CN116948046B - 人工智能辅助生成的杀虫蛋白及其应用 - Google Patents
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Abstract
本发明涉及人工智能辅助生成的杀虫蛋白及其应用，属于蛋白工程领域。本发明基于人工智能算法的活性位点预测和氨基酸序列生成，获得一批新型的杀虫蛋白。这些杀虫蛋白在制备杀虫剂或培育基因工程植物领域具有应用价值。
Description
技术领域
本发明涉及人工智能辅助生成的杀虫蛋白及其应用，属于蛋白工程领域。
背景技术
源于苏云金芽孢杆菌中的杀虫蛋白是目前转基因作物和生物农药开发过程中普遍使用的目的蛋白。然而随着这些商品化的杀虫剂和抗虫作物的大规模应用，害虫普遍产生了对已有杀虫蛋白的抗药性，迫切需要找到或创制出新型的杀虫蛋白。
草地贪夜蛾（学名：Spodoptera frugiperda）属于夜蛾科灰翅夜蛾属，其幼虫可大量啃食水稻、甘蔗和玉米之类禾本科以及菊科、十字花科等多种农作物，造成严重的经济损失。该物种原产于美洲热带地区，具有很强的迁徙能力。美国等转基因作物种植大国主要使用Cry1Fa、Vip3Aa蛋白培育玉米和大豆新品种防治草地贪夜蛾，然而由于该昆虫强迁徙、繁殖快的特点，越来越多的针对Cry1Fa、Vip3Aa的抗性品系已经被发现（Fatoretto J C,Michel A P, Silva Filho M C, Silva N. Adaptive potential of fall armyworm(Lepidoptera: Noctuidae) limits Bt trait durability in Brazil[J]. J. Integr.Pest Manag. 2017, 8, 17）。因此，未来的抗草地贪夜蛾作物品种需要获得与Cry1Fa和Vip3Aa完全不同的新型杀虫蛋白。
蛋白质在细胞中的功能是由其三维结构决定的。但通过实验测定蛋白质结构费时费力。随着人工智能技术的发展，蛋白结构预测已经取得了突破性的进展。这为海量研究和创制蛋白结构提供了新的机会。谷歌DeepMind研发出的AlphaFold人工智能网络已经给出了几十万蛋白质的三维结构，包括人体自身能制造的每一种蛋白质，这些成果有望为医学和药物设计领域带来更大惊喜。虽然更加精准的蛋白折叠、配体结合等细节上还有很多需要优化的空间，但通过深度学习、神经网络的人工智能算法，结合实验层面的功能验证和筛选的方式，能够实现从蛋白结构预测、实验测试、优化改进，到最终生成新的工程化功能性蛋白。
CN202311033251.5专利公开了一批人工智能辅助生成的杀虫蛋白，其中WBY-7具有较高的杀虫活性。为了进一步提高该蛋白的杀虫效果，本发明基于人工智能算法的活性位点预测和氨基酸序列生成，获得一批效果更好的杀虫蛋白。
发明内容
为了解决上述问题，本发明采用如下技术方案：
本发明提供一种人工智能辅助生成的杀虫蛋白，其特征在于，该蛋白由SEQ IDNO. 7和SEQ ID NO. 1的468-1169位融合而成。
本发明还提供一种核酸分子，其特征在于，所述的核酸分子编码上述的蛋白。
本发明还提供一种载体，其特征在于，所述载体包含上述的核酸分子。
本发明还提供一种重组细胞，其特征在于，所述重组细胞含有上述的核酸分子或上述的载体。
在一些实施方案中，上述重组细胞为原核生物细胞。
在一些实施方案中，上述重组细胞为大肠杆菌细胞。
本发明还提供上述蛋白、核酸分子、载体、重组细胞在抗虫或制备抗虫制剂或培育抗虫植物中的应用。
在一些实施方案中，上述抗虫为对草地贪夜蛾具有杀虫活性。
本发明的有益效果在于：本发明基于人工智能算法的活性区域预测和氨基酸序列生成，获得一个效果更好的杀虫蛋白，可用于开发生物农药或培育新型抗虫植物。
具体实施方式
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，术语按照相关领域普通技术人员的常规用法理解。本文所引用的所有专利文献、学术论文、行业标准及其他公开出版物等，其中的全部内容整体并入本文作为参考。
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本申请的范围。若无特别指明，实施例按照常规实验条件，如Sambrook等人的分子克隆实验手册(Sambrook J& Russell DW, Molecular cloning: a laboratory manual, 2001)，或按照制造厂商说明书建议的条件。若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1蛋白的活性区域预测和序列生成
本发明使用CN202311033251.5专利中的WBY-7蛋白（序列如SEQ ID NO. 1所示）作为模板进行活性位点分析和序列生成。由于D1和D2结构域在提高杀虫蛋白活性上具有很大的作用。因此利用AlphaFold2、RoseTTAFold在线工具预测WBY-1蛋白D1和D2结构域（1-467位）的活性区域，再利用数据库和PROTEINGAN算法预测工具建立模型，生成和设计活性区域的氨基酸序列。使用Chimera 1.17可视化比较生成蛋白结构。
通过打分排名，从WBY-7活性区域改造蛋白中筛选6个蛋白，分别命名为WBY-7.01~WBY-7.06，N端的氨基酸序列如SEQ ID NO. 2~SEQ ID NO. 7所示，C端均与SEQ ID NO. 1的468-1169位相同。
实施例2测试新生成蛋白的杀虫效果
使用蛋白表达实验系统合成这些蛋白实物，并测试它们对草地贪夜蛾的杀虫效果。
蛋白合成和杀虫效果测试方法参考CN202311033251.5专利实施例2中的内容。
测试结果如表1所示。WBY-7.06的杀虫活性比原始的WBY-7蛋白有提高，可以作为一种活性更高的新型杀虫蛋白用于生物农药和抗虫转基因植物的开发。
表1 蛋白的杀虫活性
| 蛋白名称 | 氨基酸序列 | 杀虫活性（LC501） |
| WBY-7 | SEQ ID NO. 1 | 60±14 |
| WBY-7.01 | SEQ ID NO. 2+SEQ ID NO. 1 468-1169位 | 74±25 |
| WBY-7.02 | SEQ ID NO. 3+SEQ ID NO. 1 468-1169位 | 63±17 |
| WBY-7.03 | SEQ ID NO. 4+SEQ ID NO. 1 468-1169位 | 57±26 |
| WBY-7.04 | SEQ ID NO. 5+SEQ ID NO. 1 468-1169位 | 42±22 |
| WBY-7.05 | SEQ ID NO. 6+SEQ ID NO. 1 468-1169位 | 45±16 |
| WBY-7.06 | SEQ ID NO. 7+SEQ ID NO. 1 468-1169位 | 28±12* |
1：单位ng/g；*表示与WBY-7相比有显著差异（α=0.05）。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (7)
1. 一种蛋白，其特征在于，该蛋白从N端到C端依次由SEQ ID NO. 7和SEQ ID NO. 1的468-1169位融合而成。
2.一种核酸分子，其特征在于，所述的核酸分子编码权利要求1所述的蛋白。
3.一种载体，其特征在于，所述载体包含权利要求2所述的核酸分子。
4.一种重组细胞，其特征在于，所述重组细胞含有权利要求2所述的核酸分子或权利要求3所述的载体。
5.根据权利要求4所述的重组细胞，其特征在于，所述重组细胞为原核生物细胞。
6.根据权利要求5所述的重组细胞，其特征在于，所述重组细胞为大肠杆菌细胞。
7.权利要求1所述的蛋白，或权利要求2所述的核酸分子，或权利要求3所述的载体，或权利要求4~6任一项所述的重组细胞在抗草地贪夜蛾或制备抗草地贪夜蛾制剂或培育抗草地贪夜蛾植物中的应用。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009132850A1 (en) * | 2008-05-01 | 2009-11-05 | Bayer Bioscience N.V. | Armyworm insect resistance management in transgenic plants |
| WO2021026686A1 (zh) * | 2019-08-09 | 2021-02-18 | 北京大北农生物技术有限公司 | 杀虫蛋白的用途 |
| CN116514936A (zh) * | 2023-06-29 | 2023-08-01 | 莱肯生物科技(海南)有限公司 | 一种抗虫蛋白及其制备方法和应用 |
| CN116768990A (zh) * | 2023-08-16 | 2023-09-19 | 莱肯生物科技(海南)有限公司 | 一种人工智能辅助生成的杀虫蛋白 |
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| WO2009132850A1 (en) * | 2008-05-01 | 2009-11-05 | Bayer Bioscience N.V. | Armyworm insect resistance management in transgenic plants |
| WO2021026686A1 (zh) * | 2019-08-09 | 2021-02-18 | 北京大北农生物技术有限公司 | 杀虫蛋白的用途 |
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