CN113912684B - 一种模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽及其编码基因与应用 - Google Patents
一种模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽及其编码基因与应用 Download PDFInfo
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Abstract
本发明提供一种可模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽及其编码基因与应用，该短肽DNA序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示；该短肽能与棉铃虫钙黏蛋白Bt Cry蛋白结合区特异性结合，且经克隆替换Bt Cry蛋白DomianⅡ‑Loop2区和‑Loop3区以及替换Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区，重组的Bt Cry1Ac蛋白和重组的Bt Cry1Ac蛋白抗独特型抗体均对棉铃虫和小菜蛾具有良好的杀虫活性；本发明采用抗虫蛋白靶标害虫关键受体作为靶点，通过体外高通量筛选与鉴定的方式，靶向获得具备模拟抗虫蛋白关键抗虫功能表位的短肽，目标性强，制备周期短，短肽氨基酸序列小，适合蛋白质关键表位功能替代，对Bt Cry1Ac蛋白靶标农业害虫安全性生物防控具有重要的科学意义和实用价值。
Description
技术领域
本发明涉及靶向模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽创制技术领域，特别是一种可替换Bt Cry1Ac蛋白及其抗独特型抗体关键抗虫表位的短肽(H-SC01)及其编码基因。
背景技术
Bt Cry蛋白是苏云金芽胞杆菌(Bacillus thuringiensis，Bt)在代谢过程中产生的蛋白质类伴胞晶体次生代谢产物，对多种农林害虫具有广谱特异性毒杀作用，是目前研究最成熟、应用最广的微生物农药。现已报道的Bt Cry蛋白多达数百种，其中Cry1Ab和Cry1Ac最为典型且氨基酸序列同源性高度相似、杀虫谱系几乎完全重叠，两者在应用上最为常见，特别是用于转基因抗虫作物改造，品种涉及水稻、玉米、大豆、马铃薯、番茄、棉花、烟草等全球主要大宗农作物。据国际农业生物技术应用服务组织最新统计数据显示，转BtCry蛋白基因作物推广应用范围几乎涵盖了五大洲所有农业生产大国，为全世界减少化学农药投入使用和保障农产品稳定生产作了重要贡献，蕴藏着巨大的商业价值。
然而，随着转Bt Cry蛋白基因作物长时间、大范围推广应用，其加速害虫抗药性以及可能存在的生态安全风险等问题，一直是科研关注的热点。挖掘新型安全生物源性抗虫材料，特别是探索扩大Bt Cry蛋白家族种类(提供更多新的抗虫材料)乃至可替代Bt Cry蛋白抗虫功能的生物活性材料，即可为避免靶标害虫抗药性提供潜在等效材料，已成为当今绿色生物农药领域研究的热点。
前期文献已经证实Bt Cry1Ab DomianⅡ中的Loop2区和Loop3区是与靶标害虫中肠受体结合的关键表位区域，在导致靶标昆虫死亡过程中发挥了关键的作用(“苏云金芽胞杆菌Cry1Ab毒素domainⅡ内3个Loop环对其毒性作用的影响”，邵恩斯等，农业生物技术学报，2014年第22卷第11期)，该发现也同样适用于Cry1Ac。其中针对棉铃虫中肠受体的研究，有文献表明其中一段分子量约为730bp大小的钙黏蛋白片段是与Bt Cry1Ac蛋白Loop2区和Loop3区结合的关键区域(“Roles of Midgut Cadherin from Two Moths in DifferentBacillus thuringiensis Action Mechanisms:Correlation among Toxin Binding,Cellular Toxicity,and Synergism”，Gao等，J.Agric.Food Chem，2019年总第67期)。BtCry1Ac蛋白关键抗虫功能表位和靶标害虫关键受体区域均已明确，因此这为体外人工靶向创制具有模拟Bt Cry1Ac蛋白关键抗虫功能表位的蛋白类生物材料提供了潜在可行的路径。但目前模拟Bt蛋白关键抗虫功能表位的蛋白类生物材料(如短肽)均片段较长，且只能实现定点替代，即一个模拟蛋白只能替代另一种生物材料的全功能或部分功能，从而限制了这些蛋白材料的应用。
噬菌体表面展示多肽技术是紧随分子生物学快速发展起来的外源基因与病毒核酸串联共表达的重组技术，是当前多肽创新研究和体外高通量靶向筛选研究的热点。1985年，美国科学家Simth等(“Filamentous fusion phage:novel expression vectors thatdisplay cloned antigens on the virion surface”，Science，1985年05期)首次报道将外源多肽基因克隆到丝状噬菌体DNA上，经串联共表达，外源多肽展示到噬菌体衣壳表面，实现多肽表型与其基因型统一于一个噬菌体上，由此开创了噬菌体表面展示多肽技术并获得了2018年度诺贝尔化学奖。噬菌体表面展示多肽技术实现了靶标多肽材料体外可人工定向筛选、修饰的新阶段，同时多肽基因可以依托噬菌体和宿主菌进行持续表达，使多肽制备更方便、更省时、更省力“Phage display:concept,innovations,applications andfuture”(Pande等，Biotechnol.Adv.，2010年06期)，且还能将多肽基因转移克隆到更合适的载体或表达体系中进行优化表达“Optimizing antibody expression:the nuts andbolts”(Ayyar等，Methods(San Diego,Calif.)，2017年116卷)，乃至进行进一步体外亲和成熟修饰“基因突变技术在抗体亲和力体外成熟中的应用”(刘媛等，浙江大学学报(农业与生命科学版)，2015年第1期)。
目前，利用噬菌体表面展示多肽技术筛选具体模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽技术尚未见报道。
发明内容
针对上述问题，本申请提供一种可高效靶向结合棉铃虫中肠类钙黏蛋白毒素结合区并能模拟Bt Cry1Ac及其抗独特型抗体关键抗虫功能表位的短肽材料(H-SC01)，该短肽序列片段小，可以替代多种生物材料的关键功能表位，实现全功能或部分功能替代，可用于丰富抗虫蛋白的多样性，为防控Bt Cry靶标害虫储备更多潜在可用的蛋白类生物抗虫材料。
具体来说，本发明是这样实现的：
本申请首先提供了一种可模拟Bt Cry1Ac蛋白关键抗虫功能表位的新型短肽材料，其氨基酸序列如SEQ ID NO.2所示，申请人将该短肽材料自命名为H-SC01，编码该短肽材料基因的核苷酸序列如SEQ ID NO.1所示。
该短肽材料可以应用于模拟并替代Bt Cry1Ac蛋白关键抗虫功能表位。
其次，本申请提供了一种经氨基酸序列如氨基酸序列如SEQ ID NO.2所示短肽替换Bt Cry蛋白DomianⅡ-Loop2区后的重组体I，其氨基酸序列如SEQ ID NO.6所示，重组体I的编码基因核苷酸序列如SEQ ID NO.5所示。
该重组体I可以应用于杀灭棉铃虫、小菜蛾。
第三，本申请本申请提供了一种经氨基酸序列如氨基酸序列如SEQ ID NO.2所示短肽替换Bt Cry蛋白DomianⅡ-Loop3区后的重组体II，其氨基酸序列如SEQ ID NO.8所示，重组体II的编码基因核苷酸序列如SEQ ID NO.7所示。
该重组体II可以应用于杀灭棉铃虫、小菜蛾。
第四，本申请本申请提供了一种经氨基酸序列如氨基酸序列如SEQ ID NO.2所示短肽替换Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区的重组体III，其氨基酸序列如SEQ ID NO.10所示，重组体III的编码基因核苷酸序列如SEQ ID NO.9所示。
该重组体III可以应用于杀灭棉铃虫、小菜蛾。
本申请通过随机合成短肽序列的形式，构建了一个库容量为109的噬菌体展示随机6肽库，首次设计以克隆表达的Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)作为为包被靶点，通过特异性富集筛选，从供试库中进行体外高通量靶向筛选与HaCadCR9-CR11发生高结合活性，具有模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽材料，进而将短肽替换Bt Cry1Ac蛋白及其抗独特型抗体中的关键抗虫活性区域，将构建的重组体进行室内靶标害虫的抗虫活性测定，获得具有抗虫活性的的重组体。与现有技术相比，本申请获得的短肽具有以下有益效果：
1、本发明首次锚定Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区作为包被靶点，采用多轮富集淘筛的方式筛选靶标短肽，指向性强，制备周期短，筛选效率高，短肽的氨基酸序列小，适合体外重组克隆。
2、本申请提供的短肽可以用于改造Bt Cry蛋白及其模拟物，既提高Bt Cry蛋白及其模拟物抗虫活性，也可避免靶标害虫对Bt Cry蛋白及其模拟物的抗药性。此外该短肽还有望作为增效剂直接用于辅助Bt Cry蛋白及其模拟物抗虫活性和抗虫谱系中。
3、本申请的实施例中，以模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽替换替换Bt Cry1Ac蛋白及其抗独特型抗体关键功能表位后的重组体，均具有较高抗虫活性，可作为新型抗虫蛋白用于靶标害虫的绿色防控，丰富了Bt Cry蛋白及其抗独特型抗体多样性。
附图说明
图1为ELISA测定可特异性结合Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)的噬菌体展示短肽富集效果示意图。
图2为ELISA测定靶标噬菌体展示短肽与Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)特异性结合活性检测结果示意图。
图3为靶标短肽替换Bt Cry蛋白DomianⅡ-Loop2区、-Loop3区以及Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区后的重组体对棉铃虫的致死率检测结果示意图；
图3中，CK-:为PBS溶液阴性对照；CK1+:为Bt Cry 1Ac蛋白溶液阳性对照；CK2+:为Bt Cry1Ac蛋白抗独特型人源重链单域抗体蛋白H9溶液阳性对照；重组体1:为靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop2区后的重组体蛋白溶液；重组体2：为靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop3区后的重组体蛋白溶液；重组体3：为靶标短肽(H-SC01)替换Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区后的重组体蛋白溶液。
图4为靶标短肽替换Bt Cry蛋白DomianⅡ-Loop2区、-Loop3区以及Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区后的重组体对小菜蛾的致死率检测结果示意图；
图4中，CK-:为PBS溶液阴性对照；CK1+:为Bt Cry 1Ac蛋白溶液阳性对照；CK2+:为Bt Cry1Ac蛋白抗独特型人源重链单域抗体蛋白H9溶液阳性对照；重组体1:为靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop2区后的重组体蛋白溶液；重组体2：为靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop3区后的重组体蛋白溶液；重组体3：为靶标短肽(H-SC01)替换Bt Cry1Ac蛋白抗独特型人源重链单域抗体CDR2区后的重组体蛋白溶液。
具体实施方式
实施例中所涉及的试剂和培养基配方：
(1)2×TY液体培养基：
在900ml蒸馏水中加入16g胰蛋白胨，10g酵母提取物和5g NaCl，搅拌混匀，用蒸馏水定容到1L，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(2)2×TY-AG液体培养基：
在2×TY的培养基中加入终浓度为100μg/ml氨苄青霉素和质量比为1％葡萄糖。
(5)TYE固体培养基：
在900ml蒸馏水中加入15g琼脂糖，8g NaCl，10g胰蛋白胨，5g酵母提取物，用蒸馏水定容到1L，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(6)TYE-AG固体培养基：
在TYE固体培养基中加入终浓度为100μg/ml氨苄青霉素和质量比为1％葡萄糖。
(7)PBS溶液
称取NaCl 8g，KCl 0.2g，Na2HPO4·12H2O 2.9g，KH2PO40.2 g，分别加入到蒸馏水中，充分溶解后，定容到1L。
(8)PBST溶液
在PBS溶液中加入体积比为0.05％的吐温-20。
(9)PEG/NaCl溶液：
称取20g PEG 8000，14.61g NaCl，加80ml去离子水，定容到100ml，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(10)柠檬酸盐缓冲液(CPBS,底物缓冲液，pH5.5)：
取C6H7O8(柠檬酸)21g，Na2HPO4·12H2O 71.6g，分别加入到蒸馏水中充分溶解后定容到1L。
(11)四甲基联苯胺(TMB)溶液：
称取10mg四甲基联苯胺溶于1ml二甲基亚砜中，避光，置于4℃保存备用。
(12)底物显色溶液：
10ml配方成分：9.875ml CPBS、100μl TMB溶液、25μl体积比为20％H2O2。
(13)LB液体培养基
1L体系：称取10g胰蛋白胨，5g酵母提取物，10gNaCl，以蒸馏水定容至1L，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(14)LB固体培养基
1L体系：取10g胰蛋白胨，5g酵母提取物，10gNaCl，15g琼脂粉，以蒸馏水定容至1L置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(15)IPTG溶液
1ml体系：称取0.238g(1mM)IPTG溶于1ml蒸馏水中，配置成浓度为1mM/ml的母液，于-20℃保存备用。
实施例中所涉及材料来源：
噬菌体展示随机6肽库(库容量为109)由专利申请人实验室(省部共建国家重点实验室培育基地-江苏省食品安全重点实验室)自构建，其中随机6肽基因委托通用生物系统(安徽)有限公司合成；该6肽库采用常规方法构建，即随机合成6肽基因，然后将其随机克隆至pIT2噬菌粒载体上，再导入E.coli TG1宿主菌中，即构成噬菌体展示形式的6肽库。具体步骤如下：
①委托通用生物系统(安徽)有限公司随机合成含有NcoI和NotI酶切位点的6肽基因序列。
②将合成的NcoI-6肽序列-NotI基因和提取的pIT2噬菌粒载体，按照50μL总酶切体系(包含5μL的10×NEB buffer，30μL的NcoI-6肽序列-NotI基因或pIT2噬菌粒载体，0.5μL的100×BSA buffer，2μL的NotI酶溶液，2μL的NcoI酶溶液和10.5μL ddH2O)吹打混匀后，将体系放置到37℃恒温金属浴中进行酶切过夜。第二天，取出酶切好的体系，按照PCR产物胶回收试剂盒附录的产品说明书(GenEluteTM gel extraction Kit，Sigma-USA)所描述方式进行除酶、除杂纯化，最后获得的目的片段以ddH2O溶解(可置-20℃冻存备用)。取酶切后的NcoI-6肽序列-NotI基因或pIT2噬菌粒载体片段，按照50μL总酶连体系(包含5μL的10×T4 ligase Buffer，25μL的NcoI-6肽序列-NotI基因酶切片段，5μL的pIT2噬菌粒载体酶切片段，2μL的T4 ligase和13μL
ddH2O)吹打混匀后，将体系放置到16℃恒温金属浴中进行过夜酶连。第二天，取出一管分装冻存的E.coli TG1电转感受态细胞，在手中捂化并迅速置于冰水中，将酶连体系加入到感受态细胞中(持续保持冰水预冷状态)，用移液器轻轻吹打(20～30次)混匀，接着在冰水中冰浴5min。取出冰浴混合物，迅速加入到事先冰浴预冷的商品化一次性电转杯中，并立即插入电转仪相应凹槽中以电压2500V电激100μs，取出电转杯，快速将电激后的混合物加入到800μL事先预热的2×TY液体培养基中，接着置于37℃恒温水浴锅中静置孵育40min。取出孵育体系，吸取100μL培养物涂布于TYE-AG(A和G分别表示含终浓度为100μg/mLAmp和1％Glu)固体培养基上，然后置涂布板于37℃恒温培养箱中倒置培养过夜。通过计算长出的单菌落数换算成为整个电转体系所能长出菌落数的理论值即判定为该噬菌体展示6肽库的库容量。
pIT2噬菌粒载体、E.coli TG1细菌和辅助噬菌体KM13购于英国SourceBioScience公司；
靶标短肽基因替换Bt Cry蛋白DomianⅡ-Loop2和-Loop3区以及替换BtCry1Ac蛋白抗独特型人源重链单域抗体CDR2区相应基因后的重组体全长基因序列，以及将重组体基因分别克隆到pGEX-6P-1上并化转进入E.coliBL21感受态细胞中，均委托通用生物系统(安徽)有限公司合成和克隆化转。Cry1Ac的结构为本领域公知技术，Cry1Ac的晶体结构(PDB:4ARY)可从RCSB PDB数据库(https://www.rcsb.org/)获得；也可以通过“Cry1Ac 3Dsutructure 4ARY.pdb:crystal protein[Bacillus thuringiensis]GenBank:AAA22331.1JOURNAL Gene 36(3),289-300(1985)PUBMED 3000881”获得。
Anti-M13-[HRP]Antibody,His-Trap HP亲和纯化柱购于美国GE Healthcare公司；
Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白“Roles of Midgut Cadherin from Two Moths in Different Bacillus thuringiensisAction Mechanisms:Correlation among Toxin Binding,Cellular Toxicity,andSynergism”(Gao等，J.Agric.Food Chem，2019年总第67期)和Bt Cry1Ac抗独特型抗体H9(H9不具备任何抗虫活性，其氨基酸序列如SEQ ID NO.12所示，编码基因的核苷酸序列如SEQ ID NO.11所示，H9序列首位为起始密码子)均由申请人实验室表达制备(省部共建国家重点实验室培育基地-江苏省食品安全重点实验室)；
棉铃虫和小菜蛾均由专利申请人实验室(省部共建国家重点实验室培育基地-江苏省食品安全重点实验室)饲养保存；
Bt Cry1Ac购于上海佑隆生物科技有限公司。
实施例1淘筛模拟Bt Cry1Ac蛋白关键抗虫功能表位的噬菌体展示短肽
(1)取500μl冻存的自构建的噬菌体展示随机6肽库菌液加入到200ml 2×TY-AG液体培养基中，37℃恒温培养到OD600为0.4，接着加入200μl滴度为～1012pfu/ml的KM13辅助噬菌体进行侵染，随即在37℃中孵育30min，然后以3300g离心10min，弃上清液，用100ml 2×TY-AK液体培养基重悬沉淀物；最后30℃恒温培养过夜。次日取出培养体系，以3300g离心30min，收集上清液并加入25ml PEG/NaCl溶液，冰浴1h，再以3300g离心30min，最后用5mlPBS重悬沉淀。重悬液以11600g离心10min，上清液即为扩增的噬菌体展示随机6肽库。
(2)取步骤1获得的扩增的噬菌体展示随机6肽库进行4轮富集淘筛：第1轮富集淘筛，吸取4ml浓度为100μg/ml的Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白溶液加入无菌的细胞培养瓶中，在4℃冰箱中静置包被过夜；次日，每次以1ml PBS溶液清洗细胞培养瓶，重复洗涤3次，然后加入1ml步骤(1)获得扩增的噬菌体展示随机6肽库与4ml 3％的MPBS溶液混匀的混合物，在室温下缓慢摇动1h，再静置1h，倾去培养瓶中液体；以每次1ml PBST溶液洗瓶，重复洗涤20次；最后加入1ml浓度为10mg/ml的胰蛋白酶(Trypsin)溶液洗脱特异性结合的噬菌体展示6肽材料，洗脱液即为第1轮富集淘筛的噬菌体展示6肽；第2、3、4轮富集淘筛中Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白包被的浓度分别为50、25、10μg/ml，所投入富集淘筛的噬菌体展示随机6肽库为前一轮富集淘筛获得的噬菌体6肽库，富集淘筛方法与第1轮相同，各轮次富集效果如图1所示。
取20μl第4轮富集淘筛获得的噬菌体展示随机6肽库溶液侵染1ml处于对数生长期的E.coliTG1细菌，37℃孵育1h后，涂布于TYE-AG固体培养基上，接着37℃培养过夜。次日，随机挑取单菌落，接种到含有100μl/孔2×TY-AG液体培养基的96孔板中，37℃培养过夜；接着从板孔中吸出2.5μl菌液转接到新的含有100μl/孔2×TY-AG液体培养基的96孔板中，37℃孵育2h；随即每孔加入25μl滴度为～1012pfu/ml的KM13辅助噬菌体，30℃孵育1.5h，1800g离心10min，用200μl 2×TY-AK液体培养基重悬沉淀后30℃培养过夜；次日以1800g离心30min，分别取上清液用于ELISA分析。
(3)Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白溶液，按照100μl/孔加入到96孔板中，4℃包被过夜；次日，每孔分别加入100μl步骤(2)获得的上清液，阴性对照加100μl 2×TY-AK液体培养基，37℃水浴2h；每孔用250μl PBST洗板后，每孔加入100μl 1:3000稀释的Anti-M13-[HRP]Antibody，37℃孵育2h；每孔加入100μl底物显色溶液，每孔加入100μl底物显色溶液，室温下避光显色反应15min，最后每孔加入50μl浓度为2M的H2SO4快速终止反应，用酶标仪测定OD450值。以此评价步骤(3)中筛选到的阳性噬菌体展示6肽材料对Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白的结合活性，即溶液OD450值/阴性对照OD450值大于3.0，判定为阳性，与该溶液对应的步骤(2)中的上清液即为筛选到的含有与Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白结合活性的噬菌体展示6肽上清液。
通过上述筛选和鉴定，申请人筛选到一种与Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白具有较强结合活性且能模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽材料，将其自命名为H-SC01，其所对应步骤(2)的上清液对Bt Cry1Ac靶标害虫棉铃虫中肠类钙黏蛋白毒素结合区(HaCadCR9-CR11)蛋白结合活性的ELISA检测效果如附图2所示，由图2检测结果可见，H-SC01具有较强结合活性。
分别设计上游引物(其核苷酸序列如SEQ ID NO.3所示)和下游引物(其核苷酸序列如SEQ ID NO.4所示)，测定靶标短肽H-SC01核苷酸序列如SEQ ID NO.1所示，核苷酸翻译后的氨基酸序列如SEQ ID NO.2所示。
实施例2靶标短肽(H-SC01)替换Bt Cry1Ac蛋白及其抗独特型抗体关键功能表位后的重组体蛋白可溶性表达及纯化
委托通用生物系统(安徽)有限公司，以Bt Cry1Ac和Bt Cry1Ac蛋白抗独特型人源重链单域抗体基因序列为模板，分别将Bt Cry1Ac蛋白DomianⅡ-Loop2区、Bt Cry蛋白DomianⅡ-Loop3区和Bt Cry1Ac蛋白抗独特型人源重链单域抗体H9的CDR2区的基因片等量替换为实施例获得的H-SC01短肽，获得重组体依次为重组体I、重组体II、重组体III。
重组体I的核苷酸序列如SEQ ID NO.5所示(其第1111-1128位基因被替换为H-SC01序列)，氨基酸序列如SEQ ID NO.6所示；重组体II的核苷酸序列如SEQ ID NO.7(其第位1312-1329基因被替换为H-SC01序列)所示，氨基酸序列如SEQ ID NO.8所示；重组体III的核苷酸序列如SEQ ID NO.9(其第151-168位基因被替换为H-SC01序列)所示，氨基酸序列如SEQ ID NO.10所示。
将重组体I、重组体II、重组体III分别在LB液体培养基(含终浓度为100μg/ml氨苄霉素)中25℃250rpm震荡培养至OD600 nm为0.6，然后加入IPTG(终浓度为0.8mM)诱导表达12h；次日，取各菌液，分别以3300g离心20min，收集沉淀细胞，用细胞破碎仪破碎细胞，然后5000g离心20min取上清液，过His-Trap HP affinity chromatography纯化收集各重组体蛋白(纯化方法参照论文“人源化抗Cry1B毒蛋白单链抗体的原核表达及生物学活性测定”徐重新等，南京农业大学学报，2013年3期)。最后收集的各重组体蛋白均采用超微量分光光度计将浓度定量到500μg/ml，冻存备用。
实施例3靶标短肽(H-SC01)替换Bt Cry1Ac蛋白及其抗独特型抗体关键功能表位后的重组体蛋白室内抗虫活性测定
将喂食棉铃虫和小菜蛾的人工饲料铺于24孔板，将靶标短肽(H-SC01)替换BtCry1Ac蛋白及其抗独特型抗体关键功能表位后的3种重组体蛋白分别用PBS溶液将蛋白浓度定量到20μg/ml，然后每孔200μL均匀涂布在饲料表面，晾干。各组试验均以等体积的PBS溶液作为阴性对照，以等浓度等体积的Bt Cry1Ac蛋白溶液作为阳性对照。取不同板，每孔接入1头2龄棉铃虫(或小菜蛾)，并置于温度为28℃±1℃，相对湿度80±5％，光周期(L:D)16h：8h的培养箱内饲养，每隔24h观察并记录死亡数，连续统计7天。每个处理组接30头幼虫，并重复3次试验。
结果如图3、4所示。浸药饲养108h后，靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop2区后的重组体蛋白I对供试棉铃虫和小菜蛾的校正死亡率分别达到75.3％和84.5％；靶标短肽(H-SC01)替换Bt Cry蛋白DomianⅡ-Loop3区后的重组体蛋白II对供试棉铃虫和小菜蛾的校正死亡率分别达到73.5％和25.1％；靶标短肽(H-SC01)替换Bt Cry1Ac蛋白抗独特型人源重链单域抗体(H9)CDR2区的重组体蛋白III对供试棉铃虫和小菜蛾的校正死亡率分别达到21.6％和27.3％；而阴性对照(PBS溶液阴性对照)中供试棉铃虫和小菜蛾校正死亡率分别为3.2％和5.7％，CK1+阳性对照(Bt Cry 1Ac蛋白溶液)中供试棉铃虫和小菜蛾校正死亡率分别为96.2％和93.9％；CK2+阳性对照(Bt Cry1Ac蛋白抗独特型人源重链单域抗体蛋白H9)中供试棉铃虫和小菜蛾几乎与CK-阴性对照一样，校正死亡率分别为3.5％和6.1％。
以上实验结果证明H-SC01分别替换Bt Cry1Ac的loopII和loopIII区，仍能保持良好的杀虫效果，该结果扩大了Bt Cry1Ac抗虫材料多样性，也可以为防控靶标害虫抗药性提供了潜在替代新材料。而Bt Cry1Ac蛋白抗独特型人源重链单域抗体(H9)对棉铃虫和小菜蛾均无任何抗虫活性，其CDR2区替换为H-SC01序列后的重组体蛋白III则对两种害虫表现出抗虫活性，说明该短肽促进了人源重链单域抗体抗虫活性，在材料功能上实现了抗体新材料的抗虫活性的促进作用，具有良好的应用价值和应用潜力。
以上实施例验证了的靶标短肽(H-SC01)具备模拟Bt Cry1Ac蛋白关键抗虫功能表位的功能，可用于Bt Cry1Ac蛋白及其抗独特型抗体(H9)改造，具有良好应用价值。
序列表
<110> 江苏省农业科学院
<120> 一种模拟Bt Cry1Ac蛋白关键抗虫功能表位的短肽
<141> 2021-11-23
<160> 12
<170> SIPOSequenceListing 1.0
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gccattaaga cgccaggc 18
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ile Lys Thr Pro Gly
1 5
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<212> DNA
<213> 人工序列(Artificial Sequence)
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caggaaacag ctatgac 17
<210> 4
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ctatgcggcc ccattca 17
<210> 5
<211> 1827
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggataaca atccgaacat caatgaatgc attccttata attgtttaag taaccctgaa 60
gtagaagtat taggtggaga aagaatagaa actggttaca ccccaatcga tatttccttg 120
tcgctaacgc aatttctttt gagtgaattt gttcccggtg ctggatttgt gttaggacta 180
gttgatataa tatggggaat ttttggtccc tctcaatggg acgcatttct tgtacaaatt 240
gaacagttaa ttaaccaaag aatagaagaa ttcgctagga accaagccat ttctagatta 300
gaaggactaa gcaatcttta tcaaatttac gcagaatctt ttagagagtg ggaagcagat 360
cctactaatc cagcattaag agaagagatg cgtattcaat tcaatgacat gaacagtgcc 420
cttacaaccg ctattcctct ttttgcagtt caaaattatc aagttcctct tttatcagta 480
tatgttcaag ctgcaaattt acatttatca gttttgagag atgtttcagt gtttggacaa 540
aggtggggat ttgatgccgc gactatcaat agtcgttata atgatttaac taggcttatt 600
ggcaactata cagattatgc tgtacgctgg tacaatacgg gattagaacg tgtatgggga 660
ccggattcta gagattgggt aaggtataat caatttagaa gagaattaac actaactgta 720
ttagatatcg ttgctctgtt cccgaattat gatagtagaa gatatccaat tcgaacagtt 780
tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt 840
cgaggctcgg ctcagggcat agaaagaagt attaggagtc cacatttgat ggatatactt 900
aacagtataa ccatctatac ggatgctcat aggggttatt attattggtc agggcatcaa 960
ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct atatggaact 1020
atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cgtgtataga 1080
acattatcgt ccactttata tagaagacct gccattaaga cgccaggcaa tcaacaacta 1140
tctgttcttg acgggacaga atttgcttat ggaacctcct caaatttgcc atccgctgta 1200
tacagaaaaa gcggaacggt agattcgctg gatgaaatac cgccacagaa taacaacgtg 1260
ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg ttcaggcttt 1320
agtaatagta gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1380
gaatttaata atataattgc atcggatagt attactcaaa tccctgcagt gaagggaaac 1440
tttcttttta atggttctgt aatttcagga ccaggattta ctggtgggga cttagttaga 1500
ttaaatagta gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560
ccatcgacat ctaccagata tcgagttcgt gtacggtatg cttctgtaac cccgattcac 1620
ctcaacgtta attggggtaa ttcatccatt ttttccaata cagtaccagc tacagctacg 1680
tcattagata atctacaatc aagtgatttt ggttattttg aaagtgccaa tgcttttaca 1740
tcttcattag gtaatatagt aggtgttaga aattttagtg ggactgcagg agtgataata 1800
gacagatttg aatttattcc agttact 1827
<210> 6
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Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
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Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
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Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
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Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
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Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
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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
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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 Tyr 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 Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg 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
Arg 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 Tyr 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 Ala Ile Lys Thr Pro Gly 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
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Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
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Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
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Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
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Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn
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Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly
485 490 495
Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly
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Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg
515 520 525
Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn
530 535 540
Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr
545 550 555 560
Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala
565 570 575
Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe
580 585 590
Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val
595 600 605
Thr
<210> 7
<211> 1827
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggataaca atccgaacat caatgaatgc attccttata attgtttaag taaccctgaa 60
gtagaagtat taggtggaga aagaatagaa actggttaca ccccaatcga tatttccttg 120
tcgctaacgc aatttctttt gagtgaattt gttcccggtg ctggatttgt gttaggacta 180
gttgatataa tatggggaat ttttggtccc tctcaatggg acgcatttct tgtacaaatt 240
gaacagttaa ttaaccaaag aatagaagaa ttcgctagga accaagccat ttctagatta 300
gaaggactaa gcaatcttta tcaaatttac gcagaatctt ttagagagtg ggaagcagat 360
cctactaatc cagcattaag agaagagatg cgtattcaat tcaatgacat gaacagtgcc 420
cttacaaccg ctattcctct ttttgcagtt caaaattatc aagttcctct tttatcagta 480
tatgttcaag ctgcaaattt acatttatca gttttgagag atgtttcagt gtttggacaa 540
aggtggggat ttgatgccgc gactatcaat agtcgttata atgatttaac taggcttatt 600
ggcaactata cagattatgc tgtacgctgg tacaatacgg gattagaacg tgtatgggga 660
ccggattcta gagattgggt aaggtataat caatttagaa gagaattaac actaactgta 720
ttagatatcg ttgctctgtt cccgaattat gatagtagaa gatatccaat tcgaacagtt 780
tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt 840
cgaggctcgg ctcagggcat agaaagaagt attaggagtc cacatttgat ggatatactt 900
aacagtataa ccatctatac ggatgctcat aggggttatt attattggtc agggcatcaa 960
ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct atatggaact 1020
atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cgtgtataga 1080
acattatcgt ccactttata tagaagacct tttaatatag ggataaataa tcaacaacta 1140
tctgttcttg acgggacaga atttgcttat ggaacctcct caaatttgcc atccgctgta 1200
tacagaaaaa gcggaacggt agattcgctg gatgaaatac cgccacagaa taacaacgtg 1260
ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg tgccattaag 1320
acgccaggca gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1380
gaatttaata atataattgc atcggatagt attactcaaa tccctgcagt gaagggaaac 1440
tttcttttta atggttctgt aatttcagga ccaggattta ctggtgggga cttagttaga 1500
ttaaatagta gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560
ccatcgacat ctaccagata tcgagttcgt gtacggtatg cttctgtaac cccgattcac 1620
ctcaacgtta attggggtaa ttcatccatt ttttccaata cagtaccagc tacagctacg 1680
tcattagata atctacaatc aagtgatttt ggttattttg aaagtgccaa tgcttttaca 1740
tcttcattag gtaatatagt aggtgttaga aattttagtg ggactgcagg agtgataata 1800
gacagatttg aatttattcc agttact 1827
<210> 8
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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 Tyr 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 Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg 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
Arg 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 Tyr 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 Ala Ile Lys Thr Pro Gly 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 Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn
465 470 475 480
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly
485 490 495
Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly
500 505 510
Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg
515 520 525
Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn
530 535 540
Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr
545 550 555 560
Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala
565 570 575
Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe
580 585 590
Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val
595 600 605
Thr
<210> 9
<211> 369
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgcaggtgc agctgttgga gtctggggga ggcttggtac agcctggggg gtccctgcgt 60
ctctcctgtg cagcctccgg agttaccgtt accaatcaca atatgagctg ggtccgccag 120
gctccaggga agggtctaga gtgggtatca gccattaaga cgccaggcgg tagcacatac 180
tacgcagact ccgtgaaggg ccggttcacc atctcccgtg acaattccaa gaacacgctg 240
tatctgcaaa tgaacagcct gcgtgccgag gacaccgcgg tatattattg cgcgagttgg 300
attttgcttc ctattgcgcc caagaacttg gactattggg gtcagggaac cctggtcacc 360
gtctcgagc 369
<210> 10
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Val Thr Asn
20 25 30
His Asn Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Ala Ile Lys Thr Pro Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ser Trp Ile Leu Leu Pro Ile Ala Pro Lys Asn Leu Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 11
<211> 369
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgcaggtgc agctgttgga gtctggggga ggcttggtac agcctggggg gtccctgcgt 60
ctctcctgtg cagcctccgg agttaccgtt accaatcaca atatgagctg ggtccgccag 120
gctccaggga agggtctaga gtgggtatca ggacgtacgt tagacctggg tagcacatac 180
tacgcagact ccgtgaaggg ccggttcacc atctcccgtg acaattccaa gaacacgctg 240
tatctgcaaa tgaacagcct gcgtgccgag gacaccgcgg tatattattg cgcgagttgg 300
attttgcttc ctattgcgcc caagaacttg gactattggg gtcagggaac cctggtcacc 360
gtctcgagc 369
<210> 12
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Val Thr Asn
20 25 30
His Asn Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Gly Arg Thr Leu Asp Leu Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ser Trp Ile Leu Leu Pro Ile Ala Pro Lys Asn Leu Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
Claims (4)
1.一种氨基酸序列如SEQ ID NO.6所示的重组体I。
2.如权利要求1所述重组体I在杀灭棉铃虫、小菜蛾中的应用。
3. 一种氨基酸序列如SEQ ID NO.8所示的重组体II。
4.如权利要求3所述重组体II在杀灭棉铃虫中的应用。
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