CN106589134B - 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用 - Google Patents

嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用 Download PDF

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CN106589134B
CN106589134B CN201610996972.XA CN201610996972A CN106589134B CN 106589134 B CN106589134 B CN 106589134B CN 201610996972 A CN201610996972 A CN 201610996972A CN 106589134 B CN106589134 B CN 106589134B
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仪宏
冯惠勇
李天明
刘金雷
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Abstract

本发明涉及生物技术，具体而言，本发明涉及一种嵌合蛋白pAgoE及其构建方法和应用，同时，本发明还涉及一种使用向导的嵌合蛋白pAgoE及其构建方法和应用，以及基于嵌合蛋白pAgoE的具有基因组靶向编辑功能的嵌合蛋白。所述的嵌合蛋白pAgoE含有具有基因组靶向定位功能的pAgo结构域，以及与所述pAgo结构域连接的、具有基因组切割或修饰活性的效应结构域E。本发明的嵌合蛋白pAgoE，是将具有基因组靶向定位功能的pAgo结构域，以及具有基因组切割或修饰活性等催化活性的效应结构域融合，构建了嵌合蛋白(pAgoE)，该嵌合蛋白pAgoE同时保持了pAgo结构域中原有的具有靶向定位功能的基因组(DNA)的靶向结合能力以及效应结构域E的基因组催化或修饰能力，以便为效应结构域E发挥作用提供空间位置保障基础。

Description

嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白 pAgoE及构建方法、应用
技术领域
本发明涉及生物技术，具体而言，本发明涉及一种嵌合蛋白pAgoE及其构建方法和应用，同时，本发明还涉及一种使用向导的嵌合蛋白pAgoE及其构建方法和应用，以及基于嵌合蛋白pAgoE的具有基因组靶向编辑功能的嵌合蛋白。
技术背景
靶向基因组编辑技术及靶向基因组修饰技术是一类高效率基因组改写技术的总称，它与一般意义上的基因重组技术的主要优势在于：基因组靶向精度高、基因编辑效率高、应用范围广泛、可以操作哺乳动物细胞。靶向基因组编辑技术及靶向基因组修饰技术可以针对微生物、植物、动物(包括哺乳动物)的细胞或个体，开展基因组及表观组的功能研究、遗传改造；进一步地，靶向基因组编辑技术及靶向基因组修饰技术可以在一定程度上实现物种定制。
基因编辑技术的发展，主要历经了ZFN、TALEN、CRISPR三代技术***的演化，时至今日，CRISPR‐CAS9技术被广泛运用。上述ZFN和TALEN技术有共同点，他们都是采用了嵌合蛋白策略，嵌合蛋白中负责DNA识别的结构域经过人工设计编辑之后，可以靶向基因组中特定的DNA序列，从而实现基因组定位；嵌合蛋白的催化结构域(CD)，选自ⅡS型限制性核酸内切酶FokⅠ的切割活性结构域，可以实现对靶向位点的切割，造成基因组双链断裂(DSB)，迫使细胞启动同源介导的修复(HDR)、或者非同源末端连接(NHEJ)来挽救基因组的完整性。NHEJ修复保真度低，有可能造成DSB附近的碱基缺失、置换等错误，从而引入突变或者失活基因。在同源序列充当修复模板的前提下，通过HDR修复可以实现外源基因的精确导入基因组。但是，ZFN和TALEN技术依赖对蛋白质结构域开展编辑的成本高、效率低。
基于CRISPR的基因组编辑几年来取得重大进展和广泛应用，已经积累和形成了大量的公开发表文献。CRISPR原本是细菌进化过程中演化出来的防御外来DNA入侵的初级可获得性免疫***，自2012年起，它被迅速开发成新一代高效率基因编辑技术，其中CAS9蛋白可以俘获一个人工设计的单链向导RNA(sgRNA)，利用此sgRNA对基因组特定的靶向区域进行识别并结合，之后，CAS9蛋白自身的两个核酸酶活性位点切割DNA，造成双链断裂(DSB)并引发NHEJ或HDR，实现基因编辑。
Agronautes蛋白是一种普遍存在于从低等原核细菌到高等生物包括哺乳动物的一种有进化谱系的核酸酶。与短链寡核苷酸互作并实现对相应互补核酸序列的向导性靶向结合，是Agronaute的一个普遍的生物学特征。在高等细胞中Agronautes蛋白参与了ncRNA相关的许多重要生理过程，例如RNAi。
近几年来，来自原核微生物的pArgonaute(pAgo)蛋白的相关研究也取得了有趣的进展，形成了一些公开发表的文献，但是与CRISPR相比较，pAgo的研究目前文献并不够丰富。
已经公开的发表的学术论文主要有：2009年8月25日，Kira S Makarova等依据生物信息学的深度挖掘，预测了来自原核生物的pArgonaute很可能是一种对抗外来遗传物质入侵的原始防御***。2013年9月12日，Ivan Olovnikov等报告了来自Rhodobactersphaeroides的RsAgo能够通过对转录组“采样”而识别监控外来DNA。2014年3月13日，DaanC.Swarts等报道了来自嗜热菌Thermus thermophilus的TtAgo)以13–25nt的5’磷酸化的gDNA为向导切割靶向DNA，并将这种宿主防御***称之为DNA向导的DNA干扰，简称DNAi。2015年4月29日，Daan C.Swarts等在线发表了来自古细菌Pyrococcus furiosus的PfAgo，该PfAgo利用5’磷酸化的单链DNA为向导，能够靶向切割单链或双链DNA，起到防御外来DNA入侵的作用。2016年4月12日，Emine Kayaa等报道了来自Marinitoga piezophila的与CRISPR相关的MpAgo，该MpAgo以15到40nt的5’羟基化单链RNA为向导，能够识别和切割所靶向的单链DNA或者单链RNA。2016年5月2日，Feng Gao等报道了来自嗜盐微生物Natronobacterium gregoryi的NgAgo，该论文声称以24nt的5’磷酸化的单链DNA为向导的该NgAgo，能够在常温下造成哺乳动物细胞靶向基因组位点的双链断裂(DSB)并引发高效率的基因组编辑，然而该论文遭遇到了严重的可重复性危机。2016年6月21日，TomohiroMiyoshi等以
Figure GDA0001167618950000021
的分辨率解析了来自Rhodobacter sphaeroides的RsAgo的结构，该RsAgo识别18nt的gRNA。根据有限的研究报告，一些pAgo在异源表达时可能因自我靶向而造成表达困难，例如RsAgo。
已经公开的专利文献主要有：发明人为VAN DER OOST,John的WO/2014/189628A1专利公开了DNA向导的DNA干扰技术，但是并没有在常温下采用来自嗜热原核微生物Thermus thermophilus的TtAgo完成细胞内基因组编辑的实施例。发明人为VALTON,Julien的WO2015/140347A1专利，公开了利用DNA向导的Argonaute干扰***(DAIS)工程化哺乳动物基因组的技术，采用序列优化之后可以在30℃到40℃工作的TtAgo，以一个TtAgo加两个对向的5’磷酸化的单链向导DNA的方式、实施了包括T细胞在内哺乳动物细胞基因组编辑。发明人为DOUDNA,Jennifer的WO2015/157534A1专利公开了利用来自原核微生物Marinitoga piezophila的MpAgo进行单链靶标核酸(DNA或者RNA)切割的技术方法，只有体外实验验证(in vitro)实施例、缺乏活体细胞内(in vivo)的实施例。
这些文献表明，pAgo可能具有与CRISPR类似的防御外来DNA及病毒入侵的功能，其中，一部分pAgo以5’端磷酸化的ssDNA为向导参与了DNAi，例如TtAgo、AfAgo、PfAgo等；少数的pAgo在宿主的CRISPR区域存在，并且以5’端羟基化的ssRNA为向导，能够靶向切割单链的DNA或RNA。
从蛋白质结构上分析，pAgo蛋白具有相对保守的结构域，一般地，从pAgo蛋白的氮端到碳端分别是N‐terminal、L1、PAZ、L2、MID、PIWI结构域。其中PAZ结构域主要是向导寡核苷酸识别和结合域，缺失PAZ域的pAgo不能够独自识别和结合短链向导寡核苷酸。对于原核Agronaute而言，最为保守的区域是中间至碳端的L2、MID、PIWI。
一部分pAgo的PIWI结构域保持了核酸酶活性，例如来自高温微生物Thermusthermophilus的TtAgo、以及Pyrococcus furiosus的PfAgo，已有的生物信息学软件可以分析和预测pAgo的PIWI结构域的活性位点。自然界还存在另一类的pAgo，其PIWI结构域却失去了核酸酶活性，例如来自Rhodobacter sphaeroides的RsAgo。
已有的研究结果表明，在原微生物宿主中保留PAZ结构域并且保持PIWI核酸酶活性的Argonaute以DNAi的方式扮演了防御病毒等外来入侵DNA的角色；而在原微生物宿主中保留PAZ结构域但PIWI丧失核酸酶活性的Argonaute则很可能以尚不十分明确的方式和机制参与了对外来入侵DNA的防御，甚至，以持续取样宿主全部DNA的方式对过度活跃的基因元件进行监控。
发明内容
有鉴于此，本发明旨在提供一种嵌合蛋白pAgoE，以能够靶向切割或修饰基因组。
为实现上述目的，本发明的嵌合蛋白pAgoE，含有具有基因组靶向定位功能的pAgo结构域，以及与所述pAgo结构域连接的、具有基因组切割或修饰活性的效应结构域E。
进一步的，所述效应结构域E采用具有核酸酶样DNA切割活性(Nuclease‐like DNAcleavage activity)的、用于基因组靶向编辑的结构域N，以构成pAgoE‐N型的嵌合蛋白pAgoE。
进一步的，所述效应结构域N来自核酸酶、转座酶、位点特异重组酶。
进一步的，所述效应结构域N来自酶学编号为EC 3.1.21.4的ⅡS型限制性核酸内切酶。
进一步的，所述效应结构域N采用Planomicrobium okeanokoites的、GenBank登记号为AAA24934.1的ⅡS型限制性核酸内切酶FOKⅠ的第383到第579个氨基酸。
进一步的，所述效应结构域E含有具有基因组或表观组修饰活性(Modificationactivity)的、用于基因组靶向修饰的结构域M。
进一步的，所述效应结构域M采用具有DNA核苷酸修饰活性(NucleotideModification activity)的结构域NM，可用于基因组靶向核苷酸修饰或基因组靶向加速进化。
进一步的，所述结构域NM采用酶学编号为EC 3.5.4.5的AID、或酶学编号为EC3.2.2.21的MAG1。
进一步的，所述结构域M采用具有表观组修饰活性(Epigenome Modificationactivity)的结构域EM，可用于表观组编辑。
进一步的，所述结构域EM采用酶学编号为EC 1.14.11.n2的TET1、或酶学编号为EC2.1.1.37的DBMt3a/b。
进一步的，所述pAgo结构域与任意一种使用向导的天然原核Argonaute蛋白的氨基酸序列同源性大于30％。
进一步的，所述pAgo结构域采用PIWI结构域中核酸酶活性失活的pAgo。
进一步的，所述pAgo结构域采用来自Rhodobacter sphaeroides ATCC 17025的、NCBI序列号为ABP72561.1的RsAgo，以构建得到RsAgoE型的嵌合蛋白pAgoE。
进一步的，所述pAgo结构域采用PIWI结构域中有核酸酶活性的天然pAgo。
进一步的，所述pAgo结构域采用来自Marinitoga piezophila的、NCBI序列号为WP_014295921.1的MpAgo，以构建得到MpAgoE型的嵌合蛋白pAgoE。
进一步的，所述pAgo结构域采用PIWI结构域有核酸酶活性的天然pAgo的人工失活突变体。
进一步的，所述pAgo结构域采用MpAgo的PIWI结构域核酸酶活性失活的人工突变体dMpAgo，以构建得到dMpAgoE型的嵌合蛋白pAgoE。
进一步的，所述人工突变体dMpAgo来自MpAgo的以下四个位点中的至少一个的失活：D446、E482、D516、N624。
进一步的，所述人工突变体dMpAgo含有以下四个丙氨酸替代突变位点中的至少一个：D446A、E482A、D516A、N624A。
进一步的，所述pAgo结构域采用NgAgo的PIWI结构域核酸酶失活的人工突变体dNgAgo，以构建得到的dNgAgoE型的嵌合蛋白pAgoE。
进一步的，所述人工突变体dNgAgo来自NgAgo的以下四个位点中的至少一个的失活：D663、D738、D863、S665。
进一步的，所述人工突变体dNgAgo含有以下四个丙氨酸替代突变位点中的至少一个：D663A、D738A、D863A、S665A。
采用本发明的技术方案，其效果如下：
本发明的嵌合蛋白pAgoE，是将具有基因组靶向定位功能的pAgo结构域，以及具有基因组切割或修饰活性等催化活性的效应结构域Effector Domain(以下简称E)融合，构建了“Argonaute—效应域”嵌合蛋白(pAgoE)，该嵌合蛋白pAgoE同时保持了pAgo结构域中原有的具有靶向定位功能的基因组(DNA)的靶向结合能力以及效应结构域E的基因组催化或修饰能力，以便为效应结构域E发挥作用提供空间位置保障基础。
本发明同时提供了一种嵌合蛋白pAgoE的构建方法，该方法是将具有基因组靶向定位功能的pAgo结构域，以及具有基因组切割或修饰活性的效应结构域E连接。
进一步的，所述pAgo结构域与所述效应结构域E采用人工连接。
进一步的，所述pAgo结构域与所述效应结构域E共价键连接。
进一步的，所述pAgo结构域与所述效应结构域E之间形成碳端和氮端之间的连接。
进一步的，所述pAgo结构域的氮端与效应结构域E的碳端连接，以构建E‐pAgo型的pAgoE；或所述pAgo结构域的碳端与效应结构域E的氮端连接，构建pAgo‐E型的pAgoE；或所述pAgo结构域的碳端和氮端分别与效应结构域E的氮端和碳端连接，构建E1‐pAgo‐E2型的pAgoE，其中，E1与E2相同或不同。
进一步的，所述pAgoE被构建成分体形式的嵌合蛋白(split‐pAgoE)，分体之间以非共价键的方式连接。
进一步的，所述嵌合蛋白pAgoE的所述pAgo结构域与所述效应结构域E之间，采用Mxe GryA Intein构建，以形成split‐pAgoE型的嵌合蛋白pAgoE。
本发明同时涉及一种嵌合蛋白pAgoE的应用，所述嵌合蛋白pAgoE使用向导，靶向编辑或修饰基因组。
此外，本发明的另一个目的是提供了一种使用向导的嵌合蛋白pAgoE，其含有如上所述嵌合蛋白pAgoE，以及与所述嵌合蛋白pAgoE结合的、具有协助pAgoE识别和结合基因组能力的向导。
进一步的，所述向导为寡核苷酸gNA。
进一步的，所述寡核苷酸gNA依据与拟靶向基因组位点序列互补的原则而设置。
进一步的，所述寡核苷酸gNA的核苷酸序列与拟靶向区域的基因组的核苷酸序列的同源性为40％以上。
进一步的，RsAgoE型的嵌合蛋白pAgoE，使用长度为18个核苷酸的5’端磷酸化单链gRNA为gNA。
进一步的，MpAgoE型或dMpAgoE型的嵌合蛋白pAgoE，使用长度为10到40个核苷酸的5’端羟基化单链gRNA为gNA。
进一步的，dNgAgoE型的嵌合蛋白pAgoE，使用长度为24个核苷酸的5’端磷酸化单链gDNA为gNA。
本发明的使用向导的嵌合蛋白pAgoE，是基于如上的嵌合蛋白pAgoE的基础上，结合向导而成，能够使具有基因组靶向定位的原核Argonaute蛋白(pAgo)，在向导的引导下，与细胞内基因组中的靶向区域结合从而实现基因组的定位，确保效应结构域E能够在确定的空间位置下发挥作用。
具体的，如上描述中，pAgo结构域可以来自PIWI核酸酶活性减弱或失活的天然dArgonaute(简称dAgo结构域)、来自PIWI结构域中有核酸酶活性的天然pAgo、或者来自经人工改造而获得PIWI核酸酶活性减弱或失活的dArgonaute，E结构域可以来自Argonaute蛋白自身PIWI结构域之外的能够执行催化功能的蛋白，例如但不局限于：FOKⅠ等核酸酶、AID等DNA碱基修饰酶、TET1等表观组修饰相关酶。向导可以采用寡核苷酸gNA，如在单链向导寡核苷酸gNA协助下，这类嵌合蛋白pAgoE能够利用分子中的pAgo结构域靶向结合基因组的特定位点，并利用分子中的E结构域对靶向位点相关区域进行特定的催化，可用于基因组靶向编辑、基因组靶向修饰(包括单碱基编辑、表观组编辑、基因组人工进化等)。
此外，采用如上技术，可以实现常温下的应用，以及于活体真核细胞内的应用。
此外，本发明同时提供了一种使用向导的嵌合蛋白pAgoE的构建方法，该方法是将嵌合蛋白pAgoE与向导结合。
进一步的，其应用于基因组靶向编辑或基因组靶向修饰。
同时，本发明还涉及一种使用向导的嵌合蛋白pAgoE的应用方法，该方法是向导以体内表达直接转录的方式、或者转录出多聚核苷酸前体再经加工的方式实现。
进一步的，其特征在于：所述前体多聚核苷酸剪切加工***，来自酶学编号为EC3.1.‐.‐的CSY4、或者来自NCBI序列号为ABP72561.1的RsAgo。
进一步的，嵌合蛋白pAgoE、向导寡核苷酸gNA、gNA的前体加工酶中的至少一种，以实施细胞内常表达或者诱导表达的方式、或者体外制备再送入细胞内的方式，使pAgoE以结合着向导的状态出现于细胞内。
进一步的，所述细胞为活体真核细胞。
进一步的，所述活体真核细胞为活体真核微生物细胞、植物细胞、动物细胞。
进一步的，所述细胞为活体哺乳动物细胞。
进一步的，该方法在常温下进行。
本发明的另一个目的，是提供一种具有基因组靶向编辑功能的嵌合蛋白，其含有如上所述嵌合蛋白pAgoE，所述嵌合蛋白pAgoE含有具有基因组切割性的效应结构域E。
进一步的，还含有与所述嵌合蛋白pAgoE结合的、具有协助pAgoE识别和结合基因组能力的向导。
同时，本发明还涉及一种具有基因组靶向编辑功能的嵌合蛋白的应用，pAgoE‐N型的所述嵌合蛋白pAgoE，在常温下，在包括真核细胞在内的活体细胞中，进行基于同源重组修复(HDR)的基因组靶向编辑或者基于非同源末端连接(NHEJ)的基因组靶向编辑。
进一步的，使pAgoE‐N型的嵌合蛋白pAgoE、向导寡核苷酸gNA以及同源重组片段HR出现于细胞内，完成基于同源重组修复(HDR)的、包括内源基因靶向删除或外源基因靶向敲入的基因组靶向编辑。
基于如上描述，采用本发明的具有基因组靶向编辑功能的嵌合蛋白的技术方案，采用具有切割活性的效应结构域E，如选用DNA切割活性域时，可以构建嵌合蛋白pAgoE‐N，在gNA引导下该嵌合蛋白能够切割基因组靶向位点、造成基因组双链断裂(DSB)，从而引发通过非同源末端连接(NHEJ)同源重组修复(HDR)，导致靶向区域DNA序列的改变、缺失，或者在DNA同源重组片段的存在下、导致靶向区域的基因删除或外源基因***，从而完成基因编辑。选用DNA表观组修饰活性域(Epigenome Modification Domain)作为E域时，可以构建嵌合蛋白pAgoE‐EM，在gNA引导下的基因组靶向位点的表观组修饰，该嵌合蛋白执行表观组编辑。
综上所述，本发明涉及的技术方案，从整体或具体上，具有如下特点：
第一，在技术设计的整体思路上形成的嵌合蛋白pAgoE或具有向导的嵌合蛋白pAgoE，或具有基因组靶向编辑功能的嵌合蛋白，提供了一种丰富的技术平台和工具包。
第二，在具体技术上，有多种的pAgo蛋白可供选择，如包括天然的PIWI失活的pAgo、天然PIWI有活性的pAgo、天然PIWI有活性的pAgo人工失活突变体；有三种向导寡核苷酸类型的选择：5’端磷酸化的gRNA、5’端羟基化的gRNA、5’端磷酸化gDNA；可以选择外源的向导寡核苷酸、也可以选择内源化的向导寡核苷酸。
第三，可以选择由一个表达盒实现多个内源化向导的同步产生。
第四，可以选择比CRISPR‐CAS9***所采用的21个核苷酸更长的向导(例如24个核苷酸)，进一步减少脱靶效应(off‐targetting)。
第五，在蛋白质分子量方面，pAgoE总体上比较小，以dMpAgo‐Fok为例，它仅有842个氨基酸，比SpCas9以及SaCas9小，有利于解决ZFN、TALEN、CRISPR‐CAS9蛋白质太大所造成的一些问题，便于使用腺相关病毒(AAV)等递送工具实现活体内基因编辑(或基因治疗)，还便于体外组装核酸蛋白颗粒、以转染方式实现没有外源DNA参与(DNA‐free)前提下的安全编辑。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1 pAgoE嵌合蛋白结构与工作原理图；
图2 PIWI活性位点预测图；
图3 FokI的DNA切割活性域；
图4(a)质粒foki‐rsago‐pRS424‐TRP的构建图谱；
图4(b)质粒gNA‐HR‐pESCG‐LEU的构建图谱；
图4(c)gNA靶点设计与同源重组片段HR设计示意图；
图4(d)嵌合蛋白Fok‐RsAgo引发的基因敲入与敲除的PCR定位验证；
图4(e)嵌合蛋白Fok‐RsAgo获得的突变子生理表型筛选与验证；
图5(a)质粒foki‐mpago‐pRS424‐TRP的构建图谱；
图5(b)嵌合蛋白Fok‐MpAgo获得的突变子生理表型筛选与验证；
图6(a)质粒foki‐dmpago‐pRS424‐TRP的构建图谱；
图6(b)嵌合蛋白Fok‐dMpAgo引发的基因敲入与敲除的PCR定位验证；
图6(c)嵌合蛋白Fok‐dMpAgo获得的突变子测序验证；
图6(d)嵌合蛋白Fok‐dMpAgo获得的突变子生理表型筛选与验证；
图7(a)质粒foki‐dngago‐pRS424‐TRP的构建图谱；
图7(b)质粒HR‐pESCG‐LEU的构建图谱；
图7(c)5’磷酸向导DNA设计及同源重组片段HR设计示意图；
图7(d)嵌合蛋白Fok‐dNgAgo获得的突变子24孔板生理表型筛选与验证；
图8(a)质粒foki‐rsago‐foki2‐pRS424‐TRP的构建图谱；
图8(b)嵌合蛋白Fok‐RsAgo‐Fok获得的突变子生理表型筛选与验证；
图9(a)质粒foki‐dmpago‐foki2‐pRS424‐TRP的构建图谱；
图9(b)嵌合蛋白Fok‐dMpAgo‐Fok引发的基因敲入与敲除PCR定位验证；
图9(c)嵌合蛋白Fok‐dMpAgo‐Fok获得的突变子生理表型筛选与验证；
图10(a)质粒foki‐dngago‐foki2‐pRS424‐TRP的构建图谱；
图10(b)嵌合蛋白Fok‐dNgAgo‐Fok引发的基因敲入与敲除PCR定位验证；
图10(c)嵌合蛋白Fok‐dNgAgo‐Fok获得的突变子生理表型筛选与验证；
图10(d)嵌合蛋白Fok‐dNgAgo‐Fok获得的突变子24孔板生理表型筛选与验证；
图11(a)质粒rsago‐foki2‐pRS424‐TRP的构建图谱；
图11(b)嵌合蛋白RsAgo‐Fok引发的基因敲入与敲除PCR定位验证；
图11(c)嵌合蛋白RsAgo‐Fok获得的突变子生理表型筛选与验证；
图12(a)质粒dngago‐foki2‐pRS424‐TRP的构建图谱；
图12(b)嵌合蛋白RsAgo‐Fok获得的突变子24孔板生理表型筛选与验证。
具体实施方式
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面参考附图及具体实施方式，对本发明详细描述如下：
本发明的整体设计思想，是提供了一种嵌合蛋白pAgoE，该嵌合蛋白pAgoE含有具有基因组靶向定位功能的pAgo结构域，以及与所述pAgo结构域连接的、具有基因组切割或修饰活性的效应结构域E。当效应结构域E具有切割活性时，该嵌合蛋白pAgoE可以应用在基因编辑上；当效应结构域E具有修饰活性时，该嵌合蛋白pAgoE可以应用在基因修饰上。
进一步的，基于该嵌合蛋白pAgoE，结合向导gNA，以形成靶向定位下的DNA的编辑或修饰的具体应用结构，其结合下的应用简图如图1所示。
基于如上整体设计思想下，具体的应用示例如下：
利用PSI‐Blast，在NCBI数据库中找到了398个候选pAgo，见表1。
表1 NCBI数据库中查到的398个候选pAgo
Figure GDA0001167618950000111
Figure GDA0001167618950000121
Figure GDA0001167618950000131
Figure GDA0001167618950000141
Figure GDA0001167618950000151
Figure GDA0001167618950000161
Figure GDA0001167618950000171
Figure GDA0001167618950000181
Figure GDA0001167618950000191
Figure GDA0001167618950000201
分析上述398个pAgo的宿主情况，将TtAgo等43个源自嗜热菌的pAgo剔除，得到355个候选pAgo，再剔除氨基酸个数小于300的pAgo，得到350个候选pAgo，见表2。
表2剔除嗜热菌的pAgo和氨基酸个数小于300的pAgo后得到的350个候选pAgo
Figure GDA0001167618950000202
Figure GDA0001167618950000211
Figure GDA0001167618950000221
Figure GDA0001167618950000231
Figure GDA0001167618950000241
Figure GDA0001167618950000251
Figure GDA0001167618950000261
Figure GDA0001167618950000271
Figure GDA0001167618950000281
搜寻上述350个候选pAgo，将不同种属间的、氨基酸序列同源性大于99％的pAgo组合并，留下代表性的pAgo，并将以Microcystis aeruginosa为代表的同一个物种的不同基因序列号或蛋白序列号比对，留下代表性序列号，得到319个候选pAgo，见表3。
表3合并不同种属间的序列同源性大于99％的pAgo之后的319个候选pAgo
Figure GDA0001167618950000282
Figure GDA0001167618950000291
Figure GDA0001167618950000301
Figure GDA0001167618950000311
Figure GDA0001167618950000321
Figure GDA0001167618950000331
Figure GDA0001167618950000341
Figure GDA0001167618950000351
表3中所列出的319个候选pAgo中，包括PIWI结构域具有核酸酶活性的activepAgo，也包括PIWI结构域不具有核酸酶活性的dead pAgo。利用生物信息学分析预测了其中的55个active pAgo的活性位点，结果见图2。
实验实施例所用的微生物菌种、基础培养基及筛选培养基、培养条件：
(a)大肠杆菌Escherichia coli DH5α等：LB培养基，37℃培养。
(b)双倍体酿酒酵母Saccharomyces cerevisiae SC4(His-，Leu-，Trp-，Ura-)：YNB培养基、YPD培养基，30℃，固体平板培养，液体摇瓶培养时，采用30mL/250mL三角瓶，摇瓶转速180rpm。
实施例所用的实验方法
下列实施例中所涉及到的酿酒酵母的微生物学操作技术与流程以及质粒构建、转化、PCR验证、基因测序验证等分子生物学操作技术与流程，除以下额外说明的情况之外，均采用常规方法进行，这些常规方法是生命科学与生物技术领域经受过专业教育或培训的人员所熟悉和掌握的基本技术，参见萨姆布鲁克(Sambrook，J.)等著、黄培堂等译、科学出版社2002年8月出版的《分子克隆实验指南》(上、下册)第三版，本说明书不再具体加以描述。
为了快速评估酿酒酵母基因编辑效率，选用其编码产物为精氨酸转运蛋白的can1基因作为模式靶标，这个靶标也是哈佛大学医学院乔治丘奇教授课题组在2013年发表CRISPR首次用于酿酒酵母基因编辑论文中所采用的模式靶标(doi：10.1093/nar/gkt135)。can1基因作为模式靶标可以快速筛选表型，其基本原理是：如果can1基因突变，则丧失将细胞外的精氨酸转运进入细胞的能力，该突变株可以通过自我合成精氨酸而继续存活，同时，因为上述精氨酸转运能力的缺失，这类突变株也不能够运输刀豆氨酸这个有毒的精氨酸类似物，因而可以在含有刀豆氨酸的培养基中存活下来并正常生长；与此相对应的是，can1基因未被编辑(未突变)的出发菌株，却将因为具备精氨酸转运能力而将刀豆氨酸中毒，不能够在含有刀豆氨酸的培养基中存活下来。为了提高刀豆氨酸评估can1基因编辑的准确性，以下实施例中，本发明中相关实施例对刀豆氨酸法评估can1基因编辑的方法学做了进一步改进，主要包括：1、培养基中的刀豆氨酸浓度由一般方法中的60ug/ml提高到100ug/ml，以便排除假阳性。为了更好地排除假阳性，新方法中，不直接在刀豆氨酸平板上筛选基因编辑转化子，对于各种pAgo引发的基因编辑，这个改进是尤其重要的，因为天然的pAgo极有可能只引发所靶向DNA的单链断裂、并随后出现瞬时的单链编辑的可能性，这类可能的瞬时单链编辑突变株，在60ug/ml提高到100ug/ml的筛选压力下，有可能进一步演化为双链完全编辑的真突变株，而无筛选压力的真实情况下，这类可能的瞬时单链编辑突变株一般总是回复突变为完全的CAN基因野生型。因此，在多数情况下，在完成对酿酒酵母的化学转化或电转化操作及必要的孵育培养之后，尽量避免在刀豆氨酸平板上做涂布筛选操作，而是仅仅采用营养缺陷型筛选方案，在无刀豆氨酸的平板上筛选出携带着pAgoE、gNA、HR等相关基因元件的目的质粒的转化子，之后，将这些无刀豆氨酸平板上的单克隆稀释成菌悬液并实施影印操作，筛选表型突变株。
在以上改进基础上，以酿酒酵母can1基因为模式基因，建立了3个生理表型快速筛选的实验流程与方法，具体如下：
(a)平板微滴影印法。以每个微滴代表一个传统的涂布平板，以便在每一个平板上开展多个实验验证筛选。具体步骤如下：将待检验菌悬液或者待检验菌株的液体培养物取500ul，置于在96孔板中，稀释到OD600值0.10，依此为基准菌浓，再依次稀释10‐1、10‐2、10‐3、10‐4四个稀释度，分别影印到含有100mg/mL刀豆氨酸和不含有刀豆氨酸的LEU‐TRP‐的YNB筛选培养基的平板上，能在含有100mg/mL刀豆氨酸筛选培养基的平板上生长的为突变子，并能通过转化子的单菌落的个数，以不含有刀豆氨酸的对照平板的单菌落数据为分母，计算基因编辑效率。
(b)24孔板微培养评估法。为了判定和快速评估较低重组率的情况，建立了该方法：挑取单菌落至80μL的NaCl溶液中，混悬，取35μL菌液分别注入含有和不含有100mg/mL刀豆氨酸的LEU‐TRP‐的YNB固体选培养基的24孔板上，能在含刀豆氨酸的24孔板固体培养基上生长的即为突变子。经稀释涂布标定每μL菌液的cfu数值，根据在含刀豆氨酸的24孔板固体培养基上生长的菌落数，估算基因编辑效率。
(c)平板微滴计数培养法。以每个微滴代表一个传统的涂布平板，以便在每一个平板上开展多个实验验证筛选。但是不同于上述的平板微滴影印法，不再分别影印到含有100mg/mL刀豆氨酸和不含有刀豆氨酸的LEU‐TRP‐的YNB筛选培养基的平板上，而是以标定每微升的cfu数值，根据在含刀豆氨酸的固体培养基上生长的菌落数，估算基因编辑效率。
实施例中所采用的内源化的5’‐PO4‐gRNA设计原理
RsAgoE使用的18个核苷酸的5’‐PO4‐gRNA gNA，细胞内源的这种向导寡核苷酸可以依赖RsAgoE中的RsAgo结构域对高丰度转录后的5’羟基RNA的特定加工，该加工过程对被加工的RNA是有碱基偏好性的：其5’端开始的、由5’到3’方向的第1位核苷酸是U、第2位核苷酸是U或者C。
所用的基础质粒见表4：
表4实施例所采用的基础质粒列表
Figure GDA0001167618950000381
所构建的pAgoE表达质粒、携带gNA表达盒及同源重组片段的质粒，见表5：
表5实施例所构建嵌合蛋白表达质粒、携带gNA表达盒及同源重组片段的质粒表
Figure GDA0001167618950000382
所构建的几种代表性嵌合蛋白pAgoE所使用的gNA情况，见表6：
表6代表性pAgoE的pAgo的来源与所使用的gNA情况分类表
Figure GDA0001167618950000391
所构建的工程菌菌株见表7：
表7实施例所构建工程菌菌株表
Figure GDA0001167618950000392
实施例一：嵌合蛋白Fok‐RsAgo的构建与基因组编辑。
从表三的319个候选pAgo中，选用RsAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo型pAgoE(以下简称Fok‐RsAgo)，采用该Fok‐RsAgo与携带相应的gNA及HR片段的质粒配合，转化酵母并继代培养，检测基因编辑情况。
1、Fok‐RsAgo表达质粒的构建及表达：
根据Rhodobacter sphaeroides ATCC 17025的RsAgo蛋白(NCBI登录号：ABP72561.1)的氨基酸序列如SEQ ID NO:1，按照酵母的碱基偏好性，设计了编码RsAgo蛋白的rsago基因序列，并采用重叠延伸PCR技术、进行了该基因序列的人工合成及T克隆，获得rsago‐pEASY。根据Planomicrobium okeanokoites的内切核酸酶FokI蛋白(NCBI登录号:AAA24934.1)的氨基酸序列。选取了FokⅠ蛋白C端的从第383个氨基酸到第579个氨基酸的fokiC的DNA切割活性结构域(Nuclease域)作为pAgoE中的E域(简称Fok域)，如图3，按照酵母的碱基偏好性设计了编码该结构域的基因序列。采用重叠延伸PCR技术、进行了人工合成及T克隆，获得foki‐pEASY。选用GGGGS作为linker，利用Overlapping PCR技术融合启动子TEF、NLS、foki、linker、rsago和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐rsago‐pRS424‐TRP(如图4a所示)，所构建的嵌合蛋白Fok‐RsAgo的氨基酸序列见SEQ ID NO:4，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐RsAgo的基因序列见SEQ ID NO:13。将foki‐rsago‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐RsAgo嵌合蛋白的底盘细胞SC4‐FRs。
2、gNA表达盒及同源臂HR克隆质粒的构建
参照NCBI中酿酒酵母模式菌株Saccharomyces cerevisiae288c的基因组信息，选取编码精氨酸透酶CAN1的基因can1为靶点基因，在编码链选取含有TT的30nt序列5’‐CAAAGACATATTGGTATGATTGCCCTTGGT与SNR52启动子及SUP43FLANK序列融合，再融合30nt靶位点上游长583bp同源序列、含终止密码子的20bp的供体DNA和30nt靶位点下游长596bp同源序列，连接于pESCG‐LEU质粒骨架，构建成gNA表达盒和同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU(如图4b)。在非编码链选取内含TT的30nt序列5’‐CCTTCATCTTCATCACCTATGCCATCCTCC与SNR52启动子及SUP43FLANK序列融合，再融合30nt靶位点上游长583bp同源序列、20bp的供体DNA和30nt靶位点下游长596bp序列，得到同源重组片段(HR),连接于pESCG‐LEU质粒骨架，构建成gNA表达盒和同源臂克隆质粒gNA30R‐HR‐pESCG‐LEU(见图4b)。靶点设计与同源重组片段HR设计见图4c。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐RsAgo的SC4‐FRs细胞
制备本实施例步骤1获得的SC4‐FRs的感受态细胞，化学转化法转化本实施例步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。具体包括：将细胞培养到OD600＝0.5‐1.0，离心收获细胞，用1×TE缓冲液洗细胞一次，用30μL 1×LiAc/0.5×TE重悬，室温静置10min，加入1μg质粒gNA30F‐HR‐pESCG‐LEU和3μg HR片段DNA(或质粒gNA30R‐HR‐pESCG‐LEU和3μg HR片段DNA)和3μL ss‐DNA(10.0mg/mL)，加入250μL 1×LiAc/40％PEG3350/1×TE，混合均匀，30℃孵育培养30min，加入30μL DMSO，混合均匀，42℃热激7min，离心，弃去上清，1mL 1×TE洗一次，离心，弃去上清，30μL菌液涂在LEU‐TRP‐的YNB筛选培养基的平板上，30℃，培养72小时。
4、突变子分子生物学验证
将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子，混合，制成菌悬液，接种于LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，摇床培养，每48小时继代一次，累计培养至264小时后，将培养物划线分离出单菌落，挑单菌落，96孔板微培养20小时，各取5μL菌液，分组混合，稀释，采用匀浆器震荡裂解，用定位验证引物：CANYYF:5’‐GATTGATAACTAGGCTGACGGGGT和CNXDYR:5’‐GCAGTGATACCAACTAGTTCAGTACCT验证***的失活基因的存在，用敲除验证引物：CNAYWF:5’‐AGGATGGCATAGGTGATGAAGAT CNXDYR:5’‐GCAGTGATACCAACTAGTTCAGTACCT验证被敲除基因。验证结果如图4d，gNA30F实验组：84个中有21个为突变子。有63个为WT。重组率为25％。
5、突变子的生理表型筛选与验证
采用平板微滴影印法，分别将上述步骤4进行的继代培养中的第192h、216h、240h、264h的培养物取样500μL，稀释到OD值0.1，再依次按照10‐1、10‐2、10‐3、10‐4共四个稀释度进行稀释，分别影印到含有100mg/mL刀豆氨酸和不含有刀豆氨酸的LEU‐TRP‐的YNB培养基平板上，评估基因编辑效率，结果如图4e。从264小时的培养物情况分析，在10‐4稀释度下，含有100mg/mL刀豆氨酸筛选培养基的平板上生长出3个突变子，在不含刀豆氨酸平板上生长出10个，估算基因编辑效率30％，与分子验证计算的基因编辑效率基本吻合。
实施例二：嵌合蛋白Fok‐MpAgo的构建与基因组编辑。
从表三的319个候选pAgo中，选用MpAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo型pAgoE(以下简称Fok‐MpAgo)，采用该Fok‐MpAgo与携带相应的gNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐MpAgo表达质粒的构建及表达：
根据Marinitoga piezophila的MpAgo蛋白(NCBI登录号：WP_014295921.1)的氨基酸序列如SEQ ID NO:2，按照酵母的碱基偏好性，设计了编码MpAgo蛋白的mpago基因序列，并采用重叠延伸PCR技术、进行了该基因序列的人工合成及T克隆，获得mpago‐pEASY。利用实施例一步骤1所获得的foki‐pEASY，选用GGGGS作为linker，利用overlapping PCR技术融合启动子TEF、NLS、foki、linker、mpago和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐mpago‐pRS424‐TRP(如图5a所示)，所构建的嵌合蛋白Fok‐MpAgo的氨基酸序列见SEQ ID NO:5，按酵母碱基偏好性设计的编码嵌合蛋白Fok‐MpAgo的基因序列见SEQ ID NO:14。将foki‐mpago‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐MpAgo融合蛋白的底盘细胞SC4‐FMp。
2、gNA表达盒及同源臂克隆质粒的构建
参照NCBI中酿酒酵母模式菌株Saccharomyces cerevisiae288c的基因组信息，选取编码精氨酸透酶CAN1的基因can1为靶点基因，在编码链选取30nt序列5’‐CAAAGACATATTGGTATGATTGCCCTTGGT与SNR52启动子及SUP43FLANK序列融合，再融合30nt靶位点上游长583bp同源序列、含终止密码子的20bp的供体DNA和30nt靶位点下游长596bp同源序列，连接于pESCG‐LEU质粒骨架，构建成gNA表达盒和同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU(如图4b)。在非编码链选取30nt序列5’‐CCTTCATCTTCATCACCTATGCCATCCTCC与SNR52启动子及SUP43FLANK序列融合，再融合30nt靶位点上游长583bp同源序列、20bp的供体DNA和长596bp30nt靶位点下游序列，得到同源重组片段(HR),连接于pESCG‐LEU质粒骨架，构建成gNA表达盒和同源臂克隆质粒gNA30R‐HR‐pESCG‐LEU(如图4b)。靶点设计与同源重组片段HR设计如图4c。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐MpAgo的SC4‐FMp细胞
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的SC4‐FMp的感受态细胞，化学转化法转化本实施例步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。
4、突变子的生理表型筛选与验证
按照实施例一步骤5所描述的方法，采用平板微滴影印法，对上述步骤3获得的液体培养物进行继代培养，至196h取样，进行生理表型筛选与验证，估算转化gNA30F‐HR‐pESCG‐LEU实验组基因编辑效率约为1％，转化gNA30R‐HR‐pESCG‐LEU实验组基因编辑效率约为20％，结果如图5b。
实施例三：嵌合蛋白Fok‐dMpAgo的构建与基因组编辑。
从表三的319个候选pAgo中，选用MpAgo的PIWI失活突变体dMpAgo作为pAgo域，选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo型pAgoE(以下简称Fok‐dMpAgo)，采用该Fok‐dMpAgo与携带相应的gNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐dMpAgo表达质粒的构建及表达：
根据Marinitoga piezophila的MpAgo蛋白(NCBI登录号：WP_014295921.1)的氨基酸序列如SEQ ID NO:2，按照酵母的碱基偏好性，设计了编码MpAgo蛋白的mpago基因序列，并采用重叠延伸PCR技术、进行了该基因序列的人工合成及T克隆，获得mpago‐pEASY。以所获得的mpago‐pEASY质粒为模板，将mpago所编码的MpAgo蛋白在446位点进行点突变，将Asp置换为Ala，T克隆突变基因，获得dmpago‐pEASY。利用实施例一步骤1所获得的foki‐pEASY，选用GGGGS作为linker，利用overlapping PCR技术融合启动子TEF、NLS、foki、linker、dmpago和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐dmpago‐pRS424‐TRP(见图6a所示)，所构建的嵌合蛋白Fok‐dMpAgo的氨基酸序列见SEQ ID NO:6，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐dMpAgo的基因序列见SEQID NO:15。将foki‐dmpago‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐dMpAgo融合蛋白的底盘细胞SC4‐FdMp。
2、gNA表达盒及同源臂HR克隆质粒的构建
按照实施例一步骤2所描述的方法，构建gNA表达盒及同源臂HR克隆质粒gNA30F‐HR‐pESCG‐LEU(见图4b)和gNA30R‐HR‐pESCG‐LEU(见图4b)。靶点的设计与同源重组片段HR的设计，见图4c。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐dMpAgo的SC4‐FdMp细胞
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的SC4‐FdMp的感受态细胞，化学转化法转化本实施例步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。
4、突变子的分子生物学验证
按照实施例一步骤4所描述的方法进行分子验证，结果如图6b。转化gNA30F‐HR‐pESCG‐LEU实验组：94个菌株中有78个为突变子，有16个为WT，重组率为82.97％。选取10个突变子测序，结果表明供体DNA序列都***既定的靶向区域，can1基因中包含靶点序列的168bp基因序列均被敲除，10个转化子都按照预期实现了靶向敲入和敲除，结果如图6c。
5、突变子的生理表型筛选与验证
按照实施例一步骤5所描述的方法，采用平板微滴影印法，将本实施例步骤4获得的液体培养物进行继代培养，第192h、216h、240h、264h的培养物取样500μL，稀释到OD值0.1，再依次按照10‐1、10‐2、10‐3、10‐4共四个稀释度进行稀释，分别影印到含有100mg/mL刀豆氨酸和不含有刀豆氨酸的LEU‐TRP‐的YNB培养基平板上，评估基因编辑效率，结果如图6d。从264小时的培养物情况分析，在10‐4稀释度下，含有100mg/mL刀豆氨酸筛选培养基的平板上生长出14个突变子，在不含刀豆氨酸平板上生长出22个，估算基因编辑效率63.6％，与分子验证计算的基因编辑效率基本吻合。
实施例四：嵌合蛋白Fok‐dNgAgo的构建与基因组编辑。
从表三的319个候选pAgo中，选用NgAgo的PIWI突变体dNgAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo型pAgoE(以下简称Fok‐dNgAgo)，采用该Fok‐dNgAgo与5’磷酸向导DNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐dNgAgo表达质粒的构建及表达：
根据Natronobacterium gregoryi的NgAgo蛋白(NCBI登录号WP_005580376.1)的氨基酸序列如SEQ ID NO:3，按照酵母的碱基偏好性设计基因序列，采用重叠延伸PCR技术，进行了该基因序列的人工合成及T克隆，获得ngago‐pEASY。以所获得的ngago‐pEASY质粒为模板，将ngago所编码的NgAgo蛋白在663位点进行点突变，将Asp置换为Ala，T克隆点突变基因，获得dngago‐pEASY。利用实施例一步骤1所获得的foki‐pEASY，选用GGGGS作为linker，利用overlapping PCR融合启动子TEF、NLS、foki、linker、dngago和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐dngago‐pRS424‐TRP(如图7a所示)，所构建的嵌合蛋白Fok‐dNgAgo的氨基酸序列见SEQ ID NO:7，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐dNgAgo的基因序列见SEQ ID NO:16。将foki‐dngago‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐dNgAgo融合蛋白的底盘细胞SC4‐FdNg。
2、5’磷酸向导DNA及同源臂HR设计
选取Saccharomyces cerevisiae 288c基因组中编码精氨酸透酶CAN1的基因can1为靶点基因，合成与编码链上24bp同源的5’磷酸化的向导DNA:Rv‐5’‐PO4‐gDNA:5’p‐CACTTCAGCGTTCTGTACTTCTCC和与非编码链上24bp同源的5’磷酸化的向导DNA：Fw‐5’‐PO4‐gDNA:5’p‐GTAAATGGCGAGGATACGTTCTCT。融合靶位点上游长583bp同源序列、含终止密码子的20bp供体DNA和30nt靶位点下游长596bp同源序列，连接于pESCG‐LEU质粒骨架，构建同源臂克隆质粒HR‐pESCG‐LEU(见图7b)。靶点设计与HR设计见图7c。
3、5’磷酸向导DNA和HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐dNgAgo的SC4‐FdNg
制备本实施例步骤1获得的SC4‐FdNg的感受态细胞，化学转化法转化上述步骤2同源臂克隆质粒HR‐pESCG‐LEU和5’磷酸化的向导DNA Rv‐5’‐PO4‐gDNA或Fw‐5’‐PO4‐gDNA。对照组只转化HR‐pESCG‐LEU质粒。具体方法：将细胞培养到OD600＝0.5‐1，收获细胞，用 1×TE缓冲液洗细胞一次，用 30μL 1×LiAc/0.5×TE重悬，室温静置10min，加入1μg质粒HR‐pESCG‐LEU、3μg HR片段DNA、2μg 5’磷酸化的向导DNA Rv‐5’‐PO4‐gDNA或Fw‐5’‐PO4‐gDNA和3μL ss‐DNA(10.0mg/ml)，加入250μL 1×LiAc/40％PEG3350/1×TE，混合均匀，30℃孵育30min，加入30μL DMSO，混合均匀，42℃热击7min，离心，弃去上清，1mL1×TE洗一次，离心，弃去上清，30μL菌液涂在LEU‐TRP‐的YNB筛选培养基的平板上，在30℃，培养72小时。
4、突变子的生理表型筛选与验证
采用24孔板微培养评估法。将本实施例步骤3获得的F‐5’‐PO4‐gRNA实验组和对照组在LEU‐TRP‐的YNB筛选培养基上长出的转化子的单菌落，进行24孔板微培养。结果见图7d。结果显示，Fok‐dNgAgo有微弱基因编辑活性：在含刀豆氨酸培养基上，对照组没有长出任何菌落，实验组24个孔中有9个孔长出16个菌落，以出发菌浓为每孔2.74X105cfu计算，估算基因编辑效率为0.24X10‐5。
实施例五：嵌合蛋白Fok‐RsAgo‐Fok的构建与基因组编辑。
从表三的319个候选pAgo中，选用RsAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo‐N型pAgoE(以下简称Fok‐RsAgo‐Fok)，采用该Fok‐RsAgo‐Fok与携带相应的gNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐RsAgo‐Fok表达质粒的构建及表达：
根据Planomicrobium okeanokoites的内切核酸酶FokI蛋白(NCBI登录号:AAA24934.1)的氨基酸序列。利用实施例一步骤1所获得的foki‐pEASY，并且按照酵母的碱基偏好性设计了与foki基因序列不同但保持所表达蛋白域的氨基酸序列一致的foki2基因序列，采用重叠延伸PCR技术、进行了人工合成及T克隆，获得了foki2‐pEASY。以实施例一所构建的rsago‐pEASY为模板，N端选用GGGGS作为linker，C端选用GGGGSGGGGS作为linker2，利用overlapping PCR融合启动子TEF、NLS、foki、linker、rsago、linker2、foki2和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐rsago‐foki2‐pRS424‐TRP(如图8a所示)，所构建的嵌合蛋白Fok‐RsAgo‐Fok的氨基酸序列见SEQ ID NO:8，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐RsAgo‐Fok的基因序列见SEQ ID NO:17。将foki‐rsago‐foki2‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐RsAgo‐Fok嵌合蛋白的底盘细胞SC4‐FRsF。
2、gNA表达盒及同源臂HR克隆质粒的构建
按照实施例一步骤2所描述的方法进行。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐RsAgo‐Fok的底盘细胞SC4‐FRsF
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的底盘细胞SC4‐FRsF的感受态细胞，化学转化法转化本实施例步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。
4、突变子的生理表型筛选与验证
采用平板微滴影印法，将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子，混合，制成菌悬液接种于LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，摇床培养，48小时取样并继代，分别于48h和72小时取样进行生理表型筛选与验证，结果显示Fok‐RsAgo‐Fok具有基因编辑活性，如图8b。
实施例六：嵌合蛋白Fok‐dMpAgo‐Fok的构建与基因组编辑。
从表三的319个候选pAgo中，选用MpAgo的PIWI失活突变体dMpAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo‐N型pAgoE(以下简称Fok‐dMpAgo‐Fok)，采用该Fok‐dMpAgo‐Fok与携带相应的gNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐dMpAgo‐Fok表达质粒的构建及表达：
按照实施例五中步骤1所描述的方法，以实施例三步骤1所获得dmpago‐pEASY为模板，N端选用GGGGS作为linker，C端选用GGGGSGGGGS作为linker2，利用overlapping PCR融合启动子TEF、NLS、foki、linker、dmpago、linker2、foki2和终止子CYC1，采用GibsonAssembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐dmpago‐foki2‐pRS424‐TRP(如图9a所示)，所构建的嵌合蛋白Fok‐dMpAgo‐Fok的氨基酸序列见SEQ ID NO:9，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐dMpAgo‐Fok的基因序列见SEQ ID NO:18。将foki‐dmpago‐foki2‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐dMpAgo‐Fok嵌合蛋白的底盘细胞SC4‐FdMpF。
2、gNA表达盒及同源臂HR克隆质粒的构建
按照实施例二步骤2所描述的方法进行。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐dMpAgo‐Fok的底盘细胞SC4‐FdMpF。
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的底盘细胞SC4‐FdMpF的感受态细胞，化学转化法转化本实施例步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。
4、突变子的分子生物学验证
按照与实施例一步骤4所描述的方法，将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子，挑取单菌落接种于96孔板LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，微培养20小时后进行分子生物学验证，结果见图9b，转化gNA30F‐HR‐pESCG‐LEU实验组：48个单克隆全部为WT，未能获得突变子。转化gNA30R‐HR‐pESCG‐LEU实验组40个中有1个突变子，有39个为WT，重组率为2.5％。
5、突变子生理表型筛选与验证
按照与实施例一步骤5所描述的方法，采用平板微滴影印法，将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子混合，制成菌悬液接种于LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，摇床培养，每48小时继代一次，分别于48h和72小时取样进行生理表型筛选与验证，结果显示Fok‐RsAgo‐Fok的gNA30F‐HR‐pESCG‐LEU实验组及gNA30R‐HR‐pESCG‐LEU实验组均检测到基因编辑活性，如图9c。
实施例七：嵌合蛋白Fok‐dNgAgo‐Fok的构建与基因组编辑。
从表三的319个候选pAgo中，选用NgAgo的PIWI失活突变体dNgAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建N‐pAgo‐N型pAgoE(以下简称Fok‐dNgAgo‐Fok)，采用该Fok‐dNgAgo‐Fok与5’磷酸向导DNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、Fok‐dNgAgo‐Fok表达质粒的构建及表达：
按照实施例五中步骤1所描述的方法，以实施例四步骤1所获得dngago‐pEASY为模板，N端选用GGGGS作为linker，C端选用GGGGSGGGGS作为linker2，利用overlapping PCR融合启动子TEF、NLS、foki、linker、dngago、linker2、foki2和终止子CYC1，采用GibsonAssembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒foki‐dngago‐foki2‐pRS424‐TRP(如图10a所示)，所构建的嵌合蛋白Fok‐dNgAgo‐Fok的氨基酸序列见SEQ IDNO:10，其按酵母碱基偏好性设计的编码嵌合蛋白Fok‐dNgAgo‐Fok的基因序列见SEQ IDNO:19。将foki‐dngago‐foki2‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达Fok‐dNgAgo‐Fok嵌合蛋白的底盘细胞SC4‐FdNgF。
2、5’磷酸向导DNA及同源臂HR设计
采用实施例三步骤2所描述的方法进行。
3、5’磷酸向导DNA和HR‐pESCG‐LEU质粒转化表达嵌合蛋白Fok‐dNgAgo‐Fok的SC4‐FdNgF
采用实施例一步骤3所描述的方法，制备本实施例步骤1获得的底盘细胞SC4‐FdNgF的感受态细胞，化学转化法转化本实施例步骤2同源臂克隆质粒HR‐pESCG‐LEU和Fw‐5’‐PO4‐gDNA或Rv‐5’‐PO4‐gDNA。对照组只转化HR‐pESCG‐LEU质粒。
4、突变子的生理表型筛选与验证
采用平板微滴计数培养法，将上诉步骤3获得的在LEU‐TRP‐YNB筛选培养基上长出的转化子，挑单菌落，混悬于70ulNaCl溶液中制备菌悬液，该菌悬液经稀释涂布标定测得菌浓为7870cfu/μL。分别取3μl菌液影印到含有100mg/mL刀豆氨酸和不含有刀豆氨酸的LEU‐TRP‐的YNB筛选培养基的平板上，能在含有100mg/mL刀豆氨酸筛选培养基的平板上生长的为突变子。Fw‐5’‐PO4‐gDNA实验组中，60个菌中有25个在刀豆氨酸平板上生长，以出发菌浓为每滴(3ul)2.36×104计，平均基因编辑效率估算为1.76×10‐5。Rv‐5’‐PO4‐gDNA实验组中，60个菌中有21个在刀豆氨酸平板上生长，估算基因编辑效率为1.48×10‐5，结果如图10c。这些转化子经PCR验证，除F‐5’‐PO4‐gRNA实验组有1个WT之外，其余均为实现了外源基因敲入和目标基因敲除的突变子，结果如图10b。
为了进一步确认该Fok‐dNgAgo‐Fok的基因编辑活性，进行24孔板微培养，评估重组率，结果见图10d，结果显示，Fok‐dNgAgo‐Fok具有基因编辑活性：在含刀豆氨酸培养基上，对照组没有长出任何菌落，实验组24个孔中有14个孔上长出共计65个菌落，经标定，出发菌浓为每孔2.54X105cfu计算，估算基因编辑效率为1.1X10‐5。
实施例八：嵌合蛋白RsAgo‐Fok的构建与基因组编辑。
从表三的319个候选pAgo中，选用RsAgo作为pAgo域；选用核酸内切酶中的FokⅠ作为E域，构建pAgo‐N型pAgoE(以下简称RsAgo‐Fok)，采用该嵌合蛋白RsAgo‐Fok与携带相应的gNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、RsAgo‐Fok表达质粒的构建及表达：
按照按照实施例一中步骤1所描述的方法，采用实施例五步骤1所获得foki2‐pEASY，以实施例一中所构建的rsago‐pEASY为模板，N端选用GGGGS作为linker，C端选用GGGGSGGGGS作为linker2，利用overlapping PCR融合启动子TEF、NLS、rsago、linker2、foki2和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒rsago‐foki2‐pRS424‐TRP(如图11a所示)，所构建的嵌合蛋白RsAgo‐Fok的氨基酸序列见SEQ ID NO:11，其按酵母碱基偏好性设计的编码嵌合蛋白RsAgo‐Fok的基因序列见SEQID NO:20。将rsago‐foki2‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达嵌合蛋白RsAgo‐Fok的底盘细胞SC4‐RsF。
2、gNA表达盒及同源臂HR克隆质粒的构建
方法过程与实施例一步骤2相同。
3、gNA‐HR‐pESCG‐LEU质粒转化表达嵌合蛋白RsAgo‐Fok的底盘细胞SC4‐RsF
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的底盘细胞SC4‐RsF的感受态细胞，化学转化法转化上述步骤2制备的gNA表达盒及同源臂克隆质粒gNA30F‐HR‐pESCG‐LEU或gNA30R‐HR‐pESCG‐LEU。
4、突变子的分子生物学验证
按照实施例一步骤4所描述的方法，将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子，挑取单菌落接种于96孔板LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，微培养20小时后进行分子生物学验证，结果见图11b。结果显示，gNA30F‐HR‐pESCG‐LEU实验组：48个菌落中有1个突变子，有47个为WT，重组率为2.08％，gNA30R‐HR‐pESCG‐LEU实验组未能检测到阳性突变子。
5、突变子的生理表型筛选与验证
按照实施例一步骤5所描述的方法，采用平板微滴影印法，将本实施例步骤3获得的在LEU‐TRP‐的YNB筛选培养基上长出的转化子，混合，制成菌悬液接种于LEU‐TRP‐的YNB液体培养基中，30℃，180rpm，摇床培养，每48小时继代一次，分别于48h和72小时取样进行生理表型筛选与验证。结果见图11c，结果显示RsAgo‐Fok的gNA30F‐HR‐pESCG‐LEU实验组及gNA30R‐HR‐pESCG‐LEU实验组均检测到基因编辑活性。
实施例九：嵌合蛋白dNgAgo‐Fok的构建与基因组编辑。
从表三的319个候选pAgo中，选用NgAgo的PIWI突变体作为pAgo域；选用核酸内切酶中的FokⅠ作为E域构建pAgo‐N型pAgoE(以下简称dNgAgo‐Fok)，采用该dNgAgo‐Fok与携带5’磷酸向导DNA及HR片段的质粒配合，转化酵母并编辑其基因组。
1、dNgAgo‐Fok表达质粒的构建及表达：
按照按照实施例一中步骤1所描述的方法，采用实施例五步骤1所获得foki2‐pEASY，以实施例三步骤1所获得的dngago‐pEASY为模板，C端选用GGGGSGGGGS作为linker2，利用overlapping PCR融合启动子TEF、NLS、dngago、linker2、foki2和终止子CYC1，采用Gibson Assembly方法，与线性质粒pRS424‐TRP的骨架连接组装成质粒dngago‐foki2‐pRS424‐TRP(如图12a所示)，所构建的嵌合蛋白dNgAgo‐Fok的氨基酸序列见SEQ ID NO:12，其按酵母碱基偏好性设计的编码嵌合蛋白dNgAgo‐Fok的基因序列见SEQ ID NO:21。将dngago‐foki2‐pRS424‐TRP利用化学法转化酿酒酵母SC4，获得表达dNgAgo‐Fok融合蛋白的底盘细胞SC4‐dNgF。
2、5’磷酸向导DNA及同源臂HR设计
按照实施例三步骤2所描述的方法进行。
3、5’磷酸向导DNA和HR‐pESCG‐LEU质粒转化表达dNgAgo‐Fok融合蛋白的SC4‐dNgF
按照实施例一步骤3所描述的方法，制备本实施例步骤1获得的底盘细胞SC4‐dNgF的感受态细胞，化学转化法转化本实施例步骤2获得的同源臂克隆质粒HR‐pESCG‐LEU和5’磷酸化的向导DNA Fw‐5’‐PO4‐gDNA或Rv‐5’‐PO4‐gDNA。对照组只转化HR‐pESCG‐LEU质粒。
4、突变子的生理表型筛选与验证
按照实施例四步骤4所描述方法，进行24孔板微培养，评估重组率，结果见12b。结果显示，dNgAgo‐Fok具有基因编辑活性：对照组在含刀豆氨酸培养基上没有长出任何菌落，实验组在含刀豆氨酸培养基上，24个孔中有22个孔长出共计320个菌落，以出发菌浓为每孔2.74X105cfu计算，估算基因编辑效率为4.8X10‐5。
专利说明书中涉及的缩略词汇总，见表8。
表8专利说明书中的缩略词一览表
Figure GDA0001167618950000521
Figure GDA0001167618950000531
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，如：如果pAgoE中的E域采用具有荧光显示功能的EGFP，则可以用于基因组示踪，如果pAgoE中的E域采用具有具有转录激活功能或抑制的模块，则可以用于转录组调控，这些凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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Ile Asp Ala Ala Ala Thr Ala Ala Gly Leu Ser His Arg Leu Leu Asn
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Ser Phe Lys Val Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile Tyr Glu
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Pro Val Asp Gly Thr Thr Arg Val Gly Val Phe Val Thr Ile Gly Met
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Arg Tyr Asp Ile Glu Ala Ser Leu Arg Asp Leu Leu Glu Ala Gly Ile
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Leu Phe Glu Glu Thr Asp Leu Ala Ser Val Asn Val Asn Asp Ala Lys
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Leu Glu Gly Ser Lys Glu Asn Phe Thr Arg Cys Leu Ser Ala Leu Leu
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Gly His Asn Tyr Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala
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Gly Tyr Arg Thr Gly Pro Arg Phe Asp Asp Ala Val Arg Arg Met Gly
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Val Gly Ile Thr Thr Val Phe Ala Gly Asp Gly Ser Tyr Leu Leu Gly
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Asn Val Ser Lys Glu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile Arg
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Glu Ser Met Thr Gly Ile Leu Arg Glu Leu Lys Lys Arg Asn Asn Trp
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Arg Pro Gly Asp Thr Val Arg Val Val Phe His Ala His Arg Pro Leu
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Lys Arg Val Asp Val Ala Ser Ile Val Phe Glu Cys Thr Arg Glu Ile
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Gly Ser Asp Gln Asn Ile Gln Met Ala Phe Val Thr Val Ser His Asp
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His Pro Phe Val Leu Ile Asp Arg Ser Glu Arg Gly Leu Glu Ala Tyr
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Lys Gly Ser Thr Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly Ala
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Ile Ser Arg Val Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser Pro
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Gln Leu Ile Lys Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu Val
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Ser Leu His Pro Asp Ser Thr Phe Lys Asp Val Asp Tyr Leu Ala Glu
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Gln Ala Leu Lys Phe Thr Ser Leu Ser Trp Arg Ser Thr Leu Pro Ala
725 730 735
Ala Thr Pro Val Thr Ile Phe Tyr Ser Glu Arg Ile Ala Glu Leu Leu
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Gly Arg Leu Lys Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu Asn Ile
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Lys Leu Lys Trp Ser Arg Trp Phe Leu
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Leu Tyr Phe Tyr Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg Asn
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Leu Ser Arg Val Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val
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Ser Glu Val Phe Ser Leu Asp Ser Asn Glu Asn Val Asn Ala His Leu
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Ile Leu Pro Lys Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser
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Ala Ile Lys Ser Gly Tyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe
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Pro Tyr Ile Ser Gly Leu Asp Gly Ile Leu Lys Ile Asp Ile Gly Asn
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Asn Gln Ile Val Glu Val Ala Tyr Pro Glu Asn Tyr Leu Phe Asn Phe
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Thr Thr Arg Asp Ala Glu Lys Tyr Gly Phe Ser Lys Glu Val His Glu
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Ile Tyr Lys Asn Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr
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Leu Gly Phe Leu Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu
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Gly Glu Ser Arg Tyr Ala Lys Asp Val Phe Lys Tyr Ser Phe Tyr Lys
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Asn Glu Gln Pro Leu Lys Ala Ile Phe Phe Phe Ser Ser Lys Lys Gln
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Phe Phe Glu Val Gln Lys Ser Leu Lys Glu Leu Phe His Asn Lys His
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Ser Val Phe Tyr Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val Glu
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Phe Leu Arg Asp Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro
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Glu Glu Phe Thr Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp
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Phe Ile Ser Tyr Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys
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Asn Thr Asn Ile Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys
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Glu Glu Ile Ile Glu Gln Ile Tyr Leu Leu Thr Arg Val Ala His Ser
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Asp Glu Asp Glu Thr Phe Ala Gly Gly Glu Pro Leu Asp His His Leu
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Asp Asp Ala Leu Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser Asp
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Ser Gly His Val Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro Glu
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Trp Thr Leu His Thr Tyr Thr Leu Thr Ala Thr Asp Gly Ala Lys Thr
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Asp Thr Glu Tyr Ala Arg Arg Thr Leu Ala Tyr Thr Val Arg Gln Glu
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Gly Val Arg Val Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr Thr
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Arg Ser Leu Trp Asp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val Leu
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Ser Lys Glu Pro Val Leu Thr Cys Asp Glu Phe Asp Leu His Glu Arg
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Tyr Asp Leu Ser Val Glu Val Gly His Ser Gly Arg Ala Tyr Leu His
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Ile Asn Phe Arg His Arg Phe Val Pro Lys Leu Thr Leu Ala Asp Ile
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Asp Asp Asp Asn Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr Arg
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Pro Arg Arg Gly His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala Thr
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Asp Ser Leu Asn Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His Arg
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Asn Asn Gln Thr Pro Ile Asn Thr Asp Leu Leu Asp Ala Ile Glu Ala
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Ala Asp Arg Arg Val Val Glu Thr Arg Arg Gln Gly His Gly Asp Asp
385 390 395 400
Ala Val Ser Phe Pro Gln Glu Leu Leu Ala Val Glu Pro Asn Thr His
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Gln Ile Lys Gln Phe Ala Ser Asp Gly Phe His Gln Gln Ala Arg Ser
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Ser Ser Glu Phe Phe Thr Gly Asn Asn Glu Gln Gln Leu Arg Leu Leu
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Tyr Glu Asn Gly Glu Ser Val Leu Thr Phe Arg Asp Gly Ala Arg Gly
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Ala His Pro Asp Glu Thr Phe Ser Lys Gly Ile Val Asn Pro Pro Glu
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Ser Phe Glu Val Ala Val Val Leu Pro Glu Gln Gln Ala Asp Thr Cys
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Lys Ala Gln Trp Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly Ala
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Pro Pro Thr Arg Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser Pro
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Glu Ser Ile Ser Leu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu Val
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Asp Ala Ala Phe Val Val Leu Pro Pro Asp Gln Glu Gly Phe Ala Asp
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Leu Ala Ser Pro Thr Glu Thr Tyr Asp Glu Leu Lys Lys Ala Leu Ala
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Asn Met Gly Ile Tyr Ser Gln Met Ala Tyr Phe Asp Arg Phe Arg Asp
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Ala Lys Ile Phe Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala Ala
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Ala Gly Gly Val Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp Ala
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Asp Met Phe Ile Gly Ile Asp Val Ser Arg Ser Tyr Pro Glu Asp Gly
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Ala Ser Gly Gln Ile Asn Ile Ala Ala Thr Ala Thr Ala Val Tyr Lys
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Asp Gly Thr Ile Leu Gly His Ser Ser Thr Arg Pro Gln Leu Gly Glu
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Lys Leu Gln Ser Thr Asp Val Arg Asp Ile Met Lys Asn Ala Ile Leu
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Gly Tyr Gln Gln Val Thr Gly Glu Ser Pro Thr His Ile Val Ile His
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Gln Thr Arg Leu Leu Ala Val Ser Asp Val Gln Tyr Asp Thr Pro Val
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Thr Phe Gly Ala Pro Glu Tyr Leu Ala Thr Arg Asp Gly Gly Gly Leu
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Pro Arg Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr Asp Ile Glu
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Thr Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln Val
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His Asn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp Gln
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Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
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Gln Ala Ala Asp Glu Met Tyr Asp Ser Asn Pro His Pro Asp Arg Arg
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Gln Leu Val Ser Asn Gly Phe Glu Val Asn Leu Pro Asp Gln Val Glu
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Val Ile Val Arg Asp Leu Pro Asp Pro Ser Lys Val Lys Glu Glu Arg
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Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala Gly Tyr Arg Thr
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Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val Tyr Pro Ser Ser Thr
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Thr Val Arg Val Val Phe His Ala His Arg Pro Leu Lys Arg Val Asp
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Val Ala Ser Ile Val Phe Glu Cys Thr Arg Glu Ile Gly Ser Asp Gln
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Asn Ile Gln Met Ala Phe Val Thr Val Ser His Asp His Pro Phe Val
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Leu Ile Asp Arg Ser Glu Arg Gly Leu Glu Ala Tyr Lys Gly Ser Thr
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Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly Ala Ile Ser Arg Val
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Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser Pro Gln Leu Ile Lys
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Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu Val Ser Leu His Pro
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Asp Ser Thr Phe Lys Asp Val Asp Tyr Leu Ala Glu Gln Ala Leu Lys
930 935 940
Phe Thr Ser Leu Ser Trp Arg Ser Thr Leu Pro Ala Ala Thr Pro Val
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Thr Ile Phe Tyr Ser Glu Arg Ile Ala Glu Leu Leu Gly Arg Leu Lys
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Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu Asn Ile Lys Leu Lys Trp
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Ser Arg Trp Phe Leu
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<313> (221)..(859)
<400> SEQ ID NO:5
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Tyr Leu Asn
210 215 220
Leu Tyr Lys Ile Asp Ile Pro Lys Lys Ile Lys Arg Leu Tyr Phe Tyr
225 230 235 240
Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg Asn Leu Ser Arg Val
245 250 255
Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile Glu Ile
260 265 270
Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys
275 280 285
Val Glu Lys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe
290 295 300
Val Lys Thr Leu Tyr Lys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp
305 310 315 320
Phe Tyr Phe Lys Lys Gly Asn Asn Phe Ile Ser Asn Ser Glu Val Phe
325 330 335
Ser Leu Asp Ser Asn Glu Asn Val Asn Ala His Leu Thr Tyr Lys Ile
340 345 350
Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile Leu Pro Lys
355 360 365
Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser
370 375 380
Gly Tyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser
385 390 395 400
Gly Leu Asp Gly Ile Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val
405 410 415
Glu Val Ala Tyr Pro Glu Asn Tyr Leu Phe Asn Phe Thr Thr Arg Asp
420 425 430
Ala Glu Lys Tyr Gly Phe Ser Lys Glu Val His Glu Ile Tyr Lys Asn
435 440 445
Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr Leu Gly Phe Leu
450 455 460
Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly Tyr
465 470 475 480
Lys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg
485 490 495
Tyr Ala Lys Asp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro
500 505 510
Leu Lys Ala Ile Phe Phe Phe Ser Ser Lys Lys Gln Phe Phe Glu Val
515 520 525
Gln Lys Ser Leu Lys Glu Leu Phe His Asn Lys His Ser Val Phe Tyr
530 535 540
Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val Glu Phe Leu Arg Asp
545 550 555 560
Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu Phe Thr
565 570 575
Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala
580 585 590
Ile Val Leu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg
595 600 605
Asn Phe Pro Asp Asn Leu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu
610 615 620
Glu Asp Ile Lys Pro Phe Ile Ile Lys Ser Tyr Val Tyr Lys Met Gly
625 630 635 640
Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile Leu Lys Lys Met Glu Asp
645 650 655
Lys Glu Lys Asn Leu Tyr Ile Gly Ile Asp Leu Ser His Asp Thr Tyr
660 665 670
Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp
675 680 685
Ile Leu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met
690 695 700
Asn Leu Asp Ile Leu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr
705 710 715 720
Ile Glu Lys Phe Asn Val Ser Pro Glu Asn Val Phe Ile Leu Arg Asp
725 730 735
Gly Arg Phe Ile Glu Asp Ile Glu Ile Ile Lys Asn Phe Ile Ser Tyr
740 745 750
Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys Asn Thr Asn Ile
755 760 765
Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu Asn
770 775 780
Thr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu
785 790 795 800
Val Lys Ile Leu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile
805 810 815
Glu Gln Ile Tyr Leu Leu Thr Arg Val Ala His Ser Thr Pro Tyr Thr
820 825 830
Asn Tyr Lys Leu Pro Tyr Pro Leu His Ile Ala Asn Lys Val Ala Leu
835 840 845
Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr
850 855
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:6
<211> 859
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (18)..(215)
<300>
<308> linker
<309> 2016-11-11
<313> (216)..(220)
<300>
<308> dMpAgo
<309> 2016-11-11
<313> (221)..(859)
<400> SEQ ID NO:6
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Tyr Leu Asn
210 215 220
Leu Tyr Lys Ile Asp Ile Pro Lys Lys Ile Lys Arg Leu Tyr Phe Tyr
225 230 235 240
Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg Asn Leu Ser Arg Val
245 250 255
Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile Glu Ile
260 265 270
Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys
275 280 285
Val Glu Lys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe
290 295 300
Val Lys Thr Leu Tyr Lys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp
305 310 315 320
Phe Tyr Phe Lys Lys Gly Asn Asn Phe Ile Ser Asn Ser Glu Val Phe
325 330 335
Ser Leu Asp Ser Asn Glu Asn Val Asn Ala His Leu Thr Tyr Lys Ile
340 345 350
Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile Leu Pro Lys
355 360 365
Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser
370 375 380
Gly Tyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser
385 390 395 400
Gly Leu Asp Gly Ile Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val
405 410 415
Glu Val Ala Tyr Pro Glu Asn Tyr Leu Phe Asn Phe Thr Thr Arg Asp
420 425 430
Ala Glu Lys Tyr Gly Phe Ser Lys Glu Val His Glu Ile Tyr Lys Asn
435 440 445
Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr Leu Gly Phe Leu
450 455 460
Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly Tyr
465 470 475 480
Lys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg
485 490 495
Tyr Ala Lys Asp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro
500 505 510
Leu Lys Ala Ile Phe Phe Phe Ser Ser Lys Lys Gln Phe Phe Glu Val
515 520 525
Gln Lys Ser Leu Lys Glu Leu Phe His Asn Lys His Ser Val Phe Tyr
530 535 540
Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val Glu Phe Leu Arg Asp
545 550 555 560
Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu Phe Thr
565 570 575
Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala
580 585 590
Ile Val Leu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg
595 600 605
Asn Phe Pro Asp Asn Leu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu
610 615 620
Glu Asp Ile Lys Pro Phe Ile Ile Lys Ser Tyr Val Tyr Lys Met Gly
625 630 635 640
Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile Leu Lys Lys Met Glu Asp
645 650 655
Lys Glu Lys Asn Leu Tyr Ile Gly Ile Ala Leu Ser His Asp Thr Tyr
660 665 670
Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp
675 680 685
Ile Leu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met
690 695 700
Asn Leu Asp Ile Leu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr
705 710 715 720
Ile Glu Lys Phe Asn Val Ser Pro Glu Asn Val Phe Ile Leu Arg Asp
725 730 735
Gly Arg Phe Ile Glu Asp Ile Glu Ile Ile Lys Asn Phe Ile Ser Tyr
740 745 750
Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys Asn Thr Asn Ile
755 760 765
Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu Asn
770 775 780
Thr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu
785 790 795 800
Val Lys Ile Leu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile
805 810 815
Glu Gln Ile Tyr Leu Leu Thr Arg Val Ala His Ser Thr Pro Tyr Thr
820 825 830
Asn Tyr Lys Leu Pro Tyr Pro Leu His Ile Ala Asn Lys Val Ala Leu
835 840 845
Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr
850 855
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:7
<211> 1107
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (18)..(215)
<300>
<308> linker
<309> 2016-11-11
<313> (216)..(220)
<300>
<308> dNgAgo
<309> 2016-11-11
<313> (221)..(1107)
<400> SEQ ID NO:7
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Thr Val Ile
210 215 220
Asp Leu Asp Ser Thr Thr Thr Ala Asp Glu Leu Thr Ser Gly His Thr
225 230 235 240
Tyr Asp Ile Ser Val Thr Leu Thr Gly Val Tyr Asp Asn Thr Asp Glu
245 250 255
Gln His Pro Arg Met Ser Leu Ala Phe Glu Gln Asp Asn Gly Glu Arg
260 265 270
Arg Tyr Ile Thr Leu Trp Lys Asn Thr Thr Pro Lys Asp Val Phe Thr
275 280 285
Tyr Asp Tyr Ala Thr Gly Ser Thr Tyr Ile Phe Thr Asn Ile Asp Tyr
290 295 300
Glu Val Lys Asp Gly Tyr Glu Asn Leu Thr Ala Thr Tyr Gln Thr Thr
305 310 315 320
Val Glu Asn Ala Thr Ala Gln Glu Val Gly Thr Thr Asp Glu Asp Glu
325 330 335
Thr Phe Ala Gly Gly Glu Pro Leu Asp His His Leu Asp Asp Ala Leu
340 345 350
Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser Asp Ser Gly His Val
355 360 365
Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro Glu Trp Thr Leu His
370 375 380
Thr Tyr Thr Leu Thr Ala Thr Asp Gly Ala Lys Thr Asp Thr Glu Tyr
385 390 395 400
Ala Arg Arg Thr Leu Ala Tyr Thr Val Arg Gln Glu Leu Tyr Thr Asp
405 410 415
His Asp Ala Ala Pro Val Ala Thr Asp Gly Leu Met Leu Leu Thr Pro
420 425 430
Glu Pro Leu Gly Glu Thr Pro Leu Asp Leu Asp Cys Gly Val Arg Val
435 440 445
Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr Thr Ala Lys Asp Arg
450 455 460
Leu Leu Ala Arg Glu Leu Val Glu Glu Gly Leu Lys Arg Ser Leu Trp
465 470 475 480
Asp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val Leu Ser Lys Glu Pro
485 490 495
Val Leu Thr Cys Asp Glu Phe Asp Leu His Glu Arg Tyr Asp Leu Ser
500 505 510
Val Glu Val Gly His Ser Gly Arg Ala Tyr Leu His Ile Asn Phe Arg
515 520 525
His Arg Phe Val Pro Lys Leu Thr Leu Ala Asp Ile Asp Asp Asp Asn
530 535 540
Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr Arg Pro Arg Arg Gly
545 550 555 560
His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala Thr Asp Ser Leu Asn
565 570 575
Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His Arg Asn Asn Gln Thr
580 585 590
Pro Ile Asn Thr Asp Leu Leu Asp Ala Ile Glu Ala Ala Asp Arg Arg
595 600 605
Val Val Glu Thr Arg Arg Gln Gly His Gly Asp Asp Ala Val Ser Phe
610 615 620
Pro Gln Glu Leu Leu Ala Val Glu Pro Asn Thr His Gln Ile Lys Gln
625 630 635 640
Phe Ala Ser Asp Gly Phe His Gln Gln Ala Arg Ser Lys Thr Arg Leu
645 650 655
Ser Ala Ser Arg Cys Ser Glu Lys Ala Gln Ala Phe Ala Glu Arg Leu
660 665 670
Asp Pro Val Arg Leu Asn Gly Ser Thr Val Glu Phe Ser Ser Glu Phe
675 680 685
Phe Thr Gly Asn Asn Glu Gln Gln Leu Arg Leu Leu Tyr Glu Asn Gly
690 695 700
Glu Ser Val Leu Thr Phe Arg Asp Gly Ala Arg Gly Ala His Pro Asp
705 710 715 720
Glu Thr Phe Ser Lys Gly Ile Val Asn Pro Pro Glu Ser Phe Glu Val
725 730 735
Ala Val Val Leu Pro Glu Gln Gln Ala Asp Thr Cys Lys Ala Gln Trp
740 745 750
Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly Ala Pro Pro Thr Arg
755 760 765
Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser Pro Glu Ser Ile Ser
770 775 780
Leu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu Val Asp Ala Ala Phe
785 790 795 800
Val Val Leu Pro Pro Asp Gln Glu Gly Phe Ala Asp Leu Ala Ser Pro
805 810 815
Thr Glu Thr Tyr Asp Glu Leu Lys Lys Ala Leu Ala Asn Met Gly Ile
820 825 830
Tyr Ser Gln Met Ala Tyr Phe Asp Arg Phe Arg Asp Ala Lys Ile Phe
835 840 845
Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala Ala Ala Gly Gly Val
850 855 860
Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp Ala Asp Met Phe Ile
865 870 875 880
Gly Ile Ala Val Ser Arg Ser Tyr Pro Glu Asp Gly Ala Ser Gly Gln
885 890 895
Ile Asn Ile Ala Ala Thr Ala Thr Ala Val Tyr Lys Asp Gly Thr Ile
900 905 910
Leu Gly His Ser Ser Thr Arg Pro Gln Leu Gly Glu Lys Leu Gln Ser
915 920 925
Thr Asp Val Arg Asp Ile Met Lys Asn Ala Ile Leu Gly Tyr Gln Gln
930 935 940
Val Thr Gly Glu Ser Pro Thr His Ile Val Ile His Arg Asp Gly Phe
945 950 955 960
Met Asn Glu Asp Leu Asp Pro Ala Thr Glu Phe Leu Asn Glu Gln Gly
965 970 975
Val Glu Tyr Asp Ile Val Glu Ile Arg Lys Gln Pro Gln Thr Arg Leu
980 985 990
Leu Ala Val Ser Asp Val Gln Tyr Asp Thr Pro Val Lys Ser Ile Ala
995 1000 1005
Ala Ile Asn Gln Asn Glu Pro Arg Ala Thr Val Ala Thr Phe Gly
1010 1015 1020
Ala Pro Glu Tyr Leu Ala Thr Arg Asp Gly Gly Gly Leu Pro Arg
1025 1030 1035
Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr Asp Ile Glu Thr
1040 1045 1050
Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln Val
1055 1060 1065
His Asn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp
1070 1075 1080
Gln Ala Ser Thr His Ala Thr Lys Gly Tyr Leu Val Gln Thr Gly
1085 1090 1095
Ala Phe Glu Ser Asn Val Gly Phe Leu
1100 1105
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:8
<211> 1205
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (18)..(215)
<300>
<308> linker
<309> 2016-11-11
<313> (216)..(220)
<300>
<308> RsAgo
<309> 2016-11-11
<313> (221)..(997)
<300>
<308> linker
<309> 2016-11-11
<313> (998)..(1007)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (1008)..(1205)
<400> SEQ ID NO:8
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Ala Pro Val
210 215 220
Gln Ala Ala Asp Glu Met Tyr Asp Ser Asn Pro His Pro Asp Arg Arg
225 230 235 240
Gln Leu Val Ser Asn Gly Phe Glu Val Asn Leu Pro Asp Gln Val Glu
245 250 255
Val Ile Val Arg Asp Leu Pro Asp Pro Ser Lys Val Lys Glu Glu Arg
260 265 270
Thr Arg Leu Met Gly Tyr Trp Phe Val His Trp Phe Asp Gly Lys Leu
275 280 285
Phe His Leu Arg Ile Lys Ala Gly Gly Pro Asn Val Asp Gly Glu His
290 295 300
Arg Ala Ile Arg Thr Ala Glu His Pro Trp Leu Leu Arg Ala Arg Leu
305 310 315 320
Asp Asp Ala Leu Glu Glu Ala Leu Pro Lys Tyr Ala Ala Val Lys Lys
325 330 335
Arg Pro Phe Thr Phe Leu Ala Gln Lys Asp Glu Leu Ile Asp Ala Ala
340 345 350
Ala Thr Ala Ala Gly Leu Ser His Arg Leu Leu Asn Ser Phe Lys Val
355 360 365
Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile Tyr Glu Pro Val Asp Gly
370 375 380
Thr Thr Arg Val Gly Val Phe Val Thr Ile Gly Met Arg Tyr Asp Ile
385 390 395 400
Glu Ala Ser Leu Arg Asp Leu Leu Glu Ala Gly Ile Asp Leu Arg Gly
405 410 415
Met Tyr Val Val Arg Arg Lys Arg Gln Pro Gly Glu Arg Gly Leu Leu
420 425 430
Gly Arg Val Arg Ala Ile Ser Asp Asp Met Val Gln Leu Phe Glu Glu
435 440 445
Thr Asp Leu Ala Ser Val Asn Val Asn Asp Ala Lys Leu Glu Gly Ser
450 455 460
Lys Glu Asn Phe Thr Arg Cys Leu Ser Ala Leu Leu Gly His Asn Tyr
465 470 475 480
Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala Gly Tyr Arg Thr
485 490 495
Gly Pro Arg Phe Asp Asp Ala Val Arg Arg Met Gly Glu Phe Leu Ala
500 505 510
Lys Lys Pro Ile Arg Leu Ala Asp Asn Ile Asn Ala Gln Val Gly Asp
515 520 525
Arg Ile Val Phe Ser Asn Glu Gly Gln Ala Arg Asn Val Arg Leu Ala
530 535 540
Pro Lys Val Glu Tyr Val Phe Asp Arg Thr Gly Ala Lys Ser Ala Glu
545 550 555 560
Tyr Ala Trp Arg Gly Leu Ser Gln Phe Gly Pro Phe Asp Arg Pro Ser
565 570 575
Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val Tyr Pro Ser Ser Thr
580 585 590
Gln Gly Lys Val Glu Asn Phe Leu Ser Ala Phe Arg Asp Gly Met Gly
595 600 605
Ser Asn Tyr Ser Gly Phe Ser Lys Gly Phe Val Asp Leu Met Gly Leu
610 615 620
Thr Lys Val Glu Phe Val Met Cys Pro Val Glu Val Ser Ser Ala Asp
625 630 635 640
Arg Asn Gly Ala His Thr Lys Tyr Asn Ser Ala Ile Glu Asp Lys Leu
645 650 655
Ala Gly Ala Gly Glu Val His Ala Gly Ile Val Val Leu Phe Glu Asp
660 665 670
His Ala Arg Leu Pro Asp Asp Arg Asn Pro Tyr Ile His Thr Lys Ser
675 680 685
Leu Leu Leu Thr Leu Gly Val Pro Thr Gln Gln Val Arg Met Pro Thr
690 695 700
Val Leu Leu Glu Pro Lys Ser Leu Gln Tyr Thr Leu Gln Asn Phe Ser
705 710 715 720
Ile Ala Thr Tyr Ala Lys Leu Asn Gly Thr Pro Trp Thr Val Asn His
725 730 735
Asp Lys Ala Ile Asn Asp Glu Leu Val Val Gly Met Gly Leu Ala Glu
740 745 750
Leu Ser Gly Ser Arg Thr Glu Lys Arg Gln Arg Phe Val Gly Ile Thr
755 760 765
Thr Val Phe Ala Gly Asp Gly Ser Tyr Leu Leu Gly Asn Val Ser Lys
770 775 780
Glu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile Arg Glu Ser Met Thr
785 790 795 800
Gly Ile Leu Arg Glu Leu Lys Lys Arg Asn Asn Trp Arg Pro Gly Asp
805 810 815
Thr Val Arg Val Val Phe His Ala His Arg Pro Leu Lys Arg Val Asp
820 825 830
Val Ala Ser Ile Val Phe Glu Cys Thr Arg Glu Ile Gly Ser Asp Gln
835 840 845
Asn Ile Gln Met Ala Phe Val Thr Val Ser His Asp His Pro Phe Val
850 855 860
Leu Ile Asp Arg Ser Glu Arg Gly Leu Glu Ala Tyr Lys Gly Ser Thr
865 870 875 880
Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly Ala Ile Ser Arg Val
885 890 895
Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser Pro Gln Leu Ile Lys
900 905 910
Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu Val Ser Leu His Pro
915 920 925
Asp Ser Thr Phe Lys Asp Val Asp Tyr Leu Ala Glu Gln Ala Leu Lys
930 935 940
Phe Thr Ser Leu Ser Trp Arg Ser Thr Leu Pro Ala Ala Thr Pro Val
945 950 955 960
Thr Ile Phe Tyr Ser Glu Arg Ile Ala Glu Leu Leu Gly Arg Leu Lys
965 970 975
Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu Asn Ile Lys Leu Lys Trp
980 985 990
Ser Arg Trp Phe Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
995 1000 1005
Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu
1010 1015 1020
Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
1025 1030 1035
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys
1040 1045 1050
Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His
1055 1060 1065
Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly
1070 1075 1080
Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser
1085 1090 1095
Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg
1100 1105 1110
Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn
1115 1120 1125
Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe
1130 1135 1140
Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu
1145 1150 1155
Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser
1160 1165 1170
Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr
1175 1180 1185
Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile
1190 1195 1200
Asn Phe
1205
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:9
<211> 1067
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (18)..(215)
<300>
<308> linker
<309> 2016-11-11
<313> (216)..(220)
<300>
<308> dMpAgo
<309> 2016-11-11
<313> (221)..(859)
<300>
<308> linker
<309> 2016-11-11
<313> (860)..(869)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (870)..(1067)
<400> SEQ ID NO:9
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Tyr Leu Asn
210 215 220
Leu Tyr Lys Ile Asp Ile Pro Lys Lys Ile Lys Arg Leu Tyr Phe Tyr
225 230 235 240
Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg Asn Leu Ser Arg Val
245 250 255
Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile Glu Ile
260 265 270
Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys
275 280 285
Val Glu Lys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe
290 295 300
Val Lys Thr Leu Tyr Lys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp
305 310 315 320
Phe Tyr Phe Lys Lys Gly Asn Asn Phe Ile Ser Asn Ser Glu Val Phe
325 330 335
Ser Leu Asp Ser Asn Glu Asn Val Asn Ala His Leu Thr Tyr Lys Ile
340 345 350
Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile Leu Pro Lys
355 360 365
Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser
370 375 380
Gly Tyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser
385 390 395 400
Gly Leu Asp Gly Ile Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val
405 410 415
Glu Val Ala Tyr Pro Glu Asn Tyr Leu Phe Asn Phe Thr Thr Arg Asp
420 425 430
Ala Glu Lys Tyr Gly Phe Ser Lys Glu Val His Glu Ile Tyr Lys Asn
435 440 445
Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr Leu Gly Phe Leu
450 455 460
Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly Tyr
465 470 475 480
Lys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg
485 490 495
Tyr Ala Lys Asp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro
500 505 510
Leu Lys Ala Ile Phe Phe Phe Ser Ser Lys Lys Gln Phe Phe Glu Val
515 520 525
Gln Lys Ser Leu Lys Glu Leu Phe His Asn Lys His Ser Val Phe Tyr
530 535 540
Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val Glu Phe Leu Arg Asp
545 550 555 560
Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu Phe Thr
565 570 575
Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala
580 585 590
Ile Val Leu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg
595 600 605
Asn Phe Pro Asp Asn Leu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu
610 615 620
Glu Asp Ile Lys Pro Phe Ile Ile Lys Ser Tyr Val Tyr Lys Met Gly
625 630 635 640
Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile Leu Lys Lys Met Glu Asp
645 650 655
Lys Glu Lys Asn Leu Tyr Ile Gly Ile Ala Leu Ser His Asp Thr Tyr
660 665 670
Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp
675 680 685
Ile Leu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met
690 695 700
Asn Leu Asp Ile Leu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr
705 710 715 720
Ile Glu Lys Phe Asn Val Ser Pro Glu Asn Val Phe Ile Leu Arg Asp
725 730 735
Gly Arg Phe Ile Glu Asp Ile Glu Ile Ile Lys Asn Phe Ile Ser Tyr
740 745 750
Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys Asn Thr Asn Ile
755 760 765
Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu Asn
770 775 780
Thr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu
785 790 795 800
Val Lys Ile Leu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile
805 810 815
Glu Gln Ile Tyr Leu Leu Thr Arg Val Ala His Ser Thr Pro Tyr Thr
820 825 830
Asn Tyr Lys Leu Pro Tyr Pro Leu His Ile Ala Asn Lys Val Ala Leu
835 840 845
Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr Gly Gly Gly Gly Ser
850 855 860
Gly Gly Gly Gly Ser Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu
865 870 875 880
Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr
885 890 895
Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu
900 905 910
Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly
915 920 925
Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val
930 935 940
Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser
945 950 955 960
Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr
965 970 975
Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp
980 985 990
Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val
995 1000 1005
Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu
1010 1015 1020
Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu
1025 1030 1035
Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu
1040 1045 1050
Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe
1055 1060 1065
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:10
<211> 1315
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (18)..(215)
<300>
<308> linker
<309> 2016-11-11
<313> (216)..(220)
<300>
<308> dNgAgo
<309> 2016-11-11
<313> (221)..(1107)
<300>
<308> linker
<309> 2016-11-11
<313> (1108)..(1117)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (1118)..(1315)
<400> SEQ ID NO:10
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
20 25 30
Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile
35 40 45
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val
50 55 60
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
65 70 75 80
Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile
85 90 95
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn
100 105 110
Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn
115 120 125
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr
130 135 140
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
145 150 155 160
Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn
165 170 175
Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu
180 185 190
Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
195 200 205
Asn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Thr Val Ile
210 215 220
Asp Leu Asp Ser Thr Thr Thr Ala Asp Glu Leu Thr Ser Gly His Thr
225 230 235 240
Tyr Asp Ile Ser Val Thr Leu Thr Gly Val Tyr Asp Asn Thr Asp Glu
245 250 255
Gln His Pro Arg Met Ser Leu Ala Phe Glu Gln Asp Asn Gly Glu Arg
260 265 270
Arg Tyr Ile Thr Leu Trp Lys Asn Thr Thr Pro Lys Asp Val Phe Thr
275 280 285
Tyr Asp Tyr Ala Thr Gly Ser Thr Tyr Ile Phe Thr Asn Ile Asp Tyr
290 295 300
Glu Val Lys Asp Gly Tyr Glu Asn Leu Thr Ala Thr Tyr Gln Thr Thr
305 310 315 320
Val Glu Asn Ala Thr Ala Gln Glu Val Gly Thr Thr Asp Glu Asp Glu
325 330 335
Thr Phe Ala Gly Gly Glu Pro Leu Asp His His Leu Asp Asp Ala Leu
340 345 350
Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser Asp Ser Gly His Val
355 360 365
Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro Glu Trp Thr Leu His
370 375 380
Thr Tyr Thr Leu Thr Ala Thr Asp Gly Ala Lys Thr Asp Thr Glu Tyr
385 390 395 400
Ala Arg Arg Thr Leu Ala Tyr Thr Val Arg Gln Glu Leu Tyr Thr Asp
405 410 415
His Asp Ala Ala Pro Val Ala Thr Asp Gly Leu Met Leu Leu Thr Pro
420 425 430
Glu Pro Leu Gly Glu Thr Pro Leu Asp Leu Asp Cys Gly Val Arg Val
435 440 445
Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr Thr Ala Lys Asp Arg
450 455 460
Leu Leu Ala Arg Glu Leu Val Glu Glu Gly Leu Lys Arg Ser Leu Trp
465 470 475 480
Asp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val Leu Ser Lys Glu Pro
485 490 495
Val Leu Thr Cys Asp Glu Phe Asp Leu His Glu Arg Tyr Asp Leu Ser
500 505 510
Val Glu Val Gly His Ser Gly Arg Ala Tyr Leu His Ile Asn Phe Arg
515 520 525
His Arg Phe Val Pro Lys Leu Thr Leu Ala Asp Ile Asp Asp Asp Asn
530 535 540
Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr Arg Pro Arg Arg Gly
545 550 555 560
His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala Thr Asp Ser Leu Asn
565 570 575
Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His Arg Asn Asn Gln Thr
580 585 590
Pro Ile Asn Thr Asp Leu Leu Asp Ala Ile Glu Ala Ala Asp Arg Arg
595 600 605
Val Val Glu Thr Arg Arg Gln Gly His Gly Asp Asp Ala Val Ser Phe
610 615 620
Pro Gln Glu Leu Leu Ala Val Glu Pro Asn Thr His Gln Ile Lys Gln
625 630 635 640
Phe Ala Ser Asp Gly Phe His Gln Gln Ala Arg Ser Lys Thr Arg Leu
645 650 655
Ser Ala Ser Arg Cys Ser Glu Lys Ala Gln Ala Phe Ala Glu Arg Leu
660 665 670
Asp Pro Val Arg Leu Asn Gly Ser Thr Val Glu Phe Ser Ser Glu Phe
675 680 685
Phe Thr Gly Asn Asn Glu Gln Gln Leu Arg Leu Leu Tyr Glu Asn Gly
690 695 700
Glu Ser Val Leu Thr Phe Arg Asp Gly Ala Arg Gly Ala His Pro Asp
705 710 715 720
Glu Thr Phe Ser Lys Gly Ile Val Asn Pro Pro Glu Ser Phe Glu Val
725 730 735
Ala Val Val Leu Pro Glu Gln Gln Ala Asp Thr Cys Lys Ala Gln Trp
740 745 750
Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly Ala Pro Pro Thr Arg
755 760 765
Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser Pro Glu Ser Ile Ser
770 775 780
Leu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu Val Asp Ala Ala Phe
785 790 795 800
Val Val Leu Pro Pro Asp Gln Glu Gly Phe Ala Asp Leu Ala Ser Pro
805 810 815
Thr Glu Thr Tyr Asp Glu Leu Lys Lys Ala Leu Ala Asn Met Gly Ile
820 825 830
Tyr Ser Gln Met Ala Tyr Phe Asp Arg Phe Arg Asp Ala Lys Ile Phe
835 840 845
Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala Ala Ala Gly Gly Val
850 855 860
Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp Ala Asp Met Phe Ile
865 870 875 880
Gly Ile Ala Val Ser Arg Ser Tyr Pro Glu Asp Gly Ala Ser Gly Gln
885 890 895
Ile Asn Ile Ala Ala Thr Ala Thr Ala Val Tyr Lys Asp Gly Thr Ile
900 905 910
Leu Gly His Ser Ser Thr Arg Pro Gln Leu Gly Glu Lys Leu Gln Ser
915 920 925
Thr Asp Val Arg Asp Ile Met Lys Asn Ala Ile Leu Gly Tyr Gln Gln
930 935 940
Val Thr Gly Glu Ser Pro Thr His Ile Val Ile His Arg Asp Gly Phe
945 950 955 960
Met Asn Glu Asp Leu Asp Pro Ala Thr Glu Phe Leu Asn Glu Gln Gly
965 970 975
Val Glu Tyr Asp Ile Val Glu Ile Arg Lys Gln Pro Gln Thr Arg Leu
980 985 990
Leu Ala Val Ser Asp Val Gln Tyr Asp Thr Pro Val Lys Ser Ile Ala
995 1000 1005
Ala Ile Asn Gln Asn Glu Pro Arg Ala Thr Val Ala Thr Phe Gly
1010 1015 1020
Ala Pro Glu Tyr Leu Ala Thr Arg Asp Gly Gly Gly Leu Pro Arg
1025 1030 1035
Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr Asp Ile Glu Thr
1040 1045 1050
Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln Val
1055 1060 1065
His Asn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp
1070 1075 1080
Gln Ala Ser Thr His Ala Thr Lys Gly Tyr Leu Val Gln Thr Gly
1085 1090 1095
Ala Phe Glu Ser Asn Val Gly Phe Leu Gly Gly Gly Gly Ser Gly
1100 1105 1110
Gly Gly Gly Ser Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu
1115 1120 1125
Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu
1130 1135 1140
Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg
1145 1150 1155
Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly
1160 1165 1170
Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala
1175 1180 1185
Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp
1190 1195 1200
Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala
1205 1210 1215
Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys
1220 1225 1230
His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val
1235 1240 1245
Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn
1250 1255 1260
Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn
1265 1270 1275
Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met
1280 1285 1290
Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe
1295 1300 1305
Asn Asn Gly Glu Ile Asn Phe
1310 1315
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:11
<211> 1002
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> RsAgo
<309> 2016-11-11
<313> (18)..(794)
<300>
<308> linker
<309> 2016-11-11
<313> (795)..(804)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (805)..(1002)
<400> SEQ ID NO:11
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Met Ala Pro Val Gln Ala Ala Asp Glu Met Tyr Asp Ser Asn Pro
20 25 30
His Pro Asp Arg Arg Gln Leu Val Ser Asn Gly Phe Glu Val Asn Leu
35 40 45
Pro Asp Gln Val Glu Val Ile Val Arg Asp Leu Pro Asp Pro Ser Lys
50 55 60
Val Lys Glu Glu Arg Thr Arg Leu Met Gly Tyr Trp Phe Val His Trp
65 70 75 80
Phe Asp Gly Lys Leu Phe His Leu Arg Ile Lys Ala Gly Gly Pro Asn
85 90 95
Val Asp Gly Glu His Arg Ala Ile Arg Thr Ala Glu His Pro Trp Leu
100 105 110
Leu Arg Ala Arg Leu Asp Asp Ala Leu Glu Glu Ala Leu Pro Lys Tyr
115 120 125
Ala Ala Val Lys Lys Arg Pro Phe Thr Phe Leu Ala Gln Lys Asp Glu
130 135 140
Leu Ile Asp Ala Ala Ala Thr Ala Ala Gly Leu Ser His Arg Leu Leu
145 150 155 160
Asn Ser Phe Lys Val Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile Tyr
165 170 175
Glu Pro Val Asp Gly Thr Thr Arg Val Gly Val Phe Val Thr Ile Gly
180 185 190
Met Arg Tyr Asp Ile Glu Ala Ser Leu Arg Asp Leu Leu Glu Ala Gly
195 200 205
Ile Asp Leu Arg Gly Met Tyr Val Val Arg Arg Lys Arg Gln Pro Gly
210 215 220
Glu Arg Gly Leu Leu Gly Arg Val Arg Ala Ile Ser Asp Asp Met Val
225 230 235 240
Gln Leu Phe Glu Glu Thr Asp Leu Ala Ser Val Asn Val Asn Asp Ala
245 250 255
Lys Leu Glu Gly Ser Lys Glu Asn Phe Thr Arg Cys Leu Ser Ala Leu
260 265 270
Leu Gly His Asn Tyr Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu
275 280 285
Ala Gly Tyr Arg Thr Gly Pro Arg Phe Asp Asp Ala Val Arg Arg Met
290 295 300
Gly Glu Phe Leu Ala Lys Lys Pro Ile Arg Leu Ala Asp Asn Ile Asn
305 310 315 320
Ala Gln Val Gly Asp Arg Ile Val Phe Ser Asn Glu Gly Gln Ala Arg
325 330 335
Asn Val Arg Leu Ala Pro Lys Val Glu Tyr Val Phe Asp Arg Thr Gly
340 345 350
Ala Lys Ser Ala Glu Tyr Ala Trp Arg Gly Leu Ser Gln Phe Gly Pro
355 360 365
Phe Asp Arg Pro Ser Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val
370 375 380
Tyr Pro Ser Ser Thr Gln Gly Lys Val Glu Asn Phe Leu Ser Ala Phe
385 390 395 400
Arg Asp Gly Met Gly Ser Asn Tyr Ser Gly Phe Ser Lys Gly Phe Val
405 410 415
Asp Leu Met Gly Leu Thr Lys Val Glu Phe Val Met Cys Pro Val Glu
420 425 430
Val Ser Ser Ala Asp Arg Asn Gly Ala His Thr Lys Tyr Asn Ser Ala
435 440 445
Ile Glu Asp Lys Leu Ala Gly Ala Gly Glu Val His Ala Gly Ile Val
450 455 460
Val Leu Phe Glu Asp His Ala Arg Leu Pro Asp Asp Arg Asn Pro Tyr
465 470 475 480
Ile His Thr Lys Ser Leu Leu Leu Thr Leu Gly Val Pro Thr Gln Gln
485 490 495
Val Arg Met Pro Thr Val Leu Leu Glu Pro Lys Ser Leu Gln Tyr Thr
500 505 510
Leu Gln Asn Phe Ser Ile Ala Thr Tyr Ala Lys Leu Asn Gly Thr Pro
515 520 525
Trp Thr Val Asn His Asp Lys Ala Ile Asn Asp Glu Leu Val Val Gly
530 535 540
Met Gly Leu Ala Glu Leu Ser Gly Ser Arg Thr Glu Lys Arg Gln Arg
545 550 555 560
Phe Val Gly Ile Thr Thr Val Phe Ala Gly Asp Gly Ser Tyr Leu Leu
565 570 575
Gly Asn Val Ser Lys Glu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile
580 585 590
Arg Glu Ser Met Thr Gly Ile Leu Arg Glu Leu Lys Lys Arg Asn Asn
595 600 605
Trp Arg Pro Gly Asp Thr Val Arg Val Val Phe His Ala His Arg Pro
610 615 620
Leu Lys Arg Val Asp Val Ala Ser Ile Val Phe Glu Cys Thr Arg Glu
625 630 635 640
Ile Gly Ser Asp Gln Asn Ile Gln Met Ala Phe Val Thr Val Ser His
645 650 655
Asp His Pro Phe Val Leu Ile Asp Arg Ser Glu Arg Gly Leu Glu Ala
660 665 670
Tyr Lys Gly Ser Thr Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly
675 680 685
Ala Ile Ser Arg Val Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser
690 695 700
Pro Gln Leu Ile Lys Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu
705 710 715 720
Val Ser Leu His Pro Asp Ser Thr Phe Lys Asp Val Asp Tyr Leu Ala
725 730 735
Glu Gln Ala Leu Lys Phe Thr Ser Leu Ser Trp Arg Ser Thr Leu Pro
740 745 750
Ala Ala Thr Pro Val Thr Ile Phe Tyr Ser Glu Arg Ile Ala Glu Leu
755 760 765
Leu Gly Arg Leu Lys Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu Asn
770 775 780
Ile Lys Leu Lys Trp Ser Arg Trp Phe Leu Gly Gly Gly Gly Ser Gly
785 790 795 800
Gly Gly Gly Ser Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys
805 810 815
Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile
820 825 830
Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu
835 840 845
Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys
850 855 860
His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly
865 870 875 880
Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly
885 890 895
Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val
900 905 910
Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp
915 920 925
Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser
930 935 940
Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His
945 950 955 960
Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile
965 970 975
Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg
980 985 990
Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe
995 1000
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:12
<211> 1112
<212> PRT
<213> 人工序列
<300>
<308> NLS
<309> 2016-11-11
<313> (1)..(17)
<300>
<308> dNgAgo
<309> 2016-11-11
<313> (18)..(904)
<300>
<308> linker
<309> 2016-11-11
<313> (905)..(914)
<300>
<308> FOKI-C
<309> 2016-11-11
<313> (915)..(1112)
<400> SEQ ID NO:12
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Met Thr Val Ile Asp Leu Asp Ser Thr Thr Thr Ala Asp Glu Leu
20 25 30
Thr Ser Gly His Thr Tyr Asp Ile Ser Val Thr Leu Thr Gly Val Tyr
35 40 45
Asp Asn Thr Asp Glu Gln His Pro Arg Met Ser Leu Ala Phe Glu Gln
50 55 60
Asp Asn Gly Glu Arg Arg Tyr Ile Thr Leu Trp Lys Asn Thr Thr Pro
65 70 75 80
Lys Asp Val Phe Thr Tyr Asp Tyr Ala Thr Gly Ser Thr Tyr Ile Phe
85 90 95
Thr Asn Ile Asp Tyr Glu Val Lys Asp Gly Tyr Glu Asn Leu Thr Ala
100 105 110
Thr Tyr Gln Thr Thr Val Glu Asn Ala Thr Ala Gln Glu Val Gly Thr
115 120 125
Thr Asp Glu Asp Glu Thr Phe Ala Gly Gly Glu Pro Leu Asp His His
130 135 140
Leu Asp Asp Ala Leu Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser
145 150 155 160
Asp Ser Gly His Val Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro
165 170 175
Glu Trp Thr Leu His Thr Tyr Thr Leu Thr Ala Thr Asp Gly Ala Lys
180 185 190
Thr Asp Thr Glu Tyr Ala Arg Arg Thr Leu Ala Tyr Thr Val Arg Gln
195 200 205
Glu Leu Tyr Thr Asp His Asp Ala Ala Pro Val Ala Thr Asp Gly Leu
210 215 220
Met Leu Leu Thr Pro Glu Pro Leu Gly Glu Thr Pro Leu Asp Leu Asp
225 230 235 240
Cys Gly Val Arg Val Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr
245 250 255
Thr Ala Lys Asp Arg Leu Leu Ala Arg Glu Leu Val Glu Glu Gly Leu
260 265 270
Lys Arg Ser Leu Trp Asp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val
275 280 285
Leu Ser Lys Glu Pro Val Leu Thr Cys Asp Glu Phe Asp Leu His Glu
290 295 300
Arg Tyr Asp Leu Ser Val Glu Val Gly His Ser Gly Arg Ala Tyr Leu
305 310 315 320
His Ile Asn Phe Arg His Arg Phe Val Pro Lys Leu Thr Leu Ala Asp
325 330 335
Ile Asp Asp Asp Asn Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr
340 345 350
Arg Pro Arg Arg Gly His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala
355 360 365
Thr Asp Ser Leu Asn Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His
370 375 380
Arg Asn Asn Gln Thr Pro Ile Asn Thr Asp Leu Leu Asp Ala Ile Glu
385 390 395 400
Ala Ala Asp Arg Arg Val Val Glu Thr Arg Arg Gln Gly His Gly Asp
405 410 415
Asp Ala Val Ser Phe Pro Gln Glu Leu Leu Ala Val Glu Pro Asn Thr
420 425 430
His Gln Ile Lys Gln Phe Ala Ser Asp Gly Phe His Gln Gln Ala Arg
435 440 445
Ser Lys Thr Arg Leu Ser Ala Ser Arg Cys Ser Glu Lys Ala Gln Ala
450 455 460
Phe Ala Glu Arg Leu Asp Pro Val Arg Leu Asn Gly Ser Thr Val Glu
465 470 475 480
Phe Ser Ser Glu Phe Phe Thr Gly Asn Asn Glu Gln Gln Leu Arg Leu
485 490 495
Leu Tyr Glu Asn Gly Glu Ser Val Leu Thr Phe Arg Asp Gly Ala Arg
500 505 510
Gly Ala His Pro Asp Glu Thr Phe Ser Lys Gly Ile Val Asn Pro Pro
515 520 525
Glu Ser Phe Glu Val Ala Val Val Leu Pro Glu Gln Gln Ala Asp Thr
530 535 540
Cys Lys Ala Gln Trp Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly
545 550 555 560
Ala Pro Pro Thr Arg Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser
565 570 575
Pro Glu Ser Ile Ser Leu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu
580 585 590
Val Asp Ala Ala Phe Val Val Leu Pro Pro Asp Gln Glu Gly Phe Ala
595 600 605
Asp Leu Ala Ser Pro Thr Glu Thr Tyr Asp Glu Leu Lys Lys Ala Leu
610 615 620
Ala Asn Met Gly Ile Tyr Ser Gln Met Ala Tyr Phe Asp Arg Phe Arg
625 630 635 640
Asp Ala Lys Ile Phe Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala
645 650 655
Ala Ala Gly Gly Val Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp
660 665 670
Ala Asp Met Phe Ile Gly Ile Ala Val Ser Arg Ser Tyr Pro Glu Asp
675 680 685
Gly Ala Ser Gly Gln Ile Asn Ile Ala Ala Thr Ala Thr Ala Val Tyr
690 695 700
Lys Asp Gly Thr Ile Leu Gly His Ser Ser Thr Arg Pro Gln Leu Gly
705 710 715 720
Glu Lys Leu Gln Ser Thr Asp Val Arg Asp Ile Met Lys Asn Ala Ile
725 730 735
Leu Gly Tyr Gln Gln Val Thr Gly Glu Ser Pro Thr His Ile Val Ile
740 745 750
His Arg Asp Gly Phe Met Asn Glu Asp Leu Asp Pro Ala Thr Glu Phe
755 760 765
Leu Asn Glu Gln Gly Val Glu Tyr Asp Ile Val Glu Ile Arg Lys Gln
770 775 780
Pro Gln Thr Arg Leu Leu Ala Val Ser Asp Val Gln Tyr Asp Thr Pro
785 790 795 800
Val Lys Ser Ile Ala Ala Ile Asn Gln Asn Glu Pro Arg Ala Thr Val
805 810 815
Ala Thr Phe Gly Ala Pro Glu Tyr Leu Ala Thr Arg Asp Gly Gly Gly
820 825 830
Leu Pro Arg Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr Asp Ile
835 840 845
Glu Thr Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln
850 855 860
Val His Asn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp
865 870 875 880
Gln Ala Ser Thr His Ala Thr Lys Gly Tyr Leu Val Gln Thr Gly Ala
885 890 895
Phe Glu Ser Asn Val Gly Phe Leu Gly Gly Gly Gly Ser Gly Gly Gly
900 905 910
Gly Ser Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser
915 920 925
Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu
930 935 940
Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys
945 950 955 960
Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu
965 970 975
Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro
980 985 990
Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr
995 1000 1005
Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu
1010 1015 1020
Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp
1025 1030 1035
Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val
1040 1045 1050
Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu
1055 1060 1065
Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu
1070 1075 1080
Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu
1085 1090 1095
Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe
1100 1105 1110
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:13
<211> 2994
<212> DNA
<213> 人工序列
<400> SEQ ID NO:13
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atggctccag ttcaagctgc tgacgaaatg tacgactcta acccacaccc agacagaaga 720
caattggttt ctaacggttt cgaagttaac ttgccagacc aagttgaagt tatcgttaga 780
gacttgccag acccatctaa ggttaaggaa gaaagaacta gattgatggg ttactggttc 840
gttcactggt tcgacggtaa gttgttccac ttgagaatca aggctggtgg tccaaacgtt 900
gacggtgaac acagagctat cagaactgct gaacacccat ggttgttgag agctagattg 960
gacgacgctt tggaggaggc tctcccaaag tacgcggctg ttaagaagag accattcact 1020
ttcttggctc aaaaggacga attgatcgac gctgcagcta ctgcggctgg cttgtctcac 1080
agactcttga actctttcaa ggttatccca agattcgctt tgtctccaaa gatctacgaa 1140
ccagttgacg gtactactag agttggtgtt ttcgttacta tcggtatgag atacgacatc 1200
gaagcttctt tgagagactt gttggaagct ggtatcgact tgagaggtat gtacgttgtt 1260
agaagaaaga gacaaccagg tgaaagaggt ttgttgggta gagttagagc tatctctgac 1320
gacatggttc aattgttcga agaaactgac ttggcttctg ttaacgttaa cgacgctaag 1380
ttggaaggtt ctaaggaaaa cttcactaga tgtttgtctg ctttgttggg tcacaactac 1440
aagaagttgt tgaacgcttt ggacgaccaa gaagctggtt acagaactgg tccaagattc 1500
gacgacgctg ttagaagaat gggtgaattc ttggctaaga agccaatcag attggctgac 1560
aacatcaacg ctcaagttgg tgacagaatc gttttctcta acgaaggtca agctagaaac 1620
gttagattgg ctccaaaggt tgaatacgtt ttcgacagaa ctggtgctaa gtctgctgaa 1680
tacgcttgga gaggtttgtc tcaattcggt ccattcgaca gaccatcttt cgctaacaga 1740
tctccaagaa tactcgttgt gtacccaagc tctactcaag gtaaggttga aaacttcttg 1800
tctgctttca gagacggtat gggttctaac tactctggtt tctctaaggg tttcgttgac 1860
ttgatgggtt tgactaaggt tgaattcgtt atgtgtccag ttgaagtttc ttctgctgac 1920
agaaacggtg ctcacactaa gtacaactct gctatcgaag acaagttggc tggtgctggt 1980
gaagttcacg ctggtatcgt tgttttgttc gaagaccacg ctagattgcc agacgacaga 2040
aacccataca tccacactaa gtctttgttg ttgactttgg gtgttccaac tcaacaagtt 2100
agaatgccaa ctgttttgtt ggaaccaaag tctttgcaat acactttgca aaacttctct 2160
atcgctactt acgctaagtt gaacggtact ccatggactg ttaaccacga caaggctatc 2220
aacgacgaat tggttgttgg tatgggtttg gctgaattgt ctggttctag aactgaaaag 2280
agacaaagat tcgttggtat cactactgtt ttcgctggtg acggttctta cttgttgggt 2340
aacgtttcta aggaatgtga atacgaaggt tactctgacg ctatcagaga atctatgact 2400
ggtatcttga gagaattgaa gaagagaaac aactggagac caggtgacac tgttagagtt 2460
gttttccacg ctcacagacc attgaagaga gttgacgttg cttctatcgt tttcgaatgt 2520
actagagaaa tcggttctga ccaaaacatc caaatggctt tcgttactgt ttctcacgac 2580
cacccattcg ttttgatcga cagatctgaa agaggtttgg aagcttacaa gggttctact 2640
gctagaaagg gtgttttcgc tccaccaaga ggtgctatct ctagagttgg tagattgact 2700
agattgttgg ctgttaactc tccacaattg atcaagagag ctaacactcc attgccaact 2760
ccactcctcg ttagcttgca cccagactcg actttcaagg acgttgacta cctcgctgag 2820
caagctctca agtttacttc gttgtcttgg agatcgactt tgccagctgc tactccagtt 2880
actatcttct actctgaaag aatcgctgaa ttgttgggta gattgaagtc tatcccaaac 2940
tggtcttctg ctaacttgaa catcaagttg aagtggtcta gatggttctt gtaa 2994
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:14
<211> 2580
<212> DNA
<213> 人工序列
<400> SEQ ID NO:14
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atgtacttga acttgtacaa gatcgacatc ccaaagaaga tcaagagatt gtacttctac 720
aacccagaca tggaaccaaa gttgttcgct agaaacttgt ctcgtgttaa caacttcaag 780
ttccaagatt ctaacgactt ggtatggata gaaatcccag acatcgactt ccaaatcact 840
ccaaagaacg ttttccaata caaggttgaa aaggaagaaa tcatcaagga agaagaagac 900
aagaagttgt tcgttaagac tttgtacaag tacatcaaga agttgttctt ggacaacgac 960
ttctacttca agaagggtaa caacttcatc tctaactctg aagttttctc tttggactct 1020
aacgaaaacg ttaacgctca cttgacttac aagatcaaga tccacaacat ctctaacgaa 1080
tactacttgt ctatcttgcc aaagttcact ttcttgtcta aggaaccagc tttggaatct 1140
gctatcaagt ctggttactt gtacaacatc aagtctggta agtctttccc atacatctct 1200
ggtttggacg gtatcttgaa gatcgacatc ggtaacaacc aaatcgttga agttgcttac 1260
ccagaaaact acttgttcaa cttcactact agagacgctg aaaagtacgg tttctctaag 1320
gaagttcacg aaatctacaa gaacaaggtt ttcgaaggtt tcaagaagat cccaaagact 1380
ctcggcttct tgaacaagat aactaacttg aacgaaaact accaattgaa ggacggttac 1440
aagatcttca tcaacgttat ctacaagttc aagaacggtg aatctcgtta cgctaaggac 1500
gttttcaagt actctttcta caagaacgaa caaccattga aggctatctt cttcttctct 1560
tctaagaagc aattcttcga agttcaaaag tctttgaagg aattgttcca caacaagcac 1620
tctgttttct acagagctgc tgctgaattg ggtttctcta aggttgaatt cttgagagac 1680
tctaagacta agtcttctgc tttcttgtac aacccagaag aattcactgt taagaacact 1740
gaattcatca accaaatcga agacaacgtt atggctatcg ttttgttgga caagtacatc 1800
ggtaacatcg acccattggt tagaaacttc ccagacaact tgatcttgca accaatcttg 1860
aaggaaaagt tggaagacat caagccattc atcatcaagt cttacgttta caagatgggt 1920
aacttcatcc cagaatgtaa gccattcatc ttgaagaaga tggaagacaa ggaaaagaac 1980
ttgtacatcg gtatcgactt gtctcacgac acttacgcta gaaagactaa cttgtgtatc 2040
gctgctgttg acaacactgg tgacatcttg tacatcggta agcacaagaa cttggaattg 2100
aacgaaaaga tgaacttgga catcttggaa aaggaataca tcaaggcttt cgaaaagtac 2160
atcgaaaagt tcaacgtttc tccagagaac gtattcatcc tcagagacgg tagattcatc 2220
gaagacatcg aaatcatcaa gaacttcatc tcttacaacg acactaagta cactttggtt 2280
gaagttaaca agaacactaa catcaactct tacgacgact tgaaggaatg gatcatcaag 2340
ttggacgaaa acacttacat ctactaccca aagactttct tgaaccaaaa gggtgttgaa 2400
gttaagatct tggaaaacaa cactgactac actatcgaag aaatcatcga acaaatctac 2460
ttgttgacta gagttgctca ctctactcca tacactaact acaagttgcc atacccattg 2520
cacatcgcta acaaggttgc tttgactgac tacgaatgga agttgtacat cccatactaa 2580
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:15
<211> 2580
<212> DNA
<213> 人工序列
<400> SEQ ID NO:15
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atgtacttga acttgtacaa gatcgacatc ccaaagaaga tcaagagatt gtacttctac 720
aacccagaca tggaaccaaa gttgttcgct agaaacttgt ctcgtgttaa caacttcaag 780
ttccaagatt ctaacgactt ggtatggata gaaatcccag acatcgactt ccaaatcact 840
ccaaagaacg ttttccaata caaggttgaa aaggaagaaa tcatcaagga agaagaagac 900
aagaagttgt tcgttaagac tttgtacaag tacatcaaga agttgttctt ggacaacgac 960
ttctacttca agaagggtaa caacttcatc tctaactctg aagttttctc tttggactct 1020
aacgaaaacg ttaacgctca cttgacttac aagatcaaga tccacaacat ctctaacgaa 1080
tactacttgt ctatcttgcc aaagttcact ttcttgtcta aggaaccagc tttggaatct 1140
gctatcaagt ctggttactt gtacaacatc aagtctggta agtctttccc atacatctct 1200
ggtttggacg gtatcttgaa gatcgacatc ggtaacaacc aaatcgttga agttgcttac 1260
ccagaaaact acttgttcaa cttcactact agagacgctg aaaagtacgg tttctctaag 1320
gaagttcacg aaatctacaa gaacaaggtt ttcgaaggtt tcaagaagat cccaaagact 1380
ctcggcttct tgaacaagat aactaacttg aacgaaaact accaattgaa ggacggttac 1440
aagatcttca tcaacgttat ctacaagttc aagaacggtg aatctcgtta cgctaaggac 1500
gttttcaagt actctttcta caagaacgaa caaccattga aggctatctt cttcttctct 1560
tctaagaagc aattcttcga agttcaaaag tctttgaagg aattgttcca caacaagcac 1620
tctgttttct acagagctgc tgctgaattg ggtttctcta aggttgaatt cttgagagac 1680
tctaagacta agtcttctgc tttcttgtac aacccagaag aattcactgt taagaacact 1740
gaattcatca accaaatcga agacaacgtt atggctatcg ttttgttgga caagtacatc 1800
ggtaacatcg acccattggt tagaaacttc ccagacaact tgatcttgca accaatcttg 1860
aaggaaaagt tggaagacat caagccattc atcatcaagt cttacgttta caagatgggt 1920
aacttcatcc cagaatgtaa gccattcatc ttgaagaaga tggaagacaa ggaaaagaac 1980
ttgtacatcg gtatcgcttt gtctcacgac acttacgcta gaaagactaa cttgtgtatc 2040
gctgctgttg acaacactgg tgacatcttg tacatcggta agcacaagaa cttggaattg 2100
aacgaaaaga tgaacttgga catcttggaa aaggaataca tcaaggcttt cgaaaagtac 2160
atcgaaaagt tcaacgtttc tccagagaac gtattcatcc tcagagacgg tagattcatc 2220
gaagacatcg aaatcatcaa gaacttcatc tcttacaacg acactaagta cactttggtt 2280
gaagttaaca agaacactaa catcaactct tacgacgact tgaaggaatg gatcatcaag 2340
ttggacgaaa acacttacat ctactaccca aagactttct tgaaccaaaa gggtgttgaa 2400
gttaagatct tggaaaacaa cactgactac actatcgaag aaatcatcga acaaatctac 2460
ttgttgacta gagttgctca ctctactcca tacactaact acaagttgcc atacccattg 2520
cacatcgcta acaaggttgc tttgactgac tacgaatgga agttgtacat cccatactaa 2580
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:16
<211> 3324
<212> DNA
<213> 人工序列
<400> SEQ ID NO:16
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atgactgtta tcgacttgga ctctactact actgctgacg aattgacttc tggtcacact 720
tacgacatct ctgttacttt gactggtgtt tacgacaaca ctgacgaaca acacccaaga 780
atgtcgttgg cgttcgaaca agacaatggt gaaagaagat acatcacttt gtggaagaac 840
actacaccaa aggacgtgtt cacgtacgac tacgctactg gttctactta catcttcact 900
aacatcgact acgaagttaa ggacggttac gaaaacttga ctgctactta ccaaactact 960
gttgaaaacg ctactgctca agaagttggt actactgacg aagacgaaac tttcgctggt 1020
ggtgaaccat tggaccacca cttggacgac gctttgaacg aaactccaga cgacgctgaa 1080
actgaatctg actctggcca cgttatgact agcttcgctt ctagagacca attgccagaa 1140
tggactttgc acacttacac tttgactgct actgacggtg ctaagactga cactgaatac 1200
gctagaagaa ctttggctta cactgttaga caagaattgt acactgacca cgacgctgct 1260
cccgttgcta ctgacggctt gatgttgttg actccagaac cattgggtga aactccattg 1320
gacttggact gtggtgttag agttgaagct gacgaaacta gaactttgga ctacactact 1380
gctaaggaca gattgttggc tagagaattg gttgaagaag gtttgaagag atctttgtgg 1440
gacgactact tggttagagg tatcgacgaa gttttgtcta aggaaccagt tttgacttgt 1500
gacgaattcg acttgcacga aagatacgac ttgtctgttg aagttggtca ctctggtaga 1560
gcttacttgc acatcaactt cagacacaga ttcgttccaa agttgacttt ggctgacatc 1620
gacgacgaca acatctaccc aggtttgaga gttaagacta cttacagacc aagaagaggt 1680
cacatcgttt ggggtttgag agacgaatgt gctactgact ctttgaacac tttgggtaac 1740
caatctgttg ttgcttacca cagaaacaac caaactccaa tcaacactga cttgttggac 1800
gctatcgaag ctgctgacag aagagttgtt gaaactagaa gacaaggtca cggtgacgac 1860
gctgtttctt tcccacaaga attgttggct gttgaaccaa acactcacca aatcaagcaa 1920
ttcgcttctg acggtttcca ccaacaagct agatctaaga ctagattgtc tgcttctaga 1980
tgttctgaaa aggctcaagc tttcgctgaa agattggacc cagttagatt gaacggttct 2040
actgttgaat tctcttctga attcttcact ggtaacaacg aacaacaatt gagattgttg 2100
tacgaaaacg gtgaatctgt tttgactttc agagacggtg ctagaggtgc tcacccagac 2160
gaaactttct ctaagggtat cgttaaccca ccagaatctt tcgaagttgc ggttgtgctg 2220
ccagaacaac aagctgacac ttgtaaggct caatgggaca ctatggctga cttgttgaac 2280
caagctggtg ctccaccaac tagatctgaa actgttcaat acgacgcttt ctcttctcca 2340
gaatctatct ctttgaacgt tgctggtgct atcgacccat ctgaagttga cgctgctttc 2400
gttgttttgc caccagacca agaaggtttc gctgacttgg cttctccaac tgaaacttac 2460
gacgaattga agaaggcttt ggctaacatg ggtatctact ctcaaatggc ttacttcgac 2520
agattcagag acgctaagat cttctacact agaaacgttg ctttgggttt gttggctgct 2580
gctggtggtg ttgctttcac tactgaacac gctatgccag gtgacgctga catgttcatc 2640
ggcatagccg taagcagatc ttacccagaa gacggtgctt ctggtcaaat caacatcgct 2700
gctactgcta ctgctgttta caaggacggt actatcttgg gtcactcttc tactagacca 2760
caattgggtg aaaagttgca atctactgac gttagagaca tcatgaagaa cgctatcttg 2820
ggttaccaac aagttactgg tgaatctcca actcacatcg ttatccacag agacggtttc 2880
atgaacgaag acttggaccc agctactgaa ttcttgaacg aacaaggtgt tgaatacgac 2940
atcgttgaaa tcagaaagca accacaaact agattgttgg ctgtttctga cgttcaatac 3000
gacactccag ttaagtctat cgctgctatc aaccaaaacg aaccaagagc tacagttgct 3060
acattcggtg ctccagagta cttggctact agagacggtg gtggtttgcc aagaccaatc 3120
caaatcgaaa gagttgctgg tgaaactgac atcgaaactt tgactagaca agtttacttg 3180
ttgtctcaat ctcacatcca agttcacaac tctactgcta gattgccaat cactactgct 3240
tacgctgacc aagcttctac tcacgctact aagggttact tggttcaaac tggtgctttc 3300
gaatctaacg ttggtttctt gtaa 3324
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:17
<211> 3618
<212> DNA
<213> 人工序列
<400> SEQ ID NO:17
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atggctccag ttcaagctgc tgacgaaatg tacgactcta acccacaccc agacagaaga 720
caattggttt ctaacggttt cgaagttaac ttgccagacc aagttgaagt tatcgttaga 780
gacttgccag acccatctaa ggttaaggaa gaaagaacta gattgatggg ttactggttc 840
gttcactggt tcgacggtaa gttgttccac ttgagaatca aggctggtgg tccaaacgtt 900
gacggtgaac acagagctat cagaactgct gaacacccat ggttgttgag agctagattg 960
gacgacgctt tggaggaggc tctcccaaag tacgcggctg ttaagaagag accattcact 1020
ttcttggctc aaaaggacga attgatcgac gctgcagcta ctgcggctgg cttgtctcac 1080
agactcttga actctttcaa ggttatccca agattcgctt tgtctccaaa gatctacgaa 1140
ccagttgacg gtactactag agttggtgtt ttcgttacta tcggtatgag atacgacatc 1200
gaagcttctt tgagagactt gttggaagct ggtatcgact tgagaggtat gtacgttgtt 1260
agaagaaaga gacaaccagg tgaaagaggt ttgttgggta gagttagagc tatctctgac 1320
gacatggttc aattgttcga agaaactgac ttggcttctg ttaacgttaa cgacgctaag 1380
ttggaaggtt ctaaggaaaa cttcactaga tgtttgtctg ctttgttggg tcacaactac 1440
aagaagttgt tgaacgcttt ggacgaccaa gaagctggtt acagaactgg tccaagattc 1500
gacgacgctg ttagaagaat gggtgaattc ttggctaaga agccaatcag attggctgac 1560
aacatcaacg ctcaagttgg tgacagaatc gttttctcta acgaaggtca agctagaaac 1620
gttagattgg ctccaaaggt tgaatacgtt ttcgacagaa ctggtgctaa gtctgctgaa 1680
tacgcttgga gaggtttgtc tcaattcggt ccattcgaca gaccatcttt cgctaacaga 1740
tctccaagaa tactcgttgt gtacccaagc tctactcaag gtaaggttga aaacttcttg 1800
tctgctttca gagacggtat gggttctaac tactctggtt tctctaaggg tttcgttgac 1860
ttgatgggtt tgactaaggt tgaattcgtt atgtgtccag ttgaagtttc ttctgctgac 1920
agaaacggtg ctcacactaa gtacaactct gctatcgaag acaagttggc tggtgctggt 1980
gaagttcacg ctggtatcgt tgttttgttc gaagaccacg ctagattgcc agacgacaga 2040
aacccataca tccacactaa gtctttgttg ttgactttgg gtgttccaac tcaacaagtt 2100
agaatgccaa ctgttttgtt ggaaccaaag tctttgcaat acactttgca aaacttctct 2160
atcgctactt acgctaagtt gaacggtact ccatggactg ttaaccacga caaggctatc 2220
aacgacgaat tggttgttgg tatgggtttg gctgaattgt ctggttctag aactgaaaag 2280
agacaaagat tcgttggtat cactactgtt ttcgctggtg acggttctta cttgttgggt 2340
aacgtttcta aggaatgtga atacgaaggt tactctgacg ctatcagaga atctatgact 2400
ggtatcttga gagaattgaa gaagagaaac aactggagac caggtgacac tgttagagtt 2460
gttttccacg ctcacagacc attgaagaga gttgacgttg cttctatcgt tttcgaatgt 2520
actagagaaa tcggttctga ccaaaacatc caaatggctt tcgttactgt ttctcacgac 2580
cacccattcg ttttgatcga cagatctgaa agaggtttgg aagcttacaa gggttctact 2640
gctagaaagg gtgttttcgc tccaccaaga ggtgctatct ctagagttgg tagattgact 2700
agattgttgg ctgttaactc tccacaattg atcaagagag ctaacactcc attgccaact 2760
ccactcctcg ttagcttgca cccagactcg actttcaagg acgttgacta cctcgctgag 2820
caagctctca agtttacttc gttgtcttgg agatcgactt tgccagctgc tactccagtt 2880
actatcttct actctgaaag aatcgctgaa ttgttgggta gattgaagtc tatcccaaac 2940
tggtcttctg ctaacttgaa catcaagttg aagtggtcta gatggttctt gggaggtggg 3000
ggatctggtg gaggtgggtc atcctcccag ctggttaagt ccgagctgga ggagaagaag 3060
tccgagctgc gccacaagct gaagtacgtt ccacacgagt acatcgagct gatcgagatc 3120
gctcgcaact ccacccagga ccgcatcctg gagatgaagg ttatggagtt cttcatgaag 3180
gtttacggct accgcggcaa gcacctgggc ggctcccgca agccagacgg cgctatctac 3240
accgttggct ccccaatcga ctacggcgtt atcgttgaca ccaaggctta ctccggcggc 3300
tacaacctgc caatcggcca ggctgacgag atgcagcgct acgttgagga gaaccagacc 3360
cgcaacaagc acatcaaccc aaacgagtgg tggaaggttt acccatcctc cgttaccgag 3420
ttcaagttcc tgttcgtttc cggccacttc aagggcaact acaaggctca gctgacccgc 3480
ctgaaccaca tcaccaactg caacggcgct gttctgtccg ttgaggagct gctgatcggc 3540
ggcgagatga tcaaggctgg caccctgacc ctggaggagg ttcgccgcaa gttcaacaac 3600
ggcgagatca acttctaa 3618
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:18
<211> 3204
<212> DNA
<213> 人工序列
<400> SEQ ID NO:18
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atgtacttga acttgtacaa gatcgacatc ccaaagaaga tcaagagatt gtacttctac 720
aacccagaca tggaaccaaa gttgttcgct agaaacttgt ctcgtgttaa caacttcaag 780
ttccaagatt ctaacgactt ggtatggata gaaatcccag acatcgactt ccaaatcact 840
ccaaagaacg ttttccaata caaggttgaa aaggaagaaa tcatcaagga agaagaagac 900
aagaagttgt tcgttaagac tttgtacaag tacatcaaga agttgttctt ggacaacgac 960
ttctacttca agaagggtaa caacttcatc tctaactctg aagttttctc tttggactct 1020
aacgaaaacg ttaacgctca cttgacttac aagatcaaga tccacaacat ctctaacgaa 1080
tactacttgt ctatcttgcc aaagttcact ttcttgtcta aggaaccagc tttggaatct 1140
gctatcaagt ctggttactt gtacaacatc aagtctggta agtctttccc atacatctct 1200
ggtttggacg gtatcttgaa gatcgacatc ggtaacaacc aaatcgttga agttgcttac 1260
ccagaaaact acttgttcaa cttcactact agagacgctg aaaagtacgg tttctctaag 1320
gaagttcacg aaatctacaa gaacaaggtt ttcgaaggtt tcaagaagat cccaaagact 1380
ctcggcttct tgaacaagat aactaacttg aacgaaaact accaattgaa ggacggttac 1440
aagatcttca tcaacgttat ctacaagttc aagaacggtg aatctcgtta cgctaaggac 1500
gttttcaagt actctttcta caagaacgaa caaccattga aggctatctt cttcttctct 1560
tctaagaagc aattcttcga agttcaaaag tctttgaagg aattgttcca caacaagcac 1620
tctgttttct acagagctgc tgctgaattg ggtttctcta aggttgaatt cttgagagac 1680
tctaagacta agtcttctgc tttcttgtac aacccagaag aattcactgt taagaacact 1740
gaattcatca accaaatcga agacaacgtt atggctatcg ttttgttgga caagtacatc 1800
ggtaacatcg acccattggt tagaaacttc ccagacaact tgatcttgca accaatcttg 1860
aaggaaaagt tggaagacat caagccattc atcatcaagt cttacgttta caagatgggt 1920
aacttcatcc cagaatgtaa gccattcatc ttgaagaaga tggaagacaa ggaaaagaac 1980
ttgtacatcg gtatcgcttt gtctcacgac acttacgcta gaaagactaa cttgtgtatc 2040
gctgctgttg acaacactgg tgacatcttg tacatcggta agcacaagaa cttggaattg 2100
aacgaaaaga tgaacttgga catcttggaa aaggaataca tcaaggcttt cgaaaagtac 2160
atcgaaaagt tcaacgtttc tccagagaac gtattcatcc tcagagacgg tagattcatc 2220
gaagacatcg aaatcatcaa gaacttcatc tcttacaacg acactaagta cactttggtt 2280
gaagttaaca agaacactaa catcaactct tacgacgact tgaaggaatg gatcatcaag 2340
ttggacgaaa acacttacat ctactaccca aagactttct tgaaccaaaa gggtgttgaa 2400
gttaagatct tggaaaacaa cactgactac actatcgaag aaatcatcga acaaatctac 2460
ttgttgacta gagttgctca ctctactcca tacactaact acaagttgcc atacccattg 2520
cacatcgcta acaaggttgc tttgactgac tacgaatgga agttgtacat cccatacgga 2580
ggtgggggat ctggtggagg tgggtcatcc tcccagctgg ttaagtccga gctggaggag 2640
aagaagtccg agctgcgcca caagctgaag tacgttccac acgagtacat cgagctgatc 2700
gagatcgctc gcaactccac ccaggaccgc atcctggaga tgaaggttat ggagttcttc 2760
atgaaggttt acggctaccg cggcaagcac ctgggcggct cccgcaagcc agacggcgct 2820
atctacaccg ttggctcccc aatcgactac ggcgttatcg ttgacaccaa ggcttactcc 2880
ggcggctaca acctgccaat cggccaggct gacgagatgc agcgctacgt tgaggagaac 2940
cagacccgca acaagcacat caacccaaac gagtggtgga aggtttaccc atcctccgtt 3000
accgagttca agttcctgtt cgtttccggc cacttcaagg gcaactacaa ggctcagctg 3060
acccgcctga accacatcac caactgcaac ggcgctgttc tgtccgttga ggagctgctg 3120
atcggcggcg agatgatcaa ggctggcacc ctgaccctgg aggaggttcg ccgcaagttc 3180
aacaacggcg agatcaactt ctaa 3204
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:19
<211> 3948
<212> DNA
<213> 人工序列
<400> SEQ ID NO:19
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc ctcttctcaa 60
ttggttaagt ctgaattgga agaaaagaag tctgaattga gacacaagtt gaagtacgtt 120
ccacacgaat acatcgaatt gatcgaaatc gctagaaact ctactcaaga cagaatcttg 180
gaaatgaagg ttatggaatt tttcatgaag gtttacggtt acagaggtaa gcacttgggt 240
ggttctcgta agccagacgg tgctatctac actgttggtt ctccaatcga ctacggtgtt 300
atcgttgaca ctaaggctta ctctggtggt tacaacttgc caatcggtca agctgacgaa 360
atgcaaagat acgttgaaga aaaccaaact agaaacaagc acatcaaccc aaacgaatgg 420
tggaaggttt acccatcttc tgttactgaa tttaagttct tgttcgtttc tggtcacttc 480
aagggtaact acaaggctca attgactaga ttgaaccaca tcactaactg taacggtgct 540
gttttgtctg ttgaagaatt gttgatcggt ggtgaaatga tcaaggctgg tactttgact 600
ttggaagaag ttagaagaaa gttcaacaac ggtgaaatca acttcggtgg tggtggttct 660
atgactgtta tcgacttgga ctctactact actgctgacg aattgacttc tggtcacact 720
tacgacatct ctgttacttt gactggtgtt tacgacaaca ctgacgaaca acacccaaga 780
atgtcgttgg cgttcgaaca agacaatggt gaaagaagat acatcacttt gtggaagaac 840
actacaccaa aggacgtgtt cacgtacgac tacgctactg gttctactta catcttcact 900
aacatcgact acgaagttaa ggacggttac gaaaacttga ctgctactta ccaaactact 960
gttgaaaacg ctactgctca agaagttggt actactgacg aagacgaaac tttcgctggt 1020
ggtgaaccat tggaccacca cttggacgac gctttgaacg aaactccaga cgacgctgaa 1080
actgaatctg actctggcca cgttatgact agcttcgctt ctagagacca attgccagaa 1140
tggactttgc acacttacac tttgactgct actgacggtg ctaagactga cactgaatac 1200
gctagaagaa ctttggctta cactgttaga caagaattgt acactgacca cgacgctgct 1260
cccgttgcta ctgacggctt gatgttgttg actccagaac cattgggtga aactccattg 1320
gacttggact gtggtgttag agttgaagct gacgaaacta gaactttgga ctacactact 1380
gctaaggaca gattgttggc tagagaattg gttgaagaag gtttgaagag atctttgtgg 1440
gacgactact tggttagagg tatcgacgaa gttttgtcta aggaaccagt tttgacttgt 1500
gacgaattcg acttgcacga aagatacgac ttgtctgttg aagttggtca ctctggtaga 1560
gcttacttgc acatcaactt cagacacaga ttcgttccaa agttgacttt ggctgacatc 1620
gacgacgaca acatctaccc aggtttgaga gttaagacta cttacagacc aagaagaggt 1680
cacatcgttt ggggtttgag agacgaatgt gctactgact ctttgaacac tttgggtaac 1740
caatctgttg ttgcttacca cagaaacaac caaactccaa tcaacactga cttgttggac 1800
gctatcgaag ctgctgacag aagagttgtt gaaactagaa gacaaggtca cggtgacgac 1860
gctgtttctt tcccacaaga attgttggct gttgaaccaa acactcacca aatcaagcaa 1920
ttcgcttctg acggtttcca ccaacaagct agatctaaga ctagattgtc tgcttctaga 1980
tgttctgaaa aggctcaagc tttcgctgaa agattggacc cagttagatt gaacggttct 2040
actgttgaat tctcttctga attcttcact ggtaacaacg aacaacaatt gagattgttg 2100
tacgaaaacg gtgaatctgt tttgactttc agagacggtg ctagaggtgc tcacccagac 2160
gaaactttct ctaagggtat cgttaaccca ccagaatctt tcgaagttgc ggttgtgctg 2220
ccagaacaac aagctgacac ttgtaaggct caatgggaca ctatggctga cttgttgaac 2280
caagctggtg ctccaccaac tagatctgaa actgttcaat acgacgcttt ctcttctcca 2340
gaatctatct ctttgaacgt tgctggtgct atcgacccat ctgaagttga cgctgctttc 2400
gttgttttgc caccagacca agaaggtttc gctgacttgg cttctccaac tgaaacttac 2460
gacgaattga agaaggcttt ggctaacatg ggtatctact ctcaaatggc ttacttcgac 2520
agattcagag acgctaagat cttctacact agaaacgttg ctttgggttt gttggctgct 2580
gctggtggtg ttgctttcac tactgaacac gctatgccag gtgacgctga catgttcatc 2640
ggcatagccg taagcagatc ttacccagaa gacggtgctt ctggtcaaat caacatcgct 2700
gctactgcta ctgctgttta caaggacggt actatcttgg gtcactcttc tactagacca 2760
caattgggtg aaaagttgca atctactgac gttagagaca tcatgaagaa cgctatcttg 2820
ggttaccaac aagttactgg tgaatctcca actcacatcg ttatccacag agacggtttc 2880
atgaacgaag acttggaccc agctactgaa ttcttgaacg aacaaggtgt tgaatacgac 2940
atcgttgaaa tcagaaagca accacaaact agattgttgg ctgtttctga cgttcaatac 3000
gacactccag ttaagtctat cgctgctatc aaccaaaacg aaccaagagc tacagttgct 3060
acattcggtg ctccagagta cttggctact agagacggtg gtggtttgcc aagaccaatc 3120
caaatcgaaa gagttgctgg tgaaactgac atcgaaactt tgactagaca agtttacttg 3180
ttgtctcaat ctcacatcca agttcacaac tctactgcta gattgccaat cactactgct 3240
tacgctgacc aagcttctac tcacgctact aagggttact tggttcaaac tggtgctttc 3300
gaatctaacg ttggtttctt gggaggtggg ggatctggtg gaggtgggtc atcctcccag 3360
ctggttaagt ccgagctgga ggagaagaag tccgagctgc gccacaagct gaagtacgtt 3420
ccacacgagt acatcgagct gatcgagatc gctcgcaact ccacccagga ccgcatcctg 3480
gagatgaagg ttatggagtt cttcatgaag gtttacggct accgcggcaa gcacctgggc 3540
ggctcccgca agccagacgg cgctatctac accgttggct ccccaatcga ctacggcgtt 3600
atcgttgaca ccaaggctta ctccggcggc tacaacctgc caatcggcca ggctgacgag 3660
atgcagcgct acgttgagga gaaccagacc cgcaacaagc acatcaaccc aaacgagtgg 3720
tggaaggttt acccatcctc cgttaccgag ttcaagttcc tgttcgtttc cggccacttc 3780
aagggcaact acaaggctca gctgacccgc ctgaaccaca tcaccaactg caacggcgct 3840
gttctgtccg ttgaggagct gctgatcggc ggcgagatga tcaaggctgg caccctgacc 3900
ctggaggagg ttcgccgcaa gttcaacaac ggcgagatca acttctaa 3948
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:20
<211> 3009
<212> DNA
<213> 人工序列
<400> SEQ ID NO:20
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc catggctcca 60
gttcaagctg ctgacgaaat gtacgactct aacccacacc cagacagaag acaattggtt 120
tctaacggtt tcgaagttaa cttgccagac caagttgaag ttatcgttag agacttgcca 180
gacccatcta aggttaagga agaaagaact agattgatgg gttactggtt cgttcactgg 240
ttcgacggta agttgttcca cttgagaatc aaggctggtg gtccaaacgt tgacggtgaa 300
cacagagcta tcagaactgc tgaacaccca tggttgttga gagctagatt ggacgacgct 360
ttggaggagg ctctcccaaa gtacgcggct gttaagaaga gaccattcac tttcttggct 420
caaaaggacg aattgatcga cgctgcagct actgcggctg gcttgtctca cagactcttg 480
aactctttca aggttatccc aagattcgct ttgtctccaa agatctacga accagttgac 540
ggtactacta gagttggtgt tttcgttact atcggtatga gatacgacat cgaagcttct 600
ttgagagact tgttggaagc tggtatcgac ttgagaggta tgtacgttgt tagaagaaag 660
agacaaccag gtgaaagagg tttgttgggt agagttagag ctatctctga cgacatggtt 720
caattgttcg aagaaactga cttggcttct gttaacgtta acgacgctaa gttggaaggt 780
tctaaggaaa acttcactag atgtttgtct gctttgttgg gtcacaacta caagaagttg 840
ttgaacgctt tggacgacca agaagctggt tacagaactg gtccaagatt cgacgacgct 900
gttagaagaa tgggtgaatt cttggctaag aagccaatca gattggctga caacatcaac 960
gctcaagttg gtgacagaat cgttttctct aacgaaggtc aagctagaaa cgttagattg 1020
gctccaaagg ttgaatacgt tttcgacaga actggtgcta agtctgctga atacgcttgg 1080
agaggtttgt ctcaattcgg tccattcgac agaccatctt tcgctaacag atctccaaga 1140
atactcgttg tgtacccaag ctctactcaa ggtaaggttg aaaacttctt gtctgctttc 1200
agagacggta tgggttctaa ctactctggt ttctctaagg gtttcgttga cttgatgggt 1260
ttgactaagg ttgaattcgt tatgtgtcca gttgaagttt cttctgctga cagaaacggt 1320
gctcacacta agtacaactc tgctatcgaa gacaagttgg ctggtgctgg tgaagttcac 1380
gctggtatcg ttgttttgtt cgaagaccac gctagattgc cagacgacag aaacccatac 1440
atccacacta agtctttgtt gttgactttg ggtgttccaa ctcaacaagt tagaatgcca 1500
actgttttgt tggaaccaaa gtctttgcaa tacactttgc aaaacttctc tatcgctact 1560
tacgctaagt tgaacggtac tccatggact gttaaccacg acaaggctat caacgacgaa 1620
ttggttgttg gtatgggttt ggctgaattg tctggttcta gaactgaaaa gagacaaaga 1680
ttcgttggta tcactactgt tttcgctggt gacggttctt acttgttggg taacgtttct 1740
aaggaatgtg aatacgaagg ttactctgac gctatcagag aatctatgac tggtatcttg 1800
agagaattga agaagagaaa caactggaga ccaggtgaca ctgttagagt tgttttccac 1860
gctcacagac cattgaagag agttgacgtt gcttctatcg ttttcgaatg tactagagaa 1920
atcggttctg accaaaacat ccaaatggct ttcgttactg tttctcacga ccacccattc 1980
gttttgatcg acagatctga aagaggtttg gaagcttaca agggttctac tgctagaaag 2040
ggtgttttcg ctccaccaag aggtgctatc tctagagttg gtagattgac tagattgttg 2100
gctgttaact ctccacaatt gatcaagaga gctaacactc cattgccaac tccactcctc 2160
gttagcttgc acccagactc gactttcaag gacgttgact acctcgctga gcaagctctc 2220
aagtttactt cgttgtcttg gagatcgact ttgccagctg ctactccagt tactatcttc 2280
tactctgaaa gaatcgctga attgttgggt agattgaagt ctatcccaaa ctggtcttct 2340
gctaacttga acatcaagtt gaagtggtct agatggttct tgggaggtgg gggatctggt 2400
ggaggtgggt catcctccca gctggttaag tccgagctgg aggagaagaa gtccgagctg 2460
cgccacaagc tgaagtacgt tccacacgag tacatcgagc tgatcgagat cgctcgcaac 2520
tccacccagg accgcatcct ggagatgaag gttatggagt tcttcatgaa ggtttacggc 2580
taccgcggca agcacctggg cggctcccgc aagccagacg gcgctatcta caccgttggc 2640
tccccaatcg actacggcgt tatcgttgac accaaggctt actccggcgg ctacaacctg 2700
ccaatcggcc aggctgacga gatgcagcgc tacgttgagg agaaccagac ccgcaacaag 2760
cacatcaacc caaacgagtg gtggaaggtt tacccatcct ccgttaccga gttcaagttc 2820
ctgttcgttt ccggccactt caagggcaac tacaaggctc agctgacccg cctgaaccac 2880
atcaccaact gcaacggcgc tgttctgtcc gttgaggagc tgctgatcgg cggcgagatg 2940
atcaaggctg gcaccctgac cctggaggag gttcgccgca agttcaacaa cggcgagatc 3000
aacttctaa 3009
SEQUENCE LISTING
<110> 仪宏
<120> 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
<130>
<160> 21
<170> PatentIn version 3.3
<210> SEQ ID NO:21
<211> 3339
<212> DNA
<213> 人工序列
<400> SEQ ID NO:21
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc catgactgtt 60
atcgacttgg actctactac tactgctgac gaattgactt ctggtcacac ttacgacatc 120
tctgttactt tgactggtgt ttacgacaac actgacgaac aacacccaag aatgtcgttg 180
gcgttcgaac aagacaatgg tgaaagaaga tacatcactt tgtggaagaa cactacacca 240
aaggacgtgt tcacgtacga ctacgctact ggttctactt acatcttcac taacatcgac 300
tacgaagtta aggacggtta cgaaaacttg actgctactt accaaactac tgttgaaaac 360
gctactgctc aagaagttgg tactactgac gaagacgaaa ctttcgctgg tggtgaacca 420
ttggaccacc acttggacga cgctttgaac gaaactccag acgacgctga aactgaatct 480
gactctggcc acgttatgac tagcttcgct tctagagacc aattgccaga atggactttg 540
cacacttaca ctttgactgc tactgacggt gctaagactg acactgaata cgctagaaga 600
actttggctt acactgttag acaagaattg tacactgacc acgacgctgc tcccgttgct 660
actgacggct tgatgttgtt gactccagaa ccattgggtg aaactccatt ggacttggac 720
tgtggtgtta gagttgaagc tgacgaaact agaactttgg actacactac tgctaaggac 780
agattgttgg ctagagaatt ggttgaagaa ggtttgaaga gatctttgtg ggacgactac 840
ttggttagag gtatcgacga agttttgtct aaggaaccag ttttgacttg tgacgaattc 900
gacttgcacg aaagatacga cttgtctgtt gaagttggtc actctggtag agcttacttg 960
cacatcaact tcagacacag attcgttcca aagttgactt tggctgacat cgacgacgac 1020
aacatctacc caggtttgag agttaagact acttacagac caagaagagg tcacatcgtt 1080
tggggtttga gagacgaatg tgctactgac tctttgaaca ctttgggtaa ccaatctgtt 1140
gttgcttacc acagaaacaa ccaaactcca atcaacactg acttgttgga cgctatcgaa 1200
gctgctgaca gaagagttgt tgaaactaga agacaaggtc acggtgacga cgctgtttct 1260
ttcccacaag aattgttggc tgttgaacca aacactcacc aaatcaagca attcgcttct 1320
gacggtttcc accaacaagc tagatctaag actagattgt ctgcttctag atgttctgaa 1380
aaggctcaag ctttcgctga aagattggac ccagttagat tgaacggttc tactgttgaa 1440
ttctcttctg aattcttcac tggtaacaac gaacaacaat tgagattgtt gtacgaaaac 1500
ggtgaatctg ttttgacttt cagagacggt gctagaggtg ctcacccaga cgaaactttc 1560
tctaagggta tcgttaaccc accagaatct ttcgaagttg cggttgtgct gccagaacaa 1620
caagctgaca cttgtaaggc tcaatgggac actatggctg acttgttgaa ccaagctggt 1680
gctccaccaa ctagatctga aactgttcaa tacgacgctt tctcttctcc agaatctatc 1740
tctttgaacg ttgctggtgc tatcgaccca tctgaagttg acgctgcttt cgttgttttg 1800
ccaccagacc aagaaggttt cgctgacttg gcttctccaa ctgaaactta cgacgaattg 1860
aagaaggctt tggctaacat gggtatctac tctcaaatgg cttacttcga cagattcaga 1920
gacgctaaga tcttctacac tagaaacgtt gctttgggtt tgttggctgc tgctggtggt 1980
gttgctttca ctactgaaca cgctatgcca ggtgacgctg acatgttcat cggcatagcc 2040
gtaagcagat cttacccaga agacggtgct tctggtcaaa tcaacatcgc tgctactgct 2100
actgctgttt acaaggacgg tactatcttg ggtcactctt ctactagacc acaattgggt 2160
gaaaagttgc aatctactga cgttagagac atcatgaaga acgctatctt gggttaccaa 2220
caagttactg gtgaatctcc aactcacatc gttatccaca gagacggttt catgaacgaa 2280
gacttggacc cagctactga attcttgaac gaacaaggtg ttgaatacga catcgttgaa 2340
atcagaaagc aaccacaaac tagattgttg gctgtttctg acgttcaata cgacactcca 2400
gttaagtcta tcgctgctat caaccaaaac gaaccaagag ctacagttgc tacattcggt 2460
gctccagagt acttggctac tagagacggt ggtggtttgc caagaccaat ccaaatcgaa 2520
agagttgctg gtgaaactga catcgaaact ttgactagac aagtttactt gttgtctcaa 2580
tctcacatcc aagttcacaa ctctactgct agattgccaa tcactactgc ttacgctgac 2640
caagcttcta ctcacgctac taagggttac ttggttcaaa ctggtgcttt cgaatctaac 2700
gttggtttct tgggaggtgg gggatctggt ggaggtgggt catcctccca gctggttaag 2760
tccgagctgg aggagaagaa gtccgagctg cgccacaagc tgaagtacgt tccacacgag 2820
tacatcgagc tgatcgagat cgctcgcaac tccacccagg accgcatcct ggagatgaag 2880
gttatggagt tcttcatgaa ggtttacggc taccgcggca agcacctggg cggctcccgc 2940
aagccagacg gcgctatcta caccgttggc tccccaatcg actacggcgt tatcgttgac 3000
accaaggctt actccggcgg ctacaacctg ccaatcggcc aggctgacga gatgcagcgc 3060
tacgttgagg agaaccagac ccgcaacaag cacatcaacc caaacgagtg gtggaaggtt 3120
tacccatcct ccgttaccga gttcaagttc ctgttcgttt ccggccactt caagggcaac 3180
tacaaggctc agctgacccg cctgaaccac atcaccaact gcaacggcgc tgttctgtcc 3240
gttgaggagc tgctgatcgg cggcgagatg atcaaggctg gcaccctgac cctggaggag 3300
gttcgccgca agttcaacaa cggcgagatc aacttctaa 3339

Claims (31)

1.一种嵌合蛋白pAgoE，其特征在于：所述的嵌合蛋白pAgoE含有具有基因组靶向定位功能的pAgo结构域，以及与所述pAgo结构域连接的、具有基因组切割或修饰活性的效应结构域E；
所述pAgo结构域与任意一种使用向导的天然原核Argonaute蛋白的氨基酸序列同源性大于30％，且：
所述pAgo结构域采用来自Rhodobacter sphaeroides ATCC 17025的、NCBI序列号为ABP72561.1的RsAgo，以构建得到RsAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用来自Marinitoga piezophila的、NCBI序列号为WP_014295921.1的MpAgo，以构建得到MpAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用MpAgo的PIWI结构域核酸酶活性失活的人工突变体dMpAgo，以构建得到dMpAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用NgAgo的PIWI结构域核酸酶失活的人工突变体dNgAgo，以构建得到的dNgAgoE型的嵌合蛋白pAgoE；
其中，构建得到的RsAgoE型的嵌合蛋白pAgoE的氨基酸序列为：
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala SerSer Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys LeuLys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr GlnAsp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr ArgGly Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly SerPro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn LeuPro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg AsnLys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu PheLys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr ArgLeu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu IleGly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys PheAsn Asn Gly Glu Ile Asn Phe Gly Gly Gly Gly Ser Met Ala Pro Val Gln Ala AlaAsp Glu Met Tyr Asp Ser Asn Pro His Pro Asp Arg Arg Gln Leu Val Ser Asn GlyPhe Glu Val Asn Leu Pro Asp Gln Val Glu Val Ile Val Arg Asp Leu Pro Asp ProSer Lys Val Lys Glu Glu Arg Thr Arg Leu Met Gly Tyr Trp Phe Val His Trp PheAsp Gly Lys Leu Phe His Leu Arg Ile Lys Ala Gly Gly Pro Asn Val Asp Gly GluHis Arg Ala Ile Arg Thr Ala Glu His Pro Trp Leu Leu Arg Ala Arg Leu Asp AspAla Leu Glu Glu Ala Leu Pro Lys Tyr Ala Ala Val Lys Lys Arg Pro Phe Thr PheLeu Ala Gln Lys Asp Glu Leu Ile Asp Ala Ala Ala Thr Ala Ala Gly Leu Ser HisArg Leu Leu Asn Ser Phe Lys Val Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile TyrGlu Pro Val Asp Gly Thr Thr Arg Val Gly Val Phe Val Thr Ile Gly Met Arg TyrAsp Ile Glu Ala Ser Leu Arg Asp Leu Leu Glu Ala Gly Ile Asp Leu Arg Gly MetTyr Val Val Arg Arg Lys Arg Gln Pro Gly Glu Arg Gly Leu Leu Gly Arg Val ArgAla Ile Ser Asp Asp Met Val Gln Leu Phe Glu Glu Thr Asp Leu Ala Ser Val AsnVal Asn Asp Ala Lys Leu Glu Gly Ser Lys Glu Asn Phe Thr Arg Cys Leu Ser AlaLeu Leu Gly His Asn Tyr Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala GlyTyr Arg Thr Gly Pro Arg Phe Asp Asp Ala Val Arg Arg Met Gly Glu Phe Leu AlaLys Lys Pro Ile Arg Leu Ala Asp Asn Ile Asn Ala Gln Val Gly Asp Arg Ile ValPhe Ser Asn Glu Gly Gln Ala Arg Asn Val Arg Leu Ala Pro Lys Val Glu Tyr ValPhe Asp Arg Thr Gly Ala Lys Ser Ala Glu Tyr Ala Trp Arg Gly Leu Ser Gln PheGly Pro Phe Asp Arg Pro Ser Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val TyrPro Ser Ser Thr Gln Gly Lys Val Glu Asn Phe Leu Ser Ala Phe Arg Asp Gly MetGly Ser Asn Tyr Ser Gly Phe Ser Lys Gly Phe Val Asp Leu Met Gly Leu Thr LysVal Glu Phe Val Met Cys Pro Val Glu Val Ser Ser Ala Asp Arg Asn Gly Ala HisThr Lys Tyr Asn Ser Ala Ile Glu Asp Lys Leu Ala Gly Ala Gly Glu Val His AlaGly Ile Val Val Leu Phe Glu Asp His Ala Arg Leu Pro Asp Asp Arg Asn Pro TyrIle His Thr Lys Ser Leu Leu Leu Thr Leu Gly Val Pro Thr Gln Gln Val Arg MetPro Thr Val Leu Leu Glu Pro Lys Ser Leu Gln Tyr Thr Leu Gln Asn Phe Ser IleAla Thr Tyr Ala Lys Leu Asn Gly Thr Pro Trp Thr Val Asn His Asp Lys Ala IleAsn Asp Glu Leu Val Val Gly Met Gly Leu Ala Glu Leu Ser Gly Ser Arg Thr GluLys Arg Gln Arg Phe Val Gly Ile Thr Thr Val Phe Ala Gly Asp Gly Ser Tyr LeuLeu Gly Asn Val Ser Lys Glu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile Arg GluSer Met Thr Gly Ile Leu Arg Glu Leu Lys Lys Arg Asn Asn Trp Arg Pro Gly AspThr Val Arg Val Val Phe His Ala His Arg Pro Leu Lys Arg Val Asp Val Ala SerIle Val Phe Glu Cys Thr Arg Glu Ile Gly Ser Asp Gln Asn Ile Gln Met Ala PheVal Thr Val Ser His Asp His Pro Phe Val Leu Ile Asp Arg Ser Glu Arg Gly LeuGlu Ala Tyr Lys Gly Ser Thr Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly AlaIle Ser Arg Val Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser Pro Gln Leu IleLys Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu Val Ser Leu His Pro Asp SerThr Phe Lys Asp Val Asp Tyr Leu Ala Glu Gln Ala Leu Lys Phe Thr Ser Leu SerTrp Arg Ser Thr Leu Pro Ala Ala Thr Pro Val Thr Ile Phe Tyr Ser Glu Arg IleAla Glu Leu Leu Gly Arg Leu Lys Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu AsnIle Lys Leu Lys Trp Ser Arg Trp Phe Leu；
或者，构建得到的RsAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Ala Pro Val Gln Ala Ala Asp Glu Met Tyr Asp SerAsn Pro His Pro Asp Arg Arg Gln Leu Val Ser Asn Gly Phe Glu Val Asn Leu ProAsp Gln Val Glu Val Ile Val Arg Asp Leu Pro Asp Pro Ser Lys Val Lys Glu GluArg Thr Arg Leu Met Gly Tyr Trp Phe Val His Trp Phe Asp Gly Lys Leu Phe HisLeu Arg Ile Lys Ala Gly Gly Pro Asn Val Asp Gly Glu His Arg Ala Ile Arg ThrAla Glu His Pro Trp Leu Leu Arg Ala Arg Leu Asp Asp Ala Leu Glu Glu Ala LeuPro Lys Tyr Ala Ala Val Lys Lys Arg Pro Phe Thr Phe Leu Ala Gln Lys Asp GluLeu Ile Asp Ala Ala Ala Thr Ala Ala Gly Leu Ser His Arg Leu Leu Asn Ser PheLys Val Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile Tyr Glu Pro Val Asp Gly ThrThr Arg Val Gly Val Phe Val Thr Ile Gly Met Arg Tyr Asp Ile Glu Ala Ser LeuArg Asp Leu Leu Glu Ala Gly Ile Asp Leu Arg Gly Met Tyr Val Val Arg Arg LysArg Gln Pro Gly Glu Arg Gly Leu Leu Gly Arg Val Arg Ala Ile Ser Asp Asp MetVal Gln Leu Phe Glu Glu Thr Asp Leu Ala Ser Val Asn Val Asn Asp Ala Lys LeuGlu Gly Ser Lys Glu Asn Phe Thr Arg Cys Leu Ser Ala Leu Leu Gly His Asn TyrLys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala Gly Tyr Arg Thr Gly Pro ArgPhe Asp Asp Ala Val Arg Arg Met Gly Glu Phe Leu Ala Lys Lys Pro Ile Arg LeuAla Asp Asn Ile Asn Ala Gln Val Gly Asp Arg Ile Val Phe Ser Asn Glu Gly GlnAla Arg Asn Val Arg Leu Ala Pro Lys Val Glu Tyr Val Phe Asp Arg Thr Gly AlaLys Ser Ala Glu Tyr Ala Trp Arg Gly Leu Ser Gln Phe Gly Pro Phe Asp Arg ProSer Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val Tyr Pro Ser Ser Thr Gln GlyLys Val Glu Asn Phe Leu Ser Ala Phe Arg Asp Gly Met Gly Ser Asn Tyr Ser GlyPhe Ser Lys Gly Phe Val Asp Leu Met Gly Leu Thr Lys Val Glu Phe Val Met CysPro Val Glu Val Ser Ser Ala Asp Arg Asn Gly Ala His Thr Lys Tyr Asn Ser AlaIle Glu Asp Lys Leu Ala Gly Ala Gly Glu Val His Ala Gly Ile Val Val Leu PheGlu Asp His Ala Arg Leu Pro Asp Asp Arg Asn Pro Tyr Ile His Thr Lys Ser LeuLeu Leu Thr Leu Gly Val Pro Thr Gln Gln Val Arg Met Pro Thr Val Leu Leu GluPro Lys Ser Leu Gln Tyr Thr Leu Gln Asn Phe Ser Ile Ala Thr Tyr Ala Lys LeuAsn Gly Thr Pro Trp Thr Val Asn His Asp Lys Ala Ile Asn Asp Glu Leu Val ValGly Met Gly Leu Ala Glu Leu Ser Gly Ser Arg Thr Glu Lys Arg Gln Arg Phe ValGly Ile Thr Thr Val Phe Ala Gly Asp Gly Ser Tyr Leu Leu Gly Asn Val Ser LysGlu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile Arg Glu Ser Met Thr Gly Ile LeuArg Glu Leu Lys Lys Arg Asn Asn Trp Arg Pro Gly Asp Thr Val Arg Val Val PheHis Ala His Arg Pro Leu Lys Arg Val Asp Val Ala Ser Ile Val Phe Glu Cys ThrArg Glu Ile Gly Ser Asp Gln Asn Ile Gln Met Ala Phe Val Thr Val Ser His AspHis Pro Phe Val Leu Ile Asp Arg Ser Glu Arg Gly Leu Glu Ala Tyr Lys Gly SerThr Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly Ala Ile Ser Arg Val Gly ArgLeu Thr Arg Leu Leu Ala Val Asn Ser Pro Gln Leu Ile Lys Arg Ala Asn Thr ProLeu Pro Thr Pro Leu Leu Val Ser Leu His Pro Asp Ser Thr Phe Lys Asp Val AspTyr Leu Ala Glu Gln Ala Leu Lys Phe Thr Ser Leu Ser Trp Arg Ser Thr Leu ProAla Ala Thr Pro Val Thr Ile Phe Tyr Ser Glu Arg Ile Ala Glu Leu Leu Gly ArgLeu Lys Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu Asn Ile Lys Leu Lys Trp SerArg Trp Phe Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Gln Leu ValLys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val ProHis Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile LeuGlu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His LeuGly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp TyrGly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly GlnAla Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile AsnPro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu PheVal Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His IleThr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu MetIle Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly GluIle Asn Phe；
或者，构建得到的RsAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Met Ala Pro Val Gln Ala AlaAsp Glu Met Tyr Asp Ser Asn Pro His Pro Asp Arg Arg Gln Leu Val Ser Asn GlyPhe Glu Val Asn Leu Pro Asp Gln Val Glu Val Ile Val Arg Asp Leu Pro Asp ProSer Lys Val Lys Glu Glu Arg Thr Arg Leu Met Gly Tyr Trp Phe Val His Trp PheAsp Gly Lys Leu Phe His Leu Arg Ile Lys Ala Gly Gly Pro Asn Val Asp Gly GluHis Arg Ala Ile Arg Thr Ala Glu His Pro Trp Leu Leu Arg Ala Arg Leu Asp AspAla Leu Glu Glu Ala Leu Pro Lys Tyr Ala Ala Val Lys Lys Arg Pro Phe Thr PheLeu Ala Gln Lys Asp Glu Leu Ile Asp Ala Ala Ala Thr Ala Ala Gly Leu Ser HisArg Leu Leu Asn Ser Phe Lys Val Ile Pro Arg Phe Ala Leu Ser Pro Lys Ile TyrGlu Pro Val Asp Gly Thr Thr Arg Val Gly Val Phe Val Thr Ile Gly Met Arg TyrAsp Ile Glu Ala Ser Leu Arg Asp Leu Leu Glu Ala Gly Ile Asp Leu Arg Gly MetTyr Val Val Arg Arg Lys Arg Gln Pro Gly Glu Arg Gly Leu Leu Gly Arg Val ArgAla Ile Ser Asp Asp Met Val Gln Leu Phe Glu Glu Thr Asp Leu Ala Ser Val AsnVal Asn Asp Ala Lys Leu Glu Gly Ser Lys Glu Asn Phe Thr Arg Cys Leu Ser AlaLeu Leu Gly His Asn Tyr Lys Lys Leu Leu Asn Ala Leu Asp Asp Gln Glu Ala GlyTyr Arg Thr Gly Pro Arg Phe Asp Asp Ala Val Arg Arg Met Gly Glu Phe Leu AlaLys Lys Pro Ile Arg Leu Ala Asp Asn Ile Asn Ala Gln Val Gly Asp Arg Ile ValPhe Ser Asn Glu Gly Gln Ala Arg Asn Val Arg Leu Ala Pro Lys Val Glu Tyr ValPhe Asp Arg Thr Gly Ala Lys Ser Ala Glu Tyr Ala Trp Arg Gly Leu Ser Gln PheGly Pro Phe Asp Arg Pro Ser Phe Ala Asn Arg Ser Pro Arg Ile Leu Val Val TyrPro Ser Ser Thr Gln Gly Lys Val Glu Asn Phe Leu Ser Ala Phe Arg Asp Gly MetGly Ser Asn Tyr Ser Gly Phe Ser Lys Gly Phe Val Asp Leu Met Gly Leu Thr LysVal Glu Phe Val Met Cys Pro Val Glu Val Ser Ser Ala Asp Arg Asn Gly Ala HisThr Lys Tyr Asn Ser Ala Ile Glu Asp Lys Leu Ala Gly Ala Gly Glu Val His AlaGly Ile Val Val Leu Phe Glu Asp His Ala Arg Leu Pro Asp Asp Arg Asn Pro TyrIle His Thr Lys Ser Leu Leu Leu Thr Leu Gly Val Pro Thr Gln Gln Val Arg MetPro Thr Val Leu Leu Glu Pro Lys Ser Leu Gln Tyr Thr Leu Gln Asn Phe Ser IleAla Thr Tyr Ala Lys Leu Asn Gly Thr Pro Trp Thr Val Asn His Asp Lys Ala IleAsn Asp Glu Leu Val Val Gly Met Gly Leu Ala Glu Leu Ser Gly Ser Arg Thr GluLys Arg Gln Arg Phe Val Gly Ile Thr Thr Val Phe Ala Gly Asp Gly Ser Tyr LeuLeu Gly Asn Val Ser Lys Glu Cys Glu Tyr Glu Gly Tyr Ser Asp Ala Ile Arg GluSer Met Thr Gly Ile Leu Arg Glu Leu Lys Lys Arg Asn Asn Trp Arg Pro Gly AspThr Val Arg Val Val Phe His Ala His Arg Pro Leu Lys Arg Val Asp Val Ala SerIle Val Phe Glu Cys Thr Arg Glu Ile Gly Ser Asp Gln Asn Ile Gln Met Ala PheVal Thr Val Ser His Asp His Pro Phe Val Leu Ile Asp Arg Ser Glu Arg Gly LeuGlu Ala Tyr Lys Gly Ser Thr Ala Arg Lys Gly Val Phe Ala Pro Pro Arg Gly AlaIle Ser Arg Val Gly Arg Leu Thr Arg Leu Leu Ala Val Asn Ser Pro Gln Leu IleLys Arg Ala Asn Thr Pro Leu Pro Thr Pro Leu Leu Val Ser Leu His Pro Asp SerThr Phe Lys Asp Val Asp Tyr Leu Ala Glu Gln Ala Leu Lys Phe Thr Ser Leu SerTrp Arg Ser Thr Leu Pro Ala Ala Thr Pro Val Thr Ile Phe Tyr Ser Glu Arg IleAla Glu Leu Leu Gly Arg Leu Lys Ser Ile Pro Asn Trp Ser Ser Ala Asn Leu AsnIle Lys Leu Lys Trp Ser Arg Trp Phe Leu Gly Gly Gly Gly Ser Gly Gly Gly GlySer Ser Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg HisLys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn SerThr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr GlyTyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr ValGly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly TyrAsn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln ThrArg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val ThrGlu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln LeuThr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu LeuLeu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg ArgLys Phe Asn Asn Gly Glu Ile Asn Phe；
其中，构建得到的MpAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Tyr Leu Asn Leu Tyr Lys Ile Asp Ile Pro Lys LysIle Lys Arg Leu Tyr Phe Tyr Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg AsnLeu Ser Arg Val Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile GluIle Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys Val GluLys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe Val Lys Thr Leu TyrLys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp Phe Tyr Phe Lys Lys Gly Asn AsnPhe Ile Ser Asn Ser Glu Val Phe Ser Leu Asp Ser Asn Glu Asn Val Asn Ala HisLeu Thr Tyr Lys Ile Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile LeuPro Lys Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser GlyTyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser Gly Leu Asp GlyIle Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val Glu Val Ala Tyr Pro Glu AsnTyr Leu Phe Asn Phe Thr Thr Arg Asp Ala Glu Lys Tyr Gly Phe Ser Lys Glu ValHis Glu Ile Tyr Lys Asn Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr LeuGly Phe Leu Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly TyrLys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg Tyr Ala LysAsp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro Leu Lys Ala Ile Phe PhePhe Ser Ser Lys Lys Gln Phe Phe Glu Val Gln Lys Ser Leu Lys Glu Leu Phe HisAsn Lys His Ser Val Phe Tyr Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val GluPhe Leu Arg Asp Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu PheThr Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala Ile ValLeu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg Asn Phe Pro Asp AsnLeu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu Glu Asp Ile Lys Pro Phe Ile IleLys Ser Tyr Val Tyr Lys Met Gly Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile LeuLys Lys Met Glu Asp Lys Glu Lys Asn Leu Tyr Ile Gly Ile Asp Leu Ser His AspThr Tyr Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp IleLeu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met Asn Leu Asp IleLeu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr Ile Glu Lys Phe Asn Val SerPro Glu Asn Val Phe Ile Leu Arg Asp Gly Arg Phe Ile Glu Asp Ile Glu Ile IleLys Asn Phe Ile Ser Tyr Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys AsnThr Asn Ile Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu AsnThr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu Val Lys IleLeu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile Glu Gln Ile Tyr Leu LeuThr Arg Val Ala His Ser Thr Pro Tyr Thr Asn Tyr Lys Leu Pro Tyr Pro Leu HisIle Ala Asn Lys Val Ala Leu Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr；
其中，构建得到的dMpAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Tyr Leu Asn Leu Tyr Lys Ile Asp Ile Pro Lys LysIle Lys Arg Leu Tyr Phe Tyr Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg AsnLeu Ser Arg Val Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile GluIle Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys Val GluLys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe Val Lys Thr Leu TyrLys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp Phe Tyr Phe Lys Lys Gly Asn AsnPhe Ile Ser Asn Ser Glu Val Phe Ser Leu Asp Ser Asn Glu Asn Val Asn Ala HisLeu Thr Tyr Lys Ile Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile LeuPro Lys Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser GlyTyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser Gly Leu Asp GlyIle Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val Glu Val Ala Tyr Pro Glu AsnTyr Leu Phe Asn Phe Thr Thr Arg Asp Ala Glu Lys Tyr Gly Phe Ser Lys Glu ValHis Glu Ile Tyr Lys Asn Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr LeuGly Phe Leu Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly TyrLys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg Tyr Ala LysAsp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro Leu Lys Ala Ile Phe PhePhe Ser Ser Lys Lys Gln Phe Phe Glu Val Gln Lys Ser Leu Lys Glu Leu Phe HisAsn Lys His Ser Val Phe Tyr Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val GluPhe Leu Arg Asp Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu PheThr Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala Ile ValLeu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg Asn Phe Pro Asp AsnLeu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu Glu Asp Ile Lys Pro Phe Ile IleLys Ser Tyr Val Tyr Lys Met Gly Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile LeuLys Lys Met Glu Asp Lys Glu Lys Asn Leu Tyr Ile Gly Ile Ala Leu Ser His AspThr Tyr Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp IleLeu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met Asn Leu Asp IleLeu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr Ile Glu Lys Phe Asn Val SerPro Glu Asn Val Phe Ile Leu Arg Asp Gly Arg Phe Ile Glu Asp Ile Glu Ile IleLys Asn Phe Ile Ser Tyr Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys AsnThr Asn Ile Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu AsnThr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu Val Lys IleLeu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile Glu Gln Ile Tyr Leu LeuThr Arg Val Ala His Ser Thr Pro Tyr Thr Asn Tyr Lys Leu Pro Tyr Pro Leu HisIle Ala Asn Lys Val Ala Leu Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr；
或者，构建得到的dMpAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Tyr Leu Asn Leu Tyr Lys Ile Asp Ile Pro Lys LysIle Lys Arg Leu Tyr Phe Tyr Asn Pro Asp Met Glu Pro Lys Leu Phe Ala Arg AsnLeu Ser Arg Val Asn Asn Phe Lys Phe Gln Asp Ser Asn Asp Leu Val Trp Ile GluIle Pro Asp Ile Asp Phe Gln Ile Thr Pro Lys Asn Val Phe Gln Tyr Lys Val GluLys Glu Glu Ile Ile Lys Glu Glu Glu Asp Lys Lys Leu Phe Val Lys Thr Leu TyrLys Tyr Ile Lys Lys Leu Phe Leu Asp Asn Asp Phe Tyr Phe Lys Lys Gly Asn AsnPhe Ile Ser Asn Ser Glu Val Phe Ser Leu Asp Ser Asn Glu Asn Val Asn Ala HisLeu Thr Tyr Lys Ile Lys Ile His Asn Ile Ser Asn Glu Tyr Tyr Leu Ser Ile LeuPro Lys Phe Thr Phe Leu Ser Lys Glu Pro Ala Leu Glu Ser Ala Ile Lys Ser GlyTyr Leu Tyr Asn Ile Lys Ser Gly Lys Ser Phe Pro Tyr Ile Ser Gly Leu Asp GlyIle Leu Lys Ile Asp Ile Gly Asn Asn Gln Ile Val Glu Val Ala Tyr Pro Glu AsnTyr Leu Phe Asn Phe Thr Thr Arg Asp Ala Glu Lys Tyr Gly Phe Ser Lys Glu ValHis Glu Ile Tyr Lys Asn Lys Val Phe Glu Gly Phe Lys Lys Ile Pro Lys Thr LeuGly Phe Leu Asn Lys Ile Thr Asn Leu Asn Glu Asn Tyr Gln Leu Lys Asp Gly TyrLys Ile Phe Ile Asn Val Ile Tyr Lys Phe Lys Asn Gly Glu Ser Arg Tyr Ala LysAsp Val Phe Lys Tyr Ser Phe Tyr Lys Asn Glu Gln Pro Leu Lys Ala Ile Phe PhePhe Ser Ser Lys Lys Gln Phe Phe Glu Val Gln Lys Ser Leu Lys Glu Leu Phe HisAsn Lys His Ser Val Phe Tyr Arg Ala Ala Ala Glu Leu Gly Phe Ser Lys Val GluPhe Leu Arg Asp Ser Lys Thr Lys Ser Ser Ala Phe Leu Tyr Asn Pro Glu Glu PheThr Val Lys Asn Thr Glu Phe Ile Asn Gln Ile Glu Asp Asn Val Met Ala Ile ValLeu Leu Asp Lys Tyr Ile Gly Asn Ile Asp Pro Leu Val Arg Asn Phe Pro Asp AsnLeu Ile Leu Gln Pro Ile Leu Lys Glu Lys Leu Glu Asp Ile Lys Pro Phe Ile IleLys Ser Tyr Val Tyr Lys Met Gly Asn Phe Ile Pro Glu Cys Lys Pro Phe Ile LeuLys Lys Met Glu Asp Lys Glu Lys Asn Leu Tyr Ile Gly Ile Ala Leu Ser His AspThr Tyr Ala Arg Lys Thr Asn Leu Cys Ile Ala Ala Val Asp Asn Thr Gly Asp IleLeu Tyr Ile Gly Lys His Lys Asn Leu Glu Leu Asn Glu Lys Met Asn Leu Asp IleLeu Glu Lys Glu Tyr Ile Lys Ala Phe Glu Lys Tyr Ile Glu Lys Phe Asn Val SerPro Glu Asn Val Phe Ile Leu Arg Asp Gly Arg Phe Ile Glu Asp Ile Glu Ile IleLys Asn Phe Ile Ser Tyr Asn Asp Thr Lys Tyr Thr Leu Val Glu Val Asn Lys AsnThr Asn Ile Asn Ser Tyr Asp Asp Leu Lys Glu Trp Ile Ile Lys Leu Asp Glu AsnThr Tyr Ile Tyr Tyr Pro Lys Thr Phe Leu Asn Gln Lys Gly Val Glu Val Lys IleLeu Glu Asn Asn Thr Asp Tyr Thr Ile Glu Glu Ile Ile Glu Gln Ile Tyr Leu LeuThr Arg Val Ala His Ser Thr Pro Tyr Thr Asn Tyr Lys Leu Pro Tyr Pro Leu HisIle Ala Asn Lys Val Ala Leu Thr Asp Tyr Glu Trp Lys Leu Tyr Ile Pro Tyr GlyGly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Gln Leu Val Lys Ser Glu Leu GluGlu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile GluLeu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val MetGlu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg LysPro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val AspThr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met GlnArg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp TrpLys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His PheLys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn GlyAla Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly ThrLeu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe；
其中，构建得到的dNgAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Thr Val Ile Asp Leu Asp Ser Thr Thr Thr Ala AspGlu Leu Thr Ser Gly His Thr Tyr Asp Ile Ser Val Thr Leu Thr Gly Val Tyr AspAsn Thr Asp Glu Gln His Pro Arg Met Ser Leu Ala Phe Glu Gln Asp Asn Gly GluArg Arg Tyr Ile Thr Leu Trp Lys Asn Thr Thr Pro Lys Asp Val Phe Thr Tyr AspTyr Ala Thr Gly Ser Thr Tyr Ile Phe Thr Asn Ile Asp Tyr Glu Val Lys Asp GlyTyr Glu Asn Leu Thr Ala Thr Tyr Gln Thr Thr Val Glu Asn Ala Thr Ala Gln GluVal Gly Thr Thr Asp Glu Asp Glu Thr Phe Ala Gly Gly Glu Pro Leu Asp His HisLeu Asp Asp Ala Leu Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser Asp Ser GlyHis Val Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro Glu Trp Thr Leu His ThrTyr Thr Leu Thr Ala Thr Asp Gly Ala Lys Thr Asp Thr Glu Tyr Ala Arg Arg ThrLeu Ala Tyr Thr Val Arg Gln Glu Leu Tyr Thr Asp His Asp Ala Ala Pro Val AlaThr Asp Gly Leu Met Leu Leu Thr Pro Glu Pro Leu Gly Glu Thr Pro Leu Asp LeuAsp Cys Gly Val Arg Val Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr Thr AlaLys Asp Arg Leu Leu Ala Arg Glu Leu Val Glu Glu Gly Leu Lys Arg Ser Leu TrpAsp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val Leu Ser Lys Glu Pro Val Leu ThrCys Asp Glu Phe Asp Leu His Glu Arg Tyr Asp Leu Ser Val Glu Val Gly His SerGly Arg Ala Tyr Leu His Ile Asn Phe Arg His Arg Phe Val Pro Lys Leu Thr LeuAla Asp Ile Asp Asp Asp Asn Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr ArgPro Arg Arg Gly His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala Thr Asp Ser LeuAsn Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His Arg Asn Asn Gln Thr Pro IleAsn Thr Asp Leu Leu Asp Ala Ile Glu Ala Ala Asp Arg Arg Val Val Glu Thr ArgArg Gln Gly His Gly Asp Asp Ala Val Ser Phe Pro Gln Glu Leu Leu Ala Val GluPro Asn Thr His Gln Ile Lys Gln Phe Ala Ser Asp Gly Phe His Gln Gln Ala ArgSer Lys Thr Arg Leu Ser Ala Ser Arg Cys Ser Glu Lys Ala Gln Ala Phe Ala GluArg Leu Asp Pro Val Arg Leu Asn Gly Ser Thr Val Glu Phe Ser Ser Glu Phe PheThr Gly Asn Asn Glu Gln Gln Leu Arg Leu Leu Tyr Glu Asn Gly Glu Ser Val LeuThr Phe Arg Asp Gly Ala Arg Gly Ala His Pro Asp Glu Thr Phe Ser Lys Gly IleVal Asn Pro Pro Glu Ser Phe Glu Val Ala Val Val Leu Pro Glu Gln Gln Ala AspThr Cys Lys Ala Gln Trp Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly Ala ProPro Thr Arg Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser Pro Glu Ser Ile SerLeu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu Val Asp Ala Ala Phe Val Val LeuPro Pro Asp Gln Glu Gly Phe Ala Asp Leu Ala Ser Pro Thr Glu Thr Tyr Asp GluLeu Lys Lys Ala Leu Ala Asn Met Gly Ile Tyr Ser Gln Met Ala Tyr Phe Asp ArgPhe Arg Asp Ala Lys Ile Phe Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala AlaAla Gly Gly Val Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp Ala Asp Met PheIle Gly Ile Ala Val Ser Arg Ser Tyr Pro Glu Asp Gly Ala Ser Gly Gln Ile AsnIle Ala Ala Thr Ala Thr Ala Val Tyr Lys Asp Gly Thr Ile Leu Gly His Ser SerThr Arg Pro Gln Leu Gly Glu Lys Leu Gln Ser Thr Asp Val Arg Asp Ile Met LysAsn Ala Ile Leu Gly Tyr Gln Gln Val Thr Gly Glu Ser Pro Thr His Ile Val IleHis Arg Asp Gly Phe Met Asn Glu Asp Leu Asp Pro Ala Thr Glu Phe Leu Asn GluGln Gly Val Glu Tyr Asp Ile Val Glu Ile Arg Lys Gln Pro Gln Thr Arg Leu LeuAla Val Ser Asp Val Gln Tyr Asp Thr Pro Val Lys Ser Ile Ala Ala Ile Asn GlnAsn Glu Pro Arg Ala Thr Val Ala Thr Phe Gly Ala Pro Glu Tyr Leu Ala Thr ArgAsp Gly Gly Gly Leu Pro Arg Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr AspIle Glu Thr Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln Val HisAsn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp Gln Ala Ser Thr HisAla Thr Lys Gly Tyr Leu Val Gln Thr Gly Ala Phe Glu Ser Asn Val Gly Phe Leu；
或者，构建得到的dNgAgoE型的嵌合蛋白pAgoE的氨基酸序列为：Met Ala Pro Lys LysLys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Ser Ser Gln Leu Val Lys SerGlu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His GluTyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu MetLys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly GlySer Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly ValIle Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala AspGlu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro AsnGlu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val SerGly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr AsnCys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile LysAla Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile AsnPhe Gly Gly Gly Gly Ser Met Thr Val Ile Asp Leu Asp Ser Thr Thr Thr Ala AspGlu Leu Thr Ser Gly His Thr Tyr Asp Ile Ser Val Thr Leu Thr Gly Val Tyr AspAsn Thr Asp Glu Gln His Pro Arg Met Ser Leu Ala Phe Glu Gln Asp Asn Gly GluArg Arg Tyr Ile Thr Leu Trp Lys Asn Thr Thr Pro Lys Asp Val Phe Thr Tyr AspTyr Ala Thr Gly Ser Thr Tyr Ile Phe Thr Asn Ile Asp Tyr Glu Val Lys Asp GlyTyr Glu Asn Leu Thr Ala Thr Tyr Gln Thr Thr Val Glu Asn Ala Thr Ala Gln GluVal Gly Thr Thr Asp Glu Asp Glu Thr Phe Ala Gly Gly Glu Pro Leu Asp His HisLeu Asp Asp Ala Leu Asn Glu Thr Pro Asp Asp Ala Glu Thr Glu Ser Asp Ser GlyHis Val Met Thr Ser Phe Ala Ser Arg Asp Gln Leu Pro Glu Trp Thr Leu His ThrTyr Thr Leu Thr Ala Thr Asp Gly Ala Lys Thr Asp Thr Glu Tyr Ala Arg Arg ThrLeu Ala Tyr Thr Val Arg Gln Glu Leu Tyr Thr Asp His Asp Ala Ala Pro Val AlaThr Asp Gly Leu Met Leu Leu Thr Pro Glu Pro Leu Gly Glu Thr Pro Leu Asp LeuAsp Cys Gly Val Arg Val Glu Ala Asp Glu Thr Arg Thr Leu Asp Tyr Thr Thr AlaLys Asp Arg Leu Leu Ala Arg Glu Leu Val Glu Glu Gly Leu Lys Arg Ser Leu TrpAsp Asp Tyr Leu Val Arg Gly Ile Asp Glu Val Leu Ser Lys Glu Pro Val Leu ThrCys Asp Glu Phe Asp Leu His Glu Arg Tyr Asp Leu Ser Val Glu Val Gly His SerGly Arg Ala Tyr Leu His Ile Asn Phe Arg His Arg Phe Val Pro Lys Leu Thr LeuAla Asp Ile Asp Asp Asp Asn Ile Tyr Pro Gly Leu Arg Val Lys Thr Thr Tyr ArgPro Arg Arg Gly His Ile Val Trp Gly Leu Arg Asp Glu Cys Ala Thr Asp Ser LeuAsn Thr Leu Gly Asn Gln Ser Val Val Ala Tyr His Arg Asn Asn Gln Thr Pro IleAsn Thr Asp Leu Leu Asp Ala Ile Glu Ala Ala Asp Arg Arg Val Val Glu Thr ArgArg Gln Gly His Gly Asp Asp Ala Val Ser Phe Pro Gln Glu Leu Leu Ala Val GluPro Asn Thr His Gln Ile Lys Gln Phe Ala Ser Asp Gly Phe His Gln Gln Ala ArgSer Lys Thr Arg Leu Ser Ala Ser Arg Cys Ser Glu Lys Ala Gln Ala Phe Ala GluArg Leu Asp Pro Val Arg Leu Asn Gly Ser Thr Val Glu Phe Ser Ser Glu Phe PheThr Gly Asn Asn Glu Gln Gln Leu Arg Leu Leu Tyr Glu Asn Gly Glu Ser Val LeuThr Phe Arg Asp Gly Ala Arg Gly Ala His Pro Asp Glu Thr Phe Ser Lys Gly IleVal Asn Pro Pro Glu Ser Phe Glu Val Ala Val Val Leu Pro Glu Gln Gln Ala AspThr Cys Lys Ala Gln Trp Asp Thr Met Ala Asp Leu Leu Asn Gln Ala Gly Ala ProPro Thr Arg Ser Glu Thr Val Gln Tyr Asp Ala Phe Ser Ser Pro Glu Ser Ile SerLeu Asn Val Ala Gly Ala Ile Asp Pro Ser Glu Val Asp Ala Ala Phe Val Val LeuPro Pro Asp Gln Glu Gly Phe Ala Asp Leu Ala Ser Pro Thr Glu Thr Tyr Asp GluLeu Lys Lys Ala Leu Ala Asn Met Gly Ile Tyr Ser Gln Met Ala Tyr Phe Asp ArgPhe Arg Asp Ala Lys Ile Phe Tyr Thr Arg Asn Val Ala Leu Gly Leu Leu Ala AlaAla Gly Gly Val Ala Phe Thr Thr Glu His Ala Met Pro Gly Asp Ala Asp Met PheIle Gly Ile Ala Val Ser Arg Ser Tyr Pro Glu Asp Gly Ala Ser Gly Gln Ile AsnIle Ala Ala Thr Ala Thr Ala Val Tyr Lys Asp Gly Thr Ile Leu Gly His Ser SerThr Arg Pro Gln Leu Gly Glu Lys Leu Gln Ser Thr Asp Val Arg Asp Ile Met LysAsn Ala Ile Leu Gly Tyr Gln Gln Val Thr Gly Glu Ser Pro Thr His Ile Val IleHis Arg Asp Gly Phe Met Asn Glu Asp Leu Asp Pro Ala Thr Glu Phe Leu Asn GluGln Gly Val Glu Tyr Asp Ile Val Glu Ile Arg Lys Gln Pro Gln Thr Arg Leu LeuAla Val Ser Asp Val Gln Tyr Asp Thr Pro Val Lys Ser Ile Ala Ala Ile Asn GlnAsn Glu Pro Arg Ala Thr Val Ala Thr Phe Gly Ala Pro Glu Tyr Leu Ala Thr ArgAsp Gly Gly Gly Leu Pro Arg Pro Ile Gln Ile Glu Arg Val Ala Gly Glu Thr AspIle Glu Thr Leu Thr Arg Gln Val Tyr Leu Leu Ser Gln Ser His Ile Gln Val HisAsn Ser Thr Ala Arg Leu Pro Ile Thr Thr Ala Tyr Ala Asp Gln Ala Ser Thr HisAla Thr Lys Gly Tyr Leu Val Gln Thr Gly Ala Phe Glu Ser Asn Val Gly Phe LeuGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Gln Leu Val Lys Ser Glu LeuGlu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr IleGlu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys ValMet Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser ArgLys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile ValAsp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu MetGln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu TrpTrp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly HisPhe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys AsnGly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala GlyThr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe。
2.根据权利要求1所述的嵌合蛋白pAgoE，其特征在于：所述效应结构域E采用具有核酸酶样DNA切割活性(Nuclease-like DNA cleavage activity)的、用于基因组靶向编辑的结构域N，以构成pAgoE-N型的嵌合蛋白pAgoE。
3.根据权利要求2所述的嵌合蛋白pAgoE，其特征在于：所述效应结构域N来自核酸酶、转座酶、位点特异重组酶。
4.根据权利要求3所述的嵌合蛋白pAgoE，其特征在于：所述效应结构域N来自酶学编号为EC 3.1.21.4的ⅡS型限制性核酸内切酶。
5.根据权利要求4所述的嵌合蛋白pAg o E，其特征在于：所述效应结构域N采用Planomicrobium okeanokoites的、GenBank登记号为:AAA24934.1的ⅡS型限制性核酸内切酶FOKⅠ的第383到第579个氨基酸。
6.根据权利要求1所述的嵌合蛋白pAgoE，其特征在于：所述效应结构域E含有具有基因组或表观组修饰活性(Modification activity)的、用于基因组靶向修饰的结构域M。
7.根据权利要求6所述的嵌合蛋白pAgoE，其特征在于：所述效应结构域M采用具有DNA核苷酸修饰活性(Nucleotide Modification activity)的结构域NM，可用于基因组靶向核苷酸修饰或基因组靶向加速进化。
8.根据权利要求7所述的嵌合蛋白pAgoE，其特征在于：所述结构域NM采用酶学编号为EC 3.5.4.5的AID、或酶学编号为EC 3.2.2.21的MAG1。
9.根据权利要求6所述的嵌合蛋白pAgoE，其特征在于：所述结构域M采用具有表观组修饰活性(Epigenome Modification activity)的结构域EM，可用于表观组编辑。
10.根据权利要求9所述的嵌合蛋白pAgoE，其特征在于：所述结构域EM采用酶学编号为EC 1.14.11.n2的TET1、或酶学编号为EC 2.1.1.37的DBMt3a/b。
11.根据权利要求1所述的嵌合蛋白pAgoE，其特征在于：所述人工突变体dMpAgo来自MpAgo的以下四个位点中的至少一个的失活：D446、E482、D516、N624。
12.根据权利要求11所述的嵌合蛋白pAgoE，其特征在于：所述人工突变体dMpAgo含有以下四个丙氨酸替代突变位点中的至少一个：D446A、E482A、D516A、N624A。
13.据权利要求1所述的嵌合蛋白pAgoE，其特征在于：所述人工突变体dNgAgo来自NgAgo的以下四个位点中的至少一个的失活：D663、D738、D863、S665。
14.根据权利要求13所述的嵌合蛋白pAgoE，其特征在于：所述人工突变体dNgAgo含有以下四个丙氨酸替代突变位点中的至少一个：D663A、D738A、D863A、S665A。
15.一种嵌合蛋白pAgoE的构建方法，其特征在于：该方法是将具有基因组靶向定位功能的pAgo结构域，以及具有基因组切割或修饰活性的效应结构域E连接；
所述pAgo结构域与任意一种使用向导的天然原核Argonaute蛋白的氨基酸序列同源性大于30％，且：
所述pAgo结构域采用来自Rhodobacter sphaeroides ATCC 17025的、NCBI序列号为ABP72561.1的RsAgo，以构建得到RsAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用来自Marinitoga piezophila的、NCBI序列号为WP_014295921.1的MpAgo，以构建得到MpAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用MpAgo的PIWI结构域核酸酶活性失活的人工突变体dMpAgo，以构建得到dMpAgoE型的嵌合蛋白pAgoE；
或者，所述pAgo结构域采用NgAgo的PIWI结构域核酸酶失活的人工突变体dNgAgo，以构建得到的dNgAgoE型的嵌合蛋白pAgoE；
其中，构建得到的RsAgoE型的嵌合蛋白pAgoE具有与权利要求1中构建得到的RsAgoE型的嵌合蛋白pAgoE相同的氨基酸序列；
构建得到的MpAgoE型的嵌合蛋白pAgoE具有与权利要求1中构建得到的MpAgoE型的嵌合蛋白pAgoE相同的氨基酸序列；
构建得到的dMpAgoE型的嵌合蛋白pAgoE具有与权利要求1中构建得到的dMpAgoE型的嵌合蛋白pAgoE相同的氨基酸序列；
构建得到的dNgAgoE型的嵌合蛋白pAgoE具有与权利要求1中构建得到的dNgAgoE型的嵌合蛋白pAgoE相同的氨基酸序列。
16.根据权利要求15所述的嵌合蛋白pAgoE的构建方法，其特征在于：所述pAgo结构域与所述效应结构域E采用人工连接。
17.根据权利要求16所述的嵌合蛋白pAgoE的构建方法，其特征在于：所述pAgo结构域与所述效应结构域E共价键连接。
18.根据权利要求17所述的嵌合蛋白pAgoE的构建方法，其特征在于：所述pAgo结构域与所述效应结构域E之间形成碳端和氮端之间的连接。
19.根据权利要求18所述的嵌合蛋白pAgoE的构建方法，其特征在于：所述pAgo结构域的氮端与效应结构域E的碳端连接，以构建E-pAgo型的pAgoE；或所述pAgo结构域的碳端与效应结构域E的氮端连接，构建pAgo-E型的pAgoE；或所述pAgo结构域的碳端和氮端分别与效应结构域E的氮端和碳端连接，构建E1-pAgo-E2型的pAgoE，其中，E1与E2相同或不同。
20.根据权利要求16所述的蛋白pAgoE的构建方法，其特征在于：所述pAgoE被构建成分体形式的嵌合蛋白(split-pAgoE)，分体之间以非共价键的方式连接。
21.根据权利要求20所述的嵌合蛋白pAgoE的构建方法，其特征在于：所述嵌合蛋白pAgoE的所述pAgo结构域与所述效应结构域E之间，采用MxeGryA Intein构建，以形成split-pAgoE型的嵌合蛋白pAgoE。
22.一种使用向导的嵌合蛋白pAgoE，其特征在于：含有如权利要求1至21中任一项所述嵌合蛋白pAgoE，以及与所述嵌合蛋白pAgoE结合的、具有协助pAgoE识别和结合基因组能力的向导。
23.根据权利要求22所述的使用向导的嵌合蛋白pAgoE，其特征在于：所述向导为寡核苷酸gNA。
24.根据权利要求23所述的使用向导的嵌合蛋白pAgoE，其特征在于：所述寡核苷酸gNA依据与拟靶向基因组位点序列互补的原则而设置。
25.根据权利要求24所述的使用向导的嵌合蛋白pAgoE，其特征在于：所述寡核苷酸gNA的核苷酸序列与拟靶向区域的基因组的核苷酸序列的同源性为40％以上。
26.根据权利要求22所述的使用向导的嵌合蛋白pAgoE，其特征在于：RsAgoE型的嵌合蛋白pAgoE，使用长度为18个核苷酸的5’端磷酸化单链gRNA为gNA。
27.根据权利要求22所述的使用向导的嵌合蛋白pAgoE，其特征在于：MpAgoE型或dMpAgoE型的嵌合蛋白pAgoE，使用长度为10到40个核苷酸的5’端羟基化单链gRNA为gNA。
28.根据权利要求22所述的使用向导的嵌合蛋白pAgoE，其特征在于：dNgAgoE型的嵌合蛋白pAgoE，使用长度为24个核苷酸的5’端磷酸化单链gDNA为gNA。
29.一种使用向导的嵌合蛋白pAgoE的构建方法，其特征在于：该方法是将如权利要求1至21中任一项所述的嵌合蛋白pAgoE与向导结合。
30.一种具有基因组靶向编辑功能的嵌合蛋白，其特征在于：含有如权利要求1至14中任一项所述嵌合蛋白pAgoE，所述嵌合蛋白pAgoE含有具有基因组切割性的效应结构域E。
31.根据权利要求30所述的具有基因组靶向编辑功能的嵌合蛋白，其特征在于：还含有与所述嵌合蛋白pAgoE结合的、具有协助pAgoE识别和结合基因组能力的向导。
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