱的结果)。包含该个体替代肽的盒的这些优化过渡组成了整个MRM测定。在本披露中,对来自Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI的提供了最高的灵敏度(最强的碎片)并且具有期望的特异性的那些替代肽进行进一步选择以组成替代肽的盒,用于定量该七种靶向蛋白。表2列出了每种靶蛋白Cry1Ab、eCry3.1Ab、mCry3A、Vip3Aa20 dmEPSPS、PAT和PMI的优选替代肽,以及相应的稳定同位素标记的(SIL)肽。替代肽的盒包含要同时监测和/或定量的肽中的一种或多种。具有每种肽的特异性碎裂/过渡离子的替代肽的该盒可用于MRM测定,以定量相应的靶蛋白。
表2.特异性地检测靶蛋白的替代肽和SIL肽。
实例2-用于在转基因植物组织中检测转基因蛋白的测定
开发直接检测靶蛋白的灵敏方法对于在生物基质(诸如转基因植物的组织,例如叶、谷粒、根和花粉组织)中进行定量评估非常需要。多反应监测(MRM)质谱已成为用以通过液相色谱(LC)与串联质谱(MS/MS/)联用定量给定样品中多种蛋白质的有前途的平台。MRM测定利用蛋白水解肽的序列特异性串联MS碎裂,从而为不同的靶蛋白提供高度选择性和特异性的测量。尽管取得了这些进展,但要对低丰度蛋白或具有影响分离的特定理化性质的蛋白进行准确的定量测量仍然具有挑战性。
典型地在三重四极杆质谱仪上进行MRM测定,尽管该方法也可以在离子阱仪器中应用,在该离子阱仪器上,特征离子碎裂后,在限定质量范围内或在整个质量范围内在碎片离子上采集MS/MS数据。一系列的过渡(特征/碎片离子m/z对)与该靶向肽的保留时间组合可以构成MRM测定。为了实现最佳的MRM测定,(1)需要选择靶蛋白/肽;(2)替代肽必须产生良好的MS和MS/MS信号;(3)每个选择的肽碎片离子必须提供最佳的信号强度,并将该靶肽与该复合生物样品中存在的其他肽种类区分开。总的来说,替代肽和碎片离子为肽选择提供了高特异性,因为只记录了所需的过渡而忽略了该样品中存在的其他信号。
本领域常见的误解是MRM测定可确保特异性和灵敏度,样品制备可简化甚至省去,并且不需要色谱分离或色谱分离非常少。但是,与这种错误的认识相反,MRM测定往往会受到该样品的复杂性的极大影响,从而降低了特定靶肽的灵敏度。该特异性和灵敏度可能会受到基质效应的影响,例如叶、花粉、根、茎之间的差异,并导致离子抑制,这在MS分析过程中会发生。通常,大多数带电或可电离的分子,例如盐、离液剂、洗涤剂、聚合物、所有非挥发性离子化合物,干扰所需分析物(即肽/蛋白质)的电离,从而竞争并导致信号抑制和/或背景噪声升高。离子抑制会对几个分析参数产生负面影响,诸如检测能力、精确度和准确性。因此,为了克服本领域的MRM质谱法中的所有这些缺陷,需要开发一种用于从复合生物样品(例如转基因植物样品)中有效提取靶蛋白的方法,以富集靶蛋白和/或消除可能降低靶蛋白/肽的离子强度并影响该测定的可重复性和准确性的干扰。
总体而言,改善样品制备可能是减少基质效应和避免离子抑制的最有效方法。该方法使得实现富集选择的靶蛋白和肽而没有使干扰集中的能力,从而可以在低靶蛋白浓度下进行准确而精确的定量。
使用替代肽的盒(来自表1或表2)进行的基于MRM的测定在含有该七种蛋白中的至少一种的不同转基因事件中测量Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI(表4)。该研究中评估的转基因事件如下:Bt11(Cry1Ab和PAT);5307(eCry3.1Ab和PMI);MIR604(mCry3A和PMI);MIR162(Vip3Aa20和PMI)和GA21(dmEPSPS)。
组织提取-将12-15mg冻干的组织(叶、根、花粉、谷粒和整个植物)放入2μL裂解基质A FastPrep管(MP生物医疗公司,圣安娜,加利福尼亚州)中。然后加入1.0-1.5μL(w/v)的含0.1%RapiGest的PBS。然后在环境温度下,在具有裂解基质A(石榴石基质和1/4”陶瓷球珠)的FastPrep-24组织匀浆器(MP生物医疗公司,圣安娜,加利福尼亚州)中提取样品,进行1个循环(40s,速度设置6)。每mg冻干组织在50μl提取缓冲液(6M尿素,2M硫脲,5mM EDTA,0.1M HEPES)中从所选组织中提取蛋白
离心-组织提取后,将该样品于4℃下15,000g离心约5min。该步骤取出不溶性蛋白,例如,组蛋白和肌动蛋白,从而降低了消化前该提取物的复杂性。因此,只有可溶性蛋白才能进入该酶消化步骤。
胰蛋白酶消化-通过在均化缓冲液中稀释,将该离心步骤中上清液的总蛋白浓度调节至约0.2μg/μl。将相当于30μg的蛋白转移到孔板中。将一体积的三氟乙醇添加到该样品中,并在室温下伴随低速摇动孵育约30min。加入四体积的100mM碳酸氢铵。然后添加约12μl胰蛋白酶(0.1μg/μl)。将样品在37℃下孵育过夜。然后将样品用20%甲酸(终浓度1%)淬灭。然后加入20μl稳定同位素标记的肽。
离心-将上一步骤中的样品在4℃下15,000g离心约5min。
通过MCX进行脱盐-离心后,将该样品脱盐。该步骤在离子交换柱上进行。该脱盐步骤通过丢弃洗槽中的非目的肽来浓缩该目的肽。除了去除非目的肽外,该步骤还去除了可能干扰目的替代肽的电离和检测的盐和小分子。与本领域已知的其他方法相比,浓缩目的肽并去除干扰盐和小分子可以提高本发明的该MRM测定的灵敏度。
QTRAP-MRM-利用QTRAP 6500与NanoAcquity UPLC联用使用Halo Peptide ES-C18柱进行MRM分析。该流速为约18μl/min。溶剂A为约97/3水/DMSO+0.2%甲酸(FA),且溶剂B为约97/3乙腈(CAN)/DMSO+0.2%FA。在分析过程中,自动进样器的温度保持在约4℃。将总共8μl的样品注入保持在环境温度下的该柱上。
数据分析-使用Analyst软件(爱博才思公司,安大略省,加拿大)进行数据采集,并使用Multiquant软件(爱博才思公司)进行数据分析。
为了使用本发明的优选的标记的替代肽确定靶转基因蛋白的检测水平(LOD),将所有七种靶蛋白Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI混合在一起并添加至非转基因玉米植物的叶、根、谷粒和花粉组织中。表3-6显示了该MRM对靶蛋白的检测水平(LOD),并表明当在所有植物基质中该靶蛋白伴随存在其他转基因和非转基因蛋白时,每种标记的替代肽及其产生的过渡离子都能够选择性地检测和定量靶蛋白。对于表2的每种优选替代肽,均获得了良好的线性(r=0.988-0.998)。每种替代肽的LOD低于通过ELISA确定的定量范围,表明本发明的组合物和方法等于或优于用以定量植物中转基因蛋白的当前标准。
表3.玉米叶基质中靶蛋白的LOD。(LOD=fmol/μg总蛋白)
表4.玉米谷粒基质中靶蛋白的LOD。(LOD=fmol/μg总蛋白)
表5.玉米根基质中靶蛋白的LOD。(LOD=fmol/μg总蛋白)
表6.玉米花粉基质中靶蛋白的LOD。(LOD=fmol/μg总蛋白)
然后测试该优选的标记的替代肽(表2)及其过渡离子,以确定其特异性检测来自转基因玉米植物的叶、谷粒、根和花粉组织中靶蛋白的能力,该转基因玉米植物包含选自由以下组成的组的转基因事件:Bt11(包含Cry1Ab和PAT)、5307(包含eCry3.1Ab和PMI)、MIR604(包含mCry3A和PMI)、MIR162(包含Vip3A和PMI)和GA21(包含dmEPSPS)。针对每种转基因事件测试了该七种优选的替代肽中的每一种。表7显示了该靶蛋白的定量的结果。该结果表明,该Cry1Ab和PAT替代肽和标记的替代肽能够检测和/或定量来自包含事件Bt11的转基因玉米植物的叶、谷粒、根和花粉中的Cry1Ab和PAT。对于所测试的植物,该Cry1Ab蛋白低于花粉(见表5)组织中的LOD,而该PAT蛋白低于谷粒和花粉(见表4和5)中的LOD。该eCry3.1Ab和PMI替代肽和标记的替代肽能够从包含事件5307的转基因玉米植物的叶、谷粒、根和花粉中检测eCry3.1Ab和PMI。对于所测试的植物,该eCry3.1Ab蛋白低于花粉中的LOD(见表5)。该mCry3A和PMI替代肽和标记的替代肽能够从包含事件MIR604的转基因玉米植物的叶、谷粒、根和花粉中检测mCry3A和PMI蛋白。对于该测试的植物,该mCry3A蛋白低于花粉中的LOD(见表5)。该Vip3Aa20和PMI替代肽和标记的替代肽能够从包含事件MIR162的转基因玉米植物的叶、谷粒、根和花粉中检测Vip3Aa20和PMI蛋白。该dmEPSPS替代肽和标记的替代肽能够检测来自包含事件GA21的转基因玉米植物的叶、谷粒、根和花粉中的dmEPSPS蛋白。
为了进一步表征本发明的优选的标记的替代肽的能力,上述测定是在表达所有七种蛋白质(Cry1Ab、eCry3.1Ab、mCry3A、Vip3A、dmEPSPS、PAT和PMI)的育种堆叠上进行的。该结果表明,LC-SRM可以同时检测和定量该育种堆叠中包含的所有七种蛋白。
表1和/或表2中列出的替代肽和标记的替代肽能够检测和/或定量本发明的靶蛋白。这些肽中的每一种或这些肽的组合都是候选物,可用于该靶蛋白的定量MRM测定。
表7.转基因植物中靶蛋白的检测和定量。
nd=未测出
虽然已经结合本发明的特定实施例描述了本发明,但是应当理解,本发明的设备能够进行进一步的改良。本专利申请意在涵盖任何一般而言遵循本发明的原理的变化、用途或改编,并且包括这样的偏离本披露的内容:其在本发明所属领域的已知或惯常操作内,并且可以适用于此前所阐述的关键特征,并且遵循所附权利要求的范围。
在本说明书中提到的所有公开物和专利申请对于本发明所涉及领域的技术人员的技术水平是指示性的。所有这些公开物和专利申请均通过引用并入本文,其程度如同每个单独的公开物或专利申请被明确地并单独地指示通过引用而被并入。
序列表
<110> Syngenta Participations AG
Young, Scott
Sessler, Richard
Graser, Gerson
Guilbaud, Rudolf
Schirm, Michael
Isabelle, Maxim
<120> 用于蛋白质检测的组合物以及方法
<130> 81319-US-L-ORG-NAT-1
<160> 270
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213> 苏云金芽孢杆菌
<400> 1
Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg
1 5 10
<210> 2
<211> 11
<212> PRT
<213> 苏云金芽孢杆菌
<400> 2
Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
1 5 10
<210> 3
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 3
Thr Leu Ser Ser Thr Leu Tyr Arg
1 5
<210> 4
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 4
Asp Val Ser Val Phe Gly Gln Arg
1 5
<210> 5
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 5
Thr Tyr Pro Ile Arg
1 5
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<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 6
Thr Val Ser Gln Leu Thr Arg
1 5
<210> 7
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<212> PRT
<213> 苏云金芽孢杆菌
<400> 7
Trp Tyr Asn Thr Gly Leu Glu Arg
1 5
<210> 8
<211> 12
<212> PRT
<213> 苏云金芽孢杆菌
<400> 8
Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg
1 5 10
<210> 9
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 9
Val Trp Gly Pro Asp Ser Arg
1 5
<210> 10
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 10
Ala Pro Met Phe Ser Trp Ile His Arg
1 5
<210> 11
<211> 11
<212> PRT
<213> 苏云金芽孢杆菌
<400> 11
Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
1 5 10
<210> 12
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 12
Asn Gln Ala Ile Ser Arg
1 5
<210> 13
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 13
Ile Glu Glu Phe Ala Arg
1 5
<210> 14
<211> 12
<212> PRT
<213> 苏云金芽孢杆菌
<400> 14
Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg
1 5 10
<210> 15
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 15
Leu Ser His Val Ser Met Phe Arg
1 5
<210> 16
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<212> PRT
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<400> 16
Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg
1 5 10 15
<210> 17
<211> 16
<212> PRT
<213> 苏云金芽孢杆菌
<400> 17
Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg
1 5 10 15
<210> 18
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 18
Tyr Asn Gln Phe Arg
1 5
<210> 19
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 19
Tyr Asn Asp Leu Thr Arg
1 5
<210> 20
<211> 19
<212> PRT
<213> 苏云金芽孢杆菌
<400> 20
Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp
1 5 10 15
Ala His Arg
<210> 21
<211> 20
<212> PRT
<213> 苏云金芽孢杆菌
<400> 21
Ser Ala Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile
1 5 10 15
Pro Leu Thr Lys
20
<210> 22
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 22
Gln Gly Phe Ser His Arg
1 5
<210> 23
<211> 28
<212> PRT
<213> 苏云金芽孢杆菌
<400> 23
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg
20 25
<210> 24
<211> 19
<212> PRT
<213> 苏云金芽孢杆菌
<400> 24
Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr
1 5 10 15
Asp Ser Arg
<210> 25
<211> 34
<212> PRT
<213> 苏云金芽孢杆菌
<400> 25
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
1 5 10 15
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
20 25 30
Tyr Arg
<210> 26
<211> 20
<212> PRT
<213> 苏云金芽孢杆菌
<400> 26
Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn
1 5 10 15
Val Pro Pro Arg
20
<210> 27
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 27
Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val
1 5 10
<210> 28
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 28
Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys
1 5 10 15
<210> 29
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 29
Ile Thr Gln Leu Pro Leu Thr Lys
1 5
<210> 30
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 30
Gly Leu Asp Ser Ser Thr Thr Lys
1 5
<210> 31
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 31
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly Arg
1 5 10
<210> 32
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 32
Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu
1 5 10 15
Glu Arg
<210> 33
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 33
Ile Thr Gln Leu Pro Leu Val Lys
1 5
<210> 34
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 34
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
<210> 35
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 35
Val Tyr Ile Asp Lys
1 5
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<212> PRT
<213> 苏云金芽孢杆菌
<400> 36
Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys
1 5 10
<210> 37
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 37
Leu Ile Thr Leu Thr Cys Lys
1 5
<210> 38
<211> 11
<212> PRT
<213> 苏云金芽孢杆菌
<400> 38
Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys
1 5 10
<210> 39
<211> 13
<212> PRT
<213> 苏云金芽孢杆菌
<400> 39
Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser Ser Lys
1 5 10
<210> 40
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 40
Glu Val Leu Phe Glu Lys
1 5
<210> 41
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 41
Thr Ala Ser Glu Leu Ile Thr Lys
1 5
<210> 42
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 42
Asp Val Ser Glu Met Phe Thr Thr Lys
1 5
<210> 43
<211> 19
<212> PRT
<213> 苏云金芽孢杆菌
<400> 43
Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile Met Asn Glu His
1 5 10 15
Leu Asn Lys
<210> 44
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 44
Ile Asp Phe Thr Lys
1 5
<210> 45
<211> 14
<212> PRT
<213> 苏云金芽孢杆菌
<400> 45
Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu Ile Leu Lys
1 5 10
<210> 46
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 46
Asp Ile Met Asn Met Ile Phe Lys
1 5
<210> 47
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 47
Ala Leu Tyr Val His Lys
1 5
<210> 48
<211> 18
<212> PRT
<213> 苏云金芽孢杆菌
<400> 48
Val Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr
1 5 10 15
Ala Lys
<210> 49
<211> 10
<212> PRT
<213> 苏云金芽孢杆菌
<400> 49
Ile Thr Ser Met Leu Ser Asp Val Ile Lys
1 5 10
<210> 50
<211> 12
<212> PRT
<213> 苏云金芽孢杆菌
<400> 50
Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg
1 5 10
<210> 51
<211> 13
<212> PRT
<213> 苏云金芽孢杆菌
<400> 51
Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys
1 5 10
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<212> PRT
<213> 苏云金芽孢杆菌
<400> 52
Met Ile Val Glu Ala Lys Pro Gly His Ala Leu Ile Gly Phe Glu Ile
1 5 10 15
Ser Asn Asp Ser Ile Thr Val Leu Lys
20 25
<210> 53
<211> 12
<212> PRT
<213> 苏云金芽孢杆菌
<400> 53
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg
1 5 10
<210> 54
<211> 11
<212> PRT
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<400> 54
Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
1 5 10
<210> 55
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 55
Val Glu Ser Ser Glu Ala Glu Tyr Arg
1 5
<210> 56
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 56
Tyr Met Ser Gly Ala Lys
1 5
<210> 57
<211> 19
<212> PRT
<213> 苏云金芽孢杆菌
<400> 57
Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr Glu Leu Thr Glu
1 5 10 15
Leu Ala Lys
<210> 58
<211> 5
<212> PRT
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<400> 58
Val Tyr Glu Ala Lys
1 5
<210> 59
<211> 9
<212> PRT
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<400> 59
Leu Asp Ala Ile Asn Thr Met Leu Arg
1 5
<210> 60
<211> 8
<212> PRT
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<400> 60
Gly Lys Pro Ser Ile His Leu Lys
1 5
<210> 61
<211> 24
<212> PRT
<213> 苏云金芽孢杆菌
<400> 61
Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu
1 5 10 15
Glu Asp Tyr Gln Thr Ile Asn Lys
20
<210> 62
<211> 27
<212> PRT
<213> 苏云金芽孢杆菌
<400> 62
Asp Asn Phe Tyr Ile Glu Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly
1 5 10 15
Pro Ile Val His Phe Tyr Asp Val Ser Ile Lys
20 25
<210> 63
<211> 26
<212> PRT
<213> 苏云金芽孢杆菌
<400> 63
Leu Leu Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile
1 5 10 15
Val Phe Pro Asn Glu Tyr Val Ile Thr Lys
20 25
<210> 64
<211> 22
<212> PRT
<213> 苏云金芽孢杆菌
<400> 64
Ser Gln Asn Gly Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu
1 5 10 15
Ile Ser Pro Ser Glu Lys
20
<210> 65
<211> 14
<212> PRT
<213> 苏云金芽孢杆菌
<400> 65
Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys
1 5 10
<210> 66
<211> 23
<212> PRT
<213> 苏云金芽孢杆菌
<400> 66
Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn
1 5 10 15
Leu Asn Thr Glu Leu Ser Lys
20
<210> 67
<211> 15
<212> PRT
<213> 苏云金芽孢杆菌
<400> 67
Ile Ala Asn Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys
1 5 10 15
<210> 68
<211> 19
<212> PRT
<213> 苏云金芽孢杆菌
<400> 68
Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile Asp
1 5 10 15
Leu Asn Lys
<210> 69
<211> 14
<212> PRT
<213> 苏云金芽孢杆菌
<400> 69
Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
1 5 10
<210> 70
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 70
Gln Leu Gln Glu Ile Ser Asp Lys
1 5
<210> 71
<211> 32
<212> PRT
<213> 苏云金芽孢杆菌
<400> 71
Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
1 5 10 15
Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
20 25 30
<210> 72
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 72
Tyr Val Asn Glu Lys
1 5
<210> 73
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 73
Gln Asn Tyr Gln Val Asp Lys
1 5
<210> 74
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 74
Met Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys
1 5 10
<210> 75
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 75
Phe Pro Val Glu Asp Ala Lys
1 5
<210> 76
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 76
Glu Ile Ser Gly Thr Val Lys
1 5
<210> 77
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 77
Ile Leu Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn
1 5 10 15
Leu Leu Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg
20 25 30
<210> 78
<211> 17
<212> PRT
<213> 产绿色链霉菌
<400> 78
Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro Val Arg Pro Val Thr Gln
1 5 10 15
Ile
<210> 79
<211> 13
<212> PRT
<213> 产绿色链霉菌
<400> 79
Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys
1 5 10
<210> 80
<211> 5
<212> PRT
<213> 产绿色链霉菌
<400> 80
Met Ser Pro Glu Arg
1 5
<210> 81
<211> 12
<212> PRT
<213> 产绿色链霉菌
<400> 81
His Gly Gly Trp His Asp Val Gly Phe Trp Gln Arg
1 5 10
<210> 82
<211> 16
<212> PRT
<213> 产绿色链霉菌
<400> 82
Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Val Tyr Val Ser His Arg
1 5 10 15
<210> 83
<211> 15
<212> PRT
<213> 产绿色链霉菌
<400> 83
Thr Glu Pro Gln Thr Pro Gln Glu Trp Ile Asp Asp Leu Glu Arg
1 5 10 15
<210> 84
<211> 5
<212> PRT
<213> 产绿色链霉菌
<400> 84
Ala Ala Gly Tyr Lys
1 5
<210> 85
<211> 22
<212> PRT
<213> 产绿色链霉菌
<400> 85
Tyr Pro Trp Leu Val Ala Glu Val Glu Gly Val Val Ala Gly Ile Ala
1 5 10 15
Tyr Ala Gly Pro Trp Lys
20
<210> 86
<211> 32
<212> PRT
<213> 产绿色链霉菌
<400> 86
Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala Asp Met Ala Ala Val
1 5 10 15
Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser Thr Val Asn Phe Arg
20 25 30
<210> 87
<211> 13
<212> PRT
<213> 大肠杆菌
<400> 87
Glu Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg
1 5 10
<210> 88
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 88
Ala Leu Ala Ile Leu Lys
1 5
<210> 89
<211> 15
<212> PRT
<213> 大肠杆菌
<400> 89
Ser Ala Leu Asp Ser Gln Gln Gly Glu Pro Trp Gln Thr Ile Arg
1 5 10 15
<210> 90
<211> 25
<212> PRT
<213> 大肠杆菌
<400> 90
Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe Ile Ala
1 5 10 15
Ala Asn Glu Ser Pro Val Thr Val Lys
20 25
<210> 91
<211> 15
<212> PRT
<213> 大肠杆菌
<400> 91
Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln Pro Val Lys
1 5 10 15
<210> 92
<211> 10
<212> PRT
<213> 大肠杆菌
<400> 92
Ser Thr Leu Leu Gly Glu Ala Val Ala Lys
1 5 10
<210> 93
<211> 13
<212> PRT
<213> 大肠杆菌
<400> 93
Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys
1 5 10
<210> 94
<211> 9
<212> PRT
<213> 大肠杆菌
<400> 94
His Asn Ser Glu Ile Gly Phe Ala Lys
1 5
<210> 95
<211> 16
<212> PRT
<213> 大肠杆菌
<400> 95
Val Leu Cys Ala Ala Gln Pro Leu Ser Ile Gln Val His Pro Asn Lys
1 5 10 15
<210> 96
<211> 27
<212> PRT
<213> 大肠杆菌
<400> 96
Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro
1 5 10 15
Met Ala Glu Leu Trp Met Gly Ala His Pro Lys
20 25
<210> 97
<211> 16
<212> PRT
<213> 大肠杆菌
<400> 97
Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly Glu Glu Lys
1 5 10 15
<210> 98
<211> 24
<212> PRT
<213> 大肠杆菌
<400> 98
Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp Phe Ala Phe
1 5 10 15
Ser Leu His Asp Leu Ser Asp Lys
20
<210> 99
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 99
Gly Ile Glu Gly Ser Ile Arg
1 5
<210> 100
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 100
Glu Gly Ser Ile Arg
1 5
<210> 101
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 101
Ala Gln Leu Gly Gln Gly Val Tyr Arg
1 5
<210> 102
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 102
Gly Gln Gly Val Tyr Arg
1 5
<210> 103
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 103
Ser Ser Thr Leu Tyr Arg
1 5
<210> 104
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 104
Ser Thr Leu Tyr Arg
1 5
<210> 105
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 105
Ser Val Phe Gly Gln Arg
1 5
<210> 106
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 106
Phe Gly Gln Arg
1
<210> 107
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 107
Ser Gln Leu Thr Arg
1 5
<210> 108
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 108
Gln Leu Thr Arg
1
<210> 109
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 109
Asn Thr Gly Leu Glu Arg
1 5
<210> 110
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 110
Tyr Asn Thr Gly Leu Glu Arg
1 5
<210> 111
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 111
Pro Thr Asn Pro Ala Leu Arg
1 5
<210> 112
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 112
Asp Pro Thr Asn Pro Ala Leu Arg
1 5
<210> 113
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 113
Gly Pro Asp Ser Arg
1 5
<210> 114
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 114
Ser Trp Ile His Arg
1 5
<210> 115
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 115
Ala Thr Ile Asn Ser Arg
1 5
<210> 116
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 116
Asp Ala Ala Thr Ile Asn Ser Arg
1 5
<210> 117
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 117
Ala Ile Ser Arg
1
<210> 118
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 118
Glu Phe Ala Arg
1
<210> 119
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 119
Glu Glu Phe Ala Arg
1 5
<210> 120
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 120
Ser Asn Ser Ser Val Ser Ile Ile Arg
1 5
<210> 121
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 121
Ser Ser Val Ser Ile Ile Arg
1 5
<210> 122
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 122
Ser Met Phe Arg
1
<210> 123
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 123
Val Ser Met Phe Arg
1 5
<210> 124
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 124
Glu Asn Phe Asp Gly Ser Phe Arg
1 5
<210> 125
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 125
Gly Ser Phe Arg
1
<210> 126
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 126
Tyr Ala Glu Ser Phe Arg
1 5
<210> 127
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 127
Asn Gln Phe Arg
1
<210> 128
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 128
Asp Leu Thr Arg
1
<210> 129
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 129
Asn Asp Leu Thr Arg
1 5
<210> 130
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 130
Thr Ile Tyr Thr Asp Ala His Arg
1 5
<210> 131
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 131
Tyr Thr Asp Ala His Arg
1 5
<210> 132
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 132
Pro Leu Thr Lys
1
<210> 133
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 133
Ser Ala Glu Phe Asn Asn Ile Ile
1 5
<210> 134
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 134
Phe Ser His Arg
1
<210> 135
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 135
Gly Phe Ser His Arg
1 5
<210> 136
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 136
Glu Val Leu Gly Gly Glu Arg
1 5
<210> 137
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 137
Gly Gly Glu Arg
1
<210> 138
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 138
Phe Pro Asn Tyr Asp Ser Arg
1 5
<210> 139
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 139
Pro Asn Tyr Asp Ser Arg
1 5
<210> 140
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 140
Pro Ser Ala Val Tyr Arg
1 5
<210> 141
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 141
Ser Gly Thr Val Asp Ser Leu Asp Glu
1 5
<210> 142
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 142
Thr Asp Tyr His Ile Asp Gln Val
1 5
<210> 143
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 143
Asp Tyr His Ile Asp Gln Val
1 5
<210> 144
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 144
Thr Ser Ser Asn Gln Ile Gly Leu Lys
1 5
<210> 145
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 145
Ser Ser Asn Gln Ile Gly Leu Lys
1 5
<210> 146
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 146
Gln Leu Pro Leu Thr Lys
1 5
<210> 147
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 147
Thr Gln Leu Pro Leu Thr Lys
1 5
<210> 148
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 148
Asp Ser Ser Thr Thr Lys
1 5
<210> 149
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 149
Ser Ser Thr Thr Lys
1 5
<210> 150
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 150
Pro Tyr Asp Gly Arg
1 5
<210> 151
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 151
Gln Leu Pro Leu Val Lys
1 5
<210> 152
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 152
Thr Gln Leu Pro Leu Val Lys
1 5
<210> 153
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 153
Ala Leu Asp Ser Ser Thr Thr Lys
1 5
<210> 154
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 154
Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5
<210> 155
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 155
Tyr Ile Asp Lys
1
<210> 156
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 156
Ser Gln Phe Ile Gly Asp Lys
1 5
<210> 157
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 157
Thr Leu Thr Cys Lys
1 5
<210> 158
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 158
Ala Thr Asp Leu Ser Asn Lys
1 5
<210> 159
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 159
Leu Ala Thr Asp Leu Ser Asn Lys
1 5
<210> 160
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 160
Thr Phe Ala Thr Glu Thr Ser Ser Lys
1 5
<210> 161
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 161
Phe Ala Thr Glu Thr Ser Ser Lys
1 5
<210> 162
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 162
Leu Phe Glu Lys
1
<210> 163
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 163
Ser Glu Leu Ile Thr Lys
1 5
<210> 164
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 164
Ala Ser Glu Leu Thr Ile Lys
1 5
<210> 165
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 165
Ser Glu Met Phe Thr Thr Lys
1 5
<210> 166
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 166
Met Asn Glu His Leu Asn Lys
1 5
<210> 167
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 167
Met Asn Glu His Leu Asn Lys
1 5
<210> 168
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 168
Asp Phe Thr Lys
1
<210> 169
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 169
Thr Leu Asp Glu Ile Leu Lys
1 5
<210> 170
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 170
Leu Thr Leu Asp Glu Ile Leu Lys
1 5
<210> 171
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 171
Met Asn Met Ile Phe Lys
1 5
<210> 172
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 172
Asn Met Ile Phe Lys
1 5
<210> 173
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 173
Tyr Val His Lys
1
<210> 174
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 174
Val Asn Ile Leu
1
<210> 175
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 175
Ser Met Leu Ser Asp Val Ile Lys
1 5
<210> 176
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 176
Thr Ser Met Leu Ser Asp Val Ile Lys
1 5
<210> 177
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 177
Asp Ser Phe Ser Thr Tyr Arg
1 5
<210> 178
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 178
Leu Asp Ser Phe Ser Thr Tyr Arg
1 5
<210> 179
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 179
Ile Tyr Gly Asp Met Asp Lys
1 5
<210> 180
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 180
Val Ile Tyr Gly Asp Met Asp Lys
1 5
<210> 181
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 181
Ser Asn Asp Ser Ile Thr Val Leu Lys
1 5
<210> 182
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 182
Ser Gly Asp Ala Asn Val Arg
1 5
<210> 183
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 183
Ser Val Ser Gly Asp Ala Asn Val Arg
1 5
<210> 184
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 184
Leu Leu Asn Asp Ile Ser Gly Lys
1 5
<210> 185
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 185
Leu Asn Asp Ile Ser Gly Lys
1 5
<210> 186
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 186
Ser Ser Glu Ala Glu Tyr Arg
1 5
<210> 187
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 187
Glu Ser Ser Glu Ala Glu Tyr Arg
1 5
<210> 188
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 188
Ser Gly Ala Lys
1
<210> 189
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 189
Met Ser Gly Ala Lys
1 5
<210> 190
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 190
Thr Glu Leu Thr Glu Leu Ala Lys
1 5
<210> 191
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 191
Asp Gly Ser Pro Ala Asp Ile
1 5
<210> 192
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 192
Tyr Glu Ala Lys
1
<210> 193
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 193
Asn Thr Met Leu Arg
1 5
<210> 194
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 194
Ala Ile Asn Thr Met Leu Arg
1 5
<210> 195
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 195
Pro Ser Ile His Leu Lys
1 5
<210> 196
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 196
Asp Tyr Gln Thr Ile Asn Lys
1 5
<210> 197
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 197
Asp Asn Phe Tyr
1
<210> 198
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 198
Pro Asn Glu Tyr Val Ile Thr Lys
1 5
<210> 199
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 199
Ser Pro Ser Glu Lys
1 5
<210> 200
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 200
Leu Glu Ile Ser Pro Ser Glu Lys
1 5
<210> 201
<211> 11
<212> PRT
<213> 苏云金芽孢杆菌
<400> 201
Asp His Thr Gly Gly Val Asn Ser Gly Thr Lys
1 5 10
<210> 202
<211> 9
<212> PRT
<213> 苏云金芽孢杆菌
<400> 202
Gly Asn Leu Asn Thr Glu Leu Ser Lys
1 5
<210> 203
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 203
Asn Thr Glu Leu Ser Lys
1 5
<210> 204
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 204
Leu Asn Asp Val Asn Asn Lys
1 5
<210> 205
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 205
Asn Asp Val Asn Asn Lys
1 5
<210> 206
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 206
Asp Leu Asn Lys
1
<210> 207
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 207
Gln Ile Glu Tyr Leu Ser Lys
1 5
<210> 208
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 208
Leu Gln Ile Glu Tyr Leu Ser Lys
1 5
<210> 209
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 209
Gln Glu Ile Ser Asp Lys
1 5
<210> 210
<211> 5
<212> PRT
<213> 苏云金芽孢杆菌
<400> 210
Tyr Gln Gly Gly Arg
1 5
<210> 211
<211> 7
<212> PRT
<213> 苏云金芽孢杆菌
<400> 211
Thr Leu Tyr Gln Gly Gly Arg
1 5
<210> 212
<211> 4
<212> PRT
<213> 苏云金芽孢杆菌
<400> 212
Val Asn Glu Lys
1
<210> 213
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 213
Glu Ile Val Leu Gln Pro Ile Lys
1 5
<210> 214
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 214
Pro Val Glu Asp Ala Lys
1 5
<210> 215
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 215
Val Glu Asp Ala Lys
1 5
<210> 216
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 216
Ser Gly Thr Val Lys
1 5
<210> 217
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 217
Gly Thr Val Lys
1
<210> 218
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 218
Ile Leu Leu Leu Ala Ala
1 5
<210> 219
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 219
His Tyr Met Leu Gly Ala Leu Arg
1 5
<210> 220
<211> 6
<212> PRT
<213> 产绿色链霉菌
<400> 220
Tyr Thr His Leu Leu Lys
1 5
<210> 221
<211> 5
<212> PRT
<213> 产绿色链霉菌
<400> 221
Thr His Leu Leu Lys
1 5
<210> 222
<211> 4
<212> PRT
<213> 产绿色链霉菌
<400> 222
Ser Pro Glu Arg
1
<210> 223
<211> 5
<212> PRT
<213> 产绿色链霉菌
<400> 223
Gly Phe Trp Gln Arg
1 5
<210> 224
<211> 6
<212> PRT
<213> 产绿色链霉菌
<400> 224
Val Gly Phe Trp Gln Arg
1 5
<210> 225
<211> 8
<212> PRT
<213> 产绿色链霉菌
<400> 225
Ser Thr Val Tyr Val Ser His Arg
1 5
<210> 226
<211> 5
<212> PRT
<213> 产绿色链霉菌
<400> 226
Thr Glu Pro Gln Thr
1 5
<210> 227
<211> 4
<212> PRT
<213> 产绿色链霉菌
<400> 227
Asp Leu Glu Arg
1
<210> 228
<211> 4
<212> PRT
<213> 产绿色链霉菌
<400> 228
Ala Gly Tyr Lys
1
<210> 229
<211> 4
<212> PRT
<213> 产绿色链霉菌
<400> 229
Gly Pro Trp Lys
1
<210> 230
<211> 9
<212> PRT
<213> 产绿色链霉菌
<400> 230
Gly Ile Ala Tyr Ala Gly Pro Trp Lys
1 5
<210> 231
<211> 7
<212> PRT
<213> 产绿色链霉菌
<400> 231
Thr Ser Thr Val Asn Phe Arg
1 5
<210> 232
<211> 7
<212> PRT
<213> 大肠杆菌
<400> 232
Pro Met Asp Ala Ala Glu Arg
1 5
<210> 233
<211> 9
<212> PRT
<213> 大肠杆菌
<400> 233
Gly Ile Pro Met Asp Ala Ala Glu Arg
1 5
<210> 234
<211> 4
<212> PRT
<213> 大肠杆菌
<400> 234
Ala Ile Leu Lys
1
<210> 235
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 235
Pro Trp Gln Thr Ile Arg
1 5
<210> 236
<211> 8
<212> PRT
<213> 大肠杆菌
<400> 236
Gly Glu Pro Trp Gln Thr Ile Arg
1 5
<210> 237
<211> 9
<212> PRT
<213> 大肠杆菌
<400> 237
Ala Asn Glu Ser Pro Val Thr Val Lys
1 5
<210> 238
<211> 5
<212> PRT
<213> 大肠杆菌
<400> 238
Pro Val Thr Val Lys
1 5
<210> 239
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 239
Leu Thr Gln Pro Val Lys
1 5
<210> 240
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 240
Gly Glu Ala Val Ala Lys
1 5
<210> 241
<211> 7
<212> PRT
<213> 大肠杆菌
<400> 241
Leu Gly Glu Ala Val Ala Lys
1 5
<210> 242
<211> 8
<212> PRT
<213> 大肠杆菌
<400> 242
Gln Asn Tyr Ala Trp Gly Ser Lys
1 5
<210> 243
<211> 7
<212> PRT
<213> 大肠杆菌
<400> 243
Asn Tyr Ala Trp Gly Ser Lys
1 5
<210> 244
<211> 8
<212> PRT
<213> 大肠杆菌
<400> 244
Asn Ser Glu Ile Gly Phe Ala Lys
1 5
<210> 245
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 245
Val Leu Cys Ala Ala Gln
1 5
<210> 246
<211> 7
<212> PRT
<213> 大肠杆菌
<400> 246
Trp Met Gly Ala His Pro Lys
1 5
<210> 247
<211> 5
<212> PRT
<213> 大肠杆菌
<400> 247
Thr Ala Leu Thr Glu
1 5
<210> 248
<211> 7
<212> PRT
<213> 大肠杆菌
<400> 248
Asn Met Gln Gly Glu Glu Lys
1 5
<210> 249
<211> 8
<212> PRT
<213> 大肠杆菌
<400> 249
Leu Asn Met Gln Gly Glu Glu Lys
1 5
<210> 250
<211> 8
<212> PRT
<213> 大肠杆菌
<400> 250
Ser Leu His Asp Leu Ser Asp Lys
1 5
<210> 251
<211> 6
<212> PRT
<213> 大肠杆菌
<400> 251
His Asp Leu Ser Asp Lys
1 5
<210> 252
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 252
Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg
1 5 10
<210> 253
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 修饰的Cry3A肽
<400> 253
Ser Gly Ala Ser Val Val Ala Gly Pro Arg
1 5 10
<210> 254
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 修饰的Cry3A肽
<400> 254
Ser Val Val Ala Gly Pro Arg
1 5
<210> 255
<211> 8
<212> PRT
<213> 苏云金芽孢杆菌
<400> 255
Phe Thr Thr Gly Thr Asp Leu Lys
1 5
<210> 256
<211> 6
<212> PRT
<213> 苏云金芽孢杆菌
<400> 256
Thr Gly Thr Asp Leu Lys
1 5
<210> 257
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 突变的EPSPS
<400> 257
Ser Leu Thr Ala Ala Val Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val
1 5 10 15
Leu Asp Gly Val Pro Arg
20
<210> 258
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> dmEPSPS过渡2
<400> 258
Gly Val Pro Arg
1
<210> 259
<211> 615
<212> PRT
<213> 苏云金芽孢杆菌
<400> 259
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr
465 470 475 480
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly
485 490 495
Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg
500 505 510
Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg
515 520 525
Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg
530 535 540
Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn
545 550 555 560
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn
565 570 575
Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590
Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu
595 600 605
Val Thr Phe Glu Ala Glu Tyr
610 615
<210> 260
<211> 653
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 260
Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly
1 5 10 15
Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
20 25 30
Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe
35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr
50 55 60
Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu
65 70 75 80
Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu
85 90 95
Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110
Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His
115 120 125
Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe
130 135 140
Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe
145 150 155 160
Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175
Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile
180 185 190
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp
195 200 205
His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser
210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr
225 230 235 240
Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg
245 250 255
Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270
Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe
275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu
290 295 300
His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn
305 310 315 320
Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335
Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser
340 345 350
Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg
355 360 365
Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser
370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu
385 390 395 400
Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415
Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu
420 425 430
Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met
435 440 445
Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser
450 455 460
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro
465 470 475 480
Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly
485 490 495
Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln
500 505 510
Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr
515 520 525
Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr
530 535 540
Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met
545 550 555 560
Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe
565 570 575
Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser
580 585 590
Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu
595 600 605
Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg
610 615 620
Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly
625 630 635 640
Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val
645 650
<210> 261
<211> 598
<212> PRT
<213> 人工序列
<220>
<223> 突变的Cry3A
<400> 261
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp
530 535 540
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu
545 550 555 560
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile
565 570 575
Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
580 585 590
Glu Phe Ile Pro Val Asn
595
<210> 262
<211> 789
<212> PRT
<213> 人工序列
<220>
<223> 修饰的Vip3A
<400> 262
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe
1 5 10 15
Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
20 25 30
Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu
35 40 45
Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
50 55 60
Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn
65 70 75 80
Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln
85 90 95
Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
100 105 110
Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
115 120 125
Ile Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
130 135 140
Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val
145 150 155 160
Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile
165 170 175
Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
180 185 190
Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu
195 200 205
Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val
210 215 220
Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly
225 230 235 240
Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile
245 250 255
Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
260 265 270
Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr
275 280 285
Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
290 295 300
Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val
305 310 315 320
Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala
325 330 335
Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
340 345 350
Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
355 360 365
Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
370 375 380
Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu
385 390 395 400
Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe
405 410 415
Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
420 425 430
Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
435 440 445
Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
450 455 460
Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val
465 470 475 480
Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala
485 490 495
Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
500 505 510
Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
515 520 525
Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
530 535 540
Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr
545 550 555 560
Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His
565 570 575
Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
580 585 590
Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
595 600 605
Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
610 615 620
Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr
625 630 635 640
Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu
645 650 655
Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
660 665 670
Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
675 680 685
Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
690 695 700
Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg
705 710 715 720
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser
725 730 735
Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val
740 745 750
Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu
755 760 765
Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr
770 775 780
Asp Val Ser Ile Lys
785
<210> 263
<211> 445
<212> PRT
<213> 人工序列
<220>
<223> 双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶
<400> 263
Met Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser
1 5 10 15
Gly Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu
20 25 30
Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu
35 40 45
Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly
50 55 60
Leu Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly
65 70 75 80
Cys Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu
85 90 95
Phe Leu Gly Asn Ala Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val
100 105 110
Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg
115 120 125
Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu
130 135 140
Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg
145 150 155 160
Val Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly
165 170 175
Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu
180 185 190
Ala Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile
195 200 205
Pro Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys
210 215 220
Ala Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln
225 230 235 240
Lys Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser
245 250 255
Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr
260 265 270
Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala
275 280 285
Glu Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr Ser
290 295 300
Val Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys His Leu
305 310 315 320
Lys Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr
325 330 335
Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp
340 345 350
Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg
355 360 365
Thr Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr
370 375 380
Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr
385 390 395 400
Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala
405 410 415
Glu Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe
420 425 430
Pro Asp Tyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn
435 440 445
<210> 264
<211> 191
<212> PRT
<213> 产绿色链霉菌
<400> 264
Met Ser Pro Glu Arg Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala
1 5 10 15
Asp Met Ala Ala Val Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser
20 25 30
Thr Val Asn Phe Arg Thr Glu Pro Gln Thr Pro Gln Glu Trp Ile Asp
35 40 45
Asp Leu Glu Arg Leu Gln Asp Arg Tyr Pro Trp Leu Val Ala Glu Val
50 55 60
Glu Gly Val Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg
65 70 75 80
Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Val Tyr Val Ser His Arg
85 90 95
His Gln Arg Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys
100 105 110
Ser Met Glu Ala Gln Gly Phe Lys Ser Val Val Ala Val Ile Gly Leu
115 120 125
Pro Asn Asp Pro Ser Val Arg Leu His Glu Ala Leu Gly Tyr Thr Ala
130 135 140
Arg Gly Thr Leu Arg Ala Ala Gly Tyr Lys His Gly Gly Trp His Asp
145 150 155 160
Val Gly Phe Trp Gln Arg Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro
165 170 175
Val Arg Pro Val Thr Gln Ile Pro Asp Arg Ser Asn Ile Trp Gln
180 185 190
<210> 265
<211> 391
<212> PRT
<213> 大肠杆菌
<400> 265
Met Gln Lys Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys
1 5 10 15
Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro
20 25 30
Met Ala Glu Leu Trp Met Gly Ala His Pro Lys Ser Ser Ser Arg Val
35 40 45
Gln Asn Ala Ala Gly Asp Ile Val Ser Leu Arg Asp Val Ile Glu Ser
50 55 60
Asp Lys Ser Thr Leu Leu Gly Glu Ala Val Ala Lys Arg Phe Gly Glu
65 70 75 80
Leu Pro Phe Leu Phe Lys Val Leu Cys Ala Ala Gln Pro Leu Ser Ile
85 90 95
Gln Val His Pro Asn Lys His Asn Ser Glu Ile Gly Phe Ala Lys Glu
100 105 110
Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg Asn Tyr Lys Asp
115 120 125
Pro Asn His Lys Pro Glu Leu Val Phe Ala Leu Thr Pro Phe Leu Ala
130 135 140
Met Asn Ala Phe Arg Glu Phe Ser Glu Ile Val Ser Leu Leu Gln Pro
145 150 155 160
Val Ala Gly Ala His Pro Ala Ile Ala His Phe Leu Gln Gln Pro Asp
165 170 175
Ala Glu Arg Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly
180 185 190
Glu Glu Lys Ser Arg Ala Leu Ala Ile Leu Lys Ser Ala Leu Asp Ser
195 200 205
Gln Gln Gly Glu Pro Trp Gln Thr Ile Arg Leu Ile Ser Glu Phe Tyr
210 215 220
Pro Glu Asp Ser Gly Leu Phe Ser Pro Leu Leu Leu Asn Val Val Lys
225 230 235 240
Leu Asn Pro Gly Glu Ala Met Phe Leu Phe Ala Glu Thr Pro His Ala
245 250 255
Tyr Leu Gln Gly Val Ala Leu Glu Val Met Ala Asn Ser Asp Asn Val
260 265 270
Leu Arg Ala Gly Leu Thr Pro Lys Tyr Ile Asp Ile Pro Glu Leu Val
275 280 285
Ala Asn Val Lys Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln
290 295 300
Pro Val Lys Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp
305 310 315 320
Phe Ala Phe Ser Leu His Asp Leu Ser Asp Lys Glu Thr Thr Ile Ser
325 330 335
Gln Gln Ser Ala Ala Ile Leu Phe Cys Val Glu Gly Asp Ala Thr Leu
340 345 350
Trp Lys Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe
355 360 365
Ile Ala Ala Asn Glu Ser Pro Val Thr Val Lys Gly His Gly Arg Leu
370 375 380
Ala Arg Val Tyr Asn Lys Leu
385 390
<210> 266
<211> 391
<212> PRT
<213> 人工序列
<220>
<223> MIR604 PMI
<400> 266
Met Gln Lys Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys
1 5 10 15
Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro
20 25 30
Met Ala Glu Leu Trp Met Gly Ala His Pro Lys Ser Ser Ser Arg Val
35 40 45
Gln Asn Ala Ala Gly Asp Ile Val Ser Leu Arg Asp Ala Ile Glu Ser
50 55 60
Asp Lys Ser Thr Leu Leu Gly Glu Ala Val Ala Lys Arg Phe Gly Glu
65 70 75 80
Leu Pro Phe Leu Phe Lys Val Leu Cys Ala Ala Gln Pro Leu Ser Ile
85 90 95
Gln Val His Pro Asn Lys His Asn Ser Glu Ile Gly Phe Ala Lys Glu
100 105 110
Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg Asn Tyr Lys Asp
115 120 125
Pro Asn His Lys Pro Glu Leu Val Phe Ala Leu Thr Pro Phe Leu Ala
130 135 140
Met Asn Ala Phe Arg Glu Phe Ser Glu Ile Val Ser Leu Leu Gln Pro
145 150 155 160
Val Ala Gly Ala His Pro Ala Ile Ala His Phe Leu Gln Gln Pro Asp
165 170 175
Ala Glu Arg Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly
180 185 190
Glu Glu Lys Ser Arg Ala Leu Ala Ile Leu Lys Ser Ala Leu Asp Ser
195 200 205
Gln His Gly Glu Pro Trp Gln Thr Ile Arg Leu Ile Ser Glu Phe Tyr
210 215 220
Pro Glu Asp Ser Gly Leu Phe Ser Pro Leu Leu Leu Asn Val Val Lys
225 230 235 240
Leu Asn Pro Gly Glu Ala Met Phe Leu Phe Ala Glu Thr Pro His Ala
245 250 255
Tyr Leu Gln Gly Val Ala Leu Glu Val Met Ala Asn Ser Asp Asn Val
260 265 270
Leu Arg Ala Gly Leu Thr Pro Lys Tyr Ile Asp Ile Pro Glu Leu Val
275 280 285
Ala Asn Val Lys Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln
290 295 300
Pro Val Lys Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp
305 310 315 320
Phe Ala Phe Ser Leu His Asp Leu Ser Asp Lys Glu Thr Thr Ile Ser
325 330 335
Gln Gln Ser Ala Ala Ile Leu Phe Cys Val Glu Gly Asp Ala Thr Leu
340 345 350
Trp Lys Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe
355 360 365
Ile Ala Ala Asn Glu Ser Pro Val Thr Val Lys Gly His Gly Arg Leu
370 375 380
Ala Arg Val Tyr Asn Lys Leu
385 390
<210> 267
<211> 881
<212> PRT
<213> 人工序列
<220>
<223> 工程化杂合毒素
<400> 267
Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly
1 5 10 15
Glu Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe
20 25 30
Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala
35 40 45
Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
50 55 60
Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn
65 70 75 80
Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr
85 90 95
Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp
100 105 110
Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln
115 120 125
Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe
130 135 140
Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile
145 150 155 160
His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr
165 170 175
Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val
180 185 190
Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser
195 200 205
Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro
210 215 220
Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg
225 230 235 240
Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn
245 250 255
Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr
260 265 270
Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe
275 280 285
Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp
290 295 300
Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn
305 310 315 320
Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn
325 330 335
Gln Leu Gly Leu Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val
340 345 350
Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys
355 360 365
Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu
370 375 380
Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro
385 390 395 400
Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp
405 410 415
Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu
420 425 430
Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys
435 440 445
Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp
450 455 460
Leu Glu Ile Tyr Ser Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn
465 470 475 480
Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile
485 490 495
Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn
500 505 510
Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His
515 520 525
Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn
530 535 540
Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly
545 550 555 560
His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val
565 570 575
Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp
580 585 590
Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala
595 600 605
Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu
610 615 620
Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val
625 630 635 640
His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly
645 650 655
Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala
660 665 670
Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn
675 680 685
Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu
690 695 700
Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu
705 710 715 720
Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg
725 730 735
Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His
740 745 750
Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu
755 760 765
Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val
770 775 780
Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr
785 790 795 800
Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu
805 810 815
Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg
820 825 830
Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu
835 840 845
Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu
850 855 860
Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu
865 870 875 880
Glu
<210> 268
<211> 605
<212> PRT
<213> 苏云金芽孢杆菌
<400> 268
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn
1 5 10 15
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30
Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45
Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60
Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln
65 70 75 80
Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95
Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110
Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125
Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn
130 135 140
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val
145 150 155 160
Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175
Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn
180 185 190
Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp
210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp
225 230 235 240
Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255
Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270
Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu
275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe
290 295 300
Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu
305 310 315 320
Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335
Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350
Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365
Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380
Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile
385 390 395 400
Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415
Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro
420 425 430
Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile
450 455 460
Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr
465 470 475 480
Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495
Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe
530 535 540
Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser
545 550 555 560
Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575
Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu
595 600 605
<210> 269
<211> 123
<212> PRT
<213> 苏云金芽孢杆菌
<400> 269
Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His
1 5 10 15
Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg
20 25 30
Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala
35 40 45
Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser
50 55 60
Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala
65 70 75 80
Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile
85 90 95
Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile
100 105 110
Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser
115 120
<210> 270
<211> 354
<212> PRT
<213> 苏云金芽孢杆菌
<400> 270
Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly
1 5 10 15
Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu
20 25 30
Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe
35 40 45
Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala
50 55 60
Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser
65 70 75 80
Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn
85 90 95
Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110
Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser
115 120 125
Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln
130 135 140
Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys
145 150 155 160
Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met
165 170 175
Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp
180 185 190
Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr
195 200 205
Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His
210 215 220
Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys
225 230 235 240
Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly
245 250 255
Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys
260 265 270
Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys
275 280 285
Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp
290 295 300
Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu
305 310 315 320
Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser
325 330 335
Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala
340 345 350
Leu Leu
Claims (55)
1.一种在质谱测定中起作用以选择性地检测或定量靶转基因蛋白的标记的替代肽,所述靶转基因蛋白选自由以下组成的组:来自一种或多种转基因植物的一个或多个生物样品中的转基因蛋白和非转基因蛋白的混合物中的Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白和磷酸甘露糖异构酶(PMI)蛋白,所述替代肽包含标记和选自由以下组成的组的氨基酸序列:GSAQGIEGSIR(SEQ ID NO:1)、IVAQLGQGVYR(SEQ ID NO:2)、TLSSTLYR(SEQ IDNO:3)、DVSVFGQR(SEQ ID NO:4)、TYPIR(SEQ ID NO:5)、TVSQLTR(SEQ ID NO:6)、WYNTGLER(SEQ ID NO:7)、EWEADPTNPALR(SEQ ID NO:8)、VWGPDSR(SEQ ID NO:9)、APMFSWIHR(SEQ IDNO:10)、WGFDAATINSR(SEQ ID NO:11)、NQAISR(SEQ ID NO:12)、IEEFAR(SEQ ID NO:13)、SGFSNSSVSIIR(SEQ ID NO:14)、LSHVSMFR(SEQ ID NO:15)、EIYTNPVLENFDGSFR(SEQ ID NO:16)、LEGLSNLYQIYAESFR(SEQ ID NO:17)、YNQFR(SEQ ID NO:18)、YNDLTR(SEQ ID NO:19)、SPHLMDILNSITIYTDAHR(SEQ ID NO:20)、SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21)、QGFSHR(SEQ ID NO:22)、MDNNPNINECIPYNCLSNPEVEVLGGER(SEQ ID NO:23)、ELTLTVLDIVSLFPNYDSR(SEQ ID NO:24)、RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR(SEQ ID NO:25)、SGTVDSLDEIPPQNNNVPPR(SEQ ID NO:26)、TDVTDYHIDQV(SEQ ID NO:27)、AVNELFTSSNQIGLK(SEQ ID NO:28)、ITQLPLTK(SEQ ID NO:29)、GLDSSTTK(SEQ ID NO:30)、QCAGIRPYDGR(SEQID NO:31)、IEFVPAEVTFEAEYDLER(SEQ ID NO:32)、ITQLPLVK(SEQ ID NO:33)、MTADNNTEALDSSTTK(SEQ ID NO:34)、VYIDK(SEQ ID NO:35)、DGGISQFIGDK(SEQ ID NO:36)、LITLTCK(SEQ ID NO:37)、ELLLATDLSNK(SEQ ID NO:38)、FEELTFATETSSK(SEQ ID NO:39)、EVLFEK(SEQ ID NO:40)、TASELITK(SEQ ID NO:41)、DVSEMFTTK(SEQ ID NO:42)、LLGLADIDYTSIMNEHLNK(SEQ ID NO:43)、IDFTK(SEQ ID NO:44)、TDTGGDLTLDEILK(SEQ IDNO:45)、DIMNMIFK(SEQ ID NO:46)、ALYVHK(SEQ ID NO:47)、VNILPTLSNTFSNPNYAK(SEQ IDNO:48)、ITSMLSDVIK(SEQ ID NO:49)、QNLQLDSFSTYR(SEQ ID NO:50)、DSLSEVIYGDMDK(SEQID NO:51)、MIVEAKPGHALIGFEISNDSITVLK(SEQ ID NO:52)、VYFSVSGDANVR(SEQ ID NO:53)、NQQLLNDISGK(SEQ ID NO:54)、VESSEAEYR(SEQ ID NO:55)、YMSGAK(SEQ ID NO:56)、DGSPADILDELTELTELAK(SEQ ID NO:57)、VYEAK(SEQ IDNO:58)、LDAINTMLR(SEQ ID NO:59)、GKPSIHLK(SEQ IDNO:60)、DENTGYIHYEDTNNNLEDYQTINK(SEQ ID NO:61)、DNFYIELSQGNNLYGGPIVHFYDVSIK(SEQ ID NO:62)、LLCPDQSEQIYYTNNIVFPNEYVITK(SEQ IDNO:63)、SQNGDEAWGDNFIILEISPSEK(SEQ ID NO:64)、NAYVDHTGGVNGTK(SEQ ID NO:65)、LDGVNGSLNDLIAQGNLNTELSK(SEQ ID NO:66)、IANEQNQVLNDVNNK(SEQ ID NO:67)、YEVTANFYDSSTGEIDLNK(SEQ ID NO:68)、QNYALSLQIEYLSK(SEQ ID NO:69)、QLQEISDK(SEQID NO:70)、LLSPELINTNNWTSTGSTNISGNTLTLYQGGR(SEQ ID NO:71)、YVNEK(SEQ ID NO:72)、QNYQVDK(SEQ ID NO:73)、MAGAEEIVLQPIK(SEQ ID NO:74)、FPVEDAK(SEQ ID NO:75)、EISGTVK(SEQ ID NO:76)、ILLLAALSEGTTVVDNLLNSEDVHYMLGALR(SEQ ID NO:77)、DFELPAPPRPVRPVTQI(SEQ ID NO:78)、LGLGSTLYTHLLK(SEQ ID NO:79)、MSPER(SEQ ID NO:80)、HGGWHDVGFWQR(SEQ ID NO:81)、NAYDWTVESTVYVSHR(SEQ ID NO:82)、TEPQTPQEWIDDLER(SEQ ID NO:83)、AAGYK(SEQ ID NO:84)、YPWLVAEVEGVVAGIAYAGPWK(SEQ ID NO:85)、RPVEIRPATAADMAAVCDIVNHYIETSTVNFR(SEQ ID NO:86)、ENAAGIPMDAAER(SEQ ID NO:87)、ALAILK(SEQ ID NO:88)、SALDSQQGEPWQTIR(SEQ ID NO:89)、GSQQLQLKPGESAFIAANESPVTVK(SEQ IDNO:90)、FEAKPANQLLTQPVK(SEQ ID NO:91)、STLLGEAVAK(SEQ ID NO:92)、LINSVQNYAWGSK(SEQ ID NO:93)、HNSEIGFAK(SEQ ID NO:94)、VLCAAQPLSIQVHPNK(SEQ ID NO:95)、TALTELYGMENPSSQPMAELWMGAHPK(SEQ ID NO:96)、LSELFASLLNMQGEEK(SEQ ID NO:97)和QGAELDFPIPVDDFAFSLHDLSDK(SEQ ID NO:98)。
2.如权利要求1所述的标记的替代肽,其中通过掺入稳定的同位素标记的(SIL)氨基酸来标记所述肽。
3.如权利要求2所述的标记的替代肽,其中所述SIL氨基酸为赖氨酸、异亮氨酸、缬氨酸或精氨酸。
4.如权利要求1所述的标记的替代肽,其中所述肽选择性地检测或定量所述混合物中的Cry1Ab蛋白,并包含选自由以下组成的组的氨基酸序列:GSAQGIEGSIR(SEQ ID NO:1)、IVAQLGQGVYR(SEQ ID NO:2)、TLSSTLYR(SEQ ID NO:3)、DVSVFGQR(SEQ ID NO:4)、TYPIR(SEQ ID NO:5)、TVSQLTR(SEQ ID NO:6)、WYNTGLER(SEQ ID NO:7)、EWEADPTNPALR(SEQ IDNO:8)、VWGPDSR(SEQ ID NO:9)、APMFSWIHR(SEQ ID NO:10)、WGFDAATINSR(SEQ ID NO:11)、NQAISR(SEQ ID NO:12)、IEEFAR(SEQ ID NO:13)、SGFSNSSVSIIR(SEQ ID NO:14)、LSHVSMFR(SEQ ID NO:15)、EIYTNPVLENFDGSFR(SEQ ID NO:16)、LEGLSNLYQIYAESFR(SEQ ID NO:17)、YNQFR(SEQ ID NO:18)、YNDLTR(SEQ ID NO:19)、SPHLMDILNSITIYTDAHR(SEQ ID NO:20)、SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21)、QGFSHR(SEQ ID NO:22)、MDNNPNINECIPYNCLSNPEVEVLGGER(SEQ ID NO:23)、ELTLTVLDIVSLFPNYDSR(SEQ ID NO:24)、RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR(SEQ ID NO:25)和SGTVDSLDEIPPQNNNVPPR(SEQ IDNO:26)。
5.如权利要求4所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:GIEGSIR(SEQ ID NO:99)、EGSIR(SEQ ID NO:100)、AQLGQGVYR(SEQ ID NO:101)、GQGVYR(SEQ ID NO:102)、SSTLYR(SEQ ID NO:103)、STLYR(SEQ ID NO:104)、SVFGQR(SEQ ID NO:105)、FGQR(SEQ ID NO:106)、PIR、TY、SQLTR(SEQ ID NO:107)、QLTR(SEQ IDNO:108)、NTGLER(SEQ ID NO:109)、YNTGLER(SEQ ID NO:110)、PTNPALR(SEQ ID NO:111)、DPTNPALR(SEQ ID NO:112)、GPDSR(SEQ ID NO:113)、VW、HR、SWIHR(SEQ ID NO:114)、ATINSR(SEQ ID NO:115)、DAATINSR(SEQ ID NO:116)、AISR(SEQ ID NO:117)、ISR、EFAR(SEQ ID NO:118)、EEFAR(SEQ ID NO:119)、SNSSVSIIR(SEQ ID NO:120)、SSVSIIR(SEQ IDNO:121)、SMFR(SEQ ID NO:122)、VSMFR(SEQ ID NO:123)、ENFDGSFR(SEQ ID NO:124)、GSFR(SEQ ID NO:125)、YAESFR(SEQ ID NO:126)、LEG、NQFR(SEQ ID NO:127)、QFR、DLTR(SEQ IDNO:128)、NDLTR(SEQ ID NO:129)、TIYTDAHR(SEQ ID NO:130)、YTDAHR(SEQ ID NO:131)、PLTK(SEQ ID NO:132)、SAEFNNII(SEQ ID NO:133)、FSHR(SEQ ID NO:134)、GFSHR(SEQ IDNO:135)、EVLGGER(SEQ ID NO:136)、GGER(SEQ ID NO:137)、FPNYDSR(SEQ ID NO:138)、PNYDSR(SEQ ID NO:139)、PSAVYR(SEQ ID NO:140)、YR、PPR和SGTVDSLDE(SEQ ID NO:141)。
6.如权利要求4所述的替代肽,其中所述肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQ ID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。
7.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量eCry3.1Ab蛋白,并包含选自由以下组成的组的氨基酸序列:TDVTDYHIDQV(SEQ ID NO:27)、AVNELFTSSNQIGLK(SEQ ID NO:28)、ITQLPLTK(SEQ ID NO:29)、GLDSSTTK(SEQ ID NO:30)、QCAGIRPYDGR(SEQID NO:31)和IEFVPAEVTFEAEYDLER(SEQ ID NO:32)。
8.如权利要求7所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:TDYHIDQV(SEQ ID NO:142)、DYHIDQV(SEQ ID NO:143)、TSSNQIGLK(SEQ IDNO:144)、SSNQIGLK(SEQ ID NO:145)、QLPLTK(SEQ ID NO:146)、TQLPLTK(SEQ ID NO:147)、DSSTTK(SEQ ID NO:148)、SSTTK(SEQ ID NO:149)、PYDGR(SEQ ID NO:150)、DGR、IEF和LER。
9.如权利要求7所述的替代肽,其中所述肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。
10.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量mCry3A蛋白,并包含选自由以下组成的组的氨基酸序列:ITQLPLVK(SEQ ID NO:33)、MTADNNTEALDSSTTK(SEQ IDNO:34)和VYIDK(SEQ ID NO:35)。
11.如权利要求10所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:QLPLVK(SEQ ID NO:151)、TQLPLVK(SEQ ID NO:152)、ALDSSTTK(SEQ IDNO:153)、EALDSSTTK(SEQ ID NO:154)、YIDK(SEQ ID NO:155)和IDK。
12.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量Vip3A蛋白,并包含选自由以下组成的组的氨基酸序列:DGGISQFIGDK(SEQ ID NO:36)、LITLTCK(SEQ ID NO:37)、ELLLATDLSNK(SEQ ID NO:38)、FEELTFATETSSK(SEQ ID NO:39)、EVLFEK(SEQ ID NO:40)、TASELITK(SEQ ID NO:41)、DVSEMFTTK(SEQ ID NO:42)、LLGLADIDYTSIMNEHLNK(SEQ ID NO:43)、IDFTK(SEQ ID NO:44)、TDTGGDLTLDEILK(SEQ ID NO:45)、DIMNMIFK(SEQ ID NO:46)、ALYVHK(SEQ ID NO:47)、VNILPTLSNTFSNPNYAK(SEQ ID NO:48)、ITSMLSDVIK(SEQ ID NO:49)、QNLQLDSFSTYR(SEQ ID NO:50)、DSLSEVIYGDMDK(SEQ ID NO:51)、MIVEAKPGHALIGFEISNDSITVLK(SEQ ID NO:52)、VYFSVSGDANVR(SEQ ID NO:53)、NQQLLNDISGK(SEQ ID NO:54)、VESSEAEYR(SEQ ID NO:55)、YMSGAK(SEQ ID NO:56)、DGSPADILDELTELTELAK(SEQ ID NO:57)、VYEAK(SEQ ID NO:58)、LDAINTMLR(SEQ ID NO:59)、GKPSIHLK(SEQ ID NO:60)、DENTGYIHYEDTNNNLEDYQTINK(SEQ ID NO:61)、DNFYIELSQGNNLYGGPIVHFYDVSIK(SEQ ID NO:62)、LLCPDQSEQIYYTNNIVFPNEYVITK(SEQ IDNO:63)、SQNGDEAWGDNFIILEISPSEK(SEQ ID NO:64)、NAYVDHTGGVNGTK(SEQ ID NO:65)、LDGVNGSLNDLIAQGNLNTELSK(SEQ ID NO:66)、IANEQNQVLNDVNNK(SEQ ID NO:67)、YEVTANFYDSSTGEIDLNK(SEQ ID NO:68)、QNYALSLQIEYLSK(SEQ ID NO:69)、QLQEISDK(SEQID NO:70)、LLSPELINTNNWTSTGSTNISGNTLTLYQGGR(SEQ ID NO:71)、YVNEK(SEQ ID NO:72)和QNYQVDK(SEQ ID NO:73)。
13.如权利要求12所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:SQFIGDK(SEQ ID NO:156)、GDK、TLTCK(SEQ ID NO:157)、TCK、ATDLSNK(SEQ ID NO:158)、LATDLSNK(SEQ ID NO:159)、TFATETSSK(SEQ ID NO:160)、FATETSSK(SEQID NO:161)、FEK、LFEK(SEQ ID NO:162)、SELITK(SEQ ID NO:163)、ASELITK(SEQ ID NO:164)、SEMFTTK(SEQ ID NO:165)、DVS、IMNEHLNK(SEQ ID NO:166)、MNEHLNK(SEQ ID NO:167)、DFTK(SEQ ID NO:168)、FTK、TLDEILK(SEQ ID NO:169)、LTLDEILK(SEQ ID NO:170)、MNMIFK(SEQ ID NO:171)、NMIFK(SEQ ID NO:172)、YVHK(SEQ ID NO:173)、HK、VNI、VNIL(SEQ ID NO:174)、SMLSDVIK(SEQ ID NO:175)、TSMLSDVIK(SEQ ID NO:176)、DSFSTYR(SEQID NO:177)、LDSFSTYR(SEQ ID NO:178)、IYGDMDK(SEQ ID NO:179)、VIYGDMDK(SEQ ID NO:180)、SNDSITVLK(SEQ ID NO:181)、MIV、SGDANVR(SEQ ID NO:182)、SVSGDANVR(SEQ ID NO:183)、LLNDISGK(SEQ ID NO:184)、LNDISGK(SEQ ID NO:185)、SSEAEYR(SEQ ID NO:186)、ESSEAEYR(SEQ ID NO:187)、SGAK(SEQ ID NO:188)、MSGAK(SEQ ID NO:189)、TELTELAK(SEQID NO:190)、DGSPADI(SEQ ID NO:191)、YEAK(SEQ ID NO:192)、EAK、NTMLR(SEQ ID NO:193)、AINTMLR(SEQ ID NO:194)、PSIHLK(SEQ ID NO:195)、HLK、DYQTINK(SEQ ID NO:196)、NK、DNF、DNFY(SEQ ID NO:197)、PNEYVITK(SEQ ID NO:198)、LLC、SPSEK(SEQ ID NO:199)、LEISPSEK(SEQ ID NO:200)、NAY、DHTGGVNGTK(SEQ ID NO:201)、GNLNTELSK(SEQ ID NO:202)、NTELSK(SEQ ID NO:203)、LNDVNNK(SEQ ID NO:204)、NDVNNK(SEQ ID NO:205)、YE、DLNK(SEQ ID NO:206)、QIEYLSK(SEQ ID NO:207)、LQIEYLSK(SEQ ID NO:208)、SDK、QEISDK(SEQ ID NO:209)、YQGGR(SEQ ID NO:210)、TLYQGGR(SEQ ID NO:211)、NEK、VNEK(SEQ IDNO:212)、DK和VDK。
14.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量dmEPSPS蛋白,并包含选自由以下组成的组的氨基酸序列:MAGAEEIVLQPIK(SEQ ID NO:74)、FPVEDAK(SEQ ID NO:75)、EISGTVK(SEQ ID NO:76)和ILLLAALSEGTTVVDNLLNSEDVHYMLGALR(SEQ ID NO:77)。
15.如权利要求14所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:PIK、EIVLQPIK(SEQ ID NO:213)、PVEDAK(SEQ ID NO:214)、VEDAK(SEQ IDNO:215)、SGTVK(SEQ ID NO:216)、GTVK(SEQ ID NO:217)、ILLLAA(SEQ ID NO:218)和HYMLGALR(SEQ ID NO:219)。
16.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量PAT蛋白,并包含选自由以下组成的组的氨基酸序列:DFELPAPPRPVRPVTQI(SEQ ID NO:78)、LGLGSTLYTHLLK(SEQID NO:79)、MSPER(SEQ ID NO:80)、HGGWHDVGFWQR(SEQ ID NO:81)、NAYDWTVESTVYVSHR(SEQID NO:82)、TEPQTPQEWIDDLER(SEQ ID NO:83)、AAGYK(SEQ ID NO:84)、YPWLVAEVEGVVAGIAYAGPWK(SEQ ID NO:85)和RPVEIRPATAADMAAVCDIVNHYIETSTVNFR(SEQ IDNO:86)。
17.如权利要求16所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:DFE、DF、YTHLLK(SEQ ID NO:220)、THLLK(SEQ ID NO:221)、PER、SPER(SEQID NO:222)、GFWQR(SEQ ID NO:223)、VGFWQR(SEQ ID NO:224)、STVYVSHR(SEQ ID NO:225)、SHR、TEPQT(SEQ ID NO:226)、DLER(SEQ ID NO:227)、GYK、AGYK(SEQ ID NO:228)、GPWK(SEQ ID NO:229)、GIAYAGPWK(SEQ ID NO:230)、TSTVNFR(SEQ ID NO:231)和NFR。
18.如权利要求16所述的替代肽,其中所述肽包含氨基酸序列LGLGSTLYTHLLK(SEQ IDNO:79),并产生由氨基酸序列YTHLLK(SEQ ID NO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。
19.如权利要求1所述的替代肽,其中所述肽选择性地检测或定量PMI蛋白,并包含选自由以下组成的组的氨基酸序列:ENAAGIPMDAAER(SEQ ID NO:87)、ALAILK(SEQ ID NO:88)、SALDSQQGEPWQTIR(SEQ ID NO:89)、GSQQLQLKPGESAFIAANESPVTVK(SEQ ID NO:90)、FEAKPANQLLTQPVK(SEQ ID NO:91)、STLLGEAVAK(SEQ ID NO:92)、LINSVQNYAWGSK(SEQ IDNO:93)、HNSEIGFAK(SEQ ID NO:94)、VLCAAQPLSIQVHPNK(SEQ ID NO:95)、TALTELYGMENPSSQPMAELWMGAHPK(SEQ ID NO:96)、LSELFASLLNMQGEEK(SEQ ID NO:97)和QGAELDFPIPVDDFAFSLHDLSDK(SEQ ID NO:98)。
20.如权利要求19所述的替代肽,其中所述肽产生具有选自由以下组成的组的氨基酸序列的过渡离子:PMDAAER(SEQ ID NO:232)、GIPMDAAER(SEQ ID NO:233)、AILK(SEQ IDNO:234)、LK、PWQTIR(SEQ ID NO:235)、GEPWQTIR(SEQ ID NO:236)、ANESPVTVK(SEQ ID NO:237)、PVTVK(SEQ ID NO:238)、LTQPVK(SEQ ID NO:239)、PVK、GEAVAK(SEQ ID NO:240)、LGEAVAK(SEQ ID NO:241)、QNYAWGSK(SEQ ID NO:242)、NYAWGSK(SEQ ID NO:243)、NSEIGFAK(SEQ ID NO:244)、HN、VLCAAQ(SEQ ID NO:245)、PNK、WMGAHPK(SEQ ID NO:246)、TALTE(SEQ ID NO:247)、NMQGEEK(SEQ ID NO:248)、LNMQGEEK(SEQ ID NO:249)、SLHDLSDK(SEQ ID NO:250)和HDLSDK(SEQ ID NO:251)。
21.如权利要求19所述的替代肽,其中所述肽包含氨基酸序列SALDSQQGEPWQTIR(SEQID NO:89),并产生由氨基酸序列PWQTIR(SEQ ID NO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。
22.如权利要求4所述的替代肽,其中所述Cry1Ab蛋白包含SEQ ID NO:259的氨基酸序列。
23.如权利要求22所述的替代肽,其中所述Cry1Ab蛋白来自事件Bt11。
24.如权利要求7所述的替代肽,其中所述eCry3.1Ab蛋白包含SEQ ID NO:260的氨基酸序列。
25.如权利要求24所述的替代肽,其中所述eCry3.1Ab蛋白来自事件5307。
26.如权利要求10所述的替代肽,其中所述mCry3A蛋白包含SEQ ID NO:261的氨基酸序列。
27.如权利要求26所述的替代肽,其中所述mCry3A蛋白来自事件MIR604。
28.如权利要求12所述的替代肽,其中所述Vip3A蛋白包含SEQ ID NO:262的氨基酸序列。
29.如权利要求28所述的替代肽,其中所述Vip3A蛋白来自事件MIR162。
30.如权利要求14所述的替代肽,其中所述dmEPSPS蛋白包含SEQ ID NO:263的氨基酸序列。
31.如权利要求30所述的替代肽,其中所述dmEPSPS蛋白来自事件GA21。
32.如权利要求16所述的替代肽,其中所述PAT蛋白包含SEQ ID NO:264的氨基酸序列。
33.如权利要求32所述的替代肽,其中所述PAT蛋白来自事件Bt11、59122、TC1507、DP4114或T25。
34.如权利要求19所述的替代肽,其中所述PMI蛋白包含SEQ ID NO:265或SEQ ID NO:266的氨基酸序列。
35.如权利要求34所述的替代肽,其中所述PMI蛋白来自事件MIR162、事件MIR604、事件5307或事件3272。
36.如权利要求1-35中任一项所述的替代肽,其中所述转基因蛋白的混合物包含选自由以下组成的组的至少两种转基因蛋白:Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3A蛋白、dmEPSPS蛋白、PAT蛋白和PMI蛋白。
37.如权利要求36所述的标记的替代肽,其中所述转基因蛋白的混合物包含Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3A蛋白、dmEPSPS蛋白、PAT蛋白和PMI蛋白。
38.如权利要求37所述的标记的替代肽,其中所述转基因蛋白的混合物进一步包含选自由以下组成的组的至少一种转基因蛋白:Cry1A.105蛋白(SEQ ID NO:267)、Cry2Ab蛋白(SEQ ID NO:268)、Cry1F蛋白(SEQ ID NO:269)、Cry34蛋白(SEQ ID NO:270)和Cry35蛋白(SEQ ID NO:271)。
39.如权利要求1所述的替代肽,其中所述转基因植物选自由以下组成的组:玉米、大豆、棉花、水稻、小麦、卡诺拉油菜和茄子。
40.如权利要求39所述的替代肽,其中所述转基因玉米植物包含选自由以下组成的组的转基因玉米事件:事件Bt11、事件5307、事件MIR604和事件GA21。
41.如权利要求40所述的替代肽,其中所述转基因玉米植物包含事件Bt11、事件5307、MIR604和事件GA21。
42.如权利要求41所述的替代肽,其中所述转基因玉米植物进一步包含事件3272、事件MON89034、事件DP4114、事件1577或事件59122。
43.如权利要求1所述的替代肽,其中所述生物样品来自叶组织、种子、籽粒、花粉或根组织。
44.如权利要求43所述的替代肽,其中所述生物样品包含来自事件Bt11的Cry1Ab蛋白,来自事件5307的eCry3.1Ab蛋白,来自事件MIR604的mCry3A蛋白,来自事件GA21的EPSPS蛋白,来自Bt11、59122、DP4114、TC1507或T25的PAT蛋白,或来自事件MIR162、事件MIR604、事件5307或事件3272的PMI蛋白。
45.如权利要求44所述的替代肽,其中所述生物样品进一步包含来自事件MON89034的Cry1A.105蛋白、来自事件1507的Cry1F蛋白或来自事件59122的Cry34和Cry35蛋白。
46.一种测定盒,其包含至少两种如权利要求1所述的标记的替代肽。
47.一种同时检测或定量来自转基因植物的包含所述靶转基因蛋白和非转基因蛋白的混合物的复合生物样品中的一种或多种靶转基因蛋白的方法,所述方法包括:
a.从转基因植物中获得生物样品;
b.从所述生物样品中提取蛋白,得到包含蛋白的混合物的提取物;
c.减少步骤b的提取物中非转基因不溶性蛋白的量,得到浓缩的可溶性蛋白的提取物;
d.消化步骤c的提取物中的可溶性蛋白,得到包含肽碎片的提取物,其中所述肽碎片包含至少一种对每种靶转基因蛋白具有特异性的替代肽;
e.浓缩步骤d的提取物中的肽碎片,
f.添加如权利要求1所述的一种或多种标记的替代肽,其中每种标记的替代肽具有与所述靶转基因蛋白的每种替代肽相同的氨基酸序列,并且其中添加的标记的替代肽的数目等于所述混合物中靶转基因蛋白的数目;
g.通过减少所述混合物中非替代肽的量来浓缩所述替代肽和所述标记的替代肽;
h.通过液相色谱使步骤g的肽碎片混合物分解;
i.通过质谱分析从步骤h得到的肽碎片混合物,其中检测到标记的替代肽的过渡离子碎片指示存在衍生所述替代肽的靶转基因蛋白;以及,任选地,
j.通过比较由步骤i的过渡离子碎片产生的质谱信号与由标记的替代肽的过渡离子产生的质谱信号,计算所述生物样品中靶转基因蛋白的量。
48.如权利要求47所述的方法,其中所述靶转基因蛋白是Cry1Ab蛋白、eCry3.1Ab蛋白、mCry3A蛋白、Vip3蛋白、双突变5-烯醇丙酮酰莽草酸-3-磷酸合酶(dmEPSPS)蛋白、草丁膦乙酰转移酶(PAT)蛋白或磷酸甘露糖异构酶(PMI)蛋白。
49.如权利要求48所述的方法,其中所述靶转基因蛋白是Cry1Ab,并且所述标记的替代肽包含氨基酸序列SAEFNNIIPSSQITQIPLTK(SEQ ID NO:21),并产生由氨基酸序列PLTK(SEQID NO:132)或SAEFNNII(SEQ ID NO:133)组成的过渡离子。
50.如权利要求49所述的方法,其中通过比较由氨基酸序列PLTK(SEQ ID NO:132)组成的过渡离子碎片产生的质谱信号,对所述生物样品中的所述Cry1Ab靶蛋白进行定量。
51.如权利要求47所述的方法,其中所述靶转基因蛋白是eCry3.1Ab蛋白,并且所述标记的替代肽包含氨基酸序列TDVTDYHIDQV(SEQ ID NO:27),并产生由氨基酸序列TDYHIDQV(SEQ ID NO:142)或DYHIDQV(SEQ ID NO:143)组成的过渡离子。
52.如权利要求51所述的方法,其中通过比较由氨基酸序列组成的过渡离子碎片产生的质谱信号,对所述生物样品中的所述eCry3.1Ab转基因蛋白进行定量。
53.如权利要求38所述的方法,其中所述标记的替代肽包含氨基酸序列DGGISQFIGDK(SEQ ID NO:36),并产生由氨基酸序列SQFIGDK(SEQ ID NO:156)或GDK组成的过渡离子。
54.如权利要求38所述的方法,其中所述标记的替代肽包含氨基酸序列LGLGSTLYTHLLK(SEQ ID NO:79),并产生由氨基酸序列YTHLLK(SEQ ID NO:220)或THLLK(SEQ ID NO:221)组成的过渡离子。
55.如权利要求38所述的方法,其中所述标记的替代肽包含氨基酸序列SALDSQQGEPWQTIR(SEQ ID NO:89),并产生由氨基酸序列PWQTIR(SEQ ID NO:235)或GEPWQTIR(SEQ ID NO:236)组成的过渡离子。
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