CN114686456B - 基于双分子脱氨酶互补的碱基编辑系统及其应用 - Google Patents

基于双分子脱氨酶互补的碱基编辑系统及其应用 Download PDF

Info

Publication number
CN114686456B
CN114686456B CN202210503831.5A CN202210503831A CN114686456B CN 114686456 B CN114686456 B CN 114686456B CN 202210503831 A CN202210503831 A CN 202210503831A CN 114686456 B CN114686456 B CN 114686456B
Authority
CN
China
Prior art keywords
leu
lys
glu
gly
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210503831.5A
Other languages
English (en)
Other versions
CN114686456A (zh
Inventor
李剑峰
贺雄雷
熊翔宇
刘科辉
黎镇祥
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sun Yat Sen University
Original Assignee
Sun Yat Sen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Yat Sen University filed Critical Sun Yat Sen University
Priority to CN202210503831.5A priority Critical patent/CN114686456B/zh
Publication of CN114686456A publication Critical patent/CN114686456A/zh
Priority to PCT/CN2022/106402 priority patent/WO2023216415A1/zh
Application granted granted Critical
Publication of CN114686456B publication Critical patent/CN114686456B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

本发明涉及基于双分子脱氨酶互补的碱基编辑系统及其应用，属于基因工程技术领域。该系统主要由nCas9和核碱基脱氨酶双重互补的碱基编辑融合蛋白A、B以及向导RNA组成。与传统胞嘧啶碱基编辑系统相比，本发明公开的基于双分子脱氨酶互补的胞嘧啶碱基编辑系统，在保持高效胞嘧啶碱基编辑中靶效率的同时，极大降低了其在基因组上的Cas9依赖型和Cas9非依赖型脱靶；本发明还提供了可高效工作的基于双分子脱氨酶互补的腺嘌呤碱基编辑系统；本发明公开的基于双分子脱氨酶互补的胞嘧啶和腺嘌呤碱基编辑系统普遍适用于真核生物，从真菌、动物到植物，在作物遗传育种、动物品种改良乃至人类遗传疾病的临床治疗等领域具有广阔的应用前景。

Description

基于双分子脱氨酶互补的碱基编辑系统及其应用
技术领域
本发明涉及基于双分子脱氨酶互补的碱基编辑系统及其应用，属于基因工程技术领域。
背景技术
数量众多的作物农艺性状以及人类遗传疾病由基因组上的单核苷酸多态性(Single nucleotide polymorphisms,SNPs)决定[1,2]。由CRISPR/Cas9基因编辑技术衍生而来的碱基编辑技术(Base editing)具有不产生DNA双链断裂以及可精确实现靶位点单碱基替换等特点，极大提高了基因编辑的精确度和安全性，因而被广泛应用于基因功能研究、作物遗传改良和人类基因疾病的临床治疗等领域[3]。碱基编辑系统主要由碱基编辑器(Baseeditors,BEs)和向导RNA (single-guide RNA,sgRNA)构成。科研人员最常用的碱基编辑器BE3是将来自大鼠的胞苷脱氨酶rAPOBEC1、来自噬菌体的尿嘧啶糖苷酶抑制子(Uracilglycosylase inhibitor,UGI)与Cas9切口酶(Cas9 nickase,nCas9-D10A)融合获得[4]。在sgRNA的引导下，BE3结合于靶标基因位点处，其所融合的胞苷脱氨酶rAPOBEC1发挥脱氨作用，将靶序列中特定位置的胞嘧啶(C)碱基突变为尿嘧啶(U)碱基。在细胞内源修复机制的作用下，U碱基变为胸腺嘧啶(T) 碱基，最终达到C到T的碱基替换[4]。
然而，近年来国际上多个在水稻、小鼠等生物体中的独立研究均发现，传统碱基编辑系统(如BE3系统)会在全基因组水平上引发大量不依赖于向导RNA (或Cas9)的随机脱靶突变[5,6]。这给碱基编辑系统的实际使用，增加了难以评估的安全隐患，尤其是给临床应用带来了极大的不确定性。因此，发展高效、通用且安全的低脱靶碱基编辑系统，对基础科学研究、作物遗传改良乃至人类基因疾病的临床治疗都将大有裨益。
发明内容
本发明的目的在于提供一种新型的基于双分子脱氨酶互补的碱基编辑系统及其应用，本发明提出的基于双分子脱氨酶互补的碱基编辑系统能极大减少传统碱基编辑系统存在的全基因组随机脱靶问题，同时仍保持了高效的中靶编辑，具有广阔的应用前景。
为实现上述目的，本发明采取的技术方案为：一种双分子脱氨酶互补的碱基编辑系统，所述碱基编辑系统包括以下1)至5)中至少一项：
1)碱基编辑融合蛋白A、碱基编辑融合蛋白B和向导RNA；
2)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和向导RNA；
3)碱基编辑融合蛋白A、碱基编辑融合蛋白B、和包含编码向导RNA的核苷酸序列的表达构建体；
4)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
5)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
其中所述的碱基编辑融合蛋白A自N端到C端依次包括第一nCas9多肽片段、柔性连接肽和第一核碱基脱氨酶多肽片段；所述碱基编辑融合蛋白B自N 端到C端依次包括第二核碱基脱氨酶多肽片段、柔性连接肽和第二nCas9多肽片段；所述第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段选自相同的核碱基脱氨酶。
需要指出的是，“表达构建体”指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指可产生有功能的产物。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明所述“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者可以是能够翻译的RNA(如mRNA)。本发明的“表达构建体”可包含相同来源但以不同于天然排列方式的调控序列和感兴趣的核苷酸序列，亦包含不同来源的调控序列和感兴趣的核苷酸序列。
本申请发明人发现，在本发明所述的碱基编辑系统中，向导RNA能与碱基编辑融合蛋白A和碱基编辑融合蛋白B形成蛋白核酸复合体(RNP)，并引导 RNP靶向目标DNA序列，导致所述靶标序列中一至多个胞嘧啶碱基到胸腺嘧啶碱基，或一至多个腺嘌呤(A)碱基到鸟嘌呤(G)碱基的核苷酸取代。且其第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段均不含有完整脱氨酶序列，也不具有碱基脱氨活性，这是该系统降低脱靶的关键所在。
本申请发明人发现，在本发明所述的碱基编辑系统中，所述第一、第二核碱基脱氨酶通常是互补的，即可融合成具备完整脱氨活性的核碱基脱氨酶。
作为本发明所述碱基编辑系统的优选实施方式，所述第一nCas9多肽片段的氨基酸序列如SEQ ID NO:1所示；所述第二nCas9多肽片段的氨基酸序列如 SEQ ID NO:2所示。
作为本发明所述碱基编辑系统的优选实施方式，所述柔性连接肽包括氨基酸序列如SEQ ID NO:3所示的32aa连接肽。研究表明，所述柔性连接肽还包括 XTEN、PR、GGGGS、PRGGSGG、ARGGSGG、GS、GAP、(GGGGS)×3、 GGS和(GGS)×7。所述柔性的连接肽也可以是长1-50个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑融合蛋白A 和碱基编辑融合蛋白B的N端或C端融合有至少一个核定位序列。研究表明，所述核定位序列(NLS)通常可以与入核载体相互作用，从而可以使靶蛋白能够被运送进细胞核。一般而言，NLS由暴露于蛋白表面上的带正电的赖氨酸或精氨酸的一个或多个短序列组成，但其他类型的NLS也是已知的。NLS的非限制性实例包括氨基酸序列如：PKKKRKV或KRPAATKKAGQAKKKK。在一些具体的实施例中，本发明所述碱基编辑融合蛋白A的N端包含1个拷贝的NLS(氨基酸序列为PKKKRKV),所述碱基编辑融合蛋白B的C端包含1个拷贝的NLS (氨基酸序列为KRPAATKKAGQAKKKK)或2个拷贝的NLS(氨基酸序列为 PKKKRKV和KRPAATKKAGQAKKKK)。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑系统为胞嘧啶碱基编辑器系统；所述核碱基脱氨酶为胞苷脱氨酶；利用该系统可在向导RNA 的引导下使目标序列中的一至多个C碱基替换为T碱基。
作为本发明所述碱基编辑系统的优选实施方式，所述胞苷脱氨酶选自：由APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、APOBEC3D(A3D)、 APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、 APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID)或通过单个或多个氨基酸组合突变APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、 APOBEC3D(A3D)、APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID) 获得的胞苷脱氨酶变体构成的组。
作为本发明所述碱基编辑系统的优选实施方式，所述胞苷脱氨酶选自人源 AID高活性变体AID10(中国专利：202010285948.1)、大鼠来源的rA1、人源的hA3A或人源的hA3B。
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑融合蛋白A的第一核碱基脱氨酶多肽片段包括如SEQ ID NO:4～8所示任一氨基酸序列；所述胞嘧啶碱基编辑融合蛋白B的第二核碱基脱氨酶多肽片段包括如SEQ ID NO:9～13所示任一氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑融合蛋白B的C端融合有至少一个尿嘧啶糖苷酶抑制子(UGI)，所述UGI的氨基酸序列如如SEQ ID NO:14所示
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑系统包括以下1)至5)任一项所述的碱基编辑融合蛋白A和碱基编辑融合蛋白B的组合：
1)氨基酸序列如SEQ ID NO:15所示的碱基编辑融合蛋白A： Split-AID10-N，和氨基酸序列如SEQ ID NO:16所示的碱基编辑融合蛋白B： Split-AID10-C；
2)氨基酸序列如SEQ ID NO:17所示的碱基编辑融合蛋白A： Split-AID10-N5，和氨基酸序列如SEQ ID NO:18所示的碱基编辑融合蛋白B：Split-AID10-C4；
3)氨基酸序列如SEQ ID NO:19所示的碱基编辑融合蛋白A：Split-BE3-N，和氨基酸序列如SEQ ID NO:20所示的碱基编辑融合蛋白B：Split-BE3-C；
4)氨基酸序列如SEQ ID NO:21所示的碱基编辑融合蛋白A：Split-A3A-N，和氨基酸序列如SEQ ID NO:22所示的碱基编辑融合蛋白B：Split-A3A-C；
5)氨基酸序列如SEQ ID NO:23所示的碱基编辑融合蛋白A：Split-A3B-N，和氨基酸序列如SEQ ID NO:24所示的碱基编辑融合蛋白B：Split-A3B-C。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶AID10互补的胞嘧啶碱基编辑系统“Split-AID10”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段(氨基酸序列如SEQ ID NO:1所示)、 32aa连接肽(氨基酸序列如SEQ IDNO:3所示)以及第一核碱基多肽片段 AID10-N(氨基酸序列如SEQ ID NO:4所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:15所示，称为“Split-AID10-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段AID10-C(氨基酸序列如SEQ ID NO:9所示)、32aa 连接肽、第二nCas9多肽片段(氨基酸序列如SEQ ID NO:2所示)、1分子UGI(氨基酸序列如SEQ IDNO:14所示)和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:16所示，称为“Split-AID10-C”(如图2所示)。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶AID10互补的胞嘧啶碱基编辑系统“Split-AID10-N5-C4”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段AID10-N5(氨基酸序列如SEQ ID NO:5所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:17所示，称为“Split-AID10-N5”；所述碱基编辑融合蛋白B由第二核碱基多肽片段AID10-C4(氨基酸序列如SEQ ID NO:10所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS 从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:18所示，称为“Split-AID10-C4”(如图2所示)。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶rA1互补的胞嘧啶碱基编辑系统“Split-BE3”。其所述碱基编辑融合蛋白A 由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段 BE3-N(氨基酸序列如SEQ ID NO:6所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:19所示，称为“Split-BE3-N”；所述碱基编辑融合蛋白B 由第二核碱基多肽片段BE3-C(氨基酸序列如SEQ ID NO:11所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:20所示，称为“Split-BE3-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶hA3A互补的胞嘧啶碱基编辑系统“Split-A3A”。其所述碱基编辑融合蛋白 A由NLS多肽片段，第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段A3A-N(氨基酸序列如SEQ ID NO:7所示)依次融合而成，其氨基酸序列如 SEQ ID NO:21所示，称为“Split-A3A-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段A3A-C(氨基酸序列如SEQ ID NO:12所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:22所示，称为“Split-A3A-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶hA3B互补的胞嘧啶碱基编辑系统“Split-A3B”。其中，所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段A3B-N(氨基酸序列如SEQ IDNO:8所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:23所示，称为“Split-A3B-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段A3B-C(氨基酸序列如SEQ ID NO:13所示)、 32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:24所示，称为“Split-A3B-C”。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑系统为腺嘌呤碱基编辑器系统；所述核碱基脱氨酶为腺苷脱氨酶；利用该系统可在向导RNA 的引导下使目标序列中的一至多个A碱基替换为G碱基。
作为本发明所述碱基编辑系统的优选实施方式，所述腺苷脱氨酶为DNA依赖型腺苷脱氨酶；优选地，所述腺苷脱氨酶为单链DNA依赖型腺苷脱氨酶。
作为本发明所述碱基编辑系统的优选实施方式，所述腺苷脱氨酶包括大肠杆菌tRNA腺苷脱氨酶TadA的变体；优选地，所述变体包括TadA-7.10、TadA-8s 或TadA-8e；更优选地，所述变体为TadA-8e(国际专利：PCT/US2021/016827)。
作为本发明所述碱基编辑系统的优选实施方式，所述腺嘌呤碱基编辑融合蛋白A的第一核碱基脱氨酶多肽片段包括如SEQ ID NO:25～26所示任一氨基酸序列；所述腺嘌呤碱基编辑融合蛋白B的第二核碱基脱氨酶多肽片段包括如 SEQ ID NO:27～28所示任一氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述腺嘌呤碱基编辑系统包括以下1)至2)任一项所述的碱基编辑融合蛋白A和碱基编辑融合蛋白B组合：
1)氨基酸序列如SEQ ID NO:29所示的碱基编辑融合蛋白A： Split-ABE8e-N，和氨基酸序列如SEQ ID NO:30所示的碱基编辑融合蛋白B： Split-ABE8e-C；
2)氨基酸序列如SEQ ID NO:31所示的碱基编辑融合蛋白A： Split-ABE8e-N7，和氨基酸序列如SEQ ID NO:32所示的碱基编辑融合蛋白B： Split-ABE8e-C2。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、腺苷脱氨酶TadA-8e互补的胞嘧啶碱基编辑系统“Split-ABE8e”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段ABE8e-N(氨基酸序列如SEQ IDNO:25所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:29所示，称为“Split-ABE8e-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段ABE8e-C(氨基酸序列如SEQ IDNO:27 所示)、32aa连接肽、第二nCas9多肽片段和2分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:30所示，称为“Split-ABE8e-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、腺苷脱氨酶TadA-8e互补的胞嘧啶碱基编辑系统“Split-ABE8e-N7-C2”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段ABE8e-N7(氨基酸序列如SEQ ID NO:26所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:31所示，称为“Split-ABE8e-N7”；所述碱基编辑融合蛋白B由第二核碱基多肽片段ABE8e-C2(氨基酸序列如SEQ ID NO:28所示)、32aa连接肽、第二nCas9多肽片段和2分子NLS从N端到C 端依次融合而成，其氨基酸序列如SEQ ID NO:32所示，称为“Split-ABE8e-C2”。
作为本发明所述碱基编辑系统的优选实施方式，所述编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件可操作地连接。
作为本发明所述碱基编辑系统的优选实施方式，所述调控元件为启动子；所述启动子包括病毒35S启动子，玉米Ubi-1启动子，水稻Ubi启动子，病毒 CMV启动子，酵母TDH3启动子，酵母GAL1启动子，拟南芥卵细胞特异的 EC1.2en+EC1.1嵌合启动子，水稻U6启动子，拟南芥U6启动子或人U6启动子。
研究表明，本发明可使用的启动子的实例包括但不限于聚合酶(pol)I、pol II 或pol III启动子。pol I启动子的包括鸡RNA pol I启动子。pol II启动子包括但不限于劳斯肉瘤病毒长末端重复(RSV-LTR)启动子和猿猴病毒40(SV40)立即早期启动子。pol III启动子包括U6和H1启动子。可以使用诱导型启动子如金属硫蛋白启动子。当用于植物时，启动子可以是花椰菜花叶病毒35S启动子、玉米Ubi-1启动子、小麦U6启动子、水稻U3启动子、玉米U3启动子、水稻肌动蛋白启动子,拟南芥卵细胞特异的EC1.2en+EC1.1嵌合启动子、拟南芥U6 启动子；当用于酵母时，启动子可以是酵母ADH1启动子，酵母TDH3启动子，酵母GAL1启动子，酵母SNR52启动子；当用于人等哺乳动物时，启动子可以是病毒CMV启动子，人U3/U6启动子。
本发明还提供所述的碱基编辑系统在产生经遗传修饰的生物体中的应用，将所述碱基编辑系统导入生物体细胞，由所述向导RNA将所述碱基编辑器靶向细胞基因组中的靶序列，导致所述靶序列中至少一个C碱基被T碱基取代或至少一个A碱基被G碱基取代。
作为本发明所述应用的优选实施方式，所述生物为真核生物；所述真核生物为真菌、动物或植物。
作为本发明所述应用的优选实施方式，所述真菌包括酵母；所述动物包括人、小鼠、大鼠、猴、犬、猪、羊、牛或猫；所述植物包含单子叶植物和双子叶植物，包括并不限于拟南芥、水稻、小麦、玉米、大豆、向日葵、高粱、油菜、苜蓿、棉花、大麦、粟、甘蔗、番茄、烟草、木薯或马铃薯。
本发明的方法尤其适合于产生经遗传修饰的植物，例如作物植物。在本发明的产生经遗传修饰的植物的方法中，所述碱基编辑系统可以本领域技术人员熟知的各种方法导入植物。可用于将本发明的碱基编辑系统导入植物的方法包括但不限于：基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化、植物病毒介导的转化、花粉管通道法和子房注射法。
在本发明提供的一些优选地具体实施方式中，使用基于双分子脱氨酶互补的碱基编辑系统产生的经遗传修饰的植物体具有优良的农艺性状。如图5B所示，使用基于双分子脱氨酶互补的碱基编辑系统“Split-AID10”，结合靶向拟南芥内源基因ALS的向导RNA，使转基因拟南芥该内源靶位点上发生G13>A13的单碱基替换，从而导致该编码基因的氨基酸序列发生第122位丙氨酸到苏氨酸 (A122T)的氨基酸替换。后续的除草剂喷施实验表明，含有该A122T氨基酸替换的转基因拟南芥植株均对咪唑啉酮类除草剂产生了抗性，而不含该氨基酸替换的拟南芥植株全部枯萎致死，说明使用Split-AID10碱基编辑系统赋予了转基因拟南芥优良的除草剂抗性。
与现有技术相比，本发明的有益效果为：
(1)本发明的基于双分子脱氨酶互补的胞嘧啶碱基编辑系统相比传统胞嘧啶碱基编辑(如BE3系统)，降低了碱基编辑在基因组上的Cas9依赖型和Cas9 非依赖型(随机型)脱靶，同时仍然保持了较为高效的碱基编辑中靶活性。
(2)本发明提供的基于双分子脱氨酶互补的腺嘌呤碱基编辑系统也展现出了在几乎所有代表性真核生物的普适性，在单子叶植物如水稻，双子叶植物如拟南芥，真菌如酵母，哺乳动物如人HEK293T细胞系均展现良好的腺嘌呤碱基编辑活性。
(3)本发明的基于双分子脱氨酶互补的碱基编辑系统的开发和应用，为生物基因科学研究，作物遗传育种，尤其是人类遗传疾病的临床治疗提供了安全而有力的技术支撑。
附图说明
图1为基于双分子脱氨酶互补的碱基编辑系统的工作原理图。
图2为用于在水稻原生质体中瞬时表达碱基编辑器的载体示意图。
图3：A为在水稻原生质体中使用R-loop方法评估多个碱基编辑器的中靶效率统计图(n＝3)；B为在水稻原生质体中使用R-loop方法评估多个碱基编辑器的脱靶效率统计图(n＝3)；C为图A和图B的综合统计图(n＝12)。
图4：A为4个水稻内源靶点的中靶编辑效率柱状图；B为比较split-AID10 与两个传统碱基编辑器的Cas9依赖型脱靶编辑效率柱状图(n＝2)与脱靶位点信息表。
图5：A为split-AID10系统在3个拟南芥内源靶点的碱基编辑统计表；B 为使用split-AID10系统编辑拟南芥内源ALS基因，从而赋予转基因T1代拟南芥对于咪唑啉酮类除草剂的抗性效果图和靶位点桑格测序图。
图6：A为酿酒酵母6个靶点的编辑效率统计图(n＝3)；B为为酿酒酵母中单细胞水平全基因组范围的脱靶编辑评估，图中柱状图的值代表了多个生物学重复的平均值(ns,统计不显著；****，p值小于0.0001)。
图7：A为人胚胎肾细胞系HEK293T中6个靶点的中靶编辑效率统计图 (n＝3)；B为人胚胎肾细胞系HEK293T中6个靶点的脱靶编辑效率统计图 (n＝3)；C为人胚胎肾细胞系HEK293T中图A和图B中所有靶点的中靶检测和脱靶检测编辑水平统计图(n＝18)；D为HEK293T细胞系中4个靶点的中靶编辑效率以及每个靶点对应的Cas9依赖型脱靶编辑水平统计图(n＝3)。
图8：A为酿酒酵母内源靶点Ade1-1每个碱基C的编辑效率统计图(n＝3)； B为酿酒酵母内源靶点Ade1-3每个碱基C的编辑效率统计图(n＝3)；C为酿酒酵母内源靶点Can1-1每个碱基C的编辑效率统计图(n＝3)；D为酿酒酵母内源靶点Can1-9每个碱基C的编辑效率统计图(n＝3)。
图9：A为在拟南芥原生质体中，比较Split-ABE8e和Split-ABE8e-N7-C2 系统与2个传统腺嘌呤碱基编辑系统靶向内源基因FLS2的编辑效率统计图 (n＝2)；B和C为使用Split-ABE8e系统对转基因拟南芥(B)和转基因水稻 (C)植株中各自3个内源基因的编辑情况统计图。
图10：A、B分别展示了酿酒酵母内源靶点Can1-5和Can1-9每个碱基A 的编辑效率统计图(n＝3)；C、D分别展示了在人胚胎肾细胞系HEK293T靶点Site 1和Site 2的腺嘌呤碱基编辑情况。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在公开发表的文献中有更全面的描述[7]。下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买获得。
如图1所示，本发明提供的基于双分子脱氨酶互补的碱基编辑系统主要由三部分组成：碱基编辑融合蛋白A，碱基编辑融合蛋白B以及向导RNA。其中，所述碱基编辑融合蛋白A和碱基编辑融合蛋白B共同构成双分子脱氨酶互补的碱基编辑器。所述融合蛋白A由第一nCas9多肽片段与第一脱氨酶多肽片段通过柔性连接肽融合而成。所述融合蛋白B由第二脱氨酶多肽片段与第二nCas9 多肽片段通过柔性连接肽融合而成。所述第一，第二nCas9多肽片段可无缝融合成完整nCas9蛋白。
所述第一，第二脱氨酶多肽片段的脱氨酶选自同一核碱基脱氨酶，且其任一部分均不包含完整脱氨酶序列，也不具脱氨酶活性。在一些优选的实施方案中，如Split-AID10胞嘧啶碱基编辑系统(图2-7)，Split-BE3胞嘧啶碱基编辑系统(图6-7)，Split-A3A胞嘧啶碱基编辑系统(图8)，Split-A3B胞嘧啶碱基编辑系统(图8)和Split-ABE8e腺嘌呤碱基编辑系统(图9-10)中，所述第一，第二脱氨酶多肽片段也可无缝融合成完整的相应核碱基脱氨酶。
在一些次优选的实施方案中，如Split-AID10-N5-C4胞嘧啶碱基编辑系统(图 2)和Split-ABE8e-N7-C2腺嘌呤碱基编辑系统(图9)，其第一第二脱氨酶多肽片段有部分氨基酸序列的重叠，融合成的脱氨酶氨基酸序列长度超过原始所选核碱基脱氨酶。
其工作原理为：当向导RNA与碱基编辑融合蛋白A和B共表达时，向导 RNA引导后两者组装成完整的碱基编辑器并，三者共同组成蛋白核酸复合体 (RNPs)。随后RNPs由向导RNA靶向结合至目的基因位点处，导致所述目的基因序列中一至多个C碱基到T碱基的核苷酸取代(胞嘧啶碱基编辑系统)或一至多个A碱基到G碱基的核苷酸取代(腺嘌呤碱基编辑系统)。
本发明提供的实施例证明，前述优选的实施方案如Split-AID10胞嘧啶碱基编辑系统在植物如水稻，真菌如酵母，动物如人的HEK293T细胞系中均表现出接近背景水平的全基因组DNA脱靶，显著低于传统碱基编辑器如BE3(本发明也称“N-BE3”)，同时维持了较为高效的DNA中靶效率。前述优选的实施方案 Split-BE3胞嘧啶碱基编辑系统在代表性真核生物酵母和人的HEK293T细胞系中也表现出接近背景水平的全基因组DNA脱靶，显著低于传统碱基编辑器BE3，同时维持了较为高效的DNA中靶效率。前述优选的Split-ABE8e腺嘌呤碱基编辑系统也展示出了广泛的物种适应性，从单细胞真核生物酵母，到高等植物拟南芥和水稻，乃至人的HEK293T细胞系中均能实现对真核生物内源靶点的有效腺嘌呤碱基编辑。
实施例1在单子叶模式植物水稻中评估基于双分子脱氨酶互补的胞嘧啶碱基编辑系统的中靶效率和脱靶效率。
1、实验材料：本实施例所使用野生型水稻品种为粳稻中花11(Oryza sativaL.ssp.japonica ZH11)。
2、碱基编辑相关水稻原生质体瞬时表达载体的构建
碱基编辑器“N-BE3”(或称为“BE3”)的表达载体pHBT-rAPOBEC1-nCas9-UGI由本实验室自主构建，其所用启动子为玉米 ZmUbi-1启动子，所用终止子为NOS终止子。
基于双分子脱氨酶互补的胞嘧啶碱基编辑器“Split-AID10”由“Split-AID10-N”表达载体和“Split-AID10-C”表达载体共同构成；由上海生工生物技术有限公司(SangonBiotech.Co.,Ltd.)商业化合成编码氨基酸序列如 SEQ ID NO:15(Split-AID10-N)所述多肽片段的多核苷酸序列，随后将该产物经简单酶切连接融合至前述pHBT载体获得表达载体pHBT-Split-AID10-N；“Split-AID10-C”表达载体的构建：pHBT-Split-AID10-C载体构建过程与 pHBT-Split-AID10-N基本一致，其包含编码氨基酸序列如SEQ ID NO:16 (Split-AID10-C)所述多肽片段的多核苷酸序列。
基于双分子脱氨酶互补的胞嘧啶碱基编辑器“Split-AID10-N5-C4”由表达载体pHBT-Split-AID10-N5和表达载体pHBT-Split-AID10-C4共同构成，构建方法同“Split-AID10”，其中Split-AID10-N5的氨基酸序列如SEQ ID NO:17， Split-AID10-C4的氨基酸序列如SEQ ID NO:18。
nSaCas9表达载体的构建：植物密码子优化的SaCas9的多核苷酸序列来自德国Puchta Holger教授的馈赠(已公开论文[8])。使用购自南京诺赞生物技术有限公司(VazymeBiotech Co.,Ltd)的定点突变试剂盒(Mut Express II Fast Mutagenesis Kit V2，#C214-01)，将前述SaCas9编码氨基酸序列中的第10位天冬氨酸突变为丙氨酸，获得编码nSaCas9(D10A)的多核苷酸序列。最后通过简单酶切连接方法构建表达载体pHBT-nSaCas9。
3、水稻向导RNA瞬时表达载体的构建
参照已公开发表的论文中阐述的方法[9,10]，并基于 pUC119-OsU6apro-sgRNA[10]构建8个向导RNA表达载体 pUC119-OsAAT1-sgRNA、pUC119-OsCDC48-sgRNA、pUC119-OsNAL1-sgRNA、 pUC119-OsPDS1-sgRNA、pUC119-OsCDC48-Sa-gRNA、 pUC119-OsNRT1.1B-Sa-gRNA、pUC119-OsDEP1-Sa-gRNA和pUC119-OsAAT1-Sa-gRNA。向导RNA所包含的靶序列信息如表1所示。
表1
Figure BDA0003636470480000141
4、水稻原生质体的制备及转染
水稻原生质体制备：
1)12小时光照(32℃)/12小时黑暗(28℃)，200μmol·m-2·s-1光照，70％湿度，土培或者无菌组培8-10天龄水稻幼苗；
2)配制酶解液(1.5％纤维素酶R10，0.4％果胶酶R10，0.4M甘露醇，20mM MES pH5.7，20mM KCl，10mM CaCl2和0.1％BSA)15mL，并用针筒和0.45 μm孔径的滤膜过滤，加入至直径10cm的细胞培养皿中；
3)选取约200颗生长健康、颜色嫩绿的水稻苗，用刀片把叶鞘切成0.5-1mm 的茎段；
4)将茎段放入前述酶解液中，注意用接种环将各个茎段分散，避免粘连，并使其完全浸没于酶解液中，置于50-60rpm转速的水平摇床上，避光酶解3小时；
5)酶解完成后，加入10mL W5(154mM NaCl，125mM CaCl2，5mM KCl 和2mM MES pH5.7)溶液，用适当的力度摇晃培养皿，使原生质体释放于溶液中，此时可以看到溶液变淡绿，用孔径45μm的尼龙膜过滤酶解产物，收集滤液至圆底离心管中；
6)在水平离心机中以150g离心5分钟，然后用真空泵吸去尽可能多的上清液，淡绿色原生质体沉于圆底管底部；
7)缓慢加入10mL W5溶液，轻轻摇晃圆底管以重悬细胞，随后于冰上静置0.5-1小时；
8)冰上静置后，以150g水平离心3分钟，吸去上清，缓慢加入2mL MMg (0.4M甘露醇，15mM MgCl2和4mM MES pH5.7)溶液并轻摇圆底管以重悬原生质体，用血球计数板在显微镜下确定细胞浓度，再次加入适量MMg溶液调整细胞浓度至2×106个/mL。
水稻原生质体转染：
向2mL圆底管中依次加入300μL前述调整好细胞浓度的水稻原生质体、 30μL(66μg)质粒和330μL PEG(40％PEG4000(v/v)，0.2M甘露醇和0.1M CaCl2)，反应液轻柔地充分混匀，避光静置15分钟，随后加入1.2mL W5溶液，混匀，从而终止转染。水平离心机200g离心5分钟，吸去上清，加入150μL W5 溶液重悬，并转移至1mL WI溶液(0.5M甘露醇，20mM KCl和4mM MES pH 5.7)中。室温黑暗培养48小时，水平离心机250g离心5分钟，吸上清，淡黄色原生质体细胞聚集于管底，之后迅速冻于液氮，于-80℃冰箱保存。
5、水稻原生质体基因组中靶序列的深度测序和碱基替换分析
1)使用购自天根生化科技(北京)有限公司(TIANGEN Biotech.Co.,Ltd.) 的快捷型植物基因组DNA提取系统(DNAquick Plant System,#4992710)提取水稻原生质体DNA；
2)设计带有barcode标签序列的第一轮特异性引物扩增靶序列上下游200bp 左右的片段，在第一轮PCR产物的基础上使用通用建库引物进行第二轮建库 PCR引物信息列于表2；
3)按等摩尔比混合前述第二轮建库PCR产物，纯化后送往北京诺禾致源科技股份有限公司(Novogene Co.,Ltd.)进行扩增子测序。使用NovaSeq平台进行双端PE150测序；
4)在公共数据分析平台上(https://usegalaxy.org/)[11]根据barcode拆分前述测序数据，最后使用CRISPResso2[12]或BE-Analyzer[13]网站进行靶序列突变分析，用Excel呈现靶位点碱基替换情况。
表2引物信息
Figure BDA0003636470480000161
Figure BDA0003636470480000171
Figure BDA0003636470480000181
Figure BDA0003636470480000191
Figure BDA0003636470480000201
Figure BDA0003636470480000211
6、基于正交R-loop的高通量碱基编辑器特异性检测方法
参照已公开发表的论文中阐述的方法[14]。本实施例中选择了8个水稻内源靶点，包括4个SpCas9的向导RNA靶向的中靶位点(OsAAT1，OsCDC48， OsNAL1与OsPDS1)，与4个nSaCas9的向导RNA靶向的脱靶位点(OsCDC48, OsNRT1.1B,OsDEP1,OsAAT1)。为了模拟碱基编辑器在细胞内的真实工作情况，将前述靶位点按照SpCas9与SaCas9一一对应的方式随机分组，获得 Sp-OsAAT1与Sa-OsCDC48,Sp-OsCDC48与Sa-OsNRT1.1B,Sp-OsNAL1与 Sa-OsDEP1和Sp-OsPDS1与Sa-OsAAT1共4个组合。在每个组合的试验中，每个水稻原生质体的处理组须等量地转染碱基编辑器质粒(特别地，Split-AID10 和Split-AID10-N5-C4碱基编辑器则须转染等量pHBT-Split-AID10-N(N5)质粒和 pHBT-Split-AID10-C(C4)质粒)，SpCas9向导RNA质粒(如 pUC119-OsAAT1-sgRNA)，nSaCas9质粒(pHBT-nSaCas9)以及nSaCas9向导RNA质粒(如pUC119-OsCDC48-Sa-gRNA)。针对每个组合试验中的每个处理组，同时进行SpCas9靶点(中靶)和nSaCas9靶点(脱靶)的深度测序和分析。
7、水稻遗传转化载体的构建和水稻的遗传转化
1)碱基编辑器BE3的遗传转化载体为pH-nCas9-PBE(addgene:#98163)，其中所用启动子为ZmUbi-1启动子，所用终止子为E9终止子。靶向编辑目的基因OsSWEET11/13/14：商业化合成3个向导RNA的表达框 OsU6apro-OsSWEET14-OsU6bpro-OsSWEET13-OsU6apro-OsSWEET11，通过简单酶切连接融合进前述pH-nCas9-PBE载体中(靶序列信息列于表1)。靶向编辑目的基因OsSLR1：商业化合成单个向导RNA的表达框OsU6apro-OsSLR1,通过简单酶切连接融合进前述pH-nCas9-PBE载体中(靶序列信息列于表1)；
2)Split-AID10碱基编辑器的遗传转化载体,第一步，以pHBT-Split-AID10-N 载体为模板，经简单PCR与酶切连接获得pH-Split-AID10-N载体。第二步，首先从水稻中花11基因组上经PCR获得OsUBQ2启动子序列，再以载体 pHBT-Split-AID10-C和pH-nCas9-PBE为模板通过简单重叠PCR获得 Split-AID10-C-E9 term多核苷酸序列，通过简单PCR将前两者融合获得 Split-AID10-N的表达框OsUBQ2pro-Split-AID10-C-E9 term多核苷酸序列。最后通过简单酶切连接将前述Split-AID10-N的表达框融合进pH-Split-AID10-N载体的E9终止子之后，获得拥有2个完整表达框的pH-Split-AID10-N-C双元载体。靶向编辑目的基因OsSWEET11/13/14和OsSLR1的2个双元载体如前所述克隆至pH-PIGS-AID10中。
3)水稻遗传转化：将上述载体利用冻融转化法转入农杆菌(Agrobacteriumtumefaciens)株系EHA105感受态细胞，后续委托武汉伯远生物科技有限公司 (BioRunCo.,Ltd.)进行水稻的遗传转化工作。
8、实验结果
首先，采用前述基于正交R-loop的方法(图3A，3B)在水稻原生质体中评估2种基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10和 Split-AID10-N5C4的中靶与脱靶效率，同时选择传统碱基编辑器N-BE3作对照 (图2)。中靶编辑方面，分别针对4个SpCas9向导RNA靶点所处的基因组区域进行深度测序，分析结果显示，碱基编辑器Split-AID10和Split-AID10-N5C4 均能对水稻内源靶位点进行有效的碱基编辑，平均C到T碱基替换效率约为 4.6％和6.6％，大于传统碱基编辑器N-BE3(BE3)的3.3％(图3A，3C)。脱靶编辑方面，分别针对4个nSaCas9向导RNA靶点进行深度测序分析，结果显示 Split-AID10和Split-AID10-N5C4在4个位点的平均脱靶编辑效率(0.05％和0.8％) 均显著低于传统碱基编辑器BE3(1.6％)。尤其是碱基编辑器Split-AID10，其平均脱靶效率仅为0.05％，与空白对照的0.02％基本一致(图3B，3C)。
随后，将前述原生质体中表现最优的Split-AID10碱基编辑器与向导RNA 融合构建遗传转化载体进行水稻的遗传转化，在水稻转基因植株中评估该碱基编辑器，同时选择传统碱基编辑器N-BE3作对照。获得转基因植株后，针对不同靶基因位点设计特异性引物进行PCR，将产物送擎科生物技术有限公司 (Tsingke Biotech.Co.,Ltd.)进行桑格测序。针对4个靶点的分析结果显示，碱基编辑器Split-AID10在转基因植株中也成功进行碱基编辑，有效编辑窗口为靶序列的C5位到C13位(以PAM为21-23位)，不同位置的胞嘧啶碱基替换效率从3％到55.2％(图3A)。最后，我们使用在线分析工具CRISPR-GE (http://skl.scau.edu.cn/)分析了全部4个靶点可能的Cas9依赖型脱靶情况。以该工具脱靶得分>0.7为标准，仅在中靶位点OsSWEET13中寻找到2个可能的脱靶位点(图3B)。随后针对3种碱基编辑器BE3和Split-AID10所有在该位点成功编辑的转基因水稻植株共38株进行2个脱靶位点的桑格测序。如图3B所示， Split-AID10仅在平均50％的已编辑植株发现前述Cas9依赖型的DNA脱靶情况，显著小于BE3的100％。
综上所述，在水稻中2种基于双分子脱氨酶互补的胞嘧啶碱基编辑系统 Split-AID10和Split-AID10-N5C4均能进行有效碱基编辑，且相比传统碱基编辑器如BE3，显著降低了碱基编辑的全基因组随机脱靶概率(同“Cas9非依赖型脱靶”)，同时维持了较高的中靶编辑效率。进一步，Split-AID10在转基因水稻植株水平也展现了良好的碱基编辑中靶效率，同时发现其降低Cas9依赖型脱靶发生机率。
实施例2使用基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10也可在转基因拟南芥植株中实现高效碱基编辑。
1、实验材料
本实施例所用野生型拟南芥为Col-0生态型(Arabidopsis thaliana Col-0)。
2、拟南芥遗传转化
本实施例所用拟南芥遗传转化载体的构建参考公开发表文章[15]和实施例1 所述方法，针对2个不同靶点分别构建各自遗传转化载体(向导RNA靶序列信息见表1)。将前述载体分别通过电击转化农杆菌(Agrobacterium tumefaciens)株系GV3101，利用花粉管导入法转化拟南芥植株。具体地，将含有目标载体的 GV3101菌液，按1:100的比例接种于含卡那霉素(50mg/L)的液体LB培养基中，在28℃摇床中以转速220rpm培养2天。5000g收集菌体，弃去培养基，加入含0.05％Silwet L77的5％蔗糖溶液重悬。取已开花的拟南芥植株，倒置并使花序完全浸入农杆菌菌液中，轻轻搅动约10秒后取出，置于湿润黑暗环境中1天后，转移至正常生长环境，直至收获成熟种子。
3、转基因阳性苗筛选及基因型鉴定
将前述获得的成熟种子在无菌环境下播撒于含有50mg/L的潮霉素B (hygromycinB)的无菌1/2MS固体培养基中。10天左右，将生根的潮霉素阳性苗移至土壤中恢复培养。半个月后，使用购自购自天根生化科技(北京)有限公司的快捷型植物基因组DNA提取系统提取阳性苗的叶片基因组。设计靶基因特异性引物进行PCR，并送擎科公司进行桑格测序。
4、实验结果
如图4A所示，对2个独立内源靶点AtALS和AteTM166的转基因拟南芥基因型鉴定结果显示，Split-AID10在转基因拟南芥植株中可以成功进行有效的 C到T碱基编辑，平均编辑效率为7.7％。如图4B所示，在使用Split-AID10编辑拟南芥内源ALS基因所获得的T1代转基因植株中，其靶位点上发生预想的 G13到A13的碱基替换，从而导致其内源ALS基因编码序列上第122位丙氨酸到苏氨酸(A122T)的氨基酸取代。对2月龄的前述所有T1代转基因拟南芥植株 (不论是否发生碱基编辑)进行咪唑啉酮类除草剂(Shandong Cynda ChemicalCo.,Ltd.)的喷施，1月后发现存活植株均包含内源ALS基因上G13到A13的碱基替换，说明使用Split-AID10碱基编辑系统靶向编辑拟南芥内源基因ALS赋予了转基因拟南芥植株对咪唑啉酮类除草剂的抗性。本实施例证明Split-AID10碱基编辑系统在转基因拟南芥植株中可进行有效地C到T碱基编辑，并且能赋予拟南芥优良的农艺性状(如除草剂抗性)。
实施例3基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10和 Split-BE3在单细胞真核生物酵母中也展现出良好的中靶编辑和极低的全基因组随机脱靶。
1、实验材料
本实施例所用为酿酒酵母BY4741株系(Saccharomyces cerevisiae BY4741)。
2、碱基编辑相关酵母表达载体构建
碱基编辑相关表达载体构建参考公开发表文章[16]，获得表达载体 pGAL1-rAPOBEC1-nCas9-UGI(N-BE3),pGAL1-Split-AID10和pGAL1-Split-BE3。其中，pGAL1-Split-AID10表达载体所包含的Split-AID10-N 和Split-AID10-N氨基酸序列如前所述，pGAL1-Split-BE3表达载体所包含的 Split-BE3-N的氨基酸序列如SEQ ID NO：8所示，Split-BE3-C的氨基酸序列如SEQ ID NO：9所示。酵母U6启动子直接从基因组中PCR扩增获得，然后通过重叠PCR的策略组装向导RNA完整表达原件，完成一系列向导RNA的载体构建pGAL1-yGFP-SNR35p-sgRNAs，酵母中向导RNA靶点信息见表3。
表3酵母细胞中向导RNA靶点信息
Figure BDA0003636470480000251
Figure BDA0003636470480000261
3、酵母遗传转化和表达
所采用的酿酒酵母遗传转化参考公开发表文章[17]。通过常规醋酸锂LiOAC 化学转化法将前述载体转入酿酒酵母BY4741株系中，利用质粒上的营养缺陷基因筛选阳性转化子。具体地，从营养缺陷平板上挑取含有目标质粒的阳性转化克隆到液体缺陷培养基中，在2％葡萄糖碳源的培养基培养两天至饱和，然后按照1:1000的稀释比例接种于2％棉子糖碳源培养基培养两天至饱和，最后按照 1:10000的稀释比例接种于1％半乳糖碳源培养基培养两到三天至饱和，收集适量的菌液进行后续基因组提取。
4、酵母细胞中靶序列的深度测序及全基因组深度测序
1)对于中靶序列的深度测序，参考公开发表文章[18]进行基因组提取和靶点的扩增子建库。
2)对于全基因的深度测序，取适量半乳糖诱导的饱和菌液进行平板划线，待克隆长成后，挑取10～20个单克隆进行一代测序，鉴定对应靶点有没有编辑；将靶点已编辑的克隆接种到3mL的YPDA液体培养基中并摇菌至饱和，然后用商业化的试剂盒(HiPure YeastDNA Kit,#D3147,Guangzhou Magen Biotech.Co., Ltd.)抽提酵母菌的基因组DNA，再利用商业化的二代测序建库试剂盒完成文库构建(VAHTS Universal DNA Library Prep Kitfor Illumina,#ND607,Vazyme Biotech.Co.,Ltd.)，最后送测序公司进行双端PE150高通量测序，要求每个克隆测序覆盖深度超过100×，大约每个基因组需1G数据。
5、实验结果
如图6A所示，针对酵母基因组内源6个靶点的分析结果显示，碱基编辑器 N-BE3平均编辑效率为6.5％～94.9％，有效编辑窗口为靶序列的C3位到C10位；碱基编辑器Split-AID10平均编辑效率为56.9％～97.3％，有效编辑窗口为靶序列的C5位到C15位；碱基编辑器Split-BE3平均编辑效率为57.8％～96.8％，有效编辑窗口为靶序列的C5位到C14位。此外，与经典的碱基编辑器N端融合策略 N-BE3相比，Split-AID10和Split-BE3的编辑窗口都发生了偏移，更靠近PAM 识别区域。
如图6B所示，无论是使用脱氨酶AID10的Split-AID10系统还是使用脱氨酶rAPOBEC1的Split-BE3系统，其均能够显著降低全基因范围的脱靶效应。尽管酵母基因组非常小(只有10Mb左右)，碱基编辑器N-BE3还是导致了全基因组范围内平均可以检测到100个左右脱靶编辑事件，而Split-AID10和Split-BE3 都能够极大地降低脱靶效应，平均只能检测不到10个的脱靶编辑，与单分子碱基编辑器对照组Split-AID10-N/Split-AID10-C和Split-BE3-N/Split-BE3-C数目相当，接近本底水平。
本实施例证明双分子脱氨酶互补的碱基编辑系统在真核细胞酿酒酵母中具有高效的编辑活性，同时，能够显著地降低全基因组范围的脱靶效应，具有非常高的安全性能。
实施例4基于双分子胞苷脱氨酶互补的Split-AID10和Split-BE3系统在人胚胎肾细胞系293T中也展现出良好的中靶编辑和极低的全基因组随机脱靶。
1、实验材料
人胚胎肾细胞系293T(HEK293T)来自美国模式培养物集存库(ATCC)。
2、人碱基编辑相关载体的构建
参考已公开发表的文章[19]，核心载体整合了CMV启动子和人源U6启动子，载体骨架为pX330(Addgene#42230)，然后构建相关碱基编辑器 pCMV-rAPOBEC1-nCas9-UGI(N-BE3),pCMV-Split-AID10和pCMV-Split-BE3表达载体,其中pCMV-Split-AID10表达载体所包含的Split-AID10-N和 Split-AID10-C氨基酸序列如前所述，pCMV-Split-BE3表达载体包含的 Split-BE3-N和Split-BE3-C氨基酸序列也如前所述；本实施例对向导RNA表达载体做了如下改造，在CMV下游插入红色荧光蛋白mScarlet，用于指示细胞转染的效率，同时在U6和gRNA Scaffold之间通过BbsI酶切消化，插入对应靶点的向导RNA退火双链分子，完成向导RNA表达元件的构建，HEK293T细胞中向导RNA靶点信息见下表4。
表4人基因组中向导RNA靶序列信息
Figure BDA0003636470480000281
3、细胞培养和转染
HEK293T细胞为人胚胎肾细胞，贴壁生长，采用DMEM高糖培养基和10％胎牛血清培养，置于37℃和5％二氧化碳的培养箱培养。进行碱基编辑器质粒转染前一天，铺合适数目的细胞于48孔板中，第二天采用常规的脂质体进行细胞转染，表达48-72小时后，收集细胞并进行基因组提取。
4、HEK293T细胞中靶序列的深度测序和分析
为了更好的评估互补的双分子脱氨酶碱基编辑器的性能，本实施例设计了6 个匹配的靶点，同时进行碱基编辑器的中靶效果和脱靶效果检测。具体实施如下：以SaCas9搜索基因组合适的靶位点，然后筛选符合SpCas9相邻间隔原基序PAM的要求，获得重叠的靶序列，从而针对同一个位点进行靶向编辑的效率评估和正交的R-Loop检测。其中，R-Loop正交检测使用了pX601(Addgene #61591)作为表达载体，为了提高检测效率，表达载体pX601有适当改造，包括使用nSaCas9切口酶和增加了尿嘧啶糖基化酶抑制功能结构域UGI，正交检测的靶位点信息详见表4。
深度测序建库采用两步法策略：1)首先设计靶点特异性的结合引物进行捕获，并且在引物末端带上Illumina Nextera接头的部分序列；2)然后以第一步反应的PCR产物为模板，进行完整文库的扩增，包括8个碱基的样品条形码和P5/P7 的完整序列。最后，两轮PCR扩增的产物进行纯化、浓度测定和文库均一化混匀等一系列常规操作，送至苏州安升达公司(Genewiz Inc.,sz)进行高通量测序。
扩增子深度测序结果分析主要包含文库拆分和编辑效率计算两个部分。文库拆分利用Illumina官方的bcl2fastq软件进行，将原始下机的BCL数据根据混合样品的条形码表格挨个拆分成独立的FASTQ格式文件，包括读长1(Read1) 和读长2(Read2)；然后针对每个靶点单独进行CRISPResso2[12]分析，获得靶点附近碱基替换的矩阵，最后完成编辑效率的计算。
5、Cas9依赖性的脱靶检测
本实施例还针对4个靶点进行了Cas9蛋白依赖性的脱靶检测，通过预测软件Cas-OFFinder针对每个靶点序列进行小于6的容错搜索，从人类全基因组范围内寻找Cas9蛋白依赖性脱靶位置；然后再根据CRIPSR/Cas9系统的核心种子区域进行筛选，保留靠近PAM区域5个碱基长度没有错配的潜在脱靶位点序列；最后根据已发表的大量文献和方法，设计一套相对简易的打分算法，对潜在的脱靶位点序列特征进行分析，进一步筛选获得打分较高的位点，设计特异性的捕获引物进行后续扩增子的建库和测序。
6、实验结果
实验结果如图7所示，由图7A可知，基于胞苷脱氨酶rAPOBEC1的Split-BE3 系统和基于胞苷脱氨酶AID10的Split-AID10系统在HEK293T细胞能够成功实现靶序列C到T的碱基替换，编辑效率与常用的N-BE3碱基编辑器相当，平均编辑效率在50％左右。由图7B～D可知，Split-AID10和Split-BE3不仅能够减少 Cas9依赖性的脱靶编辑，还能够显著地降低Cas9非依赖性的脱靶风险。本实施例证明双分子脱氨酶互补的碱基编辑系统在人源HEK293T细胞中具有高效的编辑活性，同时，不仅能够减少Cas9依赖性的脱靶编辑，而且能够显著地降低全基因组范围的脱靶效应，表现出优异的安全性能，具有广阔的实际应用前景。
实施例5 Split-A3A和Split-A3B系统在真核生物中具有良好的基因编辑能力。
1、碱基编辑相关酵母表达载体构建
参照实施例3所述方法，Split-A3A系统的表达载体为pGAL1-Split-A3A-N 和pGAL1-Split-A3A-C；编码Split-A3A-N的氨基酸序列如SEQ ID NO：10所示，编码Split-A3A-C的氨基酸序列如SEQ ID NO：11所示；Split-A3B系统的表达载体为pGAL1-Split-A3B-N和pGAL1-Split-A3B-C，编码Split-A3B-N的氨基酸序列如SEQ ID NO：12所示，编码Split-A3B-C的氨基酸序列如SEQ ID NO： 13所示。
2、实验结果
如图8A-8D所示，基于胞苷脱氨酶A3A的Split-A3A系统和基于胞苷脱氨酶A3B的Split-A3B系统在代表性真核生物酵母中成功实现靶序列C到T的碱基替换。Split-A3A系统的编辑窗口为C1到C15，在编辑窗口内不同位置的C碱基的编辑效率为22.1％～84.7％；Split-A3B系统的编辑窗口为C5到C15在编辑窗口内不同位置的C碱基的编辑效率为9.5％～95.5％。
实施例6基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和 Split-ABE8e-N7-C2可在植物中实现高效腺嘌呤碱基编辑。
1、实验材料
本实施例中水稻材料如实施例1相同，拟南芥材料如实施例2相同。
2、拟南芥原生质体瞬时转化载体的构建
传统腺嘌呤碱基编辑器ABE7.10和ABE8e的拟南芥瞬时表达载体为 pHBT-ABE7.10和pHBT-ABE8e，其中所用启动子为AtUBQ10启动子，终止子为NOS终止子。前述载体均为本实验室构建，已公开发表[15]；Split-ABE8e系统和Split-ABE8e-N7-C2系统表达载体的构建参照实施例1所述Split-AID10系统以及Split-AID10-N5-C4系统的构建方法，在pHBT-PIGS-ABE8e载体的基础上获得Split-ABE8e系统的pHBT-Split-ABE8e-N载体和pHBT-Split-ABE8e-C载体以及Split-ABE8e-N7-C2系统的pHBT-Split-ABE8e-N7载体和 pHBT-Split-ABE8e-C2载体。其中，编码Split-ABE8e-N的氨基酸序列如SEQ NO: 29，编码Split-ABE8e-C的氨基酸序列如SEQ NO:30，编码Split-ABE8e-N7的氨基酸序列如SEQ NO:31，编码Split-ABE8e-C2的氨基酸序列如SEQ NO:32；适用于拟南芥的向导RNA瞬时表达载体基于Li,Z.etal.2019.Current protocols in molecular biology,https://doi.org/10.1002/cpmb.89公开发表文章所述 pUC119-AtU6-26pro-sgRNA)载体，构建pUC119-AtU6-26pro-AtFLS2-sgRNA表达载体(靶序列参见表1)。
3、拟南芥原生质体基因组中靶序列的深度测序和碱基替换分析
本实施例的拟南芥原生质体基因组中靶序列的深度测序和碱基替换分析与实施例1相同。
4、植物遗传转化载体的构建
参考实施例1所述Spit-AID10水稻遗传转化载体的构建方法构建本实施例Split-ABE8e系统适用于水稻的遗传转化载体pH-Split-ABE8e-N-C；在此基础上将商业化合成的单向导RNA表达框AtU6-26pro-AtALS/AtPDS3/ AtBAK1-sgRNA核酸片段通过简单酶切连接各自融入前述pH-Split-ABE8e-N-C 载体中，获得本实施例中靶向AtALS或AtPDS3或AtBAK1的水稻遗传转化载体(向导RNA靶序列信息见表1)。
参考实施例2所述Spit-AID10拟南芥遗传转化载体的构建方法构建本实施例Split-ABE8e系统适用于拟南芥的遗传转化载体pH-EC-Split-ABE8e-N-C；在此基础上将商业化合成的单向导RNA表达框AtU6-26pro-OsACC/OsNRT1.1B/ OsDEP1-sgRNA核酸片段通过简单酶切连接各自融入前述 pH-EC-Split-ABE8e-N-C载体中，获得本实施例中靶向OsACC或OsNRT1.1B或 OsDEP1的拟南芥遗传转化载体(向导RNA靶序列信息见表1)。
5、植物遗传转化
水稻遗传转化方法参照实施例1所述，拟南芥遗传转化方法参照实施例2 所述。
6、实验结果
为快速验证基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2的可行性，将表达Split-ABE8e系统所必须的两组分 Split-ABE8e-N表达载体和Split-ABE8e-C表达载体与靶向AtABI3-2位点的向导 RNA表达载体共同转染拟南芥原生质体，同时也将表达Split-ABE8e-N7-C2系统所必须的两组分Split-ABE8e-N7表达载体和Split-ABE8e-C2表达载体与前述向导RNA表达载体共同转染另一组原生质体，除此之外还设置了传统腺嘌呤碱基编辑器ABE7.10和ABE8e的对照组。48小时后，提取原生质体基因组DNA 并对靶位点AtFLS2进行深度测序。结果如图9A所示，使用2种新型腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2均能有效编辑拟南芥内源靶点 AtFLS2，对该靶点7位的A碱基的平均编辑效率为3.3％和3.2％，与传统碱基编辑器ABE8e的3.2％编辑效率相似，同时高于传统ABE7.10的0.3％。
为进一步确认该新型编辑系统的有效性，拟在两种代表性植物的转基因植株水平评估该碱基编辑系统的腺嘌呤碱基编辑效率。如图9B所示，在转基因拟南芥植株中，使用Spit-ABE8e系统分别靶向内源基因AtALS，AtPDS3和 AtBAK1，腺嘌呤碱基编辑效率在3.1％到35.2％之间，平均为16.6％。如图9C 所示，在转基因水稻植株中，使用Spit-ABE8e系统分别靶向内源基因OsACC， OsDEP1和OsNRT1.1B，腺嘌呤碱基编辑效率在55.1％到53.8％之间，平均为 54.7％。
本实施例提供的数据证明，基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2在植物中可以进行有效地腺嘌呤碱基编辑。特别是Split-ABE8e系统在单子叶代表性植物水稻和双子叶代表性植物拟南芥的转基因植株中均能对内源靶基因进行高效腺嘌呤碱基编辑，揭示了该系统在植物中广泛的适用性。
实施例7 Split-ABE8e系统可在酵母和人胚胎肾细胞系HEK293T中实现有效腺嘌呤碱基编辑。
1、实验材料
本实施在酵母中的实验材料与方法参考实施例3；在HEK293T细胞系中的实验材料与方法参考实施例4。
2、Split-ABE8e系统在酵母中的表达载体为pGAL1-Split-ABE8e-N和pGAL1-Split-ABE8e-C，其余对照载体为pGAL1-nSpCas9、pGAL1-N-ABE7.10 和pGAL1-N-ABE8e；Split-ABE8e系统在HEK293T细胞系中的表达载体为 pCMV-Split-ABE8e-N和pCMV-Split-ABE8e-C；前述酵母和人Split-ABE8e表达载体所包含的Split-ABE8e-N和Split-ABE8e-C的编码氨基酸序列如前所述；酵母和人细胞中向导RNA表达载体如实施例3，4所述，向导RNA靶序列信息见表3和表4。
3、实验结果
首先，将Split-ABE8e系统与靶向酵母内源Can1-8位点的向导RNA共同导入酵母细胞中，对靶点附近进行深度测序，结果如图10A所示，Split-ABE8e系统在酵母中成功对Can1-8位点进行了腺嘌呤碱基编辑，编辑窗口为A4-A13，A 碱基到G碱基的替换效率从25％-82％。图10B展示Split-ABE8e系统在酵母细胞中另一个内源位点Can1-9的成功编辑情况。随后将Split-ABE8e系统与靶向内源HEK Site 1位点的向导RNA共同转染进HEK293T细胞系中，表达3天后，提取细胞基因组，使用引物F和R对HEK Site 1进行简单PCR，所得产物进行桑格测序。结果如图10C所示，Split-ABE8e系统在HEK293T细胞系中成功实现了腺嘌呤碱基编辑，将内源靶位点HEK Site 1中A5替换为G5。图10D展示 Split-ABE8e系统在HEK293T细胞系中另一个内源位点HEK Site 2的成功编辑情况。
本实施例所展示的实验结果证明，Split-ABE8e系统在代表性单细胞生物如酵母和哺乳动物如人胚胎肾细胞系293T中均能实现对内源靶点有效地A到G 碱基替换，进一步证实了该系统广泛的适应性。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围加以限制。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
参考文献：
[1]WU J,ZENG Q,WANG Q,et al.SNP-based pool genotyping and haplotypeanalysis accelerate fine-mapping of the wheat genomic region containingstripe rust resistance gene Yr26[J].TAG Theoretical and applied geneticsTheoretische und angewandte Genetik,2018,131(7):1481-96.
[2]LANDRUM M J,LEE J M,BENSON M,et al.ClinVar:public archive ofinterpretations of clinically relevant variants[J].Nucleic Acids Res,2016,44(D1): D862-8.
[3]ANZALONE A V,KOBLAN L W,LIU D R.Genome editing with CRISPR-Casnucleases,base editors,transposases and prime editors[J].Nat Biotechnol,2020,38(7):824-44.
[4]KOMOR A C,KIM Y B,PACKER M S,et al.Programmable editing of atarget base in genomic DNA without double-stranded DNA cleavage[J].Nature,2016, 533(7603):420-4.
[5]JIN S,ZONG Y,GAO Q,et al.Cytosine,but not adenine,base editorsinduce genome-wide off-target mutations in rice[J].Science,2019,364(6437):292-5.
[6]ZUO E,SUN Y,WEI W,et al.Cytosine base editor generates substantialoff-target single-nucleotide variants in mouse embryos[J].Science,2019,364(6437): 289-92.
[7]J.R S,MANIATIS T,SAMBROOK J,et al.Molecular Cloning-A LaboratoryManual[J].2001,
[8]STEINERT J,SCHIML S,FAUSER F,et al.Highly efficient heritableplant genome engineering using Cas9 orthologues from Streptococcusthermophilus and Staphylococcus aureus[J].The Plant journal:for cell andmolecular biology,2015, 84(6):1295-305.
[9]LI J F,NORVILLE J E,AACH J,et al.Multiplex and homologousrecombination-mediated genome editing in Arabidopsis and Nicotianabenthamiana using guide RNA and Cas9[J].Nat Biotechnol,2013,31(8):688-91.
[10]LI Z,WANG F,LI J F.Targeted Transcriptional Activation in PlantsUsing a Potent Dead Cas9-Derived Synthetic Gene Activator[J].Curr Protoc MolBiol,2019, 127(1):e89.
[11]AFGAN E,BAKER D,BATUT B,et al.The Galaxy platform for accessible,reproducible and collaborative biomedical analyses:2018 update[J].NucleicAcids Res,2018,46(W1):W537-w44.
[12]CLEMENT K,REES H,CANVER M C,et al.CRISPResso2 provides accurateand rapid genome editing sequence analysis[J].Nat Biotechnol,2019,37(3): 224-6.
[13]HWANG G H,PARK J,LIM K,et al.Web-based design and analysis toolsfor CRISPR base editing[J].BMC bioinformatics,2018,19(1):542.
[14]JIN S,FEI H,ZHU Z,et al.Rationally Designed APOBEC3B CytosineBase Editors with Improved Specificity[J].Mol Cell,2020,79(5):728-40 e6.
[15]XIONG X,LI Z,LIANG J,et al.A cytosine base editor toolkit withvarying activity windows and target scopes for versatile gene manipulationin plants[J]. Nucleic Acids Res,2022,50(6):3565-80.
[16]LIU K,DENG S,YE C,et al.Mapping single-cell-resolution cellphylogeny reveals cell population dynamics during organ development[J].NatMethods,2021, 18(12):1506-14.
[17]CHEN P,WANG D,CHEN H,et al.The nonessentiality of essential genesin yeast provides therapeutic insights into a human disease[J].Genomeresearch,2016, 26(10):1355-62.
[18]TAN J,ZHANG F,KARCHER D,et al.Engineering of high-precision baseeditors for site-specific single nucleotide replacement[J].Nat Commun,2019,10(1): 439.
[19]CONG L,RAN F A,COX D,et al.Multiplex genome engineering usingCRISPR/Cas systems[J].Science,2013,339(6121):819-23。
SEQUENCE LISTING
<110> 中山大学
<120> 基于双分子脱氨酶互补的碱基编辑系统及其应用
<130> 20220509
<160> 32
<170> PatentIn version 3.3
<210> 1
<211> 1246
<212> PRT
<213> 人工合成
<400> 1
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 Ala 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
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1235 1240 1245
<210> 2
<211> 121
<212> PRT
<213> 人工合成
<400> 2
Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1 5 10 15
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
20 25 30
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
35 40 45
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His
50 55 60
Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
65 70 75 80
Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu
85 90 95
Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
100 105 110
Ile Asp Leu Ser Gln Leu Gly Gly Asp
115 120
<210> 3
<211> 32
<212> PRT
<213> 人工合成
<400> 3
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> 4
<211> 155
<212> PRT
<213> 人工合成
<400> 4
Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln Phe Lys Asn
1 5 10 15
Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val Val
20 25 30
Glu Arg Arg Asp Cys Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr Leu
35 40 45
Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr Ile
50 55 60
Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp Phe
65 70 75 80
Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp Phe
85 90 95
Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Ala Ala Arg Leu
100 105 110
Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg Leu
115 120 125
Arg Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr Phe
130 135 140
Tyr Cys Trp Asn Thr Phe Ala Glu Asn His Gly
145 150 155
<210> 5
<211> 132
<212> PRT
<213> 人工合成
<400> 5
Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln Phe Lys Asn
1 5 10 15
Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val Val
20 25 30
Glu Arg Arg Asp Cys Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr Leu
35 40 45
Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr Ile
50 55 60
Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp Phe
65 70 75 80
Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp Phe
85 90 95
Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Ala Ala Arg Leu
100 105 110
Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg Leu
115 120 125
Arg Arg Ala Gly
130
<210> 6
<211> 159
<212> PRT
<213> 人工合成
<400> 6
Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
1 5 10 15
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
20 25 30
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
35 40 45
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
50 55 60
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
65 70 75 80
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
85 90 95
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe
100 105 110
Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln
115 120 125
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
130 135 140
Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155
<210> 7
<211> 169
<212> PRT
<213> 人工合成
<400> 7
Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile
1 5 10 15
Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu
20 25 30
Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp
35 40 45
Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
50 55 60
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser
65 70 75 80
Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser
85 90 95
Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe
100 105 110
Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile
115 120 125
Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp
130 135 140
Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys
145 150 155 160
Trp Asp Thr Phe Val Asp His Gln Gly
165
<210> 8
<211> 160
<212> PRT
<213> 人工合成
<400> 8
Asp Pro Asp Thr Phe Thr Phe Asn Phe Asn Asn Asp Pro Leu Val Leu
1 5 10 15
Arg Arg Arg Gln Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn
20 25 30
Gly Thr Trp Val Leu Met Asp Gln His Met Gly Phe Leu Cys Asn Glu
35 40 45
Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg
50 55 60
Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr
65 70 75 80
Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys
85 90 95
Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu
100 105 110
Arg Ile Phe Ala Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu
115 120 125
Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr
130 135 140
Tyr Asp Glu Phe Glu Tyr Cys Trp Asp Thr Phe Val Tyr Arg Gln Gly
145 150 155 160
<210> 9
<211> 24
<212> PRT
<213> 人工合成
<400> 9
Arg Thr Phe Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu
1 5 10 15
Ser Gly Gln Leu Arg Arg Ile Leu
20
<210> 10
<211> 69
<212> PRT
<213> 人工合成
<400> 10
Arg Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg
1 5 10 15
Arg Leu Arg Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp
20 25 30
Tyr Phe Tyr Cys Trp Asn Thr Phe Ala Glu Asn His Gly Arg Thr Phe
35 40 45
Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Gly Gln
50 55 60
Leu Arg Arg Ile Leu
65
<210> 11
<211> 69
<212> PRT
<213> 人工合成
<400> 11
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
1 5 10 15
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
20 25 30
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
35 40 45
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
50 55 60
Ala Thr Gly Leu Lys
65
<210> 12
<211> 29
<212> PRT
<213> 人工合成
<400> 12
Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu
1 5 10 15
Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
20 25
<210> 13
<211> 29
<212> PRT
<213> 人工合成
<400> 13
Cys Pro Phe Gln Pro Trp Asp Gly Leu Glu Glu His Ser Gln Ala Leu
1 5 10 15
Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
20 25
<210> 14
<211> 83
<212> PRT
<213> 人工合成
<400> 14
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
1 5 10 15
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
20 25 30
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
35 40 45
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
50 55 60
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
65 70 75 80
Lys Met Leu
<210> 15
<211> 1450
<212> PRT
<213> 人工合成
<400> 15
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln
1295 1300 1305
Phe Lys Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu
1310 1315 1320
Cys Tyr Val Val Glu Arg Arg Asp Cys Ala Thr Ser Phe Ser Leu
1325 1330 1335
Asp Phe Gly Tyr Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu
1340 1345 1350
Leu Phe Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg
1355 1360 1365
Cys Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Tyr Asp
1370 1375 1380
Cys Ala Arg His Val Ala Asp Phe Leu Arg Gly Asn Pro Asn Leu
1385 1390 1395
Ser Leu Arg Ile Phe Ala Ala Arg Leu Tyr Phe Cys Glu Asp Arg
1400 1405 1410
Lys Ala Glu Pro Glu Gly Leu Arg Arg Leu Arg Arg Ala Gly Val
1415 1420 1425
Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr Phe Tyr Cys Trp Asn
1430 1435 1440
Thr Phe Ala Glu Asn His Gly
1445 1450
<210> 16
<211> 311
<212> PRT
<213> 人工合成
<400> 16
Met Arg Thr Phe Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg
1 5 10 15
Leu Ser Gly Gln Leu Arg Arg Ile Leu Ser Gly Gly Ser Ser Gly Gly
20 25 30
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
35 40 45
Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Pro Glu Asp Asn Glu Gln
50 55 60
Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile
65 70 75 80
Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn
85 90 95
Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
100 105 110
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu
115 120 125
Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
130 135 140
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln
145 150 155 160
Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
165 170 175
Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
195 200 205
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
210 215 220
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
225 230 235 240
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
245 250 255
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
260 265 270
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
275 280 285
Lys Met Leu Ser Gly Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala
290 295 300
Gly Gln Ala Lys Lys Lys Lys
305 310
<210> 17
<211> 1427
<212> PRT
<213> 人工合成
<400> 17
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln
1295 1300 1305
Phe Lys Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu
1310 1315 1320
Cys Tyr Val Val Glu Arg Arg Asp Cys Ala Thr Ser Phe Ser Leu
1325 1330 1335
Asp Phe Gly Tyr Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu
1340 1345 1350
Leu Phe Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg
1355 1360 1365
Cys Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Tyr Asp
1370 1375 1380
Cys Ala Arg His Val Ala Asp Phe Leu Arg Gly Asn Pro Asn Leu
1385 1390 1395
Ser Leu Arg Ile Phe Ala Ala Arg Leu Tyr Phe Cys Glu Asp Arg
1400 1405 1410
Lys Ala Glu Pro Glu Gly Leu Arg Arg Leu Arg Arg Ala Gly
1415 1420 1425
<210> 18
<211> 356
<212> PRT
<213> 人工合成
<400> 18
Met Arg Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu
1 5 10 15
Arg Arg Leu Arg Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys
20 25 30
Asp Tyr Phe Tyr Cys Trp Asn Thr Phe Ala Glu Asn His Gly Arg Thr
35 40 45
Phe Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Gly
50 55 60
Gln Leu Arg Arg Ile Leu Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
65 70 75 80
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
85 90 95
Gly Ser Ser Gly Gly Ser Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
100 105 110
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
115 120 125
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
130 135 140
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
145 150 155 160
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
165 170 175
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr
180 185 190
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
195 200 205
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asn Leu
245 250 255
Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu
260 265 270
Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys
275 280 285
Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp
290 295 300
Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
305 310 315 320
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu
325 330 335
Ser Gly Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala
340 345 350
Lys Lys Lys Lys
355
<210> 19
<211> 1454
<212> PRT
<213> 人工合成
<400> 19
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu
1295 1300 1305
Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro
1310 1315 1320
Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp
1325 1330 1335
Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn
1340 1345 1350
Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg
1355 1360 1365
Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser
1370 1375 1380
Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu
1385 1390 1395
Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu
1400 1405 1410
Tyr His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu
1415 1420 1425
Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser
1430 1435 1440
Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
1445 1450
<210> 20
<211> 356
<212> PRT
<213> 人工合成
<400> 20
Met Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val
1 5 10 15
Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro
20 25 30
Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr
35 40 45
Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu
50 55 60
Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
65 70 75 80
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
85 90 95
Gly Ser Ser Gly Gly Ser Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
100 105 110
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
115 120 125
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
130 135 140
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
145 150 155 160
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
165 170 175
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr
180 185 190
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
195 200 205
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asn Leu
245 250 255
Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu
260 265 270
Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys
275 280 285
Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp
290 295 300
Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
305 310 315 320
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu
325 330 335
Ser Gly Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala
340 345 350
Lys Lys Lys Lys
355
<210> 21
<211> 1411
<212> PRT
<213> 人工合成
<400> 21
Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe
1 5 10 15
Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg
20 25 30
Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile
35 40 45
Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu
50 55 60
Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro
65 70 75 80
Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro
85 90 95
Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala
100 105 110
Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg
115 120 125
Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val
130 135 140
Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu
145 150 155 160
Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser
165 170 175
Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu
180 185 190
Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser
195 200 205
Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp
210 215 220
Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn
225 230 235 240
Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala
245 250 255
Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn
260 265 270
Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr
275 280 285
Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln
290 295 300
Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
305 310 315 320
Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr
325 330 335
Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu
340 345 350
Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe
355 360 365
Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala
370 375 380
Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg
385 390 395 400
Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly
405 410 415
Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser
420 425 430
Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly
435 440 445
Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn
450 455 460
Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr
465 470 475 480
Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly
485 490 495
Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val
500 505 510
Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys
515 520 525
Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser
530 535 540
Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
545 550 555 560
Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu
565 570 575
Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg
580 585 590
Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp
595 600 605
Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
610 615 620
Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys
625 630 635 640
Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe
645 650 655
Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln
660 665 670
Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
675 680 685
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val
690 695 700
Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu
705 710 715 720
Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly
725 730 735
Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys
740 745 750
Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln
755 760 765
Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp
770 775 780
Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
785 790 795 800
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp
805 810 815
Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
820 825 830
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln
835 840 845
Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
850 855 860
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile
865 870 875 880
Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln
885 890 895
Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu
900 905 910
Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
915 920 925
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr
930 935 940
His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu
945 950 955 960
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr
965 970 975
Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile
980 985 990
Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
995 1000 1005
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg
1010 1015 1020
Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
1025 1030 1035
Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1040 1045 1050
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1055 1060 1065
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1070 1075 1080
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1085 1090 1095
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu
1100 1105 1110
Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1115 1120 1125
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp
1130 1135 1140
Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
1145 1150 1155
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1160 1165 1170
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1175 1180 1185
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1190 1195 1200
His Tyr Glu Lys Leu Lys Gly Ser Gly Gly Ser Ser Gly Gly Ser
1205 1210 1215
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
1220 1225 1230
Ser Ser Gly Gly Ser Ser Gly Gly Ser Glu Ala Ser Pro Ala Ser
1235 1240 1245
Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe
1250 1255 1260
Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val
1265 1270 1275
Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg
1280 1285 1290
Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr
1295 1300 1305
Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu
1310 1315 1320
Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser
1325 1330 1335
Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
1340 1345 1350
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala
1355 1360 1365
Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met
1370 1375 1380
Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu
1385 1390 1395
Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly
1400 1405 1410
<210> 22
<211> 316
<212> PRT
<213> 人工合成
<400> 22
Met Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala
1 5 10 15
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly
20 25 30
Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu
35 40 45
Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Pro
50 55 60
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
65 70 75 80
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
85 90 95
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
100 105 110
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
115 120 125
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
130 135 140
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
145 150 155 160
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
165 170 175
Leu Ser Gln Leu Gly Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
195 200 205
Gly Gly Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
210 215 220
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu
225 230 235 240
Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His
245 250 255
Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
260 265 270
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn
275 280 285
Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Pro Ala
290 295 300
Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
305 310 315
<210> 23
<211> 1455
<212> PRT
<213> 人工合成
<400> 23
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Asp Pro Asp Thr Phe Thr Phe Asn Phe Asn Asn Asp Pro
1295 1300 1305
Leu Val Leu Arg Arg Arg Gln Thr Tyr Leu Cys Tyr Glu Val Glu
1310 1315 1320
Arg Leu Asp Asn Gly Thr Trp Val Leu Met Asp Gln His Met Gly
1325 1330 1335
Phe Leu Cys Asn Glu Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly
1340 1345 1350
Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln
1355 1360 1365
Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp
1370 1375 1380
Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe
1385 1390 1395
Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
1400 1405 1410
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu
1415 1420 1425
Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
1430 1435 1440
Glu Tyr Cys Trp Asp Thr Phe Val Tyr Arg Gln Gly
1445 1450 1455
<210> 24
<211> 316
<212> PRT
<213> 人工合成
<400> 24
Met Cys Pro Phe Gln Pro Trp Asp Gly Leu Glu Glu His Ser Gln Ala
1 5 10 15
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly
20 25 30
Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu
35 40 45
Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Pro
50 55 60
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
65 70 75 80
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
85 90 95
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
100 105 110
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
115 120 125
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
130 135 140
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
145 150 155 160
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
165 170 175
Leu Ser Gln Leu Gly Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
195 200 205
Gly Gly Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
210 215 220
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu
225 230 235 240
Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His
245 250 255
Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
260 265 270
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn
275 280 285
Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Pro Ala
290 295 300
Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
305 310 315
<210> 25
<211> 124
<212> PRT
<213> 人工合成
<400> 25
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
115 120
<210> 26
<211> 134
<212> PRT
<213> 人工合成
<400> 26
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
130
<210> 27
<211> 41
<212> PRT
<213> 人工合成
<400> 27
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
1 5 10 15
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
20 25 30
Gln Lys Lys Ala Gln Ser Ser Ile Asn
35 40
<210> 28
<211> 67
<212> PRT
<213> 人工合成
<400> 28
Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly Ala Ala Gly Ser
1 5 10 15
Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg Val Glu Ile
20 25 30
Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Asp Phe
35 40 45
Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln Ser
50 55 60
Ser Ile Asn
65
<210> 29
<211> 1419
<212> PRT
<213> 人工合成
<400> 29
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
1295 1300 1305
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro
1310 1315 1320
Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly
1325 1330 1335
Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu
1340 1345 1350
Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
1355 1360 1365
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met
1370 1375 1380
Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe
1385 1390 1395
Gly Val Arg Asn Ser Lys Arg Gly Ala Ala Gly Ser Leu Met Asn
1400 1405 1410
Val Leu Asn Tyr Pro Gly
1415
<210> 30
<211> 222
<212> PRT
<213> 人工合成
<400> 30
Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys
1 5 10 15
Ala Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn
20 25 30
Ala Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly
35 40 45
Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
50 55 60
Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Pro Glu Asp Asn Glu
65 70 75 80
Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile
85 90 95
Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
100 105 110
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro
115 120 125
Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
130 135 140
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg
145 150 155 160
Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
165 170 175
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu
180 185 190
Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys
195 200 205
Lys Lys Lys Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
210 215 220
<210> 31
<211> 1429
<212> PRT
<213> 人工合成
<400> 31
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
725 730 735
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
740 745 750
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
755 760 765
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
770 775 780
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
785 790 795 800
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
805 810 815
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
820 825 830
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
835 840 845
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
850 855 860
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
865 870 875 880
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
885 890 895
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
900 905 910
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
915 920 925
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
930 935 940
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
945 950 955 960
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
965 970 975
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
980 985 990
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
995 1000 1005
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1010 1015 1020
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1025 1030 1035
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1040 1045 1050
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1055 1060 1065
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1070 1075 1080
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1085 1090 1095
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1100 1105 1110
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1115 1120 1125
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1130 1135 1140
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1145 1150 1155
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1160 1165 1170
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1175 1180 1185
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1190 1195 1200
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1205 1210 1215
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1235 1240 1245
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1250 1255 1260
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
1265 1270 1275
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
1280 1285 1290
Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
1295 1300 1305
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro
1310 1315 1320
Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly
1325 1330 1335
Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu
1340 1345 1350
Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg
1355 1360 1365
Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met
1370 1375 1380
Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe
1385 1390 1395
Gly Val Arg Asn Ser Lys Arg Gly Ala Ala Gly Ser Leu Met Asn
1400 1405 1410
Val Leu Asn Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu
1415 1420 1425
Gly
<210> 32
<211> 248
<212> PRT
<213> 人工合成
<400> 32
Met Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly Ala Ala Gly
1 5 10 15
Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg Val Glu
20 25 30
Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Asp
35 40 45
Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln
50 55 60
Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu
65 70 75 80
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser
85 90 95
Ser Gly Gly Ser Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
100 105 110
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu
115 120 125
Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
130 135 140
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu
145 150 155 160
Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
165 170 175
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
180 185 190
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
195 200 205
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro
210 215 220
Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Ser Gly Gly
225 230 235 240
Ser Pro Lys Lys Lys Arg Lys Val
245

Claims (5)

1.一种双分子脱氨酶互补的碱基编辑系统，其特征在于，所述碱基编辑系统包括以下1)至5)中任意一项：
1）碱基编辑融合蛋白A、碱基编辑融合蛋白B和向导RNA；
2）包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和向导RNA；
3）碱基编辑融合蛋白A、碱基编辑融合蛋白B和包含编码向导RNA的核苷酸序列的表达构建体；
4）包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
5）包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
其中所述的碱基编辑融合蛋白A自N端到C端依次包括第一nCas9多肽片段、柔性连接肽和第一核碱基脱氨酶多肽片段；所述碱基编辑融合蛋白B自N端到C端依次包括第二核碱基脱氨酶多肽片段、柔性连接肽和第二nCas9多肽片段；所述第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段选自相同的核碱基脱氨酶；
其中，所述碱基编辑系统中碱基编辑融合蛋白A和碱基编辑融合蛋白B的组合为如下1)至7)中任意一项：
1）氨基酸序列如SEQ ID NO: 15所示的碱基编辑融合蛋白A：Split-AID10-N，和氨基酸序列如SEQ ID NO: 16所示的碱基编辑融合蛋白B：Split-AID10-C；
2）氨基酸序列如SEQ ID NO: 17所示的碱基编辑融合蛋白A：Split-AID10-N5，和氨基酸序列如SEQ ID NO: 18所示的碱基编辑融合蛋白B：Split-AID10-C4；
3）氨基酸序列如SEQ ID NO: 19所示的碱基编辑融合蛋白A：Split-BE3-N，和氨基酸序列如SEQ ID NO: 20所示的碱基编辑融合蛋白B：Split-BE3-C；
4）氨基酸序列如SEQ ID NO: 21所示的碱基编辑融合蛋白A：Split-A3A-N，和氨基酸序列如SEQ ID NO: 22所示的碱基编辑融合蛋白B：Split-A3A-C；
5）氨基酸序列如SEQ ID NO: 23所示的碱基编辑融合蛋白A：Split-A3B-N，和氨基酸序列如SEQ ID NO: 24所示的碱基编辑融合蛋白B：Split-A3B-C；
6）氨基酸序列如SEQ ID NO: 29所示的碱基编辑融合蛋白A：Split-ABE8e-N，和氨基酸序列如SEQ ID NO: 30所示的碱基编辑融合蛋白B：Split-ABE8e-C；
7）氨基酸序列如SEQ ID NO: 31所示的碱基编辑融合蛋白A：Split-ABE8e-N7，和氨基酸序列如SEQ ID NO: 32所示的碱基编辑融合蛋白B：Split-ABE8e-C2。
2.根据权利要求1所述的碱基编辑系统，其特征在于，所述编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件可操作地连接。
3.根据权利要求2所述的碱基编辑系统，其特征在于，所述调控元件为启动子；所述启动子包括病毒35S启动子，玉米Ubi-1启动子，水稻Ubi启动子，病毒CMV启动子，酵母TDH3启动子，酵母GAL1启动子，拟南芥卵细胞特异的EC1.2en+EC1.1嵌合启动子，水稻U6启动子，拟南芥U6启动子，酵母U6启动子或人U6启动子。
4.根据权利要求1~3任一项所述的碱基编辑系统在产生经遗传修饰的生物体或HEK293T细胞系中的应用，其特征在于，将所述碱基编辑系统导入生物体或HEK293T细胞系，由所述向导RNA将碱基编辑器靶向细胞基因组中的靶序列，导致所述靶序列中至少一个C碱基被T碱基取代或至少一个A碱基被G碱基取代；所述碱基编辑器由权利要求1~3任一项中所述的碱基编辑融合蛋白A和碱基编辑融合蛋白B组成；所述应用为非疾病的诊断和治疗目的的应用；所述生物体为真菌、动物或植物；所述动物为小鼠、大鼠、猴、犬、猪、羊、牛或猫。
5.根据权利要求4所述的应用，其特征在于，所述真菌包括酵母；所述植物包括拟南芥、水稻、小麦、玉米、大豆、向日葵、高粱、油菜、苜蓿、棉花、大麦、粟、甘蔗、番茄、烟草、木薯或马铃薯。
CN202210503831.5A 2022-05-10 2022-05-10 基于双分子脱氨酶互补的碱基编辑系统及其应用 Active CN114686456B (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210503831.5A CN114686456B (zh) 2022-05-10 2022-05-10 基于双分子脱氨酶互补的碱基编辑系统及其应用
PCT/CN2022/106402 WO2023216415A1 (zh) 2022-05-10 2022-07-19 基于双分子脱氨酶互补的碱基编辑系统及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210503831.5A CN114686456B (zh) 2022-05-10 2022-05-10 基于双分子脱氨酶互补的碱基编辑系统及其应用

Publications (2)

Publication Number Publication Date
CN114686456A CN114686456A (zh) 2022-07-01
CN114686456B true CN114686456B (zh) 2023-02-17

Family

ID=82145309

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210503831.5A Active CN114686456B (zh) 2022-05-10 2022-05-10 基于双分子脱氨酶互补的碱基编辑系统及其应用

Country Status (2)

Country Link
CN (1) CN114686456B (zh)
WO (1) WO2023216415A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114686456B (zh) * 2022-05-10 2023-02-17 中山大学 基于双分子脱氨酶互补的碱基编辑系统及其应用

Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107722125A (zh) * 2017-09-28 2018-02-23 中山大学 一种高效的人工转录激活因子dCas9‑TV及其编码基因与应用
CN109312335A (zh) * 2016-01-11 2019-02-05 斯坦福大学托管董事会 嵌合蛋白和调节基因表达的方法
CN110835632A (zh) * 2018-08-15 2020-02-25 华东师范大学 新型碱基转换编辑系统用于基因治疗的应用
CN110835629A (zh) * 2018-08-15 2020-02-25 华东师范大学 一种新型碱基转换编辑系统的构建方法及其应用
CN110835634A (zh) * 2018-08-15 2020-02-25 华东师范大学 一种新型碱基转换编辑系统及其应用
CN111172133A (zh) * 2020-03-10 2020-05-19 上海科技大学 一种碱基编辑工具及其用途
CN111373041A (zh) * 2017-09-26 2020-07-03 伊利诺伊大学理事会 用于基因组编辑和调节转录的crispr/cas系统和方法
CN111718420A (zh) * 2019-03-19 2020-09-29 华东师范大学 一种用于基因治疗的融合蛋白及其应用
CN111748578A (zh) * 2020-07-14 2020-10-09 北大荒垦丰种业股份有限公司 植物导引模板原位合成基因编辑方法及应用
CN111801345A (zh) * 2017-07-28 2020-10-20 哈佛大学的校长及成员们 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物
WO2020214842A1 (en) * 2019-04-17 2020-10-22 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
CN112239756A (zh) * 2019-07-01 2021-01-19 科稷达隆生物技术有限公司 一组来源于植物的胞嘧啶脱氨酶和其在碱基编辑系统中的应用
CN112266418A (zh) * 2019-07-08 2021-01-26 中国科学院微生物研究所 改进的基因组编辑系统及其应用
WO2021042047A1 (en) * 2019-08-30 2021-03-04 The General Hospital Corporation C-to-g transversion dna base editors
WO2021042062A2 (en) * 2019-08-30 2021-03-04 Joung J Keith Combinatorial adenine and cytosine dna base editors
CN112654702A (zh) * 2018-09-07 2021-04-13 阿斯利康(瑞典)有限公司 改进的核酸酶的组合物和方法
CN112746083A (zh) * 2020-12-11 2021-05-04 中山大学 一种通过单碱基编辑靶基因启动子失活基因的方法
WO2021150646A1 (en) * 2020-01-25 2021-07-29 The Trustees Of The University Of Pennsylvania Compositions for small molecule control of precise base editing of target nucleic acids and methods of use thereof
WO2021175288A1 (zh) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 改进的胞嘧啶碱基编辑系统
WO2021175289A1 (zh) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 多重基因组编辑方法和系统
CN113667017A (zh) * 2021-07-28 2021-11-19 上海南方模式生物科技股份有限公司 一种可提高CRISPR/Cas9系统同源重组效率的方法和应用
WO2022060185A1 (ko) * 2020-09-18 2022-03-24 기초과학연구원 표적화된 탈아미노효소 및 이를 이용한 염기 교정
KR20220039564A (ko) * 2020-09-21 2022-03-29 고려대학교 산학협력단 신규의 개량된 염기 편집 또는 교정용 융합단백질 및 이의 용도

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3043774A1 (en) * 2016-11-14 2018-05-17 Caixia Gao A method for base editing in plants
CN111093714A (zh) * 2017-05-25 2020-05-01 通用医疗公司 使用分割型脱氨酶限制不需要的脱靶碱基编辑器脱氨
CN110157727A (zh) * 2017-12-21 2019-08-23 中国科学院遗传与发育生物学研究所 植物碱基编辑方法
CN109957569B (zh) * 2017-12-22 2022-10-25 苏州齐禾生科生物科技有限公司 基于cpf1蛋白的碱基编辑系统和方法
KR20210124280A (ko) * 2019-01-31 2021-10-14 빔 테라퓨틱스, 인크. 표적-이탈 탈아미노화가 감소된 핵염기 편집기 및 이를 이용하여 핵염기 표적 서열을 변형시키는 방법
CN114040970A (zh) * 2019-02-13 2022-02-11 比姆医疗股份有限公司 使用腺苷脱氨酶碱基编辑器编辑疾病相关基因的方法，包括遗传性疾病的治疗
US20220170027A1 (en) * 2019-02-13 2022-06-02 Beam Therapeutics Inc. Adenosine deaminase base editors and methods of using same to modify a nucleobase in a target sequence
KR20210125560A (ko) * 2019-02-13 2021-10-18 빔 테라퓨틱스, 인크. 유전성 질환의 치료를 위한 것을 포함하는, 아데노신 데아미나제 염기 편집기를 사용한 질환-관련 유전자의 스플라이스 수용체 부위 파괴
CN112979821B (zh) * 2019-12-18 2022-02-08 华东师范大学 一种提高基因编辑效率的融合蛋白及其应用
WO2021158921A2 (en) * 2020-02-05 2021-08-12 The Broad Institute, Inc. Adenine base editors and uses thereof
IL301139A (en) * 2020-09-11 2023-05-01 Lifeedit Therapeutics Inc Enzymes from secondary DNA and active fragments and their variants and methods of use
CN112266420B (zh) * 2020-10-30 2022-08-09 华南农业大学 一种植物高效胞嘧啶单碱基编辑器及其构建与应用
CN114480391B (zh) * 2022-01-26 2024-03-29 中国科学技术大学 用于提高CRISPR/Cas9系统基因编辑效率的启动子及其应用
CN114835821B (zh) * 2022-04-18 2023-12-22 上海贝斯昂科生物科技有限公司 一种高效特异实现碱基颠换的编辑系统、方法及用途
CN114686456B (zh) * 2022-05-10 2023-02-17 中山大学 基于双分子脱氨酶互补的碱基编辑系统及其应用

Patent Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109312335A (zh) * 2016-01-11 2019-02-05 斯坦福大学托管董事会 嵌合蛋白和调节基因表达的方法
CN111801345A (zh) * 2017-07-28 2020-10-20 哈佛大学的校长及成员们 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物
CN111373041A (zh) * 2017-09-26 2020-07-03 伊利诺伊大学理事会 用于基因组编辑和调节转录的crispr/cas系统和方法
CN107722125A (zh) * 2017-09-28 2018-02-23 中山大学 一种高效的人工转录激活因子dCas9‑TV及其编码基因与应用
CN110835632A (zh) * 2018-08-15 2020-02-25 华东师范大学 新型碱基转换编辑系统用于基因治疗的应用
CN110835629A (zh) * 2018-08-15 2020-02-25 华东师范大学 一种新型碱基转换编辑系统的构建方法及其应用
CN110835634A (zh) * 2018-08-15 2020-02-25 华东师范大学 一种新型碱基转换编辑系统及其应用
CN112654702A (zh) * 2018-09-07 2021-04-13 阿斯利康(瑞典)有限公司 改进的核酸酶的组合物和方法
CN111718420A (zh) * 2019-03-19 2020-09-29 华东师范大学 一种用于基因治疗的融合蛋白及其应用
WO2020214842A1 (en) * 2019-04-17 2020-10-22 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
CN112239756A (zh) * 2019-07-01 2021-01-19 科稷达隆生物技术有限公司 一组来源于植物的胞嘧啶脱氨酶和其在碱基编辑系统中的应用
CN112266418A (zh) * 2019-07-08 2021-01-26 中国科学院微生物研究所 改进的基因组编辑系统及其应用
WO2021042047A1 (en) * 2019-08-30 2021-03-04 The General Hospital Corporation C-to-g transversion dna base editors
WO2021042062A2 (en) * 2019-08-30 2021-03-04 Joung J Keith Combinatorial adenine and cytosine dna base editors
WO2021150646A1 (en) * 2020-01-25 2021-07-29 The Trustees Of The University Of Pennsylvania Compositions for small molecule control of precise base editing of target nucleic acids and methods of use thereof
WO2021175289A1 (zh) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 多重基因组编辑方法和系统
WO2021175288A1 (zh) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 改进的胞嘧啶碱基编辑系统
CN111172133A (zh) * 2020-03-10 2020-05-19 上海科技大学 一种碱基编辑工具及其用途
CN111748578A (zh) * 2020-07-14 2020-10-09 北大荒垦丰种业股份有限公司 植物导引模板原位合成基因编辑方法及应用
WO2022060185A1 (ko) * 2020-09-18 2022-03-24 기초과학연구원 표적화된 탈아미노효소 및 이를 이용한 염기 교정
KR20220039564A (ko) * 2020-09-21 2022-03-29 고려대학교 산학협력단 신규의 개량된 염기 편집 또는 교정용 융합단백질 및 이의 용도
CN112746083A (zh) * 2020-12-11 2021-05-04 中山大学 一种通过单碱基编辑靶基因启动子失活基因的方法
CN113667017A (zh) * 2021-07-28 2021-11-19 上海南方模式生物科技股份有限公司 一种可提高CRISPR/Cas9系统同源重组效率的方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants;Xiangyu Xiong等;《Nucleic Acids Research》;20220314;第50卷(第6期);第3565-3580页 *
Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors;Jordan L. Doman等;《Nature Biotechnology》;20200110;第620-631页 *
单碱基编辑技术及其在动物育种中的应用研究进展;陈璟州等;《中国畜牧杂志》;20211231;第58卷(第6期);第53-59页 *
碱基编辑系统研究最新进展及应用;徐鑫等;《生物工程学报》;20210325;第37卷(第7期);第2307-2321页 *

Also Published As

Publication number Publication date
CN114686456A (zh) 2022-07-01
WO2023216415A1 (zh) 2023-11-16

Similar Documents

Publication Publication Date Title
CN108795972B (zh) 不使用转基因标记序列分离细胞的方法
AU2013256240B2 (en) Targeted modification of malate dehydrogenase
WO2018191715A2 (en) Polypeptides with type v crispr activity and uses thereof
CN110157726B (zh) 植物基因组定点替换的方法
JP2019517261A (ja) ゲノム操作のためのハイブリッド核酸配列
JP2015500648A (ja) 所定の標的核酸配列を修飾するための組成物及び方法
CN110526993B (zh) 一种用于基因编辑的核酸构建物
CN113881652B (zh) 新型Cas酶和系统以及应用
US20210403901A1 (en) Targeted mutagenesis using base editors
WO2018098935A1 (zh) 一种用于植物基因组定点碱基替换的载体
CN107177599B (zh) 一种增强植物对镉毒害的耐受性并降低植物镉含量的编码基因与应用
US11542530B2 (en) Method for increasing efficiency of homologous recombination-based gene editing in plant
CN114686456B (zh) 基于双分子脱氨酶互补的碱基编辑系统及其应用
CN113994007B (zh) 一种核酸表达的方法
WO2019223676A1 (zh) Clals蛋白、其编码基因及它们在预测西瓜除草剂抗性中的应用
CN111549054A (zh) CRISPR/RfxCas13d抗植物RNA病毒载体及其构建方法和应用
CN109180791B (zh) 一种与植物耐旱相关的基因及其编码蛋白与应用
KR20200004382A (ko) 전이유전자성 마커 서열을 이용하지 않는 세포 단리 방법
CN111560055B (zh) 水稻基因OsLAT3在调节敌草快的吸收累积中的应用
KR20220066111A (ko) Dna 염기 편집을 위한 방법 및 조성물
US20040172689A1 (en) Rad51 polypeptides and uses thereof
CN115725533B (zh) 一种获得抗草甘膦水稻的方法及其所用双碱基融合编辑系统
CN113150072B (zh) 一种调节蛋白在细胞器中精确定位的信号肽及其编码序列和应用
CN111019968B (zh) NTS/dNTS组合在制备植物突变体中的应用
CN111019969B (zh) 一种通过优化供体dna模板来提高基因精确替换效率的方法

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant