一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用
阅读说明：本技术 一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用 (Adenine base editor fusion protein without PAM limitation and application thereof ) 是由 马旭 曹小芳 金孝华 于 2021-08-10 设计创作，主要内容包括：本发明属于生物医药领域,涉及一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用。本发明提供了一种突变体多肽,所述多肽自N端至C端依次包括SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段。含有所述突变体多肽的融合蛋白可以靶向全基因组,拓宽了基因组的可编辑范围；能够更高效率地引起A:T到G:C的碱基转换,具有很大的应用潜力,包括但不限于基因疾病致病性位点的模拟或修复；且造成转录组水平上的脱靶更低,兼顾效率高和脱靶低的突变体形式。(The invention belongs to the field of biomedicine, and relates to an adenine base editor fusion protein without PAM limitation and application thereof. The invention provides a mutant polypeptide which sequentially comprises an N-terminal fragment of SpRY (D10A), a TadA8e fragment and a C-terminal fragment of SpRY (D10A) polypeptide from an N end to a C end. The fusion protein containing the mutant polypeptide can target the whole genome, so that the editable range of the genome is widened; can cause the base conversion from A: T to G: C with higher efficiency, and has great application potential, including but not limited to the simulation or repair of gene disease pathogenicity sites; and the mutant forms with lower off-target on the transcriptome level and high efficiency and low off-target are also caused.)
技术领域
本发明属于生物医药领域，涉及一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用。
背景技术
CRISPR/Cas9系统最初发现于细菌和古细菌中，后经过优化和改造形成强大的基因编辑工具，被广泛应用于DNA的敲除、敲入、修饰等研究。CRISPR/Cas9系统由Cas9核酸酶和识别靶序列的sgRNA两部分组成，sgRNA与靶序列互补配对介导Cas9核酸酶对基因组的定向切割，造成双链DNA断裂(double strand break,DSB)后，利用细胞中的修复机制同源重组(有模板情况下)和非同源末端连接(无模板情况下)实现靶向位点的编辑[1,2]。随后，David Liu等人构建RuvC结构域失活的nickase Cas9(nCas9)，并在此基础上开发出单碱基编辑系统即胞嘧啶碱基编辑器(cytosine base editor,CBE)和腺嘌呤碱基编辑器(adenine base editor,ABE)，两种碱基编辑器在不引起DNA双链断裂的情况下可分别实现C:G到T:A、A:T到G:C的碱基转换，极大提升了单碱基编辑的效率和安全性[2,3]。
ABE由腺嘌呤脱氨酶和nCas9融合而成，根据ClinVar数据库收录的数据，与人类疾病相关的基因变异有58％是点突变，而其中47％的致病点突变可通过ABE介导的A:T到G:C的碱基转换得到修复[4]。已有大量研究显示ABE在疾病修复领域的应用价值。例如，通过病毒递送ABE和相应的sgRNA至杜氏肌营养不良的小鼠肌肉中，可修复致病基因DMD的无义突变[5]；通过脂质纳米颗粒递送mRNA形式的ABE至患酪氨酸血症的成年小鼠肝脏中，修复了致病性的剪切位点变异，恢复肝细胞中FAH的表达[6]。但ABE对于位点的编辑受到编辑窗口和PAM序列的限制，应用最为广泛的ABEmax识别的PAM序列为NGG，为进一步拓展碱基编辑器的编辑范围，识别不同PAM序列的ABE相继出现，如识别PAM序列为NG的xABE和ABE-NG[7]，其中PAM限制最为宽松的是2020年3月发表的ABEmax-SpRY，其PAM序列为NRN(R代表A、G)和NYN(Y代表C、T)[8]。ABEmax-SpRY可以靶向基因组的所有序列，但是ABEmax-SpRY编辑效率较低，且未解决ABE存在的在转录组水平上的脱靶问题，限制了该碱基编辑器的应用，需要对其进行改进和优化。
发明内容
一些实施方案中，本发明了提供了一种突变体多肽，所述多肽自N端至C端依次包括SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段。
一些实施方案中，所述SpRY(D10A)蛋白N端片段的氨基酸序列如SEQ ID NO:1所示，所述TadA8e片段的氨基酸序列如SEQ ID NO:3所示，所述SpRY(D10A)蛋白C端片段的氨基酸序列如SEQ ID NO:5所示。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述突变体多肽用于基因编辑。
一些实施方案中，所述基因编辑的编辑窗口为3～10位。
一些实施方案中，所述基因编辑的编辑窗口为8～10位。
一些实施方案中，所述突变体多肽包含如SEQ ID NO:13所示的序列。
一些实施方案中，本发明提供了一种融合蛋白，包含所述的突变体多肽。
在一些实施方案中，含有所述突变体多肽的融合蛋白可以靶向全基因组，拓宽了基因组的可编辑范围；能够更高效率地引起A:T到G:C的碱基转换，具有很大的应用潜力，包括但不限于基因疾病致病性位点的模拟或修复；一些实施例中，含有所述突变体多肽的融合蛋白拓宽了碱基编辑窗口；且造成转录组水平上的脱靶更低，兼顾效率高和脱靶低的突变体形式。
相较于现有的腺嘌呤碱基编辑器突变体，ABEmax-SpRY无PAM限制，有效提高了基因组的可靶向范围，但是其编辑活性不高。
在一些实施方案中，发明人用ABE8e中的腺嘌呤脱氨酶TadA8e代替ABEmax-SpRY中的腺嘌呤脱氨酶二聚体，构建形成8e-SpRY，8e-SpRY相比于ABEmax-SpRY不仅能够更高效率地引起碱基转换，还拓宽了碱基编辑窗口。
在一些实施方案中，发明人又在8e-SpRY的基础上构建了4种突变体，分别为CE-8e-SpRY、V106W-SpRY、8e-SpRY-HF和V106W-SpRY-HF。综合评价编辑效率和脱靶后，CE-8e-SpRY是兼顾效率高和脱靶低的突变体形式。
一些实施方案中，所述融合蛋白还包括连接肽，连接肽位于SpRY(D10A)蛋白N端片段与TadA8e片段之间，和/或位于TadA8e片段与SpRY(D10A)蛋白C端片段之间。
一些实施方案中，所述连接肽氨基酸序列如SEQ ID NO:7所示。
一些实施方案中，编码所述连接肽的核苷酸序列如SEQ ID NO:8所示。
一些实施方案中，所述融合蛋白还包括核定位信号片段。
一些实施方案中，所述核定位信号片段位于所述融合蛋白的N端和/或C端。
一些实施方案中，所述核定位信号片段的氨基酸序列如SEQ ID NO:9和/或SEQ IDNO:11所示。
一些实施方案中，所述核定位信号的核苷酸序列如SEQ ID NO:10或12所示。
一些实施方案中，所述核定位信号片段包括两个拷贝。
一些实施方案中，所述融合蛋白的氨基酸序列包含如SEQ ID NO:13所示的序列。
一些实施方案中，所述融合蛋白用于基因编辑。
一些实施方案中，所述基因编辑的编辑窗口为3～10位。
一些实施方案中，所述基因编辑的编辑窗口为8～10位。
一些实施方案中，所述突变体多肽包含如SEQ ID NO:13所示的序列。
一些实施方案中，本发明提供了一种编码所述的突变体多肽或所述的融合蛋白的多核苷酸。
一些实施方案中，所述多核苷酸为核酸构建体。
一些实施方案中，本发明提供了一种载体，所述载体包含所述的多核苷酸。
一些实施方案中，所述载体为重组表达载体。
一些实施方案中，所述载体骨架选自pCMV或其衍生质粒。
一些实施方案中，所述pCMV的衍生质粒包括ABEmax-SpRY。
一些实施方案中，所述载体包括质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，本发明提供了一种产所述载体的方法，在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得所述的载体。
一些实施方案中，所还载体包括质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA片段，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体。
一些实施方案中，所述载体为CE-8e-SpRY质粒。
一些实施方案中，本发明提供了一种sgRNA。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本发明提供了一种表达系统，所述表达系统含有所述的表达载体或其基因组中整合有外源的所述的多核苷酸。
一些实施方案中，所述表达系统表达所述的融合蛋白或其基因组中整合的外源序列表达所述的融合蛋白或所述表达系统表达含有所述的多核苷酸或其基因组中整合有外源的如上所述的多核苷酸。
一些实施方案中，所述表达系统还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本发明提供了一种宿主细胞，包含所述的多核苷酸或所述的载体或所述的表达系统。
一些实施方案中，本发明提供了一种组合物，包含所述的突变体多肽，所述的融合蛋白，所述的多核苷酸，所述的载体和所述的宿主细胞中的至少一种，或其任意组合。
一些实施方案中，所述组合物为试剂盒。
一些实施方案中，所述组合物还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本发明提供了一种任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞在制备治疗基因疾病的药物中的应用。
一些实施方案中，本发明提供了一种任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞在制备基因编辑试剂中的应用。
一些实施方案中，所述基因编辑的编辑窗口为3～10位。
一些实施方案中，所述基因编辑的编辑窗口为8～10位。
一些实施方案中，本发明提供了一种碱基编辑系统，包含任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞。
一些实施方案中，所述碱基编辑系统还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，本发明提供了一种基因编辑方法，通过所述的碱基编辑系统进行基因编辑。
一些实施方案中，所述基因编辑的编辑窗口为3～10位。
一些实施方案中，所述基因编辑的编辑窗口为8～10位。
一些实施方案中，本发明提供了一种方法，用于重组产生任一所述的突变体多肽或所述的融合蛋白，包括步骤：将所述的载体引入宿主细胞以产生转染的或感染的宿主细胞，体外培养所述转染的或感染的宿主细胞，回收细胞培养物并任选地纯化所产生的突变体多肽或融合蛋白。
一些实施方案中，本发明提供了一种所述的突变体多肽或所述的融合蛋白的制备方法，包括：(1)在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得重组表达载体；(2)转染所述重组表达载体至宿主细胞使其表达所述突变体多肽或所述融合蛋白。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY。
一些实施方案中，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA二聚体，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体。
一些实施方案中，所还载体质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，本发明提供了一种产生所述载体的方法，包括步骤：将所述载体引入至适宜的细胞系，在适宜条件下培养所述细胞系从而使所述目的载体能够产生，从所述细胞系的培养物回收所产生的质粒和任选地纯化所述质粒。
在一些实施方式中，上述的蛋白为分离的多肽。
在一些实施方式中，上述的核酸为分离的多核酸。
附图说明
图1为ABEmax-SpRY和8e-SpRY及其突变体的示意图。
图2-图7为ABEmax-SpRY和8e-SpRY在PAM为NNN时的编辑效率。
图8为ABEmax-SpRY和8e-SpRY多点编辑效率的统计结果。
图9为ABEmax-SpRY和8e-SpRY的编辑窗口。
图10-图15为8e-SpRY及其突变体在PAM为NNN时的编辑效率。
图16为8e-SpRY及其突变体多点编辑效率的统计结果。
图17为8e-SpRY及其突变体在PAM为NAN、NGN、NCN和NTN时多点编辑效率的统计结果。
图18为8e-SpRY及其突变体的编辑窗口。
图19为ABEmax-SpRY、8e-SpRY及其突变体的DNA靶向编辑效率。
图20为ABEmax-SpRY、8e-SpRY及其突变体的RNA脱靶量。
图21为ABEmax-SpRY、8e-SpRY及其突变体A-to-I的RNA脱靶示意图。
图22为PKU 728G>A细胞模型基因型的sanger测序图以及8种修复sgRNA修复效率的sanger测序图。
图23为3种有修复效果的sgRNA修复效率的柱状图。
图24为其他3种ABE突变体修复效率的sanger测序图。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
苯丙酮尿症(Phenylketonuria，简称PKU)是先天代谢性疾病的一种，本症是由于染色体基因突变导致肝脏中苯丙氨酸羟化酶(PAH)缺陷从而引起苯丙氨酸(PA)代谢障碍所致。
实施例1碱基编辑器质粒的构建
首先构建8e-SpRY以及相应的突变体。参照ClonExpress MultiS One StepCloning Kit(Vazyme,C113-01)说明书设计引物，扩增ABE8e(Addgene#138489)中的TadA8e片段，并用TadA8e替代ABEmax-SpRY(Addgene#140003)中的TadA二聚体，构建得到8e-SpRY质粒。
先将8e-SpRY中的TadA8e从原位置删除，再用TadA8e替代SpRY D10A中第1048位至1063位的氨基酸，构建得到CE-8e-SpRY质粒，从5’端到3’端的顺序依次为SpRY(D10A)N端、TadA8e和SpRY(D10A)C端，其中SpRY(D10A)N端的核苷酸序列如SEQ ID NO:2(氨基酸序列如SEQ ID NO:1所示)，TadA8e的核苷酸序列如SEQ ID NO:4(氨基酸序列如SEQ ID NO:3所示)，SpRY(D10A)C端的核苷酸序列如SEQ ID NO:6(氨基酸序列如SEQ ID NO:5所示)。
将8e-SpRY中的TadA8e进行V106W突变，得到V106W-SpRY，其中TadA8e V106W核苷酸序列如SEQ ID NO:15，SpRY D10A核苷酸序列如SEQ ID NO:16。
将8e-SpRY中的SpRY D10A进行N497A,R661A,Q695A，和Q926A突变，得到8e-SpRY-HF，其中SpRY D10A-HF核苷酸序列如SEQ ID NO:17。
将8e-SpRY-HF中的TadA8e进行V106W突变，得到V106W-SpRY-HF。
8e-SpRY及其突变体两端均携带核定位信号，核定位信号为bpNLS(核定位信号的核苷酸序列如SEQ ID NO:10所示；氨基酸序列如SEQ ID NO:9所示)或SV40NLS(核定位信号的核苷酸序列如SEQ ID NO:12所示；氨基酸序列如SEQ ID NO:11所示)。8e-SpRY及其突变体具体图示如图1所示。
(1)ABEmax-SpRY(融合蛋白)
所述ABEmax-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:67所示；其组成从N端到C端的顺序依次为bpNLS、TadA二聚体、SpRY D10A和bpNLS。一些实施例中，两端携带核定位信号也可为SV40NLS。
(2)8e-SpRY(融合蛋白)
所述8e-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:68所示；其组成从N端到C端的顺序依次为bpNLS、TadA8e、SpRY D10A和bpNLS。一些实施例中，两端携带核定位信号也可为SV40NLS。
(3)CE-8e-SpRY(融合蛋白)
所述CE-8e-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:13所示(CE-8e-SpRY融合蛋白的核苷酸序列如SEQ ID NO:14所示)，其组成为:自N端至C端依次包括bpNLS、SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段和bpNLS，在SpRY(D10A)N端片段与TadA8e片段之间、TadA8e片段与SpRY(D10A)C端片段之间具有连接肽，所述连接肽的氨基酸序列如SEQ ID NO:7所示(编码CE-8e-SpRY连接肽的核苷酸序列如SEQ ID NO:8所示)。一些实施例中，两端携带核定位信号也可为SV40NLS。
(4)V106W-SpRY(融合蛋白)
所述V106W-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:69所示；其组成从N端到C端的顺序依次为bpNLS、TadA8eV106W、SpRY D10A、和bpNLS，两端携带核定位信号也可为SV40NLS。
(5)8e-SpRY-HF(融合蛋白)
所述8e-SpRY-HF(融合蛋白)的氨基酸序列如SEQ ID NO:70所示；其组成从N端到C端的顺序依次为bpNLS、TadA8e、SpRY D10A-HF、和bpNLS，两端携带核定位信号也可为SV40NLS。
(6)V106W-SpRY-HF
所述V106W-SpRY-HF(融合蛋白)的氨基酸序列如SEQ ID NO:71所示；其组成从N端到C端的顺序依次为bpNLS、TadA8eV106W、SpRY D10A-HF、和bpNLS，两端携带核定位信号也可为SV40NLS。
实施例2
在本实施例中，利用ABEmax-SpRY、8e-SpRY及其突变体在293T细胞中进行内源位点的编辑。
2.1sgRNA质粒的构建
参考人基因组序列，根据SpRY核酸酶的PAM特征设计48条sgRNA，涵盖16种不同的PAM序列，sgRNA序列如SEQ ID NO:18-65所示，sgRNA序列5’端加入ACCG为上游序列，sgRNA反向互补序列的5’端加入AAAC为下游序列，合成oligo后上下游序列退火(程序为：95℃，5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；hold at 16℃)后与经过BsaI(NEB：R3733L)酶切后的pGL3-U6-sgRNA(Addgene#51133)载体连接。酶切体系为：pGL3-U6-sgRNA2μg；CutSmart buffer(NEB：B7204S)6μL；BsaI 1μL；ddH2O补齐到60μL，37℃酶切过夜。连接体系为：Solution I(Takara：6022Q)3μL；酶切后载体1μL；退火产物6μL，16℃连接30min后转化、挑菌、鉴定。对阳性克隆菌摇菌提取质粒(Axygene：AP-MN-P-250G)、测定浓度后备用。
2.2细胞培养与转染
HEK293T细胞(购自ATCC)接种培养于添加10％血清(Gibco:10270-106)的DMEM培养基中(Gibco：C11995500BT)，其中含1％双抗(v/v)(Gibco：15140122)。转染前一天铺24孔板，使转染时的细胞密度达到80％左右，转染前2h换液。每孔转染的质粒量分别为碱基编辑器质粒600ng，sgRNA质粒(sgRNA1-48的序列如SEQ ID NO:18～SEQ ID NO:65所示)300ng，将质粒稀释于40μL的DMEM中，将3μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于40μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有质粒和EZ的DMEM加入24孔板中，6h后用含有10％血清的完全培养基换液，转染48h后显微镜观察绿色荧光蛋白(Green fluorescent protein,GFP)的表达，流式细胞分选仪分选GFP阳性细胞。
其中GFP是pGL3-U6-sgRNA载体上的。
2.3检测编辑效率
将分选得到的GFP阳性细胞离心去上清后，加入裂解液(50mM KCl,1.5mM MgCl2,10mM Tris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K)，以GFP阳性细胞裂解液为模板，扩增靶向序列，扩增体系为：2×buffer(Vazyme：P505)25μL；dNTP 1μL；Forward Primer(10μmol/L)1μL；Reverse Primer(10pmol/L)1μL；细胞裂解产物1μL；DNA聚合酶(Vazyme：P505)0.5μL；ddH2O补齐到50μL。Forward Primer和Reverse Primer序列如SEQ ID NO:72～SEQ ID NO:167所示(分别对应sgRNA1-48)。
扩增出来的PCR产物用回收试剂盒进行纯化(Axygen：AP-PCR-250G)，具体步骤为：扩增产物中加入3倍体积的PCR-A，混匀后加入吸附柱中，12000r/min离心1min；弃废液，吸附柱中加入700μL W2(需加入指定体积的乙醇)，12000r/min离心1min；弃废-液，吸附柱中加入400μL W2(需加入指定体积的乙醇)，12000r/min离心1min；弃废液，12000r/min离心2min；开盖晾干乙醇后，加入28μL ddH2O，12000r/min离心1min进行洗脱，纯化后的PCR产物送Sanger测序或深度检测，分析编辑效果。
相关结果如图2-9所示。结果显示在所有检测位点上，涵盖NAN、NGN、NCN和NTN的PAM，8e-SpRY的编辑效率均明显高于ABEmax-SpRY；图8的多点编辑效率的统计结果显示，8e-SpRY显著改善了A到G的编辑效率。图9的编辑窗口结果显示，ABEmax-SpRY碱基编辑窗口为5-6位；8e-SpRY碱基编辑窗口为3-10位，窗口更宽。
图10-15显示在NRN(R代表A或G)、NYN(Y代表C或T)PAM下，8e-SpRY突变体编辑效率的对比结果；将8e插入到SpRY中间的CE-8e-SpRY可以很好的维持其A-to-G的编辑活性，将V106W引入Tad8e的V106W-SpRY同样未明显损伤原来的编辑活性，但是将4种突变引入SpRY的8e-SpRY-HF和V106W-SpRY-HF则使编辑活性明显下降。
图16的多点编辑效率的统计结果显示，8e-SpRY-HF和V106W-SpRY-HF显著降低了活性，CE-8e-SpRY编辑效率提高但无显著性差异，V106W-SpRY编辑效率降低同样无显著性差异。
图17在NAN、NGN、NCN和NTN的多点编辑效率的统计结果显示，CE-8e-SpRY在NGN和NTN的编辑效率有所提高，V106W-SpRY在4种PAM下的编辑效率均有所下降，但均无统计学意义。图18的编辑窗口结果显示，V106W-SpRY维持与8e-SpRY相同的编辑窗口，均为3-10位，高活编辑窗口(编辑效率大于40％)为3-9位；CE-8e-SpRY维持相同的编辑窗口即3-10位，高活编辑窗口(编辑效率大于40％)为3-10位，且在8-10位的编辑效率高于8e-SpRY。
表1.实施例2中转染细胞所用的质粒组合(1)
表2.实施例2中转染细胞所用的质粒组合(2)
实施例3
在本实施例中，对比ABEmax-SpRY、8e-SpRY及其突变体在293T细胞中的RNA脱靶情况。
3.1sgRNA的构建
用于RNA脱靶检测的sgRNA序列为5’-CTGGAACACAAAGCATAGAC-‘3(SEQ ID NO:66)，按照2.1所述的质粒构建方法进行构建。
3.2细胞培养与转染
细胞培养按照2.2所述进行，转染前一天用293T细胞铺6cm Dish，使转染时的细胞密度达到80％左右。每皿转染的质粒量为碱基编辑器质粒4μg，sgRNA质粒2μg，将质粒稀释于250μL的DMEM中，将18μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于250μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有质粒和EZ的DMEM加入6cm Dish中，6h后用含有10％血清的完全培养基换液(DMEM+10％FBS)，转染48h后显微镜观察GFP(GFP是pGL3-U6-sgRNA载体上的)表达，流式细胞分选仪分选GFP阳性细胞。取少数阳性细胞按照2.3所述检测编辑效率，其余阳性细胞提取RNA后送RNA-Seq。
3.3RNA提取
GFP阳性细胞3000r/min离心10min后弃上清，加入1mL RNAisolater Total RNAextraction Reagent(Vazyme：R401-01-AA)充分裂解细胞；加入200μL氯仿，上下剧烈混匀，室温放置3min后，4℃12000r/min离心15min；取上层水相500μL，加入500μL的异丙醇，上下颠倒混匀，4℃12000r/min离心15min；吸弃上清，加入1mL 75％的乙醇，轻轻颠倒几次清洗沉淀，4℃12000r/min离心5min；吸弃上清，开盖干燥5-10min，待乙醇完全挥发后，加入15μLRNase-Free水溶解沉淀，取1μL测浓度。取1μg RNA送RNA-Seq。
相关结果如19-21所示。图19显示在DNA靶向位点上的第8位A的编辑效率，ABEmax-SpRY、8e-SpRY及其突变体均能引起有效的编辑，其中8e-SpRY及其突变体引起的DNA靶向编辑效率相当，ABEmax-SpRY的编辑效率相对较低。图20和图21的RNA脱靶结果显示，相对于ABEmax-SpRY和8e-SpRY的其他突变体，CE-8e-SpRY有效降低了在转录组水平的脱靶编辑。
综合编辑效率检测和脱靶检测结果，发明人获得的CE-8e-SpRY碱基编辑器可以靶向全基因，同时显著提高了A-to-G的编辑效率，并且有效降低了在转录组水平上的脱靶编辑，具有很大的应用潜力。
表3.实施例3中转染细胞所用的质粒组合
实施例4 CE-8e-SpRY在修复疾病致病位点中的应用
4.1构建人PAH 728G>A细胞模型
4.1.1突变mut-sgRNA构建
参考人基因组序列，设计mut-sgRNA(SEQ ID NO:168)，按照2.1所述的质粒构建方法进行构建。
4.1.2细胞培养与转染
细胞培养按照2.2所述进行，转染前一天铺24孔板，使转染时的细胞密度达到80％左右，转染前2h换液。每孔转染的质粒量分别为碱基编辑器质粒600ng，sgRNA质粒300ng，将质粒稀释于40μL的DMEM中，将3μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于40μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有质粒和EZ的DMEM加入24孔板中，6h后用含有10％血清的完全培养基换液，转染48h后流式细胞分选仪分选GFP阳性细胞到96孔板中，每孔分选1个阳性细胞，放置于培养箱培养14天后鉴定细胞单克隆基因型。
4.1.3单克隆细胞基因型鉴定
每孔的单克隆细胞取部分细胞离心后加入裂解液(50mM KCl,1.5mM MgCl2,10mMTris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K)，以细胞裂解液为模板，扩增靶向序列，扩增体系为：2×buffer(Vazyme：P505)25μL；dNTP 1μL；ForwardPrimer(10μmol/L)1μL；Reverse Primer(10pmol/L)1μL；细胞裂解产物1μL；DNA聚合酶(Vazyme：P505)0.5μL；ddH2O补齐到50μL。Forward Primer序列为：5’-gtccctgggcagttatgtgtac-3’(SEQ ID NO:177)，Reverse Primer序列为5’-caactggtagctggaggacag-3’(SEQ ID NO:178)。扩增产物送Sanger测序后挑选PAH 728G>A纯和突变的细胞即为人PAH 728G>A细胞模型。
4.2修复PAH 728G>A突变
CE-8e-SpRY在3-10位有较高的编辑效率，且识别PAM为NNN，根据CE-8e-SpRY的编辑窗口和PAM特点，发明人围绕需要修复的致病突变设计了8条Rec-sgRNA(SEQ ID NO:169-～SEQ ID NO:176)，并按照2.1所述的质粒构建方法进行构建。按照2.2所述的细胞培养与转染方法进行转染。按照2.3所述的检测编辑效率的方法进行修复效率的检测。
结果如图22和图23所示：Mut-sgRNA成功造成728G>A的纯和突变；8条Rec-sgRNA中，Rec-sgRNA1(即图22和图23中的sg1)对728G>A的修复效率最高，Rec-sgRNA2(即图22和图23中的sg2)和Rec-sgRNA3(即图22和图23中的sg3)具有微弱的修复效果。
根据x-ABEmax，ABEmax-NG和ABEmax-SpRY的PAM特点和编辑窗口，这3种碱基编辑器的修复sgRNA为SEQ ID NO:173，按照2.1所述的质粒构建方法进行构建，按照2.2所述的细胞培养与转染方法进行转染，按照2.3所述的检测编辑效率的方法进行修复效率的检测。结果如图24所示，3种碱基编辑器对728G>A的突变位点均无明显修复效果。
本实施例说明由于CE-8e-SpRY识别的PAM为NNN，围绕需要修复的位点有多种sgRNA的选择，可经过sgRNA的筛选选择最符合修复要求的sgRNA，有效提高了可修复位点的范围和修复效果的灵活性。另外，现有的3种碱基编辑器在各自的编辑窗口内均无法修复728G>A的突变位点，发明人提供的CE-8e-SpRY在突变位点位于编辑窗口第10位时可实现有效的编辑，拓展了现有碱基编辑工具的可编辑范围，显示出独特的编辑特性。
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序列表
<110> 国家卫生健康委科学技术研究所
<120> 一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用
<160> 178
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1046
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SpRY(D10A)蛋白N端片段的氨基酸序列
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Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
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Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
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Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
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Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu Lys
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Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
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Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
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Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
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Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
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Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
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Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
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Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
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Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
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Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
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Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
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Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1010 1015 1020
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1025 1030 1035 1040
Ile Met Asn Phe Phe Lys
1045
<210> 2
<211> 3138
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码所述SpRY(D10A)蛋白N端片段的核苷酸序列
<400> 2
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgagaga 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaag 3138
<210> 3
<211> 166
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TadA8e片段的氨基酸序列
<400> 3
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly Ala
100 105 110
Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Ile Asn
165
<210> 4
<211> 498
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码所述TadA8e片段的核苷酸序列
<400> 4
tctgaggtgg agttttccca cgagtactgg atgagacatg ccctgaccct ggccaagagg 60
gcacgggatg agagggaggt gcctgtggga gccgtgctgg tgctgaacaa tagagtgatc 120
ggcgagggct ggaacagagc catcggcctg cacgacccaa cagcccatgc cgaaattatg 180
gccctgagac agggcggcct ggtcatgcag aactacagac tgattgacgc caccctgtac 240
gtgacattcg agccttgcgt gatgtgcgcc ggcgccatga tccactctag gatcggccgc 300
gtggtgtttg gcgtgaggaa ctcaaaaaga ggcgccgcag gctccctgat gaacgtgctg 360
aactaccccg gcatgaatca ccgcgtcgaa attaccgagg gaatcctggc agatgaatgt 420
gccgccctgc tgtgcgattt ctatcggatg cctagacagg tgttcaatgc tcagaagaag 480
gcccagagct ccatcaac 498
<210> 5
<211> 305
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SpRY(D10A)蛋白C端片段的氨基酸序列
<400> 5
Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1 5 10 15
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val
20 25 30
Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg
35 40 45
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
50 55 60
Pro Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val
65 70 75 80
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
85 90 95
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
100 105 110
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys
115 120 125
Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu
130 135 140
Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly
145 150 155 160
Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala
165 170 175
Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys
180 185 190
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu
195 200 205
Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
210 215 220
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
225 230 235 240
Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly
245 250 255
Ala Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln
260 265 270
Tyr Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
275 280 285
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly
290 295 300
Asp
305
<210> 6
<211> 915
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码所述SpRY(D10A)蛋白C端片段的核苷酸序列
<400> 6
gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 60
cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 120
ggcttcagca aagagtctat cagacccaag aggaacagcg ataagctgat cgccagaaag 180
aaggactggg accctaagaa gtacggcggc ttcctgtggc ccaccgtggc ctattctgtg 240
ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 300
ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 360
gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 420
ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccaagcagct gcagaaggga 480
aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 540
aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 600
cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 660
gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 720
gagcaggccg agaatatcat ccacctgttt accctgacca gactgggagc ccctagagcc 780
ttcaagtact ttgacaccac catcgacccc aagcagtaca gaagcaccaa agaggtgctg 840
gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 900
cagctgggag gtgac 915
<210> 7
<211> 32
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 融合蛋白CE-8e-SpRY含有的连接肽序列
<400> 7
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr
1 5 10 15
Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
20 25 30
<210> 8
<211> 96
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码CE-8e-SpRY连接肽的核苷酸序列
<400> 8
tctggaggat ctagcggtgg ttcctctgga agcgagacac caggcacaag cgagtccgcc 60
acaccagaga gctccggcgg ctcctccgga ggatcc 96
<210> 9
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 融合蛋白CE-8e-SpRY两端的核定位信号的氨基酸序(bpNLS)
<400> 9
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val
<210> 10
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 融合蛋白CE-8e-SpRY两端的核定位信号的核苷酸序列(bpNLS)
<400> 10
aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtc 54
<210> 11
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 核定位信号片段的氨基酸序列(SV40NLS)
<400> 11
Pro Lys Lys Lys Arg Lys Val
1 5
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 核定位信号的核苷酸序列(SV40NLS)
<400> 12
ccaaagaaga agcggaaagt c 21
<210> 13
<211> 1581
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> CE-8e-SpRY融合蛋白的氨基酸序列
<400> 13
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1010 1015 1020
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1025 1030 1035 1040
Ile Met Asn Phe Phe Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
1045 1050 1055
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
1060 1065 1070
Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp
1075 1080 1085
Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu
1090 1095 1100
Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu
1105 1110 1115 1120
Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu
1125 1130 1135
Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu
1140 1145 1150
Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala
1155 1160 1165
Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg
1170 1175 1180
Asn Ser Lys Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr
1185 1190 1195 1200
Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp
1205 1210 1215
Glu Cys Ala Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val
1220 1225 1230
Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser
1235 1240 1245
Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala
1250 1255 1260
Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Glu Thr Asn Gly
1265 1270 1275 1280
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1285 1290 1295
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1300 1305 1310
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn
1315 1320 1325
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr
1330 1335 1340
Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala
1345 1350 1355 1360
Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu
1365 1370 1375
Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile
1380 1385 1390
Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
1395 1400 1405
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys
1410 1415 1420
Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly Asn Glu Leu Ala
1425 1430 1435 1440
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu
1445 1450 1455
Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1460 1465 1470
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu
1475 1480 1485
Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1490 1495 1500
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu
1505 1510 1515 1520
Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala Pro Arg Ala
1525 1530 1535
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr Arg Ser Thr
1540 1545 1550
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1555 1560 1565
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1570 1575 1580
<210> 14
<211> 4743
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> CE-8e-SpRY融合蛋白的核苷酸序列
<400> 14
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgagaga 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagtc tggaggatct agcggtggtt cctctggaag cgagacacca 3180
ggcacaagcg agtccgccac accagagagc tccggcggct cctccggagg atcctctgag 3240
gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa gagggcacgg 3300
gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acaatagagt gatcggcgag 3360
ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat tatggccctg 3420
agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct gtacgtgaca 3480
ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatcgg ccgcgtggtg 3540
tttggcgtga ggaactcaaa aagaggcgcc gcaggctccc tgatgaacgt gctgaactac 3600
cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga atgtgccgcc 3660
ctgctgtgcg atttctatcg gatgcctaga caggtgttca atgctcagaa gaaggcccag 3720
agctccatca actccggagg atctagcgga ggctcctctg gctctgagac acctggcaca 3780
agcgagagcg caacacctga aagcagcggg ggcagcagcg gggggtcaga gacaaacggc 3840
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 3900
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 3960
gagtctatca gacccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4020
cctaagaagt acggcggctt cctgtggccc accgtggcct attctgtgct ggtggtggcc 4080
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4140
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4200
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4260
aacggccgga agagaatgct ggcctctgcc aagcagctgc agaagggaaa cgaactggcc 4320
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 4380
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 4440
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 4500
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 4560
aatatcatcc acctgtttac cctgaccaga ctgggagccc ctagagcctt caagtacttt 4620
gacaccacca tcgaccccaa gcagtacaga agcaccaaag aggtgctgga cgccaccctg 4680
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt 4740
gac 4743
<210> 15
<211> 498
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TadA8e V106W核苷酸序列
<400> 15
tctgaggtgg agttttccca cgagtactgg atgagacatg ccctgaccct ggccaagagg 60
gcacgggatg agagggaggt gcctgtggga gccgtgctgg tgctgaacaa tagagtgatc 120
ggcgagggct ggaacagagc catcggcctg cacgacccaa cagcccatgc cgaaattatg 180
gccctgagac agggcggcct ggtcatgcag aactacagac tgattgacgc caccctgtac 240
gtgacattcg agccttgcgt gatgtgcgcc ggcgccatga tccactctag gatcggccgc 300
gtggtgtttg gctggaggaa ctcaaaaaga ggcgccgcag gctccctgat gaacgtgctg 360
aactaccccg gcatgaatca ccgcgtcgaa attaccgagg gaatcctggc agatgaatgt 420
gccgccctgc tgtgcgattt ctatcggatg cctagacagg tgttcaatgc tcagaagaag 480
gcccagagct ccatcaac 498
<210> 16
<211> 4101
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> SpRY D10A核苷酸序列
<400> 16
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgagaga 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcaga cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcc tgtggcccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccaa gcagctgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccagact gggagcccct 3960
agagccttca agtactttga caccaccatc gaccccaagc agtacagaag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 17
<211> 4101
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> SpRY D10A-HF核苷酸序列
<400> 17
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgagaga 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccgcctt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcgcg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgg cgctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc gggcgatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcaga cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcc tgtggcccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccaa gcagctgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccagact gggagcccct 3960
agagccttca agtactttga caccaccatc gaccccaagc agtacagaag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA1
<400> 18
cctgcttgca agtgtcaacc 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA2
<400> 19
tacttgcgac agttccttcc 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA3
<400> 20
catttccctc tacgctcgct 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA4
<400> 21
gtgaaacacc acgcccggcc 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA5
<400> 22
cctcttctgg aaaggggtac 20
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA6
<400> 23
gacagttcct tccaattcca 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA7
<400> 24
aacaacagta cttgcgacag 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA8
<400> 25
gcagcagcct ggaaaagtac 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA9
<400> 26
gatgcagcga atgtgaaatc 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA10
<400> 27
tcatcttagg ccttcaagga 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA11
<400> 28
gagatgcagc gaatgtgaaa 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA12
<400> 29
gagcagggaa gaaggaatca 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA13
<400> 30
gaaaagtact tggggaccaa 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA14
<400> 31
ttggaactcc tgcttgcaag 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA15
<400> 32
aaggccaagc ttgcctgccc 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA16
<400> 33
aaagctgcat ccaccccccg 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA17
<400> 34
ctgctagacg acagcgcagg 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA18
<400> 35
gtaacctccc ttgaaagggg 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA19
<400> 36
ggagcatctg agtccagggg 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA20
<400> 37
ctagacgaca gcgcaggcaa 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA21
<400> 38
ccgaatgcaa aggttctctg 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA22
<400> 39
ccgtccaaag tcaaattgct 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA23
<400> 40
tggcacatag atagtaagtg 20
<210> 41
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA24
<400> 41
tggccgaatg caaaggttct 20
<210> 42
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA26
<400> 42
tagactaatt atataagcct 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA26
<400> 43
tggctaagca tcaggagttt 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA27
<400> 44
gagttccacc cgctgtgcgt 20
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA28
<400> 45
atgcaaaggt tctctgctag 20
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA29
<400> 46
gaatgcaaag gttctctgct 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA30
<400> 47
tcggccaatg gggcacaagg 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA31
<400> 48
gttacaccaa agggctagaa 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA32
<400> 49
ggttacacca aagggctaga 20
<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA33
<400> 50
taccaacaat agaggcccat 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA34
<400> 51
ctgtcaaact gtgcgtatga 20
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA35
<400> 52
accaacaata gaggcccatt 20
<210> 53
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA36
<400> 53
gtttacataa aagatcttca 20
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA37
<400> 54
ctaccaacaa tagaggccca 20
<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA38
<400> 55
acatcatcag atattctgca 20
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA39
<400> 56
gctgcaaaca agtgcagaat 20
<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA40
<400> 57
gtctcagagg atgtgttgtt 20
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA41
<400> 58
ctgcagccca agcctcagtg 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA42
<400> 59
tcctaaacca gtgtcaggga 20
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA43
<400> 60
aaagatcttc acaggctacc 20
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA44
<400> 61
ccaacaatag aggcccatta 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA45
<400> 62
aggtcctaaa ccagtgtcag 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA46
<400> 63
aatacaaata gttaagaaca 20
<210> 64
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA47
<400> 64
ctggaacaca aagcatagac 20
<210> 65
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sgRNA48
<400> 65
gaacacaaag catagactgc 20
<210> 66
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 用于RNA脱靶检测的sgRNA序列
<400> 66
ctggaacaca aagcatagac 20
<210> 67
<211> 1803
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> ABEmax-SpRY融合蛋白的氨基酸序列
<400> 67
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys
115 120 125
Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His
210 215 220
Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp
225 230 235 240
Glu Arg Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val
245 250 255
Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala
260 265 270
His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn
275 280 285
Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val
290 295 300
Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe
305 310 315 320
Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val
325 330 335
Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile
340 345 350
Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro
355 360 365
Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser
370 375 380
Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser
385 390 395 400
Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp
405 410 415
Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp
420 425 430
Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val
435 440 445
Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala
450 455 460
Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu Lys Arg
465 470 475 480
Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu
485 490 495
Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe
500 505 510
His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu
515 520 525
Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
530 535 540
Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr
545 550 555 560
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile
565 570 575
Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn
580 585 590
Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln
595 600 605
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala
610 615 620
Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile
625 630 635 640
Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile
645 650 655
Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
660 665 670
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp
675 680 685
Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe
690 695 700
Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu
705 710 715 720
Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile
725 730 735
Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu
740 745 750
Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln
755 760 765
Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu
770 775 780
Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr
785 790 795 800
Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln
805 810 815
Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu
820 825 830
Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys
835 840 845
Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr
850 855 860
Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr
865 870 875 880
Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val
885 890 895
Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe
900 905 910
Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu
915 920 925
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val
930 935 940
Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys
945 950 955 960
Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys
965 970 975
Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val
980 985 990
Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr
995 1000 1005
His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu
1010 1015 1020
Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe
1025 1030 1035 1040
Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1045 1050 1055
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly
1060 1065 1070
Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln
1075 1080 1085
Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn
1090 1095 1100
Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu
1105 1110 1115 1120
Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu
1125 1130 1135
His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu
1140 1145 1150
Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His
1155 1160 1165
Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1170 1175 1180
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu
1185 1190 1195 1200
Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu
1205 1210 1215
Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn
1220 1225 1230
Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser
1235 1240 1245
Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp
1250 1255 1260
Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys
1265 1270 1275 1280
Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1285 1290 1295
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp
1300 1305 1310
Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1315 1320 1325
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His
1330 1335 1340
Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1345 1350 1355 1360
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu
1365 1370 1375
Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile
1380 1385 1390
Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly
1395 1400 1405
Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1410 1415 1420
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu
1425 1430 1435 1440
Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
1445 1450 1455
Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg
1460 1465 1470
Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp
1475 1480 1485
Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1490 1495 1500
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser
1505 1510 1515 1520
Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1525 1530 1535
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr
1540 1545 1550
Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1555 1560 1565
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu
1570 1575 1580
Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1585 1590 1595 1600
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1605 1610 1615
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Lys
1620 1625 1630
Gln Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1635 1640 1645
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1650 1655 1660
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu
1665 1670 1675 1680
Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu
1685 1690 1695
Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg
1700 1705 1710
Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1715 1720 1725
Leu Thr Arg Leu Gly Ala Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr
1730 1735 1740
Ile Asp Pro Lys Gln Tyr Arg Ser Thr Lys Glu Val Leu Asp Ala Thr
1745 1750 1755 1760
Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1765 1770 1775
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly
1780 1785 1790
Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1795 1800
<210> 68
<211> 1605
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 8e-SpRY融合蛋白的氨基酸序列
<400> 68
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys
115 120 125
Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Arg Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
1010 1015 1020
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1025 1030 1035 1040
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
1045 1050 1055
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1060 1065 1070
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
1075 1080 1085
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
1090 1095 1100
Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu
1105 1110 1115 1120
Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1125 1130 1135
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp
1140 1145 1150
Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1155 1160 1165
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys
1170 1175 1180
Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala
1185 1190 1195 1200
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1205 1210 1215
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
1220 1225 1230
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
1250 1255 1260
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1265 1270 1275 1280
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1285 1290 1295
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1300 1305 1310
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro
1315 1320 1325
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1330 1335 1340
Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val Leu
1345 1350 1355 1360
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1365 1370 1375
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
1380 1385 1390
Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1395 1400 1405
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
1410 1415 1420
Gly Arg Lys Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly Asn
1425 1430 1435 1440
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1445 1450 1455
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1460 1465 1470
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1490 1495 1500
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1505 1510 1515 1520
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala
1525 1530 1535
Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr
1540 1545 1550
Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1555 1560 1565
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1570 1575 1580
Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys
1585 1590 1595 1600
Lys Lys Arg Lys Val
1605
<210> 69
<211> 1605
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> V106W-SpRY融合蛋白的氨基酸序列
<400> 69
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Asn Ser Lys
115 120 125
Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Arg Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
1010 1015 1020
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1025 1030 1035 1040
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
1045 1050 1055
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1060 1065 1070
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
1075 1080 1085
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
1090 1095 1100
Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu
1105 1110 1115 1120
Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1125 1130 1135
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp
1140 1145 1150
Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1155 1160 1165
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys
1170 1175 1180
Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala
1185 1190 1195 1200
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1205 1210 1215
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
1220 1225 1230
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
1250 1255 1260
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1265 1270 1275 1280
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1285 1290 1295
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1300 1305 1310
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro
1315 1320 1325
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1330 1335 1340
Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val Leu
1345 1350 1355 1360
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1365 1370 1375
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
1380 1385 1390
Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1395 1400 1405
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
1410 1415 1420
Gly Arg Lys Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly Asn
1425 1430 1435 1440
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1445 1450 1455
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1460 1465 1470
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1490 1495 1500
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1505 1510 1515 1520
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala
1525 1530 1535
Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr
1540 1545 1550
Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1555 1560 1565
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1570 1575 1580
Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys
1585 1590 1595 1600
Lys Lys Arg Lys Val
1605
<210> 70
<211> 1605
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 8e-SpRY-HF融合蛋白的氨基酸序列
<400> 70
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys
115 120 125
Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Arg Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
1010 1015 1020
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1025 1030 1035 1040
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
1045 1050 1055
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1060 1065 1070
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
1075 1080 1085
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
1090 1095 1100
Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu
1105 1110 1115 1120
Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1125 1130 1135
Leu Val Glu Thr Arg Ala Ile Thr Lys His Val Ala Gln Ile Leu Asp
1140 1145 1150
Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1155 1160 1165
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys
1170 1175 1180
Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala
1185 1190 1195 1200
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1205 1210 1215
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
1220 1225 1230
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
1250 1255 1260
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1265 1270 1275 1280
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1285 1290 1295
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1300 1305 1310
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro
1315 1320 1325
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1330 1335 1340
Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val Leu
1345 1350 1355 1360
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1365 1370 1375
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
1380 1385 1390
Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1395 1400 1405
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
1410 1415 1420
Gly Arg Lys Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly Asn
1425 1430 1435 1440
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1445 1450 1455
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1460 1465 1470
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1490 1495 1500
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1505 1510 1515 1520
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala
1525 1530 1535
Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr
1540 1545 1550
Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1555 1560 1565
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1570 1575 1580
Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys
1585 1590 1595 1600
Lys Lys Arg Lys Val
1605
<210> 71
<211> 1605
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> V106W-SpRY-HF融合蛋白的氨基酸序列
<400> 71
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Asn Ser Lys
115 120 125
Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Arg Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
1010 1015 1020
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1025 1030 1035 1040
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
1045 1050 1055
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1060 1065 1070
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
1075 1080 1085
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
1090 1095 1100
Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu
1105 1110 1115 1120
Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1125 1130 1135
Leu Val Glu Thr Arg Ala Ile Thr Lys His Val Ala Gln Ile Leu Asp
1140 1145 1150
Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1155 1160 1165
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys
1170 1175 1180
Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala
1185 1190 1195 1200
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1205 1210 1215
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
1220 1225 1230
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
1250 1255 1260
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1265 1270 1275 1280
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1285 1290 1295
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1300 1305 1310
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro
1315 1320 1325
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1330 1335 1340
Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val Leu
1345 1350 1355 1360
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1365 1370 1375
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
1380 1385 1390
Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1395 1400 1405
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
1410 1415 1420
Gly Arg Lys Arg Met Leu Ala Ser Ala Lys Gln Leu Gln Lys Gly Asn
1425 1430 1435 1440
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1445 1450 1455
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1460 1465 1470
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1490 1495 1500
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1505 1510 1515 1520
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala
1525 1530 1535
Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr
1540 1545 1550
Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1555 1560 1565
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1570 1575 1580
Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys
1585 1590 1595 1600
Lys Lys Arg Lys Val
1605
<210> 72
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg1的鉴定上游引物
<400> 72
gagtagccaa agaccatcag 20
<210> 73
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg1的鉴定下游引物
<400> 73
cttccataac aaaaggaagc 20
<210> 74
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg2的鉴定上游引物
<400> 74
tgtttttgtg gaacatggtg 20
<210> 76
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg2的鉴定下游引物
<400> 76
gaatcaggca agaaataagc 20
<210> 76
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg3的鉴定上游引物
<400> 76
gacttgtgga gatgcagcga 20
<210> 103
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg3的鉴定下游引物
<400> 103
cactcaccat gttccacaaa 20
<210> 77
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg4的鉴定上游引物
<400> 77
acagggtttt gccgtgttg 19
<210> 78
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg4的鉴定下游引物
<400> 78
gcccgttagg actgcttg 18
<210> 79
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg5的鉴定上游引物
<400> 79
cttgtggaga tgcagcgaat 20
<210> 80
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg5的鉴定下游引物
<400> 80
aagcactcac catgttccac 20
<210> 81
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg6的鉴定上游引物
<400> 81
gtggaacatg gtgagtgctt 20
<210> 82
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg6的鉴定下游引物
<400> 82
tagcagagga atcaggcaag 20
<210> 83
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg7的鉴定上游引物
<400> 83
acactgcaat gtttttgtgg 20
<210> 84
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg7的鉴定下游引物
<400> 84
aggcaagaaa taagcaactc 20
<210> 85
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg8的鉴定上游引物
<400> 85
gtggagatgc agcgaatgtg 20
<210> 86
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg8的鉴定下游引物
<400> 86
gaaaagcact caccatgttc 20
<210> 87
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg9的鉴定上游引物
<400> 87
gcttcctttt gttatggaag 20
<210> 88
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg9的鉴定下游引物
<400> 88
gatgattcct tcttccctgc 20
<210> 89
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg10的鉴定上游引物
<400> 89
atgggggtcc acactgcaat 20
<210> 90
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg10的鉴定下游引物
<400> 90
ccgaagtcac gaagtaactt 20
<210> 91
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg11的鉴定上游引物
<400> 91
gtgcttcctt ttgttatgga 20
<210> 92
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg11的鉴定下游引物
<400> 92
gattccttct tccctgctcc 20
<210> 93
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg12的鉴定上游引物
<400> 93
ctggaaaggg gtacctatta 20
<210> 94
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg12的鉴定下游引物
<400> 94
ttggaaggaa ctgtcgcaag 20
<210> 95
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg13的鉴定上游引物
<400> 95
agatcttgaa ccctcttctg 20
<210> 96
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg13的鉴定下游引物
<400> 96
gttcaaagtt ccttgaaggc 20
<210> 97
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg14的鉴定上游引物
<400> 97
gaggagtagc caaagaccat 20
<210> 98
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg14的鉴定下游引物
<400> 98
tccataacaa aaggaagcac 20
<210> 99
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg15的鉴定上游引物
<400> 99
tgaaccctct tctggaaagg 20
<210> 100
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg15的鉴定下游引物
<400> 100
ctgttgttca aagttccttg 20
<210> 101
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg16的鉴定上游引物
<400> 101
ggcgtggcag ctgatatccg 20
<210> 102
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg16的鉴定下游引物
<400> 102
gtgtttcaca aaacaggggt ggc 23
<210> 104
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg17的鉴定上游引物
<400> 104
gagcatctga gtccagggga 20
<210> 105
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg17的鉴定下游引物
<400> 105
gacagggtag acggcagttc 20
<210> 106
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg18的鉴定上游引物
<400> 106
cactccttgt gccccattgg 20
<210> 107
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg18的鉴定下游引物
<400> 107
gttgtagtag tcgcgactct 20
<210> 108
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg19的鉴定上游引物
<400> 108
cagtatccga aggcagcagc 20
<210> 109
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg19的鉴定下游引物
<400> 109
cattcggcca atggggcaca 20
<210> 110
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg20的鉴定上游引物
<400> 110
tacttagggg gcacttcgac 20
<210> 111
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg20的鉴定下游引物
<400> 111
aaggggaata ctcagcagta 20
<210> 112
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg21的鉴定上游引物
<400> 112
cgttggagca tctgagtcca 20
<210> 113
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg21的鉴定下游引物
<400> 113
gacggcagtt caagtgtccc 20
<210> 114
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg22的鉴定下游引物
<400> 114
tgttcacggg ctgcgctg 18
<210> 115
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg22的鉴定下游引物
<400> 115
ggtggatgca gctttgcgg 19
<210> 116
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg23的鉴定上游引物
<400> 116
ttaatgagtg gcatcttggc ata 23
<210> 117
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg23的鉴定下游引物
<400> 117
ttgcaccaaa atggttagta aaac 24
<210> 118
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg24的鉴定上游引物
<400> 118
tggagcatct gagtccaggg 20
<210> 119
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg24的鉴定下游引物
<400> 119
ggtagacggc agttcaagtg 20
<210> 120
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg25的鉴定上游引物
<400> 120
ctttgagatg gaatggagtt ttg 23
<210> 121
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg25的鉴定下游引物
<400> 121
agaaacatga actgtgagat gc 22
<210> 122
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg26的鉴定上游引物
<400> 122
gacacaagca gggacacg 18
<210> 123
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg26的鉴定下游引物
<400> 123
ctactctgga ggcgaaaaag g 21
<210> 124
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg27的鉴定上游引物
<400> 124
cgccaaggag ttgtgtaagg 20
<210> 125
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg27的鉴定下游引物
<400> 125
tctcgccttc tagccctttg 20
<210> 126
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg28的鉴定上游引物
<400> 126
gaacagcttc ggggggattg 20
<210> 127
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg28的鉴定下游引物
<400> 127
cttgtagaga gacagggtag 20
<210> 128
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg29的鉴定上游引物
<400> 128
gggattgcat gtacgcccca 20
<210> 129
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg29的鉴定下游引物
<400> 129
ccagtgctcc ggacttgtag 20
<210> 130
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg30的鉴定上游引物
<400> 130
ggcacttcga ccatttctga 20
<210> 131
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg30的鉴定下游引物
<400> 131
tatcttcagt gctcttgcct 20
<210> 132
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg31的鉴定上游引物
<400> 132
ccgaatgcaa aggttctctg 20
<210> 133
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg31的鉴定下游引物
<400> 133
ccagagccag tggaaagttg 20
<210> 134
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg32的鉴定上游引物
<400> 134
gtacgcccca cttttgggag 20
<210> 135
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg32的鉴定下游引物
<400> 135
gtccagtgct ccggacttgt 20
<210> 136
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg32的鉴定上游引物
<400> 136
cttcacaggc taccccctaa g 21
<210> 137
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg32的鉴定下游引物
<400> 137
gaaaattacc acatgctgtc 20
<210> 138
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg34的鉴定上游引物
<400> 138
atgtggtaat tttccagccc 20
<210> 139
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg34的鉴定下游引物
<400> 139
caagcacatg acagttaagg 20
<210> 140
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg35的鉴定上游引物
<400> 140
gatcttcaca ggctaccccc 20
<210> 141
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg35的鉴定下游引物
<400> 141
ccacatgctg tcacagttag 20
<210> 142
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg36的鉴定上游引物
<400> 142
gcagtgttta gaaggagact 20
<210> 143
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg36的鉴定下游引物
<400> 143
gttggtagaa tggcagtgca 20
<210> 144
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg37的鉴定上游引物
<400> 144
cacaggctac cccctaagtc 20
<210> 145
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg37的鉴定下游引物
<400> 145
ggctggaaaa ttaccacatg 20
<210> 146
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg38的鉴定上游引物
<400> 146
cagcatgtgg taattttcca 20
<210> 147
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg38的鉴定下游引物
<400> 147
cacatgacag ttaaggtttg 20
<210> 148
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg39的鉴定上游引物
<400> 148
caagacctgg ctgagctaac 20
<210> 149
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg39的鉴定下游引物
<400> 149
tttagtcttt caagcaggtg 20
<210> 150
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg40的鉴定上游引物
<400> 150
aattgtttga acctgggagt tg 22
<210> 151
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg40的鉴定下游引物
<400> 151
gttgatagcc tcacagaaag taac 24
<210> 152
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg41的鉴定上游引物
<400> 152
cctaaaccag tgtcagggag 20
<210> 153
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg41的鉴定下游引物
<400> 153
tgctttgtgt tccagtttcc 20
<210> 154
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg42的鉴定上游引物
<400> 154
agaaggagac ttgtgcacat 20
<210> 155
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg42的鉴定下游引物
<400> 155
attgttggta gaatggcagt 20
<210> 156
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg43的鉴定上游引物
<400> 156
gtttagaagg agacttgtgc 20
<210> 157
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg43的鉴定下游引物
<400> 157
ggtagaatgg cagtgcaata 20
<210> 158
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg44的鉴定上游引物
<400> 158
aaagatcttc acaggctacc 20
<210> 159
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg44的鉴定下游引物
<400> 159
ccagcgggct ggaaaattac 20
<210> 160
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg45的鉴定上游引物
<400> 160
ggagacttgt gcacattcta 20
<210> 161
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg45的鉴定下游引物
<400> 161
tctattgttg gtagaatggc 20
<210> 162
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg46的鉴定上游引物
<400> 162
ggccctgtaa aggaaactgg 20
<210> 163
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg46的鉴定下游引物
<400> 163
tgtttagtct ttcaagcagg 20
<210> 164
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg47的鉴定上游引物
<400> 164
atgataacaa gacctggctg 20
<210> 165
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg47的鉴定下游引物
<400> 165
tctttcaagc aggtgattac 20
<210> 166
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg48的鉴定上游引物
<400> 166
ataacaagac ctggctgagc 20
<210> 167
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sg48的鉴定下游引物
<400> 167
tcaagcaggt gattacagga 20
<210> 168
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Mut-sgRNA
<400> 168
cctccaacct gtggctggcc tg 22
<210> 169
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA1
<400> 169
tccgcctcca acctgtggct gg 22
<210> 170
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA2
<400> 170
ccgcctccaa cctgtggctg gc 22
<210> 171
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA3
<400> 171
cgcctccaac ctgtggctgg cc 22
<210> 172
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA4
<400> 172
gcctccaacc tgtggctggc ct 22
<210> 173
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA5
<400> 173
cctccaacct gtggctggcc tg 22
<210> 174
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA6
<400> 174
ctccaacctg tggctggcct gc 22
<210> 175
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA7
<400> 175
tccaacctgt ggctggcctg ct 22
<210> 176
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH Rec-sgRNA8
<400> 176
ccaacctgtg gctggcctgc tt 22
<210> 177
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH 扩增上游引物
<400> 177
gtccctgggc agttatgtgt ac 22
<210> 178
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PAH 扩增下游引物
<400> 178
caactggtag ctggaggaca g 21