CN114516920A - 用于基因编辑的cas变体 - Google Patents

用于基因编辑的cas变体 Download PDF

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CN114516920A
CN114516920A CN202210053406.0A CN202210053406A CN114516920A CN 114516920 A CN114516920 A CN 114516920A CN 202210053406 A CN202210053406 A CN 202210053406A CN 114516920 A CN114516920 A CN 114516920A
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D.R.刘
A.C.科莫尔
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Abstract

提供了用于基因编辑的CAS变体。本披露的一些方面提供对核酸的靶向编辑有用的策略、系统、试剂、方法和试剂盒，该靶向编辑包括在细胞或受试者的基因组内，例如在人类基因组内，编辑单个位点。在一些实施例中，提供了Cas9和核酸编辑酶或酶结构域，例如脱氨酶结构域的融合蛋白。在一些实施例中，提供了用于靶向核酸编辑的方法。在一些实施例中，提供了产生靶向核酸编辑蛋白，例如Cas9和核酸编辑酶或结构域的融合蛋白的试剂和试剂盒。

Description

用于基因编辑的CAS变体
相关申请
本申请是申请日为2014年12月12月，申请号：201480072550.2，发明名称为“用于基因编辑的CAS变体”的中国发明专利申请的分案申请。本申请要求在35 U.S.C.§119(e)下于2013年12月12日提交的美国临时专利申请U.S.S.N.61/915,386和于2014年4月16日提交的美国临时专利申请U.S.S.N.61/980,333的优先权；并且也要求在35 U.S.C.§120下全部于2014年7月8日提交的美国专利申请U.S.S.N.14/325,815、14/326,109、14/326,140、14/326,269、14/326,290、14/326,318和14/326,303的优先权；将其每一个通过引用结合在此。
政府支持
本发明是使用由国防高级研究计划局(DARPA)授予的拨款HR0011-11-2-0003、由美国国立卫生研究院(NIH)授予的拨款GM095501、以及由空间和海战系统中心(SPAWAR)授予的拨款N66001-12-C-4207下的美国政府资助进行的。政府具有本发明中的某些权利。
发明背景
核酸序列的靶向编辑，例如，引入特定的修饰到基因组DNA，是基因功能研究的非常有前途的方法，并且还具有对人类遗传性疾病提供新疗法的潜力。1理想的核酸编辑技术拥有三大特点：(1)高效插入所需的修饰；(2)最小的脱靶活性；和(3)被编程为在给定的核酸的任何位点(例如人类基因组内的任何位点)精确编辑的能力。2当前基因组工程工具包括设计的锌指核酸酶(ZFN)，3转录激活因子如效应核酸酶(TALEN)，4以及最近的RNA指导的DNA核酸内切酶Cas9，5在基因组中的效应序列特异性DNA切割。此可编程的切割可在切割位点通过非同源末端连接(NHEJ)导致DNA突变或在切割位点周围通过同源定向修复(HDR)导致DNA置换。6,7
当前技术的一个缺点是NHEJ和HDR都是典型地导致适度的基因编辑效率以及可以与期望的改变竞争的不需要的基因改变的随机过程。8因为原则上许多遗传性疾病可以通过在基因组中的特定位置产生特定核苷酸变化来进行处理(例如，疾病相关基因的特定密码子中从C到T的改变)，9开发可设计的方式以达到这样的精确基因编辑将代表为基于基因编辑的人类治疗提供了强大的新研究工具以及潜在的新方法。
发明概述
成簇的规律间隔短回文重复序列(CRISPR)系统是最近发现的原核适应性免疫系统，10该原核适应性免疫系统已被修改以使强大和通用的基因组工程在各种生物体和细胞系中成为可能。11CRISPR-Cas(CRISPR相关)系统是蛋白RNA复合体，该复合体使用RNA分子(sgRNA)为指导将该复合体通过碱基配对定位于靶DNA序列。12在自然系统中，Cas蛋白然后充当核酸内切酶切割靶DNA序列。13靶DNA序列必须与sgRNA互补,并且还包含“前间区序列邻近基序(protospacer-adjacent motif，PAM)”二核苷酸在互补区的3'端以使系统起作用(图1)。14在已知的Cas蛋白中，酿脓链球菌(S.Pyogenes))Cas9已被最广泛地用作基因组工程的工具。15此Cas9蛋白是含有两个不同核酸结构域的大的多结构域蛋白。可以在Cas9中引入点突变以废止核酸酶活性，产生仍然保留其以sgRNA编程的方式结合DNA的能力的死亡Cas9(dCas9)。16原则上，当与另一个蛋白或结构域融合，dCas9可以简单地通过与合适的sgRNA共表达将该蛋白靶向几乎任何DNA序列。
dCas9复合体用于基因组工程的目的的潜力是巨大的。理论上将蛋白引入被sgRNA编程的基因组的特异位点的独特的能力被发展成超越核酸酶的各种位点特异的基因组工程工具，包括转录激活因子、转录抑制因子、组蛋白修饰蛋白、整合酶和重组酶。11最近已经通过dCas9与转录激活因子融合来实施这些潜在应用的一些以提供RNA指导的转录激活因子、17,18转录抑制因子、16,19,20以及染色质修饰酶。21这些与各种sgRNA融合的简单的共表达导致靶向基因的特异表达。这些开创性的研究为用于基因组的精准操控的可易编程序列特异性效应子的设计和建造铺平了道路。
很大意义上，对人类疾病负责的80％-90％的蛋白突变起源于仅仅单个核苷酸的置换、缺失或插入。6然而，还没有开发出使以整体和直接的方式进行单核苷酸操控成为可能的基因组工程工具。目前针对单碱基基因修正的策略包括工程化的核酸酶(其依赖于创建双链断裂(DSB)，接着是随机低效的同源性定向修复(HDR))、以及DNA-RNA嵌合寡核苷酸。22较晚的策略涉及设计RNA/DNA序列从而与除了待编辑核苷酸外的基因组DNA碱基配对。通过细胞的内源修复系统识别和修理产生的错配，导致嵌合或基因组的序列的改变。这些策略都遭受了低基因编辑效率和不需要的基因改变的问题，因为它们受HDR随机性和HDR与非同源末端连接(NHEJ)之间竞争的支配。23-25HDR效率根据靶基因在基因组中的位置、26细胞周期的状态、27和细胞/组织的类型不同。28因此，开发直接的可编程的方式以类酶的效率和非随机的方式安置碱基修饰特异的类型在基因组DNA的精确位置将代表为基于基因编辑的研究工具和人类治疗提供功能强大的新方法。
本披露的一些方面提供对核酸的靶向编辑有用的策略、系统、试剂、方法和试剂盒，该靶向编辑包括在受试者的基因组(例如人类基因组)中编辑单个位点。在一些实施例中，提供了Cas9和核酸编辑酶或酶结构域(例如脱氨酶结构域)的融合蛋白。在一些实施例中，提供了用于靶向核酸编辑的方法。在一些实施例中，提供了产生靶向核酸编辑蛋白(例如Cas9和核酸编辑酶或结构域的融合蛋白)的试剂和试剂盒。
本披露的一些方面提供包括以下项的融合蛋白：(i)核酸酶非活性的Cas9结构域；和(ii)核酸编辑结构域。在一些实施例中，核酸编辑结构域是DNA编辑结构域。在一些实施例中，核酸编辑结构域是脱氨酶结构域。在一些实施例中，脱氨酶是胞苷脱氨酶。在一些实施例中，脱氨酶是载脂蛋白B mRNA编辑复合体(APOBEC)家族脱氨酶。在一些实施例中，脱氨酶是APOBEC1家族脱氨酶。在一些实施例中，脱氨酶是激活诱导的胞苷脱氨酶(AID)。在一些实施例中，脱氨酶是ACF1/ASE脱氨酶。在一些实施例中，脱氨酶是腺苷脱氨酶。在一些实施例中，脱氨酶是ADAT家族脱氨酶。在一些实施例中，核酸编辑结构域被融合至CAS9结构域的N末端。在一些实施例中，核酸编辑结构域被融合至CAS9结构域的C末端。在一些实施例中，CAS9结构域与核酸编辑结构域是通过连接体融合的。在一些实施例中，该连接体包含(GGGGS)n(SEQ ID NO:91)、(G)n、(EAAAK)n(SEQ ID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQID NO:93)基序(参见，例如，古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)。融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion ofcatalytically inactive Cas9 to FokI nuclease improves the specificity ofgenome modification)，自然生物技术(Nat.Biotechnol.)2014；32(6):577-82；将其全部内容通过引用结合在此)，或(XP)n基序，或任意这些的组合，其中n独立地是1至30之间的整数。
本披露的一些方面提供用于DNA编辑的方法。在一些实施例中，该方法包括使DNA分子与以下项接触：(a)包含核酸酶非活性的Cas9结构域和脱氨酶结构域的融合蛋白；以及(b)将(a)的融合蛋白靶向DNA链的靶核苷酸序列的sgRNA；其中所述DNA分子与处于有效量的和在适合于核苷酸碱基的脱氨基作用的条件下的所述融合蛋白和所述sgRNA接触。在一些实施例中，靶DNA序列包括与疾病或失调相关的序列，并且其中核苷酸碱基的脱氨基作用产生与疾病或失调无关的序列。在一些实施例中，该DNA序列包括与疾病或失调相关的T>C或A>G点突变，并且其中该突变的C或G碱基的脱氨基作用产生与疾病或失调无关的序列。在一些实施例中，脱氨基作用改正了与疾病或失调相关的序列中的点突变。在一些实施例中，所述与疾病或失调相关的序列编码蛋白，并且其中脱氨基作用在与疾病或失调相关的序列中引入终止密码子，导致编码蛋白的截短。在一些实施例中，脱氨基作用改正了PI3KCA基因的点突变，从而修正了H1047R和/或A3140G突变。在一些实施例中，该接触是在倾向于具有、具有或被诊断出具有疾病或失调的受试者的体内进行。在一些实施例中，该疾病或失调是基因组中与点突变或单个碱基突变相关的疾病。在一些实施例中，该疾病是遗传性疾病、癌症、代谢性疾病或溶酶体贮积病。
本披露的一些方面提供用于检测Cas9:DNA编辑结构域融合蛋白的核酸编辑活性的报告构建体。在一些实施例中，该构建体包括(a)包含用于Cas9 DNA编辑蛋白的靶位点的报告基因，其中靶向的DNA编辑导致报告基因表达的增加；以及(b)控制报告基因表达的启动子序列。在一些实施例中，该构建体进一步包括(c)编码将Cas9 DNA编辑蛋白靶向报告基因的靶位点的sgRNa的序列，其中sgRNA表达与报告基因的表达是独立的。在一些实施例中，报告基因的靶位点包括未成熟的终止密码子，并且其中通过Cas9 DNA编辑蛋白对靶向的DNA的模板链的编辑导致未成熟的终止密码子转变为编码氨基酸残基的密码子。在一些实施例中，该报告基因编码荧光素酶、荧光蛋白或抗生素抗性标记。
本披露的一些方面提供试剂盒，该试剂盒包括：核酸构建体，该构建体包含编码核酸酶非活性的Cas9序列的序列，包括克隆位点的序列，该克隆位点被定位于允许编码与Cas9编码序列同框的核酸编辑酶或酶结构域的序列的克隆，以及任选地编码连接体的序列，该连接体被定位于Cas9编码序列和克隆位点之间的。另外，在一些实施例中，试剂盒包含合适的试剂、缓冲液、和/或说明书用于将编码核酸编辑酶或酶结构域框架克隆进入核酸构建体以产生Cas9核酸编码融合蛋白。在一些实施例中，所述包括克隆位点的序列是Cas9序列的N末端。在一些实施例中，所述包括克隆位点的序列是Cas9序列的C末端。在一些实施例中，所述编码的连接体包含(GGGGS)n(SEQ ID NO:91)、(G)n、(EAAAK)n(SEQ ID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:93)基序(参见，例如，古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)，融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion of catalytically inactive Cas9 to FokI nucleaseimproves the specificity of genome modification)，自然生物技术(Nat.Biotechnol.)2014；32(6):577-82；将其全部内容通过引用结合在此)，或(XP)n基序，或任意这些的组合，其中n独立地是1至30之间的整数。
本披露的一些方面提供包括以下项的试剂盒，包含核酸酶非活性的Cas9结构域和核酸编辑酶或酶结构域的融合蛋白以及任选地，定位于Cas9结构域和核酸编辑酶或酶结构域之间的连接体。另外，在一些实施例中，该试剂盒包括合适的试剂、缓冲液和/或说明书，用于使用融合蛋白(例如，用于体外或体内DNA或RNA编辑)。在一些实施例中，该试剂盒包括关于用于核酸序列的靶向编辑的合适的sgRNA的设计和使用的说明书。
以上概述以非限制性的方式意欲说明本文披露的技术的一些实施例、优势、特征、和用途。本文披露的技术的其他实施例、优势、特征、和用途将由于详细说明、附图、实例和权利要求书而是显而易见的。
附图简要说明
图1.Cas9/sgRNA-DNA复合体。sgRNA的3’端与Cas9核酸酶形成核糖核蛋白，而sgRNA的20nt 5’端识别其互补的DNA片段。DNA结合要求3-nt的PAM序列5'与靶DNA结合。在wtCas9的情况下，双链DNA切割发生在距PAM 3nt处以产生平端(由箭头示出)。应当注意的是，泡的大小是未知的。
图2.APOBEC3G(PDB ID 3E1U)的催化结构域的晶体结构。被认为在整个家族中是保守的核心二级结构由两侧为六个α螺旋的五链β片层(箭头)组成。活性中心环(活性位点环)被认为是负责确定脱氨基特异性。负责催化活性的Zn2+被显示为球体。这些序列从上到下对应于SEQ ID NO:97-98。
图3.基于荧光素酶报告分析的设计。sgRNA将发生改变以靶向众多序列从而靶向突变的起始密码子(以下划线标出的C残基)，这些序列对应于荧光素酶基因之前和包括荧光素酶基因的区域。将在起始密码子和荧光素酶基因之间加入“缓冲”区域以包括只有A和T(显示为(ZZZ)X)的密码子。指示了夏因-达尔加诺(Shine-Dalgarno)序列。在一些实施例中，优选是保留所有C的配对碱基对以防止脱靶效应。
图4.脱氨酶试验。这些序列从上到下对应于SEQ ID NO:99-105。
图5.通过Cas9-APOBEC1融合蛋白编辑的ssDNA的SDS PAGE凝胶。
定义
如在本文和权利要求中使用的单数形式“一个/种(a、an)”和“所述/该(the)”包括单数和复数指代物，除非上下文明确地指示其他的情况。因此，例如“药剂”的提及包括单一的药剂和多种这样的药剂。
术语“Cas9”或“Cas9核酸酶”是指包括Cas9蛋白或其片段(例如包含Cas9的活性或非活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)的RNA指导的核酸酶。Cas9核酸酶有时也指casn1核酸酶或CRISPR(成簇的规律间隔短回文重复序列)相关核酸酶。CRISPR是对可移动遗传元素(病毒、转位因子和接合质粒)提供保护的适应性免疫系统。CRISPR簇包含间隔物、与先行可移动元素互补的序列和靶标侵入核酸。CRISPR簇转录并被加工成CRISPR RNA(crRNA)。在II型CRISPR系统中正确加工前crRNA要求反式编码的小RNA(tracrRNA)、内源核糖核酸酶3(rnc)和Cas9蛋白。tracrRNA用作前crRNA的核糖核酸酶3辅助加工的指导。随后，Cas9/crRNA/tracrRNA核酸内切地切割与间隔物互补的线状或环状双链DNA靶标。与crRNA不互补的靶链首先被核酸内切地切割，然后进行3’-5’核酸外切地修剪。在自然界，DNA结合与切割典型地需要蛋白和两种RNA。然而，可以设计单指导的RNA(“sgRNA”或简单地称为“gNRA”)以便于将crRNA和tracrRNA两者的方面并入单RNA种类。参见，例如，季聂克·M.(Jinek M.)、吉林斯基·K.(Chylinski K.)、方法拉·I.(FonfaraI.)、豪尔·M.(Hauer M.)、杜德纳·J.A.(Doudna J.A.)、卡彭特·E.(Charpentier E.)，科学(Science)337:816-821(2012)，将其全部内容通过引用结合在此。Cas9识别CRISPR重复序列的短基序(PAM或前间区序列邻近基序)以帮助区分自我与非自我。Cas9核酸酶序列和结构是本领域技术人员已熟知的(参见，例如，“酿脓链球菌的M1株全基因组序列(Complete genome sequence of an M1 strain of Streptococcus pyogenes)”，法拉帝·J.J.(Ferretti J.J.)、马克山W.M.(McShan W.M.)、阿杰迪克D.J.(Ajdic D.J.)、萨维奇·D.J.(Savic D.J.)、萨维奇·G.(Savic G.)、里昂·K.(Lyon K.)、普里莫斯C(Primeaux C)、索扎特S(Sezate S.)、苏沃洛夫·A.N.(Suvorov A.N.)、肯顿·S.(KentonS.)、赖·H.S.(Lai H.S.)、林·S.P.(Lin S.P.)、钱·Y.(Qian Y.)、贾·H.G.(Jia H.G.)、纳加尔·F.Z.(Najar F.Z.)、任·Q.(Ren Q.)、朱·H.(Zhu H.)、宋·L.(Song L.)、怀特·J.(White J.)、袁·X.(Yuan X.)、克利夫顿·S.W.(Clifton S.W.)、罗伊·B.A.(RoeB.A.)、麦克劳克林·R.E.(McLaughlin R.E.)，美国国家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)98:4658-4663(2001)；“通过反式编码的小RNA和宿主因子RNA酶III进行的CRISPR RNA成熟(CRISPR RNA maturation by trans-encoded small RNAand host factor RNase III)”，德特车维E.(Deltcheva E.)、吉林斯基·K.(ChylinskiK.)、夏尔马·C.M.(Sharma C.M.)、冈萨雷斯·K.(Gonzales K.)、超·Y.(Chao Y.)、皮尔扎达·Z.A.(Pirzada Z.A.)、埃克特·M.R.(Eckert M.R.)、沃格尔·J.(Vogel J.)、卡彭特·E.(Charpentier E.)，自然(Nature)471:602-607(2011)；以及“适应性细菌免疫中可编程的双RNA指导的DNA内源核酸酶(A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity)”，季聂克·M.(Jinek M.)、吉林斯基·K.(Chylinski K.)、方法拉·I.(Fonfara I.)、豪尔·M.(Hauer M.)、杜德纳·J.A.(Doudna J.A.)、卡彭特·E.(Charpentier E.)，科学(Science)337:816-821(2012)，将其每一个的全部内容通过引用结合在此。Cas9直系同源已在各种物种中描述，包括但不限于酿脓链球菌(S.pyogenes)和嗜热链球菌(S.thermophilus)。基于本披露另外的合适的Cas9核酸酶和序列对本领域的普通技术人员而言将是显而易见的，并且这样的Cas9核酸酶和序列包括来自生物有机体的Cas9序列和披露在以下文献中的位点：吉林斯基(Chylinski)、卢恩(Rhun)和卡彭特(Charpentier)，“II型CRISPR-Cas免疫系统的tracrRNA和Cas9家族(ThetracrRNA and Cas9 families of type II CRISPR-Cas immunity systems)”(2013)RNA生物学(RNA Biology)10:5，726-737；将其全部内容通过引用结合在此。在一些实施例中，Cas9核酸酶具有非活性的(例如非激活的)DNA切割结构域。
核酸酶非激活的Cas9蛋白能够可互换地被称为“dCas9”蛋白(针对核酸酶“死亡”Cas9)。产生具有非活性DNA切割结构域的Cas9蛋白(或其片段)的方法是已知的(参见，例如，季聂克(Jinek)等人，科学(Science)，337:816-821(2012)；齐(Qi)等人，“将CRISPR再利用为RNA指导的平台以用于基因表达的序列特异性控制(Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression)”(2013)，细胞(Cell)，28；152(5):1173-83，将其每一个的全部内容通过引用结合在此)。例如，Cas9的DNA切割结构域已知包括两个亚结构域：HNH核酸酶亚结构域和RuvC1亚结构域。HNH亚结构域切割与gRNA互补的链，而RuvC1亚结构域切割非互补的链。在这些亚结构域中的突变可以使Cas9的核酸酶活性沉默。例如突变D10A和H841A完全使酿脓链球菌Cas9的核酸酶失去活性(季聂克(Jinek)等人，科学(Science)，337:816-821(2012)；齐(Qi)等人，细胞(Cell)，28；152(5):1173-83(2013)。在一些实施例中，提供了包含Cas9片段的蛋白。例如，在一些实施例中，蛋白包含如下两个Cas9结构域中的一个：(1)Cas9的gRNA结合结构域；或(2)Cas9的DNA切割结构域。在一些实施例中，包含Cas9或其片段的蛋白被称为“Cas9变体”。Cas9变体与Cas9或其片段享有同源性。例如，Cas9变体与野生型Cas9有至少约70％一致性，至少约80％一致性，至少约90％一致性，至少约95％一致性，至少约96％一致性，至少约97％一致性，至少约98％一致性，至少约99％一致性，至少约99.5％一致性，或至少约99.9％一致性。在一些实施例中，Cas9变体包含Cas9的片段(例如gRNA结合结构域或DNA切割结构域)，以至于该片段与野生型Cas9的相应片段有至少约70％一致性，至少约80％一致性，至少约90％一致性，至少约95％一致性，至少约96％一致性，至少约97％一致性，至少约98％一致性，至少约99％一致性，至少约99.5％一致性，或至少约99.9％一致性。在一些实施例中，野生型Cas9对应于来自酿脓链球菌的Cas9(NCBI参考序列：NC_017053.1，SEQ ID NO:1(核苷酸)；SEQ ID NO:2(氨基酸))。
Figure BDA0003475302240000081
Figure BDA0003475302240000091
(单下划线：HNH结构域；双下划线：RuvC结构域)
在一些实施例中，野生型Cas9对应于或包含SEQ ID NO:3(核苷酸)和/或SEQ IDNO:4(氨基酸)：
Figure BDA0003475302240000092
Figure BDA0003475302240000101
(单下划线：HNH结构域；双下划线：RuvC结构域)
在一些实施例中，dCas9对应于或包含部分或全部Cas9氨基酸序列，该氨基酸序列具有使Cas9核酸酶失去活性的一个或多个突变。例如，在一些实施例中，dCas9结构域包含D10A和/或H820A突变。
dCas9(D10A和H840A)：
Figure BDA0003475302240000102
Figure BDA0003475302240000111
(单下划线：HNH结构域；双下划线：RuvC结构域)
在其他实施例中，提供了具有除D10A和H820A外的突变的dCas9变体，其(例如)产生核酸酶非激活的Cas9(dCas9)。通过实例的方式，这样的突变包括其他在D10和H820处的氨基酸置换或其他在Cas9核酸酶结构域内的置换(例如，在HNH核酸酶亚结构域和/或RuvC1亚结构域中的置换)。在一些实施例中，提供了dCas9的变体或同系物(例如，SEQ ID NO:34的变体)，这些变体或同系物与SEQ ID NO:34有至少约70％一致性，至少约80％一致性，至少约90％一致性，至少约95％一致性，至少约98％一致性，至少约99％一致性，至少约99.5％一致性，或至少约99.9％一致性。在一些实施例中，提供了dCas9的变体(例如，SEQID NO:34的变体)，这些变体具有的氨基酸序列短于或长于SEQ ID NO:34大约5个氨基酸，大约10个氨基酸，大约15个氨基酸，大约20个氨基酸，大约25个氨基酸，大约30个氨基酸，大约40个氨基酸，大约50个氨基酸，大约75个氨基酸，大约100个氨基酸或更多。
在一些实施例中，如本文提供的Cas9融合蛋白包括Cas9蛋白的全长氨基酸，例如以上提供的序列中的一个。然而，在其他实施例中，如本文提供的融合蛋白不包括全长Cas9序列，而仅仅是其片段。例如，在一些实施例中，本文提供的Cas9融合蛋白包括Cas9片段，其中该片段结合crRNA和tracrRNA或sgRNA，但是不包括功能性核酸酶结构域，例如，因为它仅仅包括核酸酶结构域的截短版本或根本没有核酸酶结构域。文本提供了适合的Cas9结构域和Cas9片段的示例性氨基酸序列，并且另外的适合的Cas9结构域和片段序列对本领域的普通技术人员而言将是显而易见的。
在一些实施例中，Cas9是指来自以下物种的Cas9：溃疡棒状杆菌(Corynebacterium ulcerans)(NCBI参考序列：NC_015683.1、NC_017317.1)；白喉棒状杆菌(Corynebacterium diphtheria)(NCBI参考序列：NC_016782.1、NC_016786.1)；Spiroplasma syrphidicola(NCBI参考序列：NC_021284.1)；中间普氏菌(Prevotellaintermedia)(NCBI参考序列：NC_017861.1)；台湾螺原体(Spiroplasma taiwanense)(NCBI参考序列：NC_021846.1)；海豚链球菌(Streptococcus iniae)(NCBI参考序列：NC_021314.1)；Belliella baltica(NCBI参考序列：NC_018010.1)；Psychroflexus torquisI(NCBI参考序列：NC_018721.1)；嗜热链球菌(Streptococcus thermophilus)(NCBI参考序列：YP_820832.1)；无害利斯特菌(Listeria innocua)(NCBI参考序列：NP_472073.1)；空肠弯曲杆菌(Campylobacter jejuni)(NCBI参考序列：YP_002344900.1)；或脑膜炎奈瑟氏菌(Neisseria meningitidis)(NCBI参考序列：YP_002342100.1)。
术语“脱氨酶”是指催化脱氨基反应的酶。在一些实施例中，该脱氨酶是胞苷脱氨酶，催化胞苷或脱氧胞苷分别水解脱氨基为尿嘧啶或脱氧尿嘧啶。
如本文使用的术语“有效量”是指足够引起期望的生物反应的生物活性剂的量。例如，在一些实施例中，核酸酶的有效量可以是指足够引起被核酸酶特异性地结合和切割的靶位点的切割的核酸酶的量。在一些实施例中，本文提供的融合蛋白的有效量，例如包含核酸酶非活性Cas9结构域和核酸编辑结构域(如脱氨酶结构域)的融合蛋白的有效量，可以是指足够引起被融合蛋白特异性地结合和编辑的靶位点的编辑的融合蛋白的量。如将被熟练的技术人员理解的，有效量的药剂，例如，融合蛋白、核酸酶、脱氨酶、重组酶、杂合蛋白、蛋白二聚体、蛋白(或蛋白二聚体)和多核苷酸的复合体、或多核苷酸，可根据各种因素不同，例如这些因素是，例如，对特定的待编辑的等位基因、基因组或靶位点，对靶向的细胞或组织，以及对所使用的试剂所期望的生物反应。
如本文使用的术语“连接体”是指化学基团或分子，连接两个分子或部分，例如融合蛋白的两个结构域，如核酸酶非活性Cas9结构域和核酸编辑结构域(如脱氨酶结构域)。在一些实施例中，连接体连接RNA可编程的核酸酶的gRNA结合结构域，包括Cas9核酸酶结构域和核酸编辑蛋白催化结构域。在一些实施例中，连接体连接dCas9和核酸编辑蛋白。典型地，连接体位于两个基团、分子或其他部分之间或两侧是两个基团、分子或其他部分，并且通过共价键连接每一个，从而连接两者。在一些实施例中，连接体是一个氨基酸或多个氨基酸(如肽或蛋白)。在一些实施例中，连接体是有机分子、基团、聚合物或化学部分。在一些实施例中，连接体是5-100个长度的氨基酸，例如5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、30-35个、35-40个、40-45个、45-50个、50-60个、60-70个、70-80个、80-90个、90-100个、100-150个、或150-200个长度的氨基酸。还考虑了较长或较短的连接体。
如本文使用的术语“突变”是指序列内残基的置换，例如核酸或氨基酸序列与其他的残基，或序列内一个或多个残基的缺失或插入。典型地本文通过识别原始残基随后是该残基在序列内的位置并且随后是新替换的残基的身份来描述突变。本文提供的进行氨基酸置换(突变)的多种方法是本领域熟知的，并且通过，例如，如下文献提供：格林(Green)和萨姆布鲁克(Sambrook)，分子克隆实验指南(Molecular Cloning:A Laboratory Manual)(第四版，纽约冷泉港冷泉港实验室出版社(Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.)(2012))中。
如本文使用的术语“核酸”和“核酸分子”是指包括核碱基和酸性部分(例如核苷、核苷酸、或核苷酸的聚合物)的化合物。典型地，聚合核酸(例如包括三个或更多个核苷酸的核酸分子)是线性分子，其中相邻的核苷酸是通过磷酸二酯键彼此连接。在一些实施例中，“核酸”是指单个核酸残基(例如核苷酸和/或核苷)。在一些实施例中，“核酸”是指包含三个或更多个单独的核苷酸残基的寡核苷酸链。如本文使用的术语“寡核苷酸”和“多核苷酸”可以互换使用，指核苷酸的聚合物(例如，一串至少三个核苷酸)。在一些实施例中，“核酸”包含RNA以及单和/或双链DNA。核酸可以是天然地发生的，例如，在基因组、转录物、mRNA、tRNA、rRNA、siRNA、snRNA、质粒、粘粒、染色体、染色单体、或其他天然存在的核酸分子的背景下。另一方面，核酸分子可以是非天然存在的分子，例如，重组DNA或RNA、人工染色体、工程化的基因组或其片段、或合成的DNA、RNA、DNA/RNA杂合体、或包括非天然存在的核苷酸或核苷。此外，术语“核酸”、“DNA”、“RNA”和/或类似术语包括核酸类似物，例如，具有除磷酸二酯骨架外的类似物。核酸可以从天然来源中纯化，使用重组表达系统产生并任选地纯化，化学合成等。在适当的地方，例如，在化学合成分子的情况下，核酸可包含核苷类似物，例如具有经化学修饰的碱基或糖和主链修饰的类似物。除非另行说明，核酸序列是以5’至3’方向存在的。在一些实施例中，核酸是或包括天然的核苷(例如腺苷、胸苷、鸟苷、胞苷、尿苷、脱氧腺苷、脱氧胸苷、脱氧鸟苷和脱氧胞苷)；核苷类似物(例如，2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷，C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤和2-硫代胞苷)；化学修饰的碱基；生物修饰的碱基(例如，甲基化的碱基)；嵌入碱基；改性糖(例如，2'-氟代核糖、核糖、2'-脱氧核糖、阿拉伯糖、和己糖)；和/或经修饰的磷酸基团(例如，硫代磷酸酯和5'-N-二亚磷酰胺键)。
如本文使用的术语“增殖性疾病”是指其中由于细胞或细胞群表现出异常升高的增殖率使细胞或组织的平衡受到干扰的任何疾病。增殖性疾病包括过度增生性疾病，如肿瘤前期增生病状和肿瘤性疾病。肿瘤性疾病的特征在于细胞的异常增殖并包括良性和恶性肿瘤。恶性肿瘤也被称为癌症。
术语“蛋白”、“肽”以及“多肽”在此可互换地使用，并且是指被肽(酰胺)键连接在一起的氨基酸残基的聚合物。该术语是指具有任何大小、结构或功能的蛋白、肽或多肽。典型地，蛋白、肽或多肽应是至少三个氨基酸长。蛋白、肽或多肽可以是指单个蛋白或蛋白的聚集体。蛋白、肽或多肽中的一个或多个氨基酸可以被修饰，例如，通过化学实体(如碳水化合物基团、羟基、磷酸盐基团、法呢基、异法呢基、脂肪酸基团、用于共轭、功能化或其他修饰的连接物，等等)的加成。蛋白、肽或多肽也可以是单个分子或可以是多分子复合体。蛋白、肽或多肽可以是天然存在的蛋白或肽的片段。蛋白、肽或多肽可以是天然存在的、重组的、或合成的或这些的任意组合。本文使用的术语“融合蛋白”是指包括来自至少两个不同蛋白的蛋白结构域的杂合多肽。一个蛋白可以位于融合蛋白的氨基末端(N末端)部分或位于羧基末端(C末端)部分，从而分别形成“氨基末端融合蛋白”或“羧基末端融合蛋白”。蛋白可以包括不同的结构域，例如，核酸结合结构域(如指导蛋白连接到靶位点的Cas9的gRNA结合结构域)和核酸切割结构域或核酸编辑蛋白的催化结构域。在一些实施例中，蛋白包括蛋白的部分(例如氨基酸序列构成核酸结合结构域)和有机的化合物(例如可以用作核酸切割剂的化合物)。在一些实施例中，蛋白与核酸如RNA处于复合体形式，或与核酸如RNA相联合。本文提供的任何蛋白可以通过本领域已知的任何方法产生。例如，本文提供的蛋白可以通过重组蛋白表达和纯化产生，其尤其适合包含肽连接体的融合蛋白。重组蛋白表达和纯化的方法是熟知的，并且包括被描述在如下文献中的那些：格林(Green)和萨姆布鲁克(Sambrook)，分子克隆实验指南(Molecular Cloning:A Laboratory Manual)(第四版，纽约冷泉港冷泉港实验室出版社(Cold Spring Harbor Laboratory Press,Cold SpringHarbor,N.Y.)(2012)，将该文献的全部内容通过引用方式结合在此。
术语“RNA可编程的核酸酶”和“RNA指导的核酸酶”在此可互换地使用，并且是指与不是切割靶标的一种或多种RNA形成(例如结合或联合)复合体的核酸酶。在一些实施例中，RNA可编程的核酸酶当与RNA形成复合体时，可以被称为核酸酶:RNA复合体。典型地，一个或多个结合的RNA被称为指导RNA(gRNA)。gRNA可以作为两个或更多RNA的复合体或作为单个RNA分子存在。作为单个RNA分子存在的gRNA可以被称为单指导RNA(sgRNA)，但是“gRNA”是可互换地使用的，是指指导作为单个分子或作为两个或更多分子的复合体存在的RNA。典型地，作为单RNA种类存在的gRNA包括两个结构域：(1)与靶核酸享有同源性的结构域(例如以及指导Cas9复合体结合至靶标)；和(2)结合Cas9蛋白的结构域。在一些实施例中，结构域(2)对应于被称为tracrRNA的序列，并包括茎环结构。例如，在一些实施例中，结构域(2)与tracrRNA是同源的，如描述在文献季聂克(Jinek)等人，科学(Science)337:816-821(2012)的图1E中，该文献的全部内容通过引用结合在此。gRNA的其他实例(例如包括结构域2的那些)可以发现于提交于2013年9月6日的美国临时专利申请U.S.S.N.61/874,682，其标题为“可切换的Cas9核酸酶及其用途(Switchable Cas9 Nucleases And Uses Thereof)”，以及提交于2013年9月6日的美国临时专利申请U.S.S.N.61/874,746，其标题为“用于功能性核酸酶的递送系统(Delivery System For Functional Nucleases)”，这些文献的每一个的全部内容通过引用以其整体结合在此。在一些实施例中，gRNA包括两个或更多结构域(1)和(2)，并可以被称为“延伸的gRNA”。例如，如本文所描述的，延伸的gRNA将，例如，结合两个或更多个Cas9蛋白并且在两个或更多个不同区域结合靶核酸。gRNA包含与靶位点互补的核苷酸序列，该核苷酸序列调节核酸酶/RNA复合体结合至所述靶位点，提供核酸酶:RNA复合体的序列特异性。在一些实施例中，RNA可编程的核酸酶是(CRISPR相关系统)Cas9内切核酸酶，例如来自酿脓链球菌(Streptococcus pyogenes)的Cas9(Csn1)(参见，例如，“酿脓链球菌的M1株的全基因组序列(Complete genome sequence of an M1 strain ofStreptococcus pyogenes)”，法拉帝·J.J.(Ferretti J.J.)、马克山W.M.(McShan W.M.)、阿杰迪克D.J.(Ajdic D.J.)、萨维奇·D.J.(Savic D.J.)、萨维奇·G.(Savic G.)、里昂·K.(Lyon K.)、普里莫斯C(Primeaux C)、索扎特S(Sezate S.)、苏沃洛夫·A.N.(SuvorovA.N.)、肯顿·S.(Kenton S.)、赖·H.S.(Lai H.S.)、林·S.P.(Lin S.P.)、钱·Y.(QianY.)、贾·H.G.(Jia H.G.)、纳加尔·F.Z.(Najar F.Z.)、任·Q.(Ren Q.)、朱·H.(ZhuH.)、宋·L.(Song L.)、怀特·J.(White J.)、袁·X.(Yuan X.)、克利夫顿·S.W.(CliftonS.W.)、罗伊·B.A.(Roe B.A.)、麦克劳克林·R.E.(McLaughlin R.E.)，美国国家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)98:4658-4663(2001)；“通过反式编码的小RNA和宿主因子RNA酶III进行的CRISPR RNA成熟(CRISPR RNA maturation by trans-encoded smallRNA and host factor RNase III)”，德特车维E.(Deltcheva E.)、吉林斯基·K.(Chylinski K.)、夏尔马·C.M.(Sharma C.M.)、冈萨雷斯·K.(Gonzales K.)、超·Y.(Chao Y.)、皮尔扎达·Z.A.(Pirzada Z.A.)、埃克特·M.R.(Eckert M.R.)、沃格尔·J.(Vogel J.)、卡彭特·E.(Charpentier E.)，自然(Nature)471:602-607(2011)；以及“适应性细菌免疫中可编程的双RNA指导的DNA内源核酸酶(A programmable dual-RNA-guidedDNA endonuclease in adaptive bacterial immunity)”，季聂克·M.(Jinek M.)、吉林斯基·K.(Chylinski K.)、方法拉·I.(Fonfara I.)、豪尔·M.(Hauer M.)、杜德纳·J.A.(Doudna J.A.)、卡彭特·E.(Charpentier E.)，科学(Science)337:816-821(2012)，将其每一个的全部内容通过引用结合在此。
因为RNA可编程的核酸酶(如Cas9)使用RNA:DNA杂合来靶向DNA切割位点，原则上这些蛋白可以靶向由指导RNA指定的任何序列。使用RNA可编程的核酸酶(例如Cas9)用于位点特异的切割(例如用于修饰基因组)的方法是本领域已知的(参见，例如，丛·L.(Cong,L.)等人，使用CRISPR/CAS系统的多元基因组工程(Multiplex genome engineering usingCRISPR/Cas systems)，科学(Science)339，819-823(2013)；玛丽·P.(Mali,P.)等人，通过Cas9进行RNA指导的人类基因组工程(RNA-guided human genome engineering viaCas9)，科学(Science)339，823-826(2013)；黄·W.Y.(Hwang,W.Y.)等人，在斑马鱼中使用CRISPR-Cas系统进行的有效基因组编辑(Efficient genome editing in zebrafishusing a CRISPR-Cas system)，自然生物技术(Nature Biotech)31，227-229(2013)；季聂克·M.(Jinek M.)等人，人类细胞中的RNA编程的基因组编辑(RNA-programmed genomeediting in human cells)，eLife 2，e00471(2013)；迪卡洛·J.E.(Dicarlo,J.E.)等人，利用CRISPR-Cas系统在酿酒酵母中进行基因组编辑(Genome engineering inSaccharomyces cerevisiae using CRISPR-Cas systems)，核酸研究(Nucleic acidsresearch)(2013)；蒋·W.(Jiang,W.)等人，利用CRISPR-Cas系统进行的细菌基因组的RNA指导的编辑(RNA-guided editing of bacterial genomes using CRISPR-Cas systems)，自然生物技术(Nature biotechnology)31:233-239(2013)，将这些文献每一个的全部内容通过引用结合在此)。
如本文使用的术语“受试者”是指单个生物有机体，例如，单个哺乳动物。在一些实施例中，该受试者是人类。在一些实施例中，该受试者是非人类哺乳动物。在一些实施例中，该受试者是非人类灵长类动物。在一些实施例中，该受试者是啮齿类动物。在一些实施例中，该受试者是绵羊、山羊、牛、猫、或狗。在一些实施例中，该受试者是脊椎动物、两栖动物、爬行动物、鱼类、昆虫、苍蝇或线虫。在一些实施例中，该受试者是研究动物。在一些实施例中，该受试者是基因工程化的，例如，基因工程化的非人类受试者。该受试者可以是雄性的或雌性的并且可以处于任何发育阶段。
术语“靶位点”是指核酸分子内的序列，该序列被脱氨酶或包含脱氨酶的融合蛋白(例如，本文提供的dCas9脱氨酶融合蛋白)脱去氨基。
如本文所述，术语“治疗(treatment、treat和treating)”是指旨在逆转、缓解、延迟疾病或失调或其一种或多种症状的发作，或抑制疾病或失调或其一种或多种症状的进展的临床干预。如本文所述，本文使用的术语“治疗(treatment、treat和treating)”是指旨在逆转、缓解、延迟疾病或失调或其一种或多种症状的发作，或抑制疾病或失调或其一种或多种症状的进展的临床干预。在一些实施例中，治疗可以在已经发展出一种或多种症状和/或已经诊断出疾病后给予。在其他实施例中，可以在没有症状时给予治疗，例如，以防止或延迟症状的发作或抑制疾病的发作或进展。例如，先于症状的发作(例如，根据症状历史和/或根据遗传或其他易感因素)向易感个体给予治疗。症状已经解决后也可以继续治疗，例如，以防止或延迟它们复发。
本发明的某些实施例的详细说明
本披露的一些方面提供包含结合至指导RNA(也被称为gRNA或sgRNA)的Cas9结构域的融合蛋白，该指导RNA反过来通过链杂合结合靶核酸序列；以及可以使核碱基(例如胞苷)脱去氨基的DNA编辑结构域(例如脱氨酶结构域)。通过脱氨酶使核碱基脱去氨基可以在各自的残基上导致点突变，其在此被称为核酸编辑。因此包含Cas9变体或结构域和DNA编辑结构域的融合蛋白可以被用于核酸序列的靶向编辑。这样的融合蛋白对于例如，用于突变的细胞或动物的产生的DNA的体外靶向编辑；对于例如，细胞(如随后被重新引入相同或另一个受试者的、从受试者获得的细胞)中体外修正遗传缺陷的靶向突变的引入；以及对于例如，在受试者疾病相关基因中修正遗传缺陷或引入失活突变的靶向突变的引入是有用的。典型地，本文描述的融合蛋白的Cas9结构域不具有任何核酸酶活性但是它是Cas9片段或dCas9蛋白或结构域。还提供了如本文描述的Cas9融合蛋白的使用的方法。
在此提供了非限制性的、示例性的核酸酶非活性Cas9结构域。一个示例性的、适合的核酸酶非活性Cas9结构域是D10A/H840A Cas9结构域突变体：
Figure BDA0003475302240000171
Figure BDA0003475302240000172
(SEQ ID NO:37；参见，例如，齐(Qi)等人，将CRISPR再利用为RNA指导的平台以用于基因表达的序列特异性控制(Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression)细胞(Cell)，2013；152(5):1173-83，将其全部内容通过引用结合在此)。
基于本披露，另外的适合的核酸酶非活性Cas9结构域对本领域的普通技术人员而言将是显而易见的。此类另外示例的适合的核酸酶非活性Cas9结构域包括但不限于D10A、D10A/D839A/H840A、和D10A/D839A/H840A/N863A突变的结构域(参见，例如，普拉桑特(Prashant)等人，用于靶标特异性筛选的CAS9转录激活因子与用于合作基因组工程化的配对切口酶(CAS9 transcriptional activators for target specificity screening andpaired nickases for cooperative genome engineering)，自然生物技术(NatureBiotechnology)，2013；31(9):833-838，将其全部内容通过引用结合在此)。
Cas9和核酸编辑酶或结构域之间的融合蛋白
本披露的一些方面提供包括以下项的融合蛋白：(i)核酸酶非活性的Cas9酶或结构域；和(ii)核酸编辑酶或结构域。在一些实施例中，核酸编辑酶或结构域是DNA编辑酶或结构域。在一些实施例中，核酸编辑酶拥有脱氨酶活性。在一些实施例中，核酸编辑酶或结构域包含或是脱氨酶结构域。在一些实施例中，脱氨酶是胞苷脱氨酶。在一些实施例中，脱氨酶是载脂蛋白B mRNA编辑复合体(APOBEC)家族脱氨酶。在一些实施例中，脱氨酶是APOBEC1家族脱氨酶。在一些实施例中，脱氨酶是激活诱导的胞苷脱氨酶(AID)。在一些实施例中，脱氨酶是ACF1/ASE脱氨酶。在一些实施例中，脱氨酶是腺苷脱氨酶。在一些实施例中，脱氨酶是ADAT家族脱氨酶。本文详细描述了一些核酸编辑酶和结构域以及包括这样的酶或结构域的Cas9融合蛋白。基于本披露，另外的适合的核酸编辑酶或结构域对本领域的普通技术人员而言将是显而易见的。
本披露提供不同构造的Cas9:核酸编辑酶/结构域融合蛋白在一些实施例中，核酸编辑酶或结构域被融合至Cas9结构域的N末端。在一些实施例中，核酸编辑酶或结构域被融合至Cas9结构域的C末端。在一些实施例中，Cas9结构域与核酸编辑酶或结构域是通过连接体融合的。在一些实施例中，该连接体包含(GGGGS)n(SEQ ID NO:91)、(G)n、(EAAAK)n(SEQID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:93)基序(参见，例如，古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)，融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion of catalytically inactive Cas9 toFokI nuclease improves the specificity of genome modification)，自然生物技术(Nat.Biotechnol.)2014；32(6):577-82；将其全部内容通过引用结合在此)，或(XP)n基序，或这些的任意组合，其中n独立地是1至30之间的整数。在一些实施例中，n独立地是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、或30，或如果存在多于一个连接体或多于一个连接体基序，n是其任意组合。另外的适合的连接体基序和连接体构造对本领域的普通技术人员而言将是显而易见的。在一些实施例中，适合的连接体基序和构造包括描述在以下文献中的那些，陈(Chen)等人，融合蛋白连接体：特性、设计和功能(Fusion protein linkers:property,design and functionality)，先进药物输送评论(Adv Drug Deliv Rev)2013；65(10):1357-69，将其全部内容通过引用结合在此)。基于本披露，另外的适合的连接体序列对本领域的普通技术人员而言将是显而易见的。
在一些实施例中，本文提供的示例性的Cas9融合蛋白的总体架构包括以下结构：
[NH2]-[核酸编辑酶或结构域]-[Cas9]-[COOH]或
[NH2]-[Cas9]-[核酸编辑酶或结构域]-[COOH]，
其中NH2是融合蛋白的N末端，并且COOH是融合蛋白的C末端。
可能存在另外的特征，例如，NLS与融合蛋白的剩下部分之间和/或核酸编辑酶或结构域与Cas9之间的一个或多个连接体序列。可能存在的其他的示例性的特征是定位序列，例如细胞核定位序列、细胞质定位序列、输出序列(例如细胞核输出序列)、或其他定位序列、以及对于融合蛋白的溶解、纯化或检测有用的序列标签。本文提供了适合的定位信号序列和蛋白标签序列，这些序列包括但不限于生物素羧化酶载体蛋白(BCCP)标签、myc标签、钙调素标签、FLAG标签、血凝素(HA)标签、多组氨酸标签(也被称为组氨酸标签或His标签)、麦芽糖结合蛋白(MBP)标签、nus标签、谷胱甘肽-S-转移酶(GST)标签、绿色荧光蛋白(GFP)标签、硫氧还蛋白标签、S-标签、Softag(例如，Softag 1、Softag 3)、strep-标签、生物素连接酶标签、FlAsH标签、V5标签、和SBP标签。另外的适合的序列对本领域的普通技术人员而言将是显而易见的。
在一些实施例中，核酸编辑酶或结构域是脱氨酶。例如，在一些实施例中，具有脱氨酶的酶或结构域的示例性的Cas9融合蛋白的总体架构包括以下结构：
[NH2]-[NLS]-[Cas9]-[脱氨酶]-[COOH]、
[NH2]-[NLS]-[脱氨酶]-[Cas9]-[COOH]、
[NH2]-[Cas9]-[脱氨酶]-[COOH]、或
[NH2]-[脱氨酶]-[Cas9]-[COOH]
其中NLS是细胞核定位信号，NH2是融合蛋白的N末端，并且COOH是融合蛋白的C末端。在一些实施例中，连接体被插在Cas9和脱氨酶之间。在一些实施例中，NLS位于脱氨酶和/或Cas9结构域的C末端。在一些实施例中，NLS位于脱氨酶和Cas9结构域之间。也可能存在另外的特征，例如序列标签。
核酸编辑酶和结构域的一个示例性的适合的类型是胞苷脱氨酶，例如APOBEC家族的。胞苷脱氨酶的载脂蛋白B mRNA编辑复合体(APOBEC)家族包含用于以受控和有益的方式启动突变形成的11个蛋白。29一个家族成员(激活诱导的胞苷脱氨酶(AID))负责以转录依赖的链偏好的方式通过将ssDNA中的胞嘧啶转换成尿嘧啶使得抗体成熟。30载脂蛋白B编辑复合体3(APOBEC3)酶通过对在反转录病毒ssDNA中的胞嘧啶的脱氨基作用为人类细胞提供针对某些HIV-1菌株的保护。31这些蛋白都要求Zn2+调节基序(His-X-Glu-X23-26-Pro-Cys-X2-4-Cys)和用于催化活性的结合水分子。在脱氨基反应中，Glu残基用作激活水分子成氢氧化锌用于亲核进攻。每一个家庭成员优选在自己特定的“热点”脱去氨基，该热点的范围从hAID的WRC(W是A或T，R是A或G)至hAPOBEC3F的TTC。32最近，APOBEC3G的催化结构域的晶体结构(图2)显示，二级结构由两侧为六个α螺旋的五链β片层构成，这被认为在整个家族中是保守的。33已经显示活性中心环负责ssDNA结合和确定“热点”身份两者。34这些酶的过量表达与基因组的不稳定和癌症相关，从而突出序列特异性靶向的重要性。35
核酸编辑酶和结构域的另一个示例性的适合的类型是腺苷脱氨酶。例如，ADAT家族腺苷脱氨酶可以融合至Cas9结构域，例如核酸酶非活性的Cas9结构域，从而产生Cas9-ADAT融合蛋白。
本披露的一些方面提供在Cas9和脱氨酶之间的融合的系统系列，脱氨酶例如是胞嘧啶脱氨酶(如APOBEC酶)或腺苷脱氨酶(如ADAT酶)，该系统系列已经被产生，从而将这些脱氨酶的酶活性引导至基因组DNA的特定位点。使用Cas9作为识别剂的优点有两重：(1)Cas9的序列特异性可以轻易地通过简单地改变sgRNA序列来改变；以及(2)Cas9通过退火dsDNA结合至它的靶序列，产生一系列单链的DNA和因而用于脱氨酶的可行的底物。已经产生出具有人类和小鼠脱氨酶结构域如AID结构域的成功的融合蛋白。还考虑了人类和小鼠AID的催化结构域和Cas9之间的各种其他融合蛋白。应当了解，其他催化结构域或来自其他脱氨酶的催化结构域也可以用于产生具有Cas9的融合蛋白，并且本披露不限于这个方面。
在一些实施例中，提供了Cas9和AID的融合蛋白。在设计Cas9融合蛋白以增加ssDNA中突变率的尝试中，小鼠和人类AID都被拴在丝状噬菌体基因V(非特异性ssDNA结合蛋白)上。在基于细胞的试验中，相比野生型酶，产生的融合蛋白显示出增强的诱变活性。这个工作证明，使用融合蛋白，这些蛋白的酶活性被保持在遗传序列中并可以成功地靶向遗传序列。36
然而，已经报道了Cas9(和甚至与它的sgRNA和靶DNA一起在复合体中的Cas9)的若干晶体结构，(参见，例如，季聂克·M(Jinek M)、蒋·F(Jiang F)、泰勒·DW(Taylor DW)、斯腾伯格·SH(Sternberg SH)、卡亚·E(Kaya E)、马·E(Ma E)、安德斯·C(Anders C)、豪尔·M(Hauer M)、周·K(Zhou K)、林·S(Lin S)、卡普兰·M(Kaplan M)、亚瓦罗内·AT(Iavarone AT)、卡彭特·E(Charpentier E)、诺加利斯·E(Nogales E)、杜德纳·JA(Doudna JA)，Cas9内切核酸酶的结构揭示RNA介导的构象活性(Structures of Cas9endonucleases reveal RNA-mediated conformational activation)，科学(Science)，2014；343(6176):1247997，PMID:24505130；以及并H(Nishimasu H)、冉·FA(Ran FA)、许·FD(Hsu PD)、科纳曼S(Konermann S)、施哈塔·SI(Shehata SI)、道来N(Dohmae)N、石谷信一·R(Ishitani R)、张·F(Zhang F)、濡木O(Nureki O)，与引导RNA和靶DNA一起在复合体中的Cas9的晶体结构(Crystal structure of Cas9 in c omplex with guide RNA andtarget DNA)，细胞(Cell).2014；156(5):935-49，PMID:24529477，将其每一个的全部内容通过引用结合在此)，在Cas9-DNA复合体中的是单链的DNA的部分是未知的(Cas9-DNA泡的大小)。然而，已经显示在具有为复合体特异地设计来干扰转录的sgRNA的dCas9系统中，转录干扰仅仅发生在sgRNA结合至非模板链时。这个结果表明在DNA-Cas9复合体中的DNA的特定部分没有被Cas9防备，并且可以潜在地被融合蛋白中的脱氨酶靶向(参见齐·LS(QiLS)、拉尔森·MH(Larson MH)、吉尔伯特·LA(Gilbert LA)、杜德纳·JA(Doudna JA)、韦斯曼·JS(Weissman JS)、阿尔金·AP(Arkin AP)、林·WA(Lim WA)，将CRISPR再利用为RNA指导的平台以用于基因表达的序列特异性控制(Repurposing CRISPR as an RNA-GuidedPlatform for Sequence-Specific Control of Gene Expression)细胞(Cell).2013；152(5):1173-83，PMID:23452860，将其全部内容以引用方式结合在此。进一步支持这个概念，使用外切核酸酶III和核酸酶P1(其仅仅在ssDNA作为底物时起作用)的足迹法试验揭示在非模板链上至少26个碱基易被这些酶消化(参见季聂克·M(Jinek M)、蒋·F(Jiang F)、泰勒·DW(Taylor DW)、斯腾伯格·SH(Sternberg SH)、卡亚·E(Kaya E)、马·E(Ma E)、安德斯·C(Anders C)、豪尔·M(Hauer M)、周·K(Zhou K)、林·S(Lin S)、卡普兰·M(KaplanM)、亚瓦罗内·AT(Iavarone AT)、卡彭特·E(Charpentier E)、诺加利斯·E(Nogales E)、杜德纳·JA(Doudna JA)，Cas9内切核酸酶的结构揭示RNA介导的构象活性(Structures ofCas9 endonucleases reveal RNA-mediated conformational activation)，科学(Science)，2014；343(6176):1247997，PMID:24505130)。还已经报道，在某些情况下，Cas9在这个易受影响的DNA段上以高达15％的频率诱导单碱基置换突变(参见蔡·SQ(TsaiSQ)、Wyvekens N、Khayter C、福登布·JA(Foden JA)、塔帕尔·V(Thapar V)、拉永·D(Reyon D)、古德温·MJ(Goodwin MJ)、阿里耶·MJ(Aryee MJ)、姜戈JK(Joung JK)，二聚CRISPR RNA指导的FokI核酸酶，用于高度特异性的基因组编辑(Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing)，自然生物技术(Nat.Biotechnol.).2014；32(6):569-76，PMID:24770325，将其全部内容以引用方式结合在此。虽然引入这些突变的机制是未知的，在所有的情况下，突变的碱基是胞嘧啶，这可能指示胞嘧啶脱氨酶的参与。总之，这些数据显然与单链的且易受其他酶影响的靶DNA的一部分一致。已经显示在具有为复合体特异地设计来干扰转录的sgRNA的dCas9系统中，转录干扰仅仅发生在sgRNA结合至非模板链时。这个结果表明在DNA-Cas9复合体中的DNA的特定部分没有被Cas9防备，并且可以潜在地被融合蛋白中的AID靶向。16相应地，根据本披露的多个方面，Cas9的N末端和C末端与脱氨酶结构域的融合是有用的。
在一些实施例中，脱氨酶结构域和Cas9结构域是通过连接体彼此融合的。可以应用脱氨酶结构域(如AID)和Cas9结构域之间的各种连接体长度和灵活性(例如，范围从非常灵活的连接体形式(GGGGS)n(SEQ ID NO:91)、(GGS)n、和(G)n至更严格的连接体形式(EAAAK)n(SEQ ID NO:5)、SGSETPGTSESATPES(SEQ ID NO:93)(参见，例如，古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)，融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion of catalytically inactive Cas9 toFokI nuclease improves the specificity of genome modification)，自然生物技术(Nat.Biotechnol.).2014；32(6):577-82；将其全部内容通过引用结合在此)和(XP)n)37，以便于达到用于特定应用的脱氨酶活性的最佳长度。
根据本披露的方面，可以融合至Cas9结构域的一些示例性的适合的核酸编辑酶和结构域(例如脱氨酶和脱氨酶结构域)提供在下文。应当了解，在一些实施例中，可以利用各自序列的活性结构域，例如，没有定位信号的结构域(细胞核定位信号、没有细胞核的输出信号、细胞质定位信号)。
人类AID：
Figure BDA0003475302240000221
(下划线：细胞核定位信号；双下划线：细胞核输出信号)
小鼠AID：
Figure BDA0003475302240000222
Figure BDA0003475302240000231
(下划线：细胞核定位信号；双下划线：细胞核输出信号)
狗AID：
Figure BDA0003475302240000232
(下划线：细胞核定位信号；双下划线：细胞核输出信号)
牛AID：
Figure BDA0003475302240000233
(下划线：细胞核定位信号；双下划线：细胞核输出信号)
小鼠APOBEC-3：
Figure BDA0003475302240000234
(斜体：核酸编辑结构域)
大鼠APOBEC-3：
Figure BDA0003475302240000235
(斜体：核酸编辑结构域)
恒河猴APOBEC-3G：
Figure BDA0003475302240000236
Figure BDA0003475302240000241
(斜体：核酸编辑结构域；下划线：细胞质定位信号)
黑猩猩APOBEC-3G：
Figure BDA0003475302240000242
(斜体：核酸编辑结构域；下划线：细胞质定位信号)
绿猴APOBEC-3G：
Figure BDA0003475302240000243
(斜体：核酸编辑结构域；下划线：细胞质定位信号)
人类APOBEC-3G：
Figure BDA0003475302240000244
(斜体：核酸编辑结构域；下划线：细胞质定位信号)
人类APOBEC-3F：
Figure BDA0003475302240000245
Figure BDA0003475302240000251
(斜体：核酸编辑结构域)
人类APOBEC-3B：
Figure BDA0003475302240000252
(斜体：核酸编辑结构域)
人类APOBEC-3C：
Figure BDA0003475302240000253
(斜体：核酸编辑结构域)
人类APOBEC-3A：
Figure BDA0003475302240000254
(斜体：核酸编辑结构域)
人类APOBEC-3H：
Figure BDA0003475302240000255
(斜体：核酸编辑结构域)
人类APOBEC-3D：
Figure BDA0003475302240000256
Figure BDA0003475302240000261
(斜体：核酸编辑结构域)
人类APOBEC-1：
MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR(SEQ ID NO:22)
小鼠APOBEC-1：
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK(SEQ ID NO:23)
大鼠APOBEC-1：
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK(SEQ ID NO:24)
人类ADAT-2：
MEAKAAPKPAASGACSVSAEETEKWMEEAMHMAKEALENTEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCRQSGKSPSEVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVEMLKTFYKQENPNAPKSKVRKKECQKS(SEQ ID NO:25)
小鼠ADAT-2：
MEEKVESTTTPDGPCVVSVQETEKWMEEAMRMAKEALENIEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCHQHGQSPSTVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVELLKTFYKQENPNAPKSKVRKKDCQKS(SEQ ID NO:26)
小鼠ADAT-1：
Figure BDA0003475302240000262
Figure BDA0003475302240000271
Figure BDA0003475302240000272
(斜体：核酸编辑结构域)
人类ADAT-1：
Figure BDA0003475302240000273
Figure BDA0003475302240000274
(斜体：核酸编辑结构域)
在一些实施例中，如本文提供的融合蛋白包括核酸编辑酶的全长氨基酸，例如以上提供的序列中的一个。然而，在其他实施例中，如本文提供的融合蛋白不包括核酸编辑酶的全长序列，仅仅是其片段。例如，在一些实施例中，如本文提供的融合蛋白包括Cas9结构域和核酸编辑酶的片段，例如，其中片段包括核酸编辑结构域。核酸编辑结构域的示例性的氨基酸序列在以上序列中示为斜体字母，并且此类结构域的另外的适合的序列对本领域的普通技术人员而言将是显而易见的。
基于本披露，另外的适合的核酸编辑酶序列，例如，可以根据本发明的诸多方面来使用的脱氨酶和结构域序列(例如可以融合至核酸酶非活性Cas9结构域)对本领域的普通技术人员而言将是显而易见的。在一些实施例中，这样的另外的酶序列包括脱氨酶或脱氨酶结构域序列，这些序列与本文提供的序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相似性。另外的适合的Cas9结构域、变体、和序列对本领域的普通技术人员而言将是显而易见的。此类另外的适合的Cas9结构域的实例包括但不限于D10A、D10A/D839A/H840A、和D10A/D839A/H840A/N863A突变的结构域(参见，例如，普拉桑特(Prashant)等人，用于靶标特异性筛选的CAS9转录激活因子与用于合作基因组工程化的配对切口酶(CAS9 transcriptional activatorsfor target specificity screening and paired nickases for cooperative genomeengineering)，自然生物技术(Nature Biotechnology)，2013；31(9):833-838，将其全部内容通过引用结合在此。
基于本披露，结合本领域的一般知识，产生包括Cas9结构域和脱氨酶结构域的融合蛋白的另外的适合的策略对本领域的普通技术人员而言将是显而易见的。基于本披露和本领域的知识，使用连接体和不使用连接体根据本披露的诸多方面产生融合蛋白的适合的策略对本领域的普通技术人员而言将是显而易见的。例如，吉尔伯特(Gilbert)等人，真核生物中CRISPR介导的模块化的RNA指导的转录调节(CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes)，细胞(Cell).2013；154(2):442-51，显示使用2NLS’s作为连接体(SPKKKRKVEAS，SEQ ID NO:29)的Cas9与VP64的C末端融合，可以应用于转录激活。玛丽(Mali)等人，用于靶标特异性筛选的CAS9转录激活因子与用于合作基因组工程化的配对切口酶(CAS9 transcriptional activators for targetspecificity screening and paired nickases for cooperative genomeengineering)，自然生物技术(Nat.Biotechnol.).2013；31(9):833-8，报道不使用连接体的与VP64的C末端融合可以应用于转录激活。并且梅德(Maeder)等人，CRISPR RNA指导的内源人类基因的激活(CRISPR RNA-guided activation of endogenous human genes)，自然方法(Nat.Methods)，2013；10:977-979，报道使用Gly4Ser(SEQ ID NO:91)连接体的与VP64的C末端融合可以用作转录激活因子。最近，dCas9-FokI核酸酶融合已经成功地产生出并且相比亲本Cas9酶展现了改进的酶特异性(参见古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)，融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion of catalytically inactive Cas9 to FokI nuclease improves thespecificity of genome modification)，自然生物技术(Nat.Biotechnol.).2014；32(6):577-82，以及参见蔡·SQ(Tsai SQ)、Wyvekens N、Khayter C、福登布·JA(Foden JA)、塔帕尔·V(Thapar V)、拉永·D(Reyon D)、古德温·MJ(Goodwin MJ)、阿里耶·MJ(Aryee MJ)、姜戈JK(Joung JK)，二聚CRISPR RNA指导的FokI核酸酶，用于高度特异性的基因组编辑(Dimeric CRISPR RNA-guided FokI nucleases for highly specific genomeediting)，自然生物技术(Nat.Biotechnol.).2014；32(6):569-76，PMID:24770325，SGSETPGTSESATPES(SEQ ID NO:93)或GGGGS(SEQ ID NO:91)连接体分别被用于FokI-dCas9融合蛋白)。
使用Cas9 DNA编辑融合蛋白来修正疾病相关突变
一些实施例提供了用于使用本文提供的Cas9 DNA编辑融合蛋白的方法。在一些实施例中，通过脱去靶核碱基(例如C残基)的氨基使用融合蛋白来在核酸中引入点突变。在一些实施例中，靶核碱基的脱氨基作用导致遗传缺陷的修正，例如导致在基因产物中引起功能丧失的点突变的修正。在一些实施例中，该遗传缺陷与疾病或失调关联，疾病或失调例如是溶酶体贮失调或代谢性疾病，如I型糖尿病。在一些实施例中，使用本文提供的方法来在编码与疾病或失调相关的基因产物的基因或等位基因中引入失活的点突变。例如，在一些实施例中，本文提供的方法应用Cas9 DNA编辑融合蛋白在致癌基因中引入失活的点突变(例如在增殖性疾病的治疗中)。在一些实施例中，失活的突变可以在编码序列中产生未成熟的终止密码子，其导致截短的基因产物的表达，例如，缺乏全长蛋白的功能的截短的蛋白。
在一些实施例中，本文提供的方法的目的是通过基因组编辑恢复机能失调基因的功能。本文提供的Cas9脱氨酶融合蛋白可以在基于基因编辑的人类体外治疗中生效，例如，通过在人类细胞培养物中修正疾病相关突变。本领域的技术人员应当了解，本文提供的融合蛋白，例如包含Cas9结构域和核酸脱氨酶结构域的融合蛋白，可以用于修正任何单点(T->C或A->G)突变。在第一种情况中，突变的C经脱氨基作用回到U修正了突变，并且在第二种情况中，与突变的G碱基配对的C经脱氨基作用，紧接着经一轮复制，修正了突变。
可以通过提供的融合蛋白在体外或体内修正的示例性的疾病相关的突变是在PI3KCA蛋白中的H1047R(A3140G)多态性。磷酸肌醇-3-激酶(催化的α亚基(PI3KCA)蛋白)的作用是磷酸化磷脂酰肌醇的肌醇环的3-OH基团。已经发现PI3KCA基因在许多不同的癌中突变，并且因此它被认为是强有力的致癌基因。50实际上，A3140G突变存在于若干NCI-60癌症细胞系，例如HCT116、SKOV3、和T47D细胞系，这些细胞系可以容易地从美国典型培养物保藏中心(ATCC)获得。51
在一些实施例中，携带待修正的突变的细胞，例如，携带点突变(例如，导致在PI3KCA蛋白中的H1047R置换的在PI3KCA基因外显子20中的A3140G点突变)的细胞，与编码Cas9脱氨酶融合蛋白的表达构建体和将融合蛋白靶向在编码PI3KCA基因中的各个突变位点的适当设计的sgRNA接触。在设计sgRNA来将融合酶靶向在PI3KCA基因中的非C残基的情况下可以进行控制实验。在人类细胞培养物中可以提取所处理的细胞的基因组DNA，并且对PI3KCA基因的相关序列进行PCR扩增和测序来评估融合蛋白的活性。
应当了解，提供的修正PI3KCA中的点突变的实例是出于说明的目的，并不意味着限制本披露。本领域的技术人员将了解本披露的DNA编辑融合蛋白可以用来修正其他的点突变以及与其他癌症和与除癌症以外的疾病(包括其他增殖性疾病)相关的突变。
成功的修正疾病相关基因和等位基因中的点突变开启了治疗学和基础研究中使用应用进行基因修正的新策略。位点特异性的单碱基修饰系统，如披露的Cas9和脱氨酶或脱氨酶结构域的融合在“逆转”基因治疗中也具有用途，其中某些基因功能故意被抑制或废止。在这些情况下，可以在体外、离体、或体内使用从Trp(TGG)、Gln(CAA和CAG)、或Arg(CGA)残基位点特异性突变为未成熟的终止密码子(TAA、TAG、TGA)来废止蛋白功能。
本披露提供用于治疗被诊断出具有与点突变相关或由点突变引起的疾病的受试者的方法，所述点突变可以通过本文提供的Cas9 DNA编辑融合蛋白来修正。例如，在一些实施例中，提供的方法包括对具有，例如，如以上所述的与PI3KCA点突变相关的癌症的疾病的受试者给予有效量的Cas9脱氨酶融合蛋白，该融合蛋白修正点突变或在疾病相关基因中引入失活的突变。在一些实施例中，该疾病是增殖性疾病。在一些实施例中，该疾病是遗传性疾病。在一些实施例中，该疾病是肿瘤性疾病。在一些实施例中，该疾病是代谢性疾病。在一些实施例中，该疾病是溶酶体贮积病。其他可以通过修正点突变或在疾病相关基因中引入失活的突变进行治疗的疾病对本领域的普通技术人员而言将是已知的，并且本披露不限于这一方面。
本披露提供用于治疗另外的疾病或失调的方法，例如与点突变相关或由点突变导致的疾病或失调，所述点突变可以通过脱氨酶介导的基因编辑修正。本文描述了一些这样的疾病，并且基于本披露可以使用本文提供的策略和融合蛋白进行治疗的另外的适合的疾病对本领域的普通技术人员而言将是显而易见的。以下列出了示例性的适合的疾病和失调。应当了解在各自序列中的特异性的位置或残基的编号依赖于特定蛋白和使用的编号方案。例如，成熟蛋白的前体和成熟蛋白本身的编号可能不同，并且不同物种的序列的不同可能影响编号。本领域的技术人员能够通过本领域熟知的方法(例如通过序列比对和同源残基的脱氨基作用)识别任何同源蛋白中和各自的编码核酸中的各自的残基。示例性的适合的疾病和失调包括但不限于囊胞性纤维症(参见，例如，施万克(Schwank)等人，通过CRISPR/Cas9在囊胞性纤维化患者的肠道干细胞组织体中进行CFTR的功能修复(Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoidsof cystic fibrosis patients)，细胞干细胞(Cell stem cell)，2013；13:653-658；以及吴(Wu)等人，通过使用CRISPR-Cas9修正小鼠中的遗传性疾病(Correction of a geneticdisease in mouse via use of CRISPR-Cas9)，细胞干细胞(Cell stem cell)，2013；13:659-662，这两个文献皆没有使用脱氨酶融合蛋白来修正遗传缺陷)；苯丙酮尿症-例如，在苯丙氨酸羟化酶基因的位置835(小鼠)或240(人)或同源残基处发生苯丙氨酸至丝氨酸的突变(T>C突变)–参见，例如，麦克唐纳(McDonald)等人，基因组学(Genomics)，1997；39:402-405；巨大血小板综合征(BSS)-例如，在血小板膜糖蛋白IX中的位置55或同源残基处发生苯丙氨酸至丝氨酸的突变，或在位置24或同源残基处发生半胱氨酸至精氨酸的突变(T>C突变)-参见，例如，诺里斯(Noris)等人，英国血液学杂志(British Journal ofHaematology)，1997；97:312-320；以及阿里(Ali)等人，血液学(Hematol.)，2014；93:381-384；表皮松解性角化过度(EHK)-例如，在角蛋白1的位置160或161(如果计数起始子甲硫氨酸)或同源残基处发生亮氨酸至脯氨酸的突变(T>C突变)-参见，例如，Chipev等人，细胞(Cell)，1992；70:821-828；也参见在www[dot]uniprot[dot]org网站的UNIPROT数据库中的登录号P04264；慢性阻塞性肺病(COPD)-例如，在α1-抗胰蛋白酶的加工形式的位置54或55(如果计数起始子甲硫氨酸)或同源残基或未加工形式的残基78或同源残基处发生亮氨酸至脯氨酸的突变(T>C突变)-参见，例如，波勒(Poller)等人，基因组学(Genomics)，1993；17:740-743，也参见UNIPROT数据库中的登录号P01011；进行性神经性腓骨肌萎缩症(Charcot-Marie-Tooth disease)4J型-例如，图4中在位置41或同源残基处发生异亮氨酸至苏氨酸的突变(T>C突变)–参见，例如，伦克(Lenk)等人，PLoS遗传学(PLoS Genetics)，2011；7:e1002104；神经母细胞瘤(NB)-例如，在半胱天冬酶-9的位置197或同源残基处发生亮氨酸至脯氨酸的突变(T>C突变)-参见，例如，昆都(Kundu)等人，3生物技术(3Biotech.)，2013；3:225-234；血管性血友病(vWD)-例如，在血管性血友病因子的加工形式的位置509或同源残基或在血管性血友病因子的未加工形式的位置1272或同源残基处发生半胱氨酸至精氨酸的突变(T>C突变)-参见，例如，拉韦涅(Lavergne)等人，英国血液学杂志(Br.J.Haematol.)，1992，也参见UNIPROT数据库中的登录号P04275；82:66-72；先天性肌强直-例如，在肌肉氯离子通道基因CLCN1的位置277或同源残基处发生半胱氨酸至精氨酸的突变(T>C突变)-参见，例如，温伯格(Weinberger)等人，生理学杂志(The Journel ofPhysiology)，2012；590:3449-3464；遗传性肾淀粉样变性-例如，在载脂蛋白AII加工形式的78位或同源残基或在未加工形式的位置101或同源残基处发生终止密码子至精氨酸突变(T>C突变)-参见，例如，矢崎(Yazaki)等人，国际肾脏(Kidney Int.)，2003；64:11-16；扩张型心肌病(DCM)–例如，在FOXD4基因的位置148或同源残基处发生色氨酸至精氨酸的突变(T>C突变)-参见，例如，米诺雷迪(Minoretti)等人，国际分子医学杂志(Int.J.ofMol.Med.)，2007；19:369-372；遗传性淋巴水肿–例如，在VEGFR3酪氨酸激酶的位置1035或同源残基处发生组氨酸至精氨酸的突变(A>G突变)-参见，例如，Irrthum等人，美国人类遗传学(Am.J.Hum.Genet.)，2000；67:295-301；家族性阿尔茨海默病–例如，在早老蛋白1(presenilin1)的位置143或同源残基处发生异亮氨酸至缬氨酸的突变(A>G突变)-参见，例如，加洛(Gallo)等人，阿尔茨海默病杂志(J.Alzheimer’s disease)，2011；25:425-431；朊病毒病–例如，在朊蛋白的位置129或同源残基处发生甲硫氨酸至缬氨酸的突变(A>G突变)-参见，例如，路易斯(Lewis)等人，普通病毒学杂志(J.of General Virology)，2006；87:2443-2449；慢性小儿神经皮肤关节综合征(CINCA)–例如，在cryopyrin蛋白的位置570或同源残基处发生酪氨酸至半胱氨酸的突变(A>G突变)-参见，例如，藤泽(Fujisawa)等人，血液(Blood)，2007；109:2903-2911；以及结蛋白相关肌病(DRM)–例如，在B晶状体蛋白的位置120或同源残基处发生精氨酸至甘氨酸的突变(A>G突变)-参见，例如，库马尔(Kumar)等人，生物化学杂志(J.Biol.Chem.)1999；274:24137-24141。所有参考文献和数据库条目的全部内容通过引用结合在此。
为了将如本文披露的Cas9:核酸编辑酶/结构域融合蛋白靶向靶位点，例如包含待编辑的点突变的位点，典型地有必要与指导RNA(如sgRNA)一起共表达Cas9:核酸编辑酶/结构域融合蛋白，这对本领域的普通技术人员而言将是显而易见的。如本文其他地方更详细的解释，指导RNA典型地包括允许Cas9结合的tracrRNA框架和赋予Cas9:核酸编辑酶/结构域融合蛋白序列特异性的指导序列。在一些实施例中，指导RNA包含结构5’-[指导序列]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:38)，其中指导序列包括与靶序列互补的序列。典型地，指导序列为20个核苷酸长。基于本披露，用于将Cas9:核酸编辑酶/结构域融合蛋白靶向特异性基因组靶位点的适合的指导RNA的序列对本领域的普通技术人员而言将是显而易见的。典型地，此类适合的指导RNA序列包括与待编辑的靶核苷酸上或下游50个核苷酸内的核酸序列互补的指导序列。以下提供了适合用于将Cas9:核酸编辑酶/结构域融合蛋白靶向特异性靶序列的一些示例性的指导RNA序列。
在磷脂酰肌醇-3-激酶催化α亚基(PI3KCA或PIK3CA)中的H1047R(A3140G)多态性(下划线是突变的核苷酸和各自的密码子的位置)：
Figure BDA0003475302240000321
(核苷酸序列-SEQ ID NO:39；蛋白序列-SEQ ID NO:40)。
用于将Cas9:核酸编辑酶/结构域融合蛋白靶向突变的A3140G残基的示例性的适合的指导序列包括但不限于：5’-aucggaauctauuuugacuc-3’(SEQ ID NO:41)；5’-ucggaaucuauuuugacucg-3’(SEQ ID NO:42)；5’-cuuagauaaaacugagcaag-3’(SEQ ID NO:43)；5’-aucuauuuugacucguucuc-3’(SEQ ID NO:44)；5’-uaaaacugagcaagaggcuu-3’(SEQID NO:45)；5’-ugguggcuggacaacaaaaa-3’(SEQ ID NO:46)；5’-gcuggacaacaaaaauggau-3’(SEQ ID NO:47)；5’-guguuaauuugucguacgua-3’(SEQ ID NO:48)。基于本披露，用于将Cas9:核酸编辑酶/结构域融合蛋白靶向突变的PI3KCA序列、靶向下文提供的任何另外的序列、或靶向与疾病相关的另外的突变的序列的另外适合的指导序列对本领域的普通技术人员而言将是显而易见的。
苯丙酮尿症在苯丙氨酸羟化酶基因的残基240处发生苯丙氨酸至丝氨酸的突变(T>C突变)(下划线是突变的核苷酸和各自的密码子的位置)：
Figure BDA0003475302240000331
(核苷酸序列-SEQ ID NO:49；蛋白序列–SEQ ID NO:50)。
巨大血小板综合征(BSS)-在血小板膜糖蛋白IX的残基24处发生半胱氨酸至精氨酸的突变(T>C突变)：
Figure BDA0003475302240000332
(核苷酸序列-SEQ ID NO:51；蛋白序列-SEQ ID NO:52)。
表皮松解性角化过度(EHK)-在角蛋白1的残基161处发生亮氨酸至脯氨酸的突变(T>C突变)：
Figure BDA0003475302240000333
(核苷酸序列-SEQ ID NO:53；蛋白序列-SEQ ID NO:54)。
慢性阻塞性肺病(COPD)-在α1-抗胰蛋白酶的残基54处发生亮氨酸至脯氨酸的突变(T>C突变)：
Figure BDA0003475302240000334
(核苷酸序列-SEQ ID NO:55；蛋白序列-SEQ ID NO:56)。
慢性阻塞性肺病(COPD)-在α1-抗胰凝乳蛋白酶的残基78处发生亮氨酸至脯氨酸的突变(T>C突变)：
Figure BDA0003475302240000341
(核苷酸序列-SEQ ID NO:89；蛋白序列-SEQ ID NO:90)。
神经母细胞瘤(NB)-在半胱天冬酶-9的残基197处发生亮氨酸至脯氨酸的突变(T>C突变)：
Figure BDA0003475302240000342
(核苷酸序列-SEQ ID NO:57；蛋白序列-SEQ ID NO:58)。
进行性神经性腓骨肌萎缩症4J型-在图4中的残基41处发生异亮氨酸至苏氨酸的突变(T>C突变)：
Figure BDA0003475302240000343
(核苷酸序列-SEQ ID NO:59；蛋白序列-SEQ ID NO:60)。
血管性血友病(vWD)–在血管性假血友病因子的残基1272处发生半胱氨酸至精氨酸的突变(T>C突变)：
Figure BDA0003475302240000344
(核苷酸序列-SEQ ID NO:61；蛋白序列-SEQ ID NO:62)。
先天性肌强直-在肌肉氯离子通道基因CLCN1的位置277处发生半胱氨酸至精氨酸的突变(T>C突变)：
Figure BDA0003475302240000345
(核苷酸序列-SEQ ID NO:63；蛋白序列-SEQ ID NO:64)。
遗传性肾淀粉样变性–在载脂蛋白AII的残基111处发生终止密码子至精氨酸的突变(T>C突变)：
Figure BDA0003475302240000351
(核苷酸序列-SEQ ID NO:65；蛋白序列-SEQ ID NO:66)。
扩张型心肌病(DCM)-在FOXD4基因的位置148处发生色氨酸至精氨酸的突变(T>C突变)：
Figure BDA0003475302240000352
(核苷酸序列-SEQ ID NO:67；蛋白序列-SEQ ID NO:68)。
遗传性淋巴水肿-在VEGFR3酪氨酸激酶的残基1035处发生组氨酸至精氨酸的突变(A>G突变)：
Figure BDA0003475302240000353
(核苷酸序列-SEQ ID NO:69；蛋白序列-SEQ ID NO:70)。
家族性阿尔茨海默病-在早老蛋白1的残基143处发生异亮氨酸至缬氨酸的突变(A>G突变)：
Figure BDA0003475302240000354
(核苷酸序列-SEQ ID NO:71；蛋白序列-SEQ ID NO:72)。
朊病毒病-在朊蛋白的残基129处发生甲硫氨酸至缬氨酸的突变(A>G突变)：
Figure BDA0003475302240000361
(核苷酸序列-SEQ ID NO:73；蛋白序列-SEQ ID NO:74)。
慢性小儿神经皮肤关节综合征(CINCA)–在cryopyrin蛋白的残基570处发生酪氨酸至半胱氨酸的突变(A>G突变)：
Figure BDA0003475302240000362
(核苷酸序列-SEQ ID NO:75；蛋白序列-SEQ ID NO:76)。
结蛋白相关性肌病(DRM)-在αB晶状体蛋白的残基120处发生精氨酸至甘氨酸的突变(A>G突变)：
Figure BDA0003475302240000363
(核苷酸序列-SEQ ID NO:77；蛋白序列-SEQ ID NO:78)。
β-地中海贫血-在血红蛋白B的残基115处发生亮氨酸至脯氨酸的突变的一个实例。
Figure BDA0003475302240000364
(核苷酸序列-SEQ ID NO:79；蛋白序列-SEQ ID NO:80)。应当了解，以上提供的序列是示例性的，并不意味着限制本披露的范围。基于本披露，疾病相关且服从Cas9:核酸编辑酶/结构域融合蛋白以及适合的指导RNA序列修正的另外的适合的点突变的序列对本领域的普通技术人员而言将是显而易见的。
报告系统
本披露的一些方面提供用于检测本文描述的融合蛋白的脱氨酶活性的报告系统。在一些实施例中，该报告系统是基于荧光素酶的试验，其中脱氨酶活性导致荧光素酶的表达。为了最小化脱氨酶结构域(例如，AID结构域)潜在的底物混乱的影响，可能会无意地被靶向以用于脱氨基作用的残基数目(例如，可能潜在地驻留在报告系统内的ssDNA上的脱靶C残基)被最小化。在一些实施例中，目的靶残基是位于不能启动翻译的荧光素酶基因的ACG突变的起始密码子中。理想的脱氨酶活性导致ACG>AUG的改变，因而使荧光素酶的翻译和脱氨酶活性的检测和定量成为可能。
在一些实施例中，为了最小化单链C残基，前导序列被插在突变的起始密码子和荧光素酶基因的起点之间，该前导序列由一串Lys(AAA)、Asn(AAT)、Leu(TTA)、Ile(ATT、ATA)、Tyr(TAT)、或Phe(TTT)残基组成。可以检测产生的突变体以确保前导序列不会负面地影响荧光素酶的表达或活性。还可以使用突变的起始密码子确定背景荧光素酶活性。
可以使用报告系统来检测许多不同的sgRNA，例如，以确定关于靶DNA序列，各自的脱氨酶(如AID酶)将靶向哪一个或哪几个残基(图3)。因为Cas9-DNA泡的大小是未知的，也可以检测靶向非模板链的sgRNA以便于评估特异性的Cas9脱氨酶融合蛋白的脱靶效应。在一些实施例中，设计这样的sgRNA以使得突变的起始密码子不会与sgRNA碱基配对。
一旦已经识别了可以可编程的将位点特异性C改变为U的融合蛋白，可以进一步描述它们活性的特征。来自荧光素酶试验的数据可以，例如，被整合进热地图，该热地图描述了关于sgRNA靶DNA，哪些核苷酸会被特异性融合蛋白靶向以用于脱氨基作用。在一些实施例中，对于每一个融合，在荧光素酶试验中导致最高活性的位置被考虑为“靶”位置，而所有其他位置被考虑为脱靶位置。
在一些实施例中，提供了具有各种APOBEC3酶的Cas9融合物或其脱氨酶结构域。在一些实施例中，提供了具有其他核酸编辑酶或催化结构域的Cas9融合蛋白，包括，例如，ssRNA编辑酶，如胞苷脱氨酶APOBEC1和ACF1/ASF，以及腺苷脱氨酶的ADAT家族，38这些核酸编辑酶或催化结构域当融合至Cas9时可以被用于ssDNA编辑活性。可以使用与以上描述的相同的报告系统和试验来检测此类融合蛋白的活性。
在一些实施例中，本文提供了报告系统，该报告系统包括包含失活的起始密码子(例如，在模板链上从3’-TAC-5’至3’-CAC-5’的突变)的报告基因。一旦靶标C成功脱去氨基，相应的mRNA将被转录为5’-AUG-3’而不是5’-GUG-3’，使得报告基因的翻译成为可能。适合的报告基因对本领域的普通技术人员而言将是显而易见的。
以上提供的对报告系统的示例性实施例的描述仅仅是出于说明性目的，并不意味着限制。本披露也包括另外的报告系统，例如，以上详细描述的各种示例性系统的变体。
实例
实例1：融合蛋白
以下提供了示例性Cas9:脱氨酶融合蛋白：
Cas9:人类AID融合物(C末端)
Figure BDA0003475302240000381
(下划线：细胞核定位信号；双下划线：细胞核输出信号；粗体：连接体序列)
Cas9:人类AID融合物(N末端)
Figure BDA0003475302240000382
Figure BDA0003475302240000391
(下划线：细胞核定位信号；粗体：连接体序列)
Cas9:小鼠AID融合物(C末端)
Figure BDA0003475302240000392
Figure BDA0003475302240000401
(下划线：细胞核定位信号；粗体：连接体序列；双下划线：细胞核输出信号)
Cas9:人类APOBEC-3G融合物(N末端)
Figure BDA0003475302240000402
(下划线：细胞核定位信号；粗体：连接体(1NLS))，
Cas9:人类APOBEC-1融合物(N末端)
Figure BDA0003475302240000403
Figure BDA0003475302240000411
(下划线：细胞核定位信号；粗体：连接体(1NLS)，(SEQ ID NO:92)
Cas9:人类ADAT1融合物(N末端)
Figure BDA0003475302240000412
Figure BDA0003475302240000421
(下划线：细胞核定位信号；粗体：连接体序列)
Cas9:人类ADAT1融合物(C末端)
Figure BDA0003475302240000422
Figure BDA0003475302240000431
Figure BDA0003475302240000432
(下划线：细胞核定位信号；粗体：连接体序列)
实例2：通过Cas9融合蛋白修正PI3K点突变
导致PI3K蛋白的H1047R氨基酸置换的在PI3KCA基因的外显子20内的A3140G点突变通过接触编码具有Cas9:AID(SEQ ID NO:30)或Cas9:APOBEC1(SEQ ID NO:92)融合蛋白的突变蛋白的核酸和在编码PI3KCA基因中将融合蛋白靶向突变位点的恰当地设计的sgRNA来修正。通过各自的外显子20序列的基因组PCR来确定A3140G点突变，例如，产生核苷酸3000-3250的PCR扩增子并且随后对PCR扩增子测序。
使表达在外显子20中包括A3140G点突变的突变的PI3K蛋白的细胞接触编码Cas9:AID(SEQ ID NO:30)或Cas9:APOBEC1(SEQ ID NO:92)融合蛋白的表达构建体和在编码PI3KCA基因的反义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA。sgRNA的序列是5’-aucggaauctauuuugacucguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu 3’(SEQ ID NO:81)；5’-ucggaaucuauuuugacucgguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:82)；5’-cuuagauaaaacugagcaagguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQID NO:83)；5’-aucuauuuugacucguucucguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuug aaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:84)；5’-uaaaacugagcaagaggcuuguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:85)；5’-ugguggcuggacaacaaaaaguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:86)；5’-gcuggacaacaaaaauggauguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3’(SEQ ID NO:87)；或5’-guguuaauuugucguacguaguuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO:88)。
Cas9:AID或Cas9:APOBEC1融合蛋白的胞嘧啶脱氨酶活性导致与突变的G3140碱基配对的胞嘧啶脱去氨基成为尿嘧啶。一轮复制以后，恢复了野生型A3140。提取所处理的细胞的基因组DNA并且使用适合的PCR引物扩增核苷酸3000-3250的PCR扩增子。通过对PCR扩增子测序来确定使用融合蛋白处理细胞后的A3140G点突变的修正。
实例3：通过Cas9融合蛋白修正早老蛋白1点突变
导致早老蛋白1(PSEN1)内I143V氨基酸置换的PSEN1基因的密码子143内的A->G点突变通过接触编码突变的PSEN1蛋白(具有Cas9:AID(SEQ ID NO:30)或Cas9:APOBEC1(SEQID NO:92)融合蛋白)的核酸和在编码PSEN1基因中将融合蛋白靶向突变位点的恰当地设计的sgRNA来修正。参见，例如，加洛(Gallo)等人，阿尔茨海默病杂志(J.Alzheimer’sdisease)，2011；25:425-431，对于与家族性阿尔茨海默病相关的示例性PSEN1 I143V突变的描述。通过各自的PSEN1序列的基因组PCR来确定A->G点突变，例如，产生外显子143周围的约100-250个核苷酸的PCR扩增子并且随后对PCR扩增子测序。
使表达突变的PSEN1蛋白的细胞接触编码Cas9:AID(SEQ ID NO:30)或Cas9:APOBEC1(SEQ ID NO:92)融合蛋白的表达构建体和在编码PSEN1基因的反义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA。Cas9:AID或Cas9:APOBEC1融合蛋白的胞嘧啶脱氨酶活性导致与密码子143中的突变的G碱基配对的胞嘧啶脱去氨基成为尿嘧啶。一轮复制以后，恢复了野生型A。提取所处理的细胞的基因组DNA并且使用适合的PCR引物扩增100-250个核苷酸的PCR扩增子。通过对PCR扩增子测序来确定使用融合蛋白处理细胞后的A->G点突变的修正。
实例4：通过Cas9融合蛋白修正α1-抗胰蛋白酶点突变
导致α1-抗胰蛋白酶蛋白内L55P氨基酸置换的α1-抗胰蛋白酶基因的密码子55内的T->C点突变通过接触编码突变的α1-抗胰蛋白酶蛋白(具有Cas9:ADAT1融合蛋白(SEQ IDNO:35或36))的核酸和在编码α1-抗胰蛋白酶基因中将融合蛋白靶向突变位点的恰当地设计的sgRNA来修正。参见，例如，波勒(Poller)等人，基因组学(Genomics)，1993；17:740-743，对于与慢性阻塞性肺病相关的示例性密码子55T->C突变的更详细的描述。通过编码密码子55的各自的α1-抗胰蛋白酶序列的基因组PCR来确定T->C点突变，例如，产生约100-250个核苷酸的PCR扩增子并且随后对PCR扩增子测序。
使表达突变的α1-抗胰蛋白酶蛋白的细胞接触编码Cas9:AID(SEQ ID NO:30)或Cas9:APOBEC1(SEQ ID NO:92)融合蛋白的表达构建体和在编码α1-抗胰蛋白酶基因的有义链中将融合蛋白靶向密码子55内突变的核苷酸的恰当地设计的sgRNA。Cas9:ADAT1融合蛋白的胞嘧啶脱氨酶活性导致突变的胞嘧啶脱去氨基成为尿嘧啶从而修正该突变。提取所处理的细胞的基因组DNA并且使用适合的PCR引物扩增100-250个核苷酸的PCR扩增子。通过对PCR扩增子测序来确定使用融合蛋白处理细胞后的α1-抗胰蛋白酶基因的密码子55内T->C点突变的修正
实例5：通过Cas9融合蛋白修正血管性血友病因子点突变
导致血管性血友病因子蛋白的C509R氨基酸置换的血管性血友病因子基因的密码子509内的T->C点突变通过接触编码突变的血管性血友病因子蛋白(具有Cas9:ADAT1融合蛋白(SEQ ID NO:35或36))的核酸和在编码血管性血友病因子基因的反义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA来修正。参见，例如，拉韦涅(Lavergne)等人，英国血液学杂志(Br.J.Haematol.)，1992；82:66-7，对于与血管性血友病(vWD)相关的示例性血管性血友病因子C509R突变的描述。通过各自的血管性血友病因子基因组序列的基因组PCR来确定T->C点突变，例如，产生外显子509周围的约100-250个核苷酸的PCR扩增子并且随后对PCR扩增子测序。
使表达突变的血管性血友病因子蛋白的细胞接触编码Cas9:ADAT1融合蛋白(SEQID NO:35或36)的表达构建体和在编码血管性血友病因子基因的有义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA。Cas9:ADAT1融合蛋白的胞嘧啶脱氨酶活性导致密码子509内突变的胞嘧啶脱去氨基成为尿嘧啶从而修正突变。提取所处理的细胞的基因组DNA并且使用适合的PCR引物扩增100-250个核苷酸的PCR扩增子。通过对PCR扩增子测序来确定使用融合蛋白处理细胞后血管性血友病因子基因的密码子509内T->C点突变的修正。实例6：通过Cas9融合蛋白修正半胱天冬酶9点突变-神经母细胞瘤
导致半胱天冬酶-9蛋白内L197P氨基酸置换的半胱天冬酶-9基因的密码子197内的T->C点突变通过接触编码突变的半胱天冬酶-9蛋白(具有Cas9:ADAT1融合蛋白(SEQ IDNO:35或36))的核酸和在编码半胱天冬酶-9基因的有义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA来修正。参见，例如，伦克(Lenk)等人，PLoS遗传学(PLoS Genetics)，2011；7:e1002104，对于与神经母细胞瘤(NB)相关的示例性半胱天冬酶-9L197P突变的描述。通过各自的半胱天冬酶-9基因组序列的基因组PCR来确定T->C点突变，例如，产生外显子197周围的约100-250个核苷酸的PCR扩增子并且随后对PCR扩增子测序。
使表达突变的半胱天冬酶-9蛋白的细胞接触编码Cas9:ADAT1融合蛋白(SEQ IDNO:35或36)的表达构建体和在编码半胱天冬酶-9基因的有义链中将融合蛋白靶向突变位点的恰当地设计的sgRNA。Cas9:ADAT1融合蛋白的胞嘧啶脱氨酶活性导致密码子197内突变的胞嘧啶脱去氨基成为尿嘧啶从而修正突变。提取所处理的细胞的基因组DNA并且使用适合的PCR引物扩增100-250个核苷酸的PCR扩增子。通过对PCR扩增子测序来确定使用融合蛋白处理细胞后的半胱天冬酶-9基因的密码子197内T->C点突变的修正。实例7：两种dCas9-APOBEC1融合蛋白的脱氨酶活性
产生了具有不同连接体的两种dCas9-APOBEC1融合蛋白：
rAPOBEC1_GGS_dCas9：
Figure BDA0003475302240000461
94)；下划线＝rAPOBEC1；双下划线＝dCas9。
rAPOBEC1_(GGS)3_dCas9：
Figure BDA0003475302240000462
Figure BDA0003475302240000471
(SEQ ID NO:95)；下划线＝rAPOBEC1；双下划线＝dCas9。
检査了两种融合蛋白的脱氨酶活性。脱氨酶试验改编自核酸研究(Nuc.AcidsRes.)2014，42，p.1095；生物化学杂志(J.Biol.Chem.)2004，279，p.53379；病毒学杂志(J.Virology)2014，88，p.3850；以及病毒学杂志(J.Virology)2006，80，p.5992，将其每一个的全部内容通过引用结合在此)。
编码融合蛋白的表达构建体被插入CMV骨架质粒中(载体质粒52970；参见古灵儿JP(Guilinger JP)、汤普森·DB(Thompson DB)、刘·DR(Liu DR)，融合无催化活性的Cas9至FokI核酸酶改善基因组修饰的特异性(Fusion of catalytically inactive Cas9 toFokI nuclease improves the specificity of genome modification)，自然生物技术(Nat.Biotechnol.)2014；32(6):577-82)。使用TNT快速耦合转录/翻译系统(TNT QuickCoupled Transcription/Translation System)(普洛麦格公司(Promega))来表达融合蛋白。90分钟后，将5μL溶解产物与5’-标记的ssDNA底物(Cy3-ATTATTATTATTCCGCGGATTTATTTATTTATTTATTTATTT，SEQ ID NO:96)和UDG(尿嘧啶DNA糖基化酶)在37℃孵育持续3小时。然后添加1M NaOH(10μL)溶液以在脱碱基位点切割DNA。见图4。将DNA在10％的TBE PAGE凝胶上分辨(图5)。还包括阴性对照(其中pUC19孵育在TNT系统中)，以及阳性对照(其中合成了用“U”代替靶标C的DNA)。图5说明两种融合蛋白都展现出脱氨酶活性。
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38.格柏·AP(Gerber AP)、凯勒·W(Keller W)，通过碱基脱氨作用进行RNA编辑：更多的酶，更多的靶标，新的奥秘(RNA editing by base deamination:more enzymes,more targets,new mysteries)，趋势生物化学科学(Trends Biochem Sci)，2001；26(6):376-84，PMID:11406411。
39.袁·L(Yuan L)、库莱克·I(Kurek I)、英格里斯J(English J)、基南·R(Keenan R)，实验室定向蛋白进化(Laboratory-directed protein evolution)，微生物分子生物学评论(Microbiol Mol Biol Rev)，2005；69(3):373-92，PMID:16148303。
40.科布·RE(Cobb RE)、孙·N(Sun N)、赵·H(Zhao H)，定向进化——强大的合成生物学工具(Directed evolution as a powerful synthetic biology tool)，方法(Methods)，2013；60(1):81-90，PMID:22465795。
41.博思坦S(Bershtein S)、陶菲克·DS(Tawfik DS)，酶的实验室进化的进展(Advances in laboratory evolution of enzymes)，化学生物学新见(Curr Opin ChemBiol)，2008；12(2):151-8，PMID:18284924。
42.希达·K(Hida K)、哈内斯·J(Hanes J)、奥斯特梅尔M(Ostermeier M)，药物和核酸递送的定向进化(Directed evolution for drug and nucleic acid delivery)，先进药物输送评论(Adv Drug Deliv Rev)，2007；59(15):1562-78，PMID:17933418。
43.艾斯维特KM(Esvelt KM)、卡尔森·JC(Carlson JC)、刘·DR(Liu DR)，生物分子持续定向进化系统(A system for the continuous directed evolution ofbiomolecules)，自然(Nature)，2011；472(7344):499-503，PMID:21478873。
44.胡斯米Y(Husimi Y)，Cellstat中噬菌体的选择与进化(Selection andevolution of bacteriophages in cellstat)，生物物理学进展(Adv Biophys)，1989；25:1-43，PMID:2696338。
45.瑞彻曼L(Riechmann L)、霍利格·P(Holliger P)，TolAC-末端结构域为大肠杆菌丝状噬菌体感染的辅助受体(The C-terminal domain of TolA is the coreceptorfor filamentous phage infection of E.coli)，细胞(Cell)，1997；90(2):351-60，PMID:9244308。
46.尼尔森FK(Nelson FK)、弗里德曼SM(Friedman SM)、史密斯GP(Smith GP)，丝状噬菌体DNA克隆载体：基因III中具有非极性删除的非感染性突变体(Filamentous phageDNA cloning vectors:a noninfective mutant with a nonpolar deletion in geneIII)，病毒学(Virology)，1981；108(2):338-50，PMID:6258292。
47.拉克杰克J(Rakonjac J)、莫德尔P(Model P)，pIII在丝状噬菌体组装中的作用(Roles of pIII in filamentous phage assembly)，分子生物学杂志(J Mol Biol)，1998；282(1):25-41。
48.史密斯GP(Smith GP)，丝状噬菌体融合：在病毒表面上显示克隆抗原的新颖表达载体(Filamentous fusion phage:novel expression vectors that display clonedantigens on the virion surface)，科学(Science)，1985；228(4705):1315-7，PMID:4001944。
49.谢里登·C(Sheridan C)，基因治疗找到其合适的位置(Gene therapy findsits niche)，自然生物技术(Nat.Biotechnol.)2011；29(2):121-8，PMID:21301435。
50.李JW(Lee JW)、宋YH(Soung YH)、金SY(Kim SY)、李HW(Lee HW)、帕克WS(ParkWS)、纳姆SW(Nam SW)、金SH(Kim SH)、李JY(Lee JY)、柳NJ(Yoo NJ)、李SH(Lee SH)，PIK3CA基因通常在乳腺癌和肝癌中突变(PIK3CA gene is frequently mutated in breastcarcinomas and hepatocellular carcinomas)，致癌基因(Oncogene)，2005；24(8):1477-80，PMID:15608678。
51.伊科得彼ON(Ikediobi ON)、戴维斯·H(Davies H)、比格内尔·G(BignellG)、艾约瑟·S(Edkins S)、史蒂文斯·C(Stevens C)、欧米拉·S(O’Meara S)、斯坦里乌斯T(Santarius T)、阿维斯·T(Avis T)、巴瑟彼S(Barthorpe S)、布拉肯伯里L(BrackenburyL)、巴克·G(Buck G)、巴特勒·A(Butler A)、克莱门茨·J(Clements J)、科尔·J(ColeJ)、迪克斯·E(Dicks E)、福布斯·S(Forbes S)、格雷·K(Gray K)、哈利迪·K(HallidayK)、哈里森·R(Harrison R)、希尔斯·K(Hills K)、欣顿·J(Hinton J)、亨特·C(HunterC)、詹金森·A(Jenkinson A)、琼斯·D(Jones D)、克斯弥渡V(Kosmidou V)、勒格·R(LuggR)、孟席斯·A(Menzies A)、米罗纳克T(Mironenko T)、帕克·A(Parker A)、佩里·J(Perry J)、雷恩·K(Raine K)、理查森·D(Richardson D)、谢泼德·R(Shepherd R)、斯莫尔·A(Small A)、史密斯·R(Smith R)、所罗门·H(Solomon H)、斯蒂芬斯·P(StephensP)、缇阙J(Teaque J)、托夫茨·C(Tofts C)、范里安·J(Varian J)、韦伯·T(Webb T)、韦斯特·S(West S)、威达S(Widaa S)、耶茨·A(Yates A)、莱因霍尔德·W(Reinhold W)、温斯坦·JN(Weinstein JN)、斯特拉顿·MR(Stratton MR)、福特瑞尔PA(Futreal PA)、伍斯特·R(Wooster R)，在NCI-60细胞系组的24个已知癌症基因的突变分析(Mutationanalysis of 24known cancer genes in the NCI-60cell line set)，分子癌症治疗学(Mol Cancer Ther)，2006；5(11):2606-12，PMID:17088437。
本文，例如，在背景、概述、详细说明、实例、和/或参考文献部分提及的所有出版物、专利、专利申请、公布物、和数据库条目(例如，序列数据库条目)以其整体通过引用结合在此，如同每个单独的出版物、专利、专利申请、公布物和数据库条目被具体和单独地通过引用结合在此。在冲突存在的情况下，将以本申请(包括本文的任何定义)为准。
等效物和范围
本领域的技术人员仅仅使用常规实验就将认识到或能够确认本文描述的实施例的许多等效物。本披露的范围不旨在限制以上说明书，而是如在所附的权利要求书中所陈述的。
冠词如“一个/种(a/an)”和“该/所述(the)”可以意为一个/种或多于一个/种，除非有相反的指明或另外从上下文明显可见。如果一组中一个、多于一个或全部成员存在，则在该组的两个或多个成员之间包括“或”的权利要求书或说明被认为是得到满足，除非有相反的指明或另外从上下文明显可见。在两个或多个组成员之间包括“或”的组的披露提供如下实施例：其中恰好存在一个组成员的实施例，其中存在多于一个组成员的实施例，和其中存在所有组成员的实施例。出于简洁的目的，那些实施例在本文没有单独地讲出来，但是应当了解本文提供了这些实施例的每一个并且可以具体地要求或拒绝这些实施例中的每一个。
应当理解的是，本发明涵盖将一个或多个权利要求项或说明书的一个或多个相关部分中的一个或多个限定、要素、条款、或说明性术语等引入另一权利要求项中的所有变化、组合和排列。例如，可以对从属于另一个权利要求的一个权利要求进行修改以便包括从属于同一基础权利要求的任何一个其他的权利要求中所发现的一个或多个限制。此外，在权利要求叙述组合物的情况下，但应当理解的是，根据本文披露的任何制造或使用的方法或根据本领域中已知的(如果存在)方法的制造或使用组合物的方法都包括在内，除非另有说明或者除非对于本领域的普通技术人员而言显然会产生矛盾或不一致性。
在将要素呈现为列表的情况下，例如以马库什组形式，应当理解的是，这些要素的每个可能的亚组也被披露，并且任何要素或要素的亚组都可以从该组中去除。也应指出，名词“包含”旨在是开放的并且允许囊括另外的要素或步骤。应当理解的是，通常，在一个/种实施例、产物或方法被指包括特定元素、特征或步骤的情况下，也提供了由或基本上由这些元素、特征或步骤组成的实施例、产物或方法。出于简洁的目的，那些实施例在本文没有单独地讲出来，但是应当了解本文提供了这些实施例的每一个并且可以具体地要求或拒绝这些实施例中的每一个。
在给出范围的情况下，包括端点。此外，应当理解的是，除非另作说明或另外从上下文和/或本领域的普通技术人员所理解的明显可见，在一些实施例中，表达为范围的值可以呈现为所陈述的范围内的任何具体值，至该范围的下限的单位十分之一，除非上下文另作清楚规定。出于简洁的目的，每个范围内的值在本文没有单独地讲出来，但是应当了解本文提供这些值的每一个并且可以具体地要求或拒绝这些值中的每一个。还应当理解的是，除非另作说明或另外从上下文和/或本领域的普通技术人员所理解的明显可见，表达为范围的值可以呈现为给定的范围内的任何亚范围，其中亚范围的端点以相同精确度被表达为该范围的下限单位的十分之一。
另外，应当理解的是本发明的任何具体的实施例可以从任何一个或多个权利要求中明确排除。在范围被给定的情况下，范围内的任何值可以从任何一个或多个权利要求中明确排除。本发明的组合物和/或方法的任何实施例、元素、特征、应用或方面可以从任何一个或多个权利要求中排除。出于简洁的目的，本文没有明确陈述其中一个或多个元素、特征、目的或方面被排除的所有实施例。
序列表
<110> 哈佛大学的校长及成员们
<120> 用于基因编辑的CAS变体
<130> H0824.70170WO00
<140> PCT/US2014/070038
<141> 2014-12-12
<150> US 14/326,318
<151> 2014-07-08
<150> US 14/326,303
<151> 2014-07-08
<150> US 14/326,290
<151> 2014-07-08
<150> US 14/326,269
<151> 2014-07-08
<150> US 14/326,140
<151> 2014-07-08
<150> US 14/326,109
<151> 2014-07-08
<150> US 14/325,815
<151> 2014-07-08
<150> US 61/980,333
<151> 2014-04-16
<150> US 61/915,386
<151> 2013-12-12
<160> 106
<170> PatentIn version 3.5
<210> 1
<211> 4104
<212> DNA
<213> 酿脓链球菌
<400> 1
atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg attataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg gcagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggcag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa atctacaatc aattatttga agaaaaccct 600
attaacgcaa gtagagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga gaaatggctt gtttgggaat 720
ctcattgctt tgtcattggg attgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatagt gaaataacta aggctcccct atcagcttca 960
atgattaagc gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgagggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggcgcct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agataggggg atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga tattcaaaaa gcacaggtgt ctggacaagg ccatagttta 2160
catgaacaga ttgctaactt agctggcagt cctgctatta aaaaaggtat tttacagact 2220
gtaaaaattg ttgatgaact ggtcaaagta atggggcata agccagaaaa tatcgttatt 2280
gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg 2340
aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt 2400
gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctacaaaa tggaagagac 2460
atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt 2520
gttccacaaa gtttcattaa agacgattca atagacaata aggtactaac gcgttctgat 2580
aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac 2640
tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg 2700
aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg 2760
gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact 2820
aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa 2880
ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac 2940
catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat 3000
ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg 3060
attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat 3120
atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct 3180
ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc 3240
acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag 3300
acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct 3360
cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat 3420
tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa 3480
gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt 3540
ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat 3600
agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa 3660
aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat 3720
tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag 3780
cataagcatt atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt 3840
ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca 3900
atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc 3960
gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa 4020
gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat 4080
ttgagtcagc taggaggtga ctga 4104
<210> 2
<211> 1367
<212> PRT
<213> 酿脓链球菌
<400> 2
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Gly Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Ala Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Ile Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Arg Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Arg Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Ser Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Ala Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Gly Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly His Ser Leu
705 710 715 720
His Glu Gln Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Ile Val Asp Glu Leu Val Lys Val Met Gly
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 Ile 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 Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 3
<211> 4212
<212> DNA
<213> 酿脓链球菌
<400> 3
atggataaaa agtattctat tggtttagac atcggcacta attccgttgg atgggctgtc 60
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 180
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 360
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 540
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 600
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 720
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 780
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 960
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 1140
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 1200
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 1380
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 1560
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 1620
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 1800
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 2760
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 2940
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 3120
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 3180
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 3360
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 3540
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 3600
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 3720
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 3780
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 3900
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 3960
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 4020
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080
gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt ctcgagcgac 4140
tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac 4200
aaggctgcag ga 4212
<210> 4
<211> 1368
<212> PRT
<213> 酿脓链球菌
<400> 4
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 5
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 5
Glu Ala Ala Ala Lys
1 5
<210> 6
<211> 198
<212> PRT
<213> 人类
<400> 6
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Thr Leu Gly Leu
195
<210> 7
<211> 198
<212> PRT
<213> 小鼠
<400> 7
Met Asp Ser Leu Leu Met Lys Gln Lys Lys Phe Leu Tyr His Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Glu
85 90 95
Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Gly Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Thr Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Met Leu Gly Phe
195
<210> 8
<211> 198
<212> PRT
<213> 狼
<400> 8
Met Asp Ser Leu Leu Met Lys Gln Arg Lys Phe Leu Tyr His Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Tyr Pro Asn Leu Ser Leu Arg Ile Phe Ala Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Glu Lys Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Thr Leu Gly Leu
195
<210> 9
<211> 199
<212> PRT
<213> 牛
<400> 9
Met Asp Ser Leu Leu Lys Lys Gln Arg Gln Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Pro Thr Ser Phe Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ala Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Tyr Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Asp Lys Glu Arg Lys Ala Glu Pro Glu Gly Leu Arg
115 120 125
Arg Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp
130 135 140
Tyr Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe
145 150 155 160
Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln
165 170 175
Leu Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp
180 185 190
Ala Phe Arg Thr Leu Gly Leu
195
<210> 10
<211> 429
<212> PRT
<213> 小鼠
<400> 10
Met Gly Pro Phe Cys Leu Gly Cys Ser His Arg Lys Cys Tyr Ser Pro
1 5 10 15
Ile Arg Asn Leu Ile Ser Gln Glu Thr Phe Lys Phe His Phe Lys Asn
20 25 30
Leu Gly Tyr Ala Lys Gly Arg Lys Asp Thr Phe Leu Cys Tyr Glu Val
35 40 45
Thr Arg Lys Asp Cys Asp Ser Pro Val Ser Leu His His Gly Val Phe
50 55 60
Lys Asn Lys Asp Asn Ile His Ala Glu Ile Cys Phe Leu Tyr Trp Phe
65 70 75 80
His Asp Lys Val Leu Lys Val Leu Ser Pro Arg Glu Glu Phe Lys Ile
85 90 95
Thr Trp Tyr Met Ser Trp Ser Pro Cys Phe Glu Cys Ala Glu Gln Ile
100 105 110
Val Arg Phe Leu Ala Thr His His Asn Leu Ser Leu Asp Ile Phe Ser
115 120 125
Ser Arg Leu Tyr Asn Val Gln Asp Pro Glu Thr Gln Gln Asn Leu Cys
130 135 140
Arg Leu Val Gln Glu Gly Ala Gln Val Ala Ala Met Asp Leu Tyr Glu
145 150 155 160
Phe Lys Lys Cys Trp Lys Lys Phe Val Asp Asn Gly Gly Arg Arg Phe
165 170 175
Arg Pro Trp Lys Arg Leu Leu Thr Asn Phe Arg Tyr Gln Asp Ser Lys
180 185 190
Leu Gln Glu Ile Leu Arg Pro Cys Tyr Ile Pro Val Pro Ser Ser Ser
195 200 205
Ser Ser Thr Leu Ser Asn Ile Cys Leu Thr Lys Gly Leu Pro Glu Thr
210 215 220
Arg Phe Cys Val Glu Gly Arg Arg Met Asp Pro Leu Ser Glu Glu Glu
225 230 235 240
Phe Tyr Ser Gln Phe Tyr Asn Gln Arg Val Lys His Leu Cys Tyr Tyr
245 250 255
His Arg Met Lys Pro Tyr Leu Cys Tyr Gln Leu Glu Gln Phe Asn Gly
260 265 270
Gln Ala Pro Leu Lys Gly Cys Leu Leu Ser Glu Lys Gly Lys Gln His
275 280 285
Ala Glu Ile Leu Phe Leu Asp Lys Ile Arg Ser Met Glu Leu Ser Gln
290 295 300
Val Thr Ile Thr Cys Tyr Leu Thr Trp Ser Pro Cys Pro Asn Cys Ala
305 310 315 320
Trp Gln Leu Ala Ala Phe Lys Arg Asp Arg Pro Asp Leu Ile Leu His
325 330 335
Ile Tyr Thr Ser Arg Leu Tyr Phe His Trp Lys Arg Pro Phe Gln Lys
340 345 350
Gly Leu Cys Ser Leu Trp Gln Ser Gly Ile Leu Val Asp Val Met Asp
355 360 365
Leu Pro Gln Phe Thr Asp Cys Trp Thr Asn Phe Val Asn Pro Lys Arg
370 375 380
Pro Phe Trp Pro Trp Lys Gly Leu Glu Ile Ile Ser Arg Arg Thr Gln
385 390 395 400
Arg Arg Leu Arg Arg Ile Lys Glu Ser Trp Gly Leu Gln Asp Leu Val
405 410 415
Asn Asp Phe Gly Asn Leu Gln Leu Gly Pro Pro Met Ser
420 425
<210> 11
<211> 429
<212> PRT
<213> 大鼠
<400> 11
Met Gly Pro Phe Cys Leu Gly Cys Ser His Arg Lys Cys Tyr Ser Pro
1 5 10 15
Ile Arg Asn Leu Ile Ser Gln Glu Thr Phe Lys Phe His Phe Lys Asn
20 25 30
Leu Arg Tyr Ala Ile Asp Arg Lys Asp Thr Phe Leu Cys Tyr Glu Val
35 40 45
Thr Arg Lys Asp Cys Asp Ser Pro Val Ser Leu His His Gly Val Phe
50 55 60
Lys Asn Lys Asp Asn Ile His Ala Glu Ile Cys Phe Leu Tyr Trp Phe
65 70 75 80
His Asp Lys Val Leu Lys Val Leu Ser Pro Arg Glu Glu Phe Lys Ile
85 90 95
Thr Trp Tyr Met Ser Trp Ser Pro Cys Phe Glu Cys Ala Glu Gln Val
100 105 110
Leu Arg Phe Leu Ala Thr His His Asn Leu Ser Leu Asp Ile Phe Ser
115 120 125
Ser Arg Leu Tyr Asn Ile Arg Asp Pro Glu Asn Gln Gln Asn Leu Cys
130 135 140
Arg Leu Val Gln Glu Gly Ala Gln Val Ala Ala Met Asp Leu Tyr Glu
145 150 155 160
Phe Lys Lys Cys Trp Lys Lys Phe Val Asp Asn Gly Gly Arg Arg Phe
165 170 175
Arg Pro Trp Lys Lys Leu Leu Thr Asn Phe Arg Tyr Gln Asp Ser Lys
180 185 190
Leu Gln Glu Ile Leu Arg Pro Cys Tyr Ile Pro Val Pro Ser Ser Ser
195 200 205
Ser Ser Thr Leu Ser Asn Ile Cys Leu Thr Lys Gly Leu Pro Glu Thr
210 215 220
Arg Phe Cys Val Glu Arg Arg Arg Val His Leu Leu Ser Glu Glu Glu
225 230 235 240
Phe Tyr Ser Gln Phe Tyr Asn Gln Arg Val Lys His Leu Cys Tyr Tyr
245 250 255
His Gly Val Lys Pro Tyr Leu Cys Tyr Gln Leu Glu Gln Phe Asn Gly
260 265 270
Gln Ala Pro Leu Lys Gly Cys Leu Leu Ser Glu Lys Gly Lys Gln His
275 280 285
Ala Glu Ile Leu Phe Leu Asp Lys Ile Arg Ser Met Glu Leu Ser Gln
290 295 300
Val Ile Ile Thr Cys Tyr Leu Thr Trp Ser Pro Cys Pro Asn Cys Ala
305 310 315 320
Trp Gln Leu Ala Ala Phe Lys Arg Asp Arg Pro Asp Leu Ile Leu His
325 330 335
Ile Tyr Thr Ser Arg Leu Tyr Phe His Trp Lys Arg Pro Phe Gln Lys
340 345 350
Gly Leu Cys Ser Leu Trp Gln Ser Gly Ile Leu Val Asp Val Met Asp
355 360 365
Leu Pro Gln Phe Thr Asp Cys Trp Thr Asn Phe Val Asn Pro Lys Arg
370 375 380
Pro Phe Trp Pro Trp Lys Gly Leu Glu Ile Ile Ser Arg Arg Thr Gln
385 390 395 400
Arg Arg Leu His Arg Ile Lys Glu Ser Trp Gly Leu Gln Asp Leu Val
405 410 415
Asn Asp Phe Gly Asn Leu Gln Leu Gly Pro Pro Met Ser
420 425
<210> 12
<211> 370
<212> PRT
<213> 食蟹猴
<400> 12
Met Val Glu Pro Met Asp Pro Arg Thr Phe Val Ser Asn Phe Asn Asn
1 5 10 15
Arg Pro Ile Leu Ser Gly Leu Asn Thr Val Trp Leu Cys Cys Glu Val
20 25 30
Lys Thr Lys Asp Pro Ser Gly Pro Pro Leu Asp Ala Lys Ile Phe Gln
35 40 45
Gly Lys Val Tyr Ser Lys Ala Lys Tyr His Pro Glu Met Arg Phe Leu
50 55 60
Arg Trp Phe His Lys Trp Arg Gln Leu His His Asp Gln Glu Tyr Lys
65 70 75 80
Val Thr Trp Tyr Val Ser Trp Ser Pro Cys Thr Arg Cys Ala Asn Ser
85 90 95
Val Ala Thr Phe Leu Ala Lys Asp Pro Lys Val Thr Leu Thr Ile Phe
100 105 110
Val Ala Arg Leu Tyr Tyr Phe Trp Lys Pro Asp Tyr Gln Gln Ala Leu
115 120 125
Arg Ile Leu Cys Gln Lys Arg Gly Gly Pro His Ala Thr Met Lys Ile
130 135 140
Met Asn Tyr Asn Glu Phe Gln Asp Cys Trp Asn Lys Phe Val Asp Gly
145 150 155 160
Arg Gly Lys Pro Phe Lys Pro Arg Asn Asn Leu Pro Lys His Tyr Thr
165 170 175
Leu Leu Gln Ala Thr Leu Gly Glu Leu Leu Arg His Leu Met Asp Pro
180 185 190
Gly Thr Phe Thr Ser Asn Phe Asn Asn Lys Pro Trp Val Ser Gly Gln
195 200 205
His Glu Thr Tyr Leu Cys Tyr Lys Val Glu Arg Leu His Asn Asp Thr
210 215 220
Trp Val Pro Leu Asn Gln His Arg Gly Phe Leu Arg Asn Gln Ala Pro
225 230 235 240
Asn Ile His Gly Phe Pro Lys Gly Arg His Ala Glu Leu Cys Phe Leu
245 250 255
Asp Leu Ile Pro Phe Trp Lys Leu Asp Gly Gln Gln Tyr Arg Val Thr
260 265 270
Cys Phe Thr Ser Trp Ser Pro Cys Phe Ser Cys Ala Gln Glu Met Ala
275 280 285
Lys Phe Ile Ser Asn Asn Glu His Val Ser Leu Cys Ile Phe Ala Ala
290 295 300
Arg Ile Tyr Asp Asp Gln Gly Arg Tyr Gln Glu Gly Leu Arg Ala Leu
305 310 315 320
His Arg Asp Gly Ala Lys Ile Ala Met Met Asn Tyr Ser Glu Phe Glu
325 330 335
Tyr Cys Trp Asp Thr Phe Val Asp Arg Gln Gly Arg Pro Phe Gln Pro
340 345 350
Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg
355 360 365
Ala Ile
370
<210> 13
<211> 384
<212> PRT
<213> 黑猩猩
<400> 13
Met Lys Pro His Phe Arg Asn Pro Val Glu Arg Met Tyr Gln Asp Thr
1 5 10 15
Phe Ser Asp Asn Phe Tyr Asn Arg Pro Ile Leu Ser His Arg Asn Thr
20 25 30
Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Gly Pro Ser Arg Pro Pro
35 40 45
Leu Asp Ala Lys Ile Phe Arg Gly Gln Val Tyr Ser Lys Leu Lys Tyr
50 55 60
His Pro Glu Met Arg Phe Phe His Trp Phe Ser Lys Trp Arg Lys Leu
65 70 75 80
His Arg Asp Gln Glu Tyr Glu Val Thr Trp Tyr Ile Ser Trp Ser Pro
85 90 95
Cys Thr Lys Cys Thr Arg Asp Val Ala Thr Phe Leu Ala Glu Asp Pro
100 105 110
Lys Val Thr Leu Thr Ile Phe Val Ala Arg Leu Tyr Tyr Phe Trp Asp
115 120 125
Pro Asp Tyr Gln Glu Ala Leu Arg Ser Leu Cys Gln Lys Arg Asp Gly
130 135 140
Pro Arg Ala Thr Met Lys Ile Met Asn Tyr Asp Glu Phe Gln His Cys
145 150 155 160
Trp Ser Lys Phe Val Tyr Ser Gln Arg Glu Leu Phe Glu Pro Trp Asn
165 170 175
Asn Leu Pro Lys Tyr Tyr Ile Leu Leu His Ile Met Leu Gly Glu Ile
180 185 190
Leu Arg His Ser Met Asp Pro Pro Thr Phe Thr Ser Asn Phe Asn Asn
195 200 205
Glu Leu Trp Val Arg Gly Arg His Glu Thr Tyr Leu Cys Tyr Glu Val
210 215 220
Glu Arg Leu His Asn Asp Thr Trp Val Leu Leu Asn Gln Arg Arg Gly
225 230 235 240
Phe Leu Cys Asn Gln Ala Pro His Lys His Gly Phe Leu Glu Gly Arg
245 250 255
His Ala Glu Leu Cys Phe Leu Asp Val Ile Pro Phe Trp Lys Leu Asp
260 265 270
Leu His Gln Asp Tyr Arg Val Thr Cys Phe Thr Ser Trp Ser Pro Cys
275 280 285
Phe Ser Cys Ala Gln Glu Met Ala Lys Phe Ile Ser Asn Asn Lys His
290 295 300
Val Ser Leu Cys Ile Phe Ala Ala Arg Ile Tyr Asp Asp Gln Gly Arg
305 310 315 320
Cys Gln Glu Gly Leu Arg Thr Leu Ala Lys Ala Gly Ala Lys Ile Ser
325 330 335
Ile Met Thr Tyr Ser Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp
340 345 350
His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Glu Glu His Ser
355 360 365
Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
370 375 380
<210> 14
<211> 377
<212> PRT
<213> Chlorocebus aethiops
<400> 14
Met Asn Pro Gln Ile Arg Asn Met Val Glu Gln Met Glu Pro Asp Ile
1 5 10 15
Phe Val Tyr Tyr Phe Asn Asn Arg Pro Ile Leu Ser Gly Arg Asn Thr
20 25 30
Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Asp Pro Ser Gly Pro Pro
35 40 45
Leu Asp Ala Asn Ile Phe Gln Gly Lys Leu Tyr Pro Glu Ala Lys Asp
50 55 60
His Pro Glu Met Lys Phe Leu His Trp Phe Arg Lys Trp Arg Gln Leu
65 70 75 80
His Arg Asp Gln Glu Tyr Glu Val Thr Trp Tyr Val Ser Trp Ser Pro
85 90 95
Cys Thr Arg Cys Ala Asn Ser Val Ala Thr Phe Leu Ala Glu Asp Pro
100 105 110
Lys Val Thr Leu Thr Ile Phe Val Ala Arg Leu Tyr Tyr Phe Trp Lys
115 120 125
Pro Asp Tyr Gln Gln Ala Leu Arg Ile Leu Cys Gln Glu Arg Gly Gly
130 135 140
Pro His Ala Thr Met Lys Ile Met Asn Tyr Asn Glu Phe Gln His Cys
145 150 155 160
Trp Asn Glu Phe Val Asp Gly Gln Gly Lys Pro Phe Lys Pro Arg Lys
165 170 175
Asn Leu Pro Lys His Tyr Thr Leu Leu His Ala Thr Leu Gly Glu Leu
180 185 190
Leu Arg His Val Met Asp Pro Gly Thr Phe Thr Ser Asn Phe Asn Asn
195 200 205
Lys Pro Trp Val Ser Gly Gln Arg Glu Thr Tyr Leu Cys Tyr Lys Val
210 215 220
Glu Arg Ser His Asn Asp Thr Trp Val Leu Leu Asn Gln His Arg Gly
225 230 235 240
Phe Leu Arg Asn Gln Ala Pro Asp Arg His Gly Phe Pro Lys Gly Arg
245 250 255
His Ala Glu Leu Cys Phe Leu Asp Leu Ile Pro Phe Trp Lys Leu Asp
260 265 270
Asp Gln Gln Tyr Arg Val Thr Cys Phe Thr Ser Trp Ser Pro Cys Phe
275 280 285
Ser Cys Ala Gln Lys Met Ala Lys Phe Ile Ser Asn Asn Lys His Val
290 295 300
Ser Leu Cys Ile Phe Ala Ala Arg Ile Tyr Asp Asp Gln Gly Arg Cys
305 310 315 320
Gln Glu Gly Leu Arg Thr Leu His Arg Asp Gly Ala Lys Ile Ala Val
325 330 335
Met Asn Tyr Ser Glu Phe Glu Tyr Cys Trp Asp Thr Phe Val Asp Arg
340 345 350
Gln Gly Arg Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln
355 360 365
Ala Leu Ser Gly Arg Leu Arg Ala Ile
370 375
<210> 15
<211> 384
<212> PRT
<213> 人类
<400> 15
Met Lys Pro His Phe Arg Asn Thr Val Glu Arg Met Tyr Arg Asp Thr
1 5 10 15
Phe Ser Tyr Asn Phe Tyr Asn Arg Pro Ile Leu Ser Arg Arg Asn Thr
20 25 30
Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Gly Pro Ser Arg Pro Pro
35 40 45
Leu Asp Ala Lys Ile Phe Arg Gly Gln Val Tyr Ser Glu Leu Lys Tyr
50 55 60
His Pro Glu Met Arg Phe Phe His Trp Phe Ser Lys Trp Arg Lys Leu
65 70 75 80
His Arg Asp Gln Glu Tyr Glu Val Thr Trp Tyr Ile Ser Trp Ser Pro
85 90 95
Cys Thr Lys Cys Thr Arg Asp Met Ala Thr Phe Leu Ala Glu Asp Pro
100 105 110
Lys Val Thr Leu Thr Ile Phe Val Ala Arg Leu Tyr Tyr Phe Trp Asp
115 120 125
Pro Asp Tyr Gln Glu Ala Leu Arg Ser Leu Cys Gln Lys Arg Asp Gly
130 135 140
Pro Arg Ala Thr Met Lys Ile Met Asn Tyr Asp Glu Phe Gln His Cys
145 150 155 160
Trp Ser Lys Phe Val Tyr Ser Gln Arg Glu Leu Phe Glu Pro Trp Asn
165 170 175
Asn Leu Pro Lys Tyr Tyr Ile Leu Leu His Ile Met Leu Gly Glu Ile
180 185 190
Leu Arg His Ser Met Asp Pro Pro Thr Phe Thr Phe Asn Phe Asn Asn
195 200 205
Glu Pro Trp Val Arg Gly Arg His Glu Thr Tyr Leu Cys Tyr Glu Val
210 215 220
Glu Arg Met His Asn Asp Thr Trp Val Leu Leu Asn Gln Arg Arg Gly
225 230 235 240
Phe Leu Cys Asn Gln Ala Pro His Lys His Gly Phe Leu Glu Gly Arg
245 250 255
His Ala Glu Leu Cys Phe Leu Asp Val Ile Pro Phe Trp Lys Leu Asp
260 265 270
Leu Asp Gln Asp Tyr Arg Val Thr Cys Phe Thr Ser Trp Ser Pro Cys
275 280 285
Phe Ser Cys Ala Gln Glu Met Ala Lys Phe Ile Ser Lys Asn Lys His
290 295 300
Val Ser Leu Cys Ile Phe Thr Ala Arg Ile Tyr Asp Asp Gln Gly Arg
305 310 315 320
Cys Gln Glu Gly Leu Arg Thr Leu Ala Glu Ala Gly Ala Lys Ile Ser
325 330 335
Ile Met Thr Tyr Ser Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp
340 345 350
His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser
355 360 365
Gln Asp Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Glu Asn
370 375 380
<210> 16
<211> 373
<212> PRT
<213> 人类
<400> 16
Met Lys Pro His Phe Arg Asn Thr Val Glu Arg Met Tyr Arg Asp Thr
1 5 10 15
Phe Ser Tyr Asn Phe Tyr Asn Arg Pro Ile Leu Ser Arg Arg Asn Thr
20 25 30
Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Gly Pro Ser Arg Pro Arg
35 40 45
Leu Asp Ala Lys Ile Phe Arg Gly Gln Val Tyr Ser Gln Pro Glu His
50 55 60
His Ala Glu Met Cys Phe Leu Ser Trp Phe Cys Gly Asn Gln Leu Pro
65 70 75 80
Ala Tyr Lys Cys Phe Gln Ile Thr Trp Phe Val Ser Trp Thr Pro Cys
85 90 95
Pro Asp Cys Val Ala Lys Leu Ala Glu Phe Leu Ala Glu His Pro Asn
100 105 110
Val Thr Leu Thr Ile Ser Ala Ala Arg Leu Tyr Tyr Tyr Trp Glu Arg
115 120 125
Asp Tyr Arg Arg Ala Leu Cys Arg Leu Ser Gln Ala Gly Ala Arg Val
130 135 140
Lys Ile Met Asp Asp Glu Glu Phe Ala Tyr Cys Trp Glu Asn Phe Val
145 150 155 160
Tyr Ser Glu Gly Gln Pro Phe Met Pro Trp Tyr Lys Phe Asp Asp Asn
165 170 175
Tyr Ala Phe Leu His Arg Thr Leu Lys Glu Ile Leu Arg Asn Pro Met
180 185 190
Glu Ala Met Tyr Pro His Ile Phe Tyr Phe His Phe Lys Asn Leu Arg
195 200 205
Lys Ala Tyr Gly Arg Asn Glu Ser Trp Leu Cys Phe Thr Met Glu Val
210 215 220
Val Lys His His Ser Pro Val Ser Trp Lys Arg Gly Val Phe Arg Asn
225 230 235 240
Gln Val Asp Pro Glu Thr His Cys His Ala Glu Arg Cys Phe Leu Ser
245 250 255
Trp Phe Cys Asp Asp Ile Leu Ser Pro Asn Thr Asn Tyr Glu Val Thr
260 265 270
Trp Tyr Thr Ser Trp Ser Pro Cys Pro Glu Cys Ala Gly Glu Val Ala
275 280 285
Glu Phe Leu Ala Arg His Ser Asn Val Asn Leu Thr Ile Phe Thr Ala
290 295 300
Arg Leu Tyr Tyr Phe Trp Asp Thr Asp Tyr Gln Glu Gly Leu Arg Ser
305 310 315 320
Leu Ser Gln Glu Gly Ala Ser Val Glu Ile Met Gly Tyr Lys Asp Phe
325 330 335
Lys Tyr Cys Trp Glu Asn Phe Val Tyr Asn Asp Asp Glu Pro Phe Lys
340 345 350
Pro Trp Lys Gly Leu Lys Tyr Asn Phe Leu Phe Leu Asp Ser Lys Leu
355 360 365
Gln Glu Ile Leu Glu
370
<210> 17
<211> 382
<212> PRT
<213> 人类
<400> 17
Met Asn Pro Gln Ile Arg Asn Pro Met Glu Arg Met Tyr Arg Asp Thr
1 5 10 15
Phe Tyr Asp Asn Phe Glu Asn Glu Pro Ile Leu Tyr Gly Arg Ser Tyr
20 25 30
Thr Trp Leu Cys Tyr Glu Val Lys Ile Lys Arg Gly Arg Ser Asn Leu
35 40 45
Leu Trp Asp Thr Gly Val Phe Arg Gly Gln Val Tyr Phe Lys Pro Gln
50 55 60
Tyr His Ala Glu Met Cys Phe Leu Ser Trp Phe Cys Gly Asn Gln Leu
65 70 75 80
Pro Ala Tyr Lys Cys Phe Gln Ile Thr Trp Phe Val Ser Trp Thr Pro
85 90 95
Cys Pro Asp Cys Val Ala Lys Leu Ala Glu Phe Leu Ser Glu His Pro
100 105 110
Asn Val Thr Leu Thr Ile Ser Ala Ala Arg Leu Tyr Tyr Tyr Trp Glu
115 120 125
Arg Asp Tyr Arg Arg Ala Leu Cys Arg Leu Ser Gln Ala Gly Ala Arg
130 135 140
Val Thr Ile Met Asp Tyr Glu Glu Phe Ala Tyr Cys Trp Glu Asn Phe
145 150 155 160
Val Tyr Asn Glu Gly Gln Gln Phe Met Pro Trp Tyr Lys Phe Asp Glu
165 170 175
Asn Tyr Ala Phe Leu His Arg Thr Leu Lys Glu Ile Leu Arg Tyr Leu
180 185 190
Met Asp Pro Asp Thr Phe Thr Phe Asn Phe Asn Asn Asp Pro Leu Val
195 200 205
Leu Arg Arg Arg Gln Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp
210 215 220
Asn Gly Thr Trp Val Leu Met Asp Gln His Met Gly Phe Leu Cys Asn
225 230 235 240
Glu Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu
245 250 255
Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile
260 265 270
Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly
275 280 285
Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg
290 295 300
Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys
305 310 315 320
Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met
325 330 335
Thr Tyr Asp Glu Phe Glu Tyr Cys Trp Asp Thr Phe Val Tyr Arg Gln
340 345 350
Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Glu Glu His Ser Gln Ala
355 360 365
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
370 375 380
<210> 18
<211> 190
<212> PRT
<213> 人类
<400> 18
Met Asn Pro Gln Ile Arg Asn Pro Met Lys Ala Met Tyr Pro Gly Thr
1 5 10 15
Phe Tyr Phe Gln Phe Lys Asn Leu Trp Glu Ala Asn Asp Arg Asn Glu
20 25 30
Thr Trp Leu Cys Phe Thr Val Glu Gly Ile Lys Arg Arg Ser Val Val
35 40 45
Ser Trp Lys Thr Gly Val Phe Arg Asn Gln Val Asp Ser Glu Thr His
50 55 60
Cys His Ala Glu Arg Cys Phe Leu Ser Trp Phe Cys Asp Asp Ile Leu
65 70 75 80
Ser Pro Asn Thr Lys Tyr Gln Val Thr Trp Tyr Thr Ser Trp Ser Pro
85 90 95
Cys Pro Asp Cys Ala Gly Glu Val Ala Glu Phe Leu Ala Arg His Ser
100 105 110
Asn Val Asn Leu Thr Ile Phe Thr Ala Arg Leu Tyr Tyr Phe Gln Tyr
115 120 125
Pro Cys Tyr Gln Glu Gly Leu Arg Ser Leu Ser Gln Glu Gly Val Ala
130 135 140
Val Glu Ile Met Asp Tyr Glu Asp Phe Lys Tyr Cys Trp Glu Asn Phe
145 150 155 160
Val Tyr Asn Asp Asn Glu Pro Phe Lys Pro Trp Lys Gly Leu Lys Thr
165 170 175
Asn Phe Arg Leu Leu Lys Arg Arg Leu Arg Glu Ser Leu Gln
180 185 190
<210> 19
<211> 199
<212> PRT
<213> 人类
<400> 19
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 20
<211> 200
<212> PRT
<213> 人类
<400> 20
Met Ala Leu Leu Thr Ala Glu Thr Phe Arg Leu Gln Phe Asn Asn Lys
1 5 10 15
Arg Arg Leu Arg Arg Pro Tyr Tyr Pro Arg Lys Ala Leu Leu Cys Tyr
20 25 30
Gln Leu Thr Pro Gln Asn Gly Ser Thr Pro Thr Arg Gly Tyr Phe Glu
35 40 45
Asn Lys Lys Lys Cys His Ala Glu Ile Cys Phe Ile Asn Glu Ile Lys
50 55 60
Ser Met Gly Leu Asp Glu Thr Gln Cys Tyr Gln Val Thr Cys Tyr Leu
65 70 75 80
Thr Trp Ser Pro Cys Ser Ser Cys Ala Trp Glu Leu Val Asp Phe Ile
85 90 95
Lys Ala His Asp His Leu Asn Leu Gly Ile Phe Ala Ser Arg Leu Tyr
100 105 110
Tyr His Trp Cys Lys Pro Gln Gln Lys Gly Leu Arg Leu Leu Cys Gly
115 120 125
Ser Gln Val Pro Val Glu Val Met Gly Phe Pro Lys Phe Ala Asp Cys
130 135 140
Trp Glu Asn Phe Val Asp His Glu Lys Pro Leu Ser Phe Asn Pro Tyr
145 150 155 160
Lys Met Leu Glu Glu Leu Asp Lys Asn Ser Arg Ala Ile Lys Arg Arg
165 170 175
Leu Glu Arg Ile Lys Ile Pro Gly Val Arg Ala Gln Gly Arg Tyr Met
180 185 190
Asp Ile Leu Cys Asp Ala Glu Val
195 200
<210> 21
<211> 386
<212> PRT
<213> 人类
<400> 21
Met Asn Pro Gln Ile Arg Asn Pro Met Glu Arg Met Tyr Arg Asp Thr
1 5 10 15
Phe Tyr Asp Asn Phe Glu Asn Glu Pro Ile Leu Tyr Gly Arg Ser Tyr
20 25 30
Thr Trp Leu Cys Tyr Glu Val Lys Ile Lys Arg Gly Arg Ser Asn Leu
35 40 45
Leu Trp Asp Thr Gly Val Phe Arg Gly Pro Val Leu Pro Lys Arg Gln
50 55 60
Ser Asn His Arg Gln Glu Val Tyr Phe Arg Phe Glu Asn His Ala Glu
65 70 75 80
Met Cys Phe Leu Ser Trp Phe Cys Gly Asn Arg Leu Pro Ala Asn Arg
85 90 95
Arg Phe Gln Ile Thr Trp Phe Val Ser Trp Asn Pro Cys Leu Pro Cys
100 105 110
Val Val Lys Val Thr Lys Phe Leu Ala Glu His Pro Asn Val Thr Leu
115 120 125
Thr Ile Ser Ala Ala Arg Leu Tyr Tyr Tyr Arg Asp Arg Asp Trp Arg
130 135 140
Trp Val Leu Leu Arg Leu His Lys Ala Gly Ala Arg Val Lys Ile Met
145 150 155 160
Asp Tyr Glu Asp Phe Ala Tyr Cys Trp Glu Asn Phe Val Cys Asn Glu
165 170 175
Gly Gln Pro Phe Met Pro Trp Tyr Lys Phe Asp Asp Asn Tyr Ala Ser
180 185 190
Leu His Arg Thr Leu Lys Glu Ile Leu Arg Asn Pro Met Glu Ala Met
195 200 205
Tyr Pro His Ile Phe Tyr Phe His Phe Lys Asn Leu Leu Lys Ala Cys
210 215 220
Gly Arg Asn Glu Ser Trp Leu Cys Phe Thr Met Glu Val Thr Lys His
225 230 235 240
His Ser Ala Val Phe Arg Lys Arg Gly Val Phe Arg Asn Gln Val Asp
245 250 255
Pro Glu Thr His Cys His Ala Glu Arg Cys Phe Leu Ser Trp Phe Cys
260 265 270
Asp Asp Ile Leu Ser Pro Asn Thr Asn Tyr Glu Val Thr Trp Tyr Thr
275 280 285
Ser Trp Ser Pro Cys Pro Glu Cys Ala Gly Glu Val Ala Glu Phe Leu
290 295 300
Ala Arg His Ser Asn Val Asn Leu Thr Ile Phe Thr Ala Arg Leu Cys
305 310 315 320
Tyr Phe Trp Asp Thr Asp Tyr Gln Glu Gly Leu Cys Ser Leu Ser Gln
325 330 335
Glu Gly Ala Ser Val Lys Ile Met Gly Tyr Lys Asp Phe Val Ser Cys
340 345 350
Trp Lys Asn Phe Val Tyr Ser Asp Asp Glu Pro Phe Lys Pro Trp Lys
355 360 365
Gly Leu Gln Thr Asn Phe Arg Leu Leu Lys Arg Arg Leu Arg Glu Ile
370 375 380
Leu Gln
385
<210> 22
<211> 236
<212> PRT
<213> 人类
<400> 22
Met Thr Ser Glu Lys Gly Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro Trp Glu Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Ala Cys Leu Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg
35 40 45
Lys Ile Trp Arg Ser Ser Gly Lys Asn Thr Thr Asn His Val Glu Val
50 55 60
Asn Phe Ile Lys Lys Phe Thr Ser Glu Arg Asp Phe His Pro Ser Met
65 70 75 80
Ser Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys
85 90 95
Ser Gln Ala Ile Arg Glu Phe Leu Ser Arg His Pro Gly Val Thr Leu
100 105 110
Val Ile Tyr Val Ala Arg Leu Phe Trp His Met Asp Gln Gln Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Arg Ala Ser Glu Tyr Tyr His Cys Trp Arg Asn Phe Val Asn Tyr Pro
145 150 155 160
Pro Gly Asp Glu Ala His Trp Pro Gln Tyr Pro Pro Leu Trp Met Met
165 170 175
Leu Tyr Ala Leu Glu Leu His Cys Ile Ile Leu Ser Leu Pro Pro Cys
180 185 190
Leu Lys Ile Ser Arg Arg Trp Gln Asn His Leu Thr Phe Phe Arg Leu
195 200 205
His Leu Gln Asn Cys His Tyr Gln Thr Ile Pro Pro His Ile Leu Leu
210 215 220
Ala Thr Gly Leu Ile His Pro Ser Val Ala Trp Arg
225 230 235
<210> 23
<211> 229
<212> PRT
<213> 小鼠
<400> 23
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Val Trp Arg His Thr Ser Gln Asn Thr Ser Asn His Val Glu Val
50 55 60
Asn Phe Leu Glu Lys Phe Thr Thr Glu Arg Tyr Phe Arg Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg His Pro Tyr Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Thr Asp Gln Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Tyr Cys Tyr Cys Trp Arg Asn Phe Val Asn Tyr Pro
145 150 155 160
Pro Ser Asn Glu Ala Tyr Trp Pro Arg Tyr Pro His Leu Trp Val Lys
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Lys Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Thr Leu Gln Thr Cys His Tyr Gln Arg Ile Pro Pro His Leu Leu Trp
210 215 220
Ala Thr Gly Leu Lys
225
<210> 24
<211> 229
<212> PRT
<213> 大鼠
<400> 24
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys
225
<210> 25
<211> 191
<212> PRT
<213> 人类
<400> 25
Met Glu Ala Lys Ala Ala Pro Lys Pro Ala Ala Ser Gly Ala Cys Ser
1 5 10 15
Val Ser Ala Glu Glu Thr Glu Lys Trp Met Glu Glu Ala Met His Met
20 25 30
Ala Lys Glu Ala Leu Glu Asn Thr Glu Val Pro Val Gly Cys Leu Met
35 40 45
Val Tyr Asn Asn Glu Val Val Gly Lys Gly Arg Asn Glu Val Asn Gln
50 55 60
Thr Lys Asn Ala Thr Arg His Ala Glu Met Val Ala Ile Asp Gln Val
65 70 75 80
Leu Asp Trp Cys Arg Gln Ser Gly Lys Ser Pro Ser Glu Val Phe Glu
85 90 95
His Thr Val Leu Tyr Val Thr Val Glu Pro Cys Ile Met Cys Ala Ala
100 105 110
Ala Leu Arg Leu Met Lys Ile Pro Leu Val Val Tyr Gly Cys Gln Asn
115 120 125
Glu Arg Phe Gly Gly Cys Gly Ser Val Leu Asn Ile Ala Ser Ala Asp
130 135 140
Leu Pro Asn Thr Gly Arg Pro Phe Gln Cys Ile Pro Gly Tyr Arg Ala
145 150 155 160
Glu Glu Ala Val Glu Met Leu Lys Thr Phe Tyr Lys Gln Glu Asn Pro
165 170 175
Asn Ala Pro Lys Ser Lys Val Arg Lys Lys Glu Cys Gln Lys Ser
180 185 190
<210> 26
<211> 191
<212> PRT
<213> 小鼠
<400> 26
Met Glu Glu Lys Val Glu Ser Thr Thr Thr Pro Asp Gly Pro Cys Val
1 5 10 15
Val Ser Val Gln Glu Thr Glu Lys Trp Met Glu Glu Ala Met Arg Met
20 25 30
Ala Lys Glu Ala Leu Glu Asn Ile Glu Val Pro Val Gly Cys Leu Met
35 40 45
Val Tyr Asn Asn Glu Val Val Gly Lys Gly Arg Asn Glu Val Asn Gln
50 55 60
Thr Lys Asn Ala Thr Arg His Ala Glu Met Val Ala Ile Asp Gln Val
65 70 75 80
Leu Asp Trp Cys His Gln His Gly Gln Ser Pro Ser Thr Val Phe Glu
85 90 95
His Thr Val Leu Tyr Val Thr Val Glu Pro Cys Ile Met Cys Ala Ala
100 105 110
Ala Leu Arg Leu Met Lys Ile Pro Leu Val Val Tyr Gly Cys Gln Asn
115 120 125
Glu Arg Phe Gly Gly Cys Gly Ser Val Leu Asn Ile Ala Ser Ala Asp
130 135 140
Leu Pro Asn Thr Gly Arg Pro Phe Gln Cys Ile Pro Gly Tyr Arg Ala
145 150 155 160
Glu Glu Ala Val Glu Leu Leu Lys Thr Phe Tyr Lys Gln Glu Asn Pro
165 170 175
Asn Ala Pro Lys Ser Lys Val Arg Lys Lys Asp Cys Gln Lys Ser
180 185 190
<210> 27
<211> 499
<212> PRT
<213> 小鼠
<400> 27
Met Trp Thr Ala Asp Glu Ile Ala Gln Leu Cys Tyr Ala His Tyr Asn
1 5 10 15
Val Arg Leu Pro Lys Gln Gly Lys Pro Glu Pro Asn Arg Glu Trp Thr
20 25 30
Leu Leu Ala Ala Val Val Lys Ile Gln Ala Ser Ala Asn Gln Ala Cys
35 40 45
Asp Ile Pro Glu Lys Glu Val Gln Val Thr Lys Glu Val Val Ser Met
50 55 60
Gly Thr Gly Thr Lys Cys Ile Gly Gln Ser Lys Met Arg Glu Ser Gly
65 70 75 80
Asp Ile Leu Asn Asp Ser His Ala Glu Ile Ile Ala Arg Arg Ser Phe
85 90 95
Gln Arg Tyr Leu Leu His Gln Leu His Leu Ala Ala Val Leu Lys Glu
100 105 110
Asp Ser Ile Phe Val Pro Gly Thr Gln Arg Gly Leu Trp Arg Leu Arg
115 120 125
Pro Asp Leu Ser Phe Val Phe Phe Ser Ser His Thr Pro Cys Gly Asp
130 135 140
Ala Ser Ile Ile Pro Met Leu Glu Phe Glu Glu Gln Pro Cys Cys Pro
145 150 155 160
Val Ile Arg Ser Trp Ala Asn Asn Ser Pro Val Gln Glu Thr Glu Asn
165 170 175
Leu Glu Asp Ser Lys Asp Lys Arg Asn Cys Glu Asp Pro Ala Ser Pro
180 185 190
Val Ala Lys Lys Met Arg Leu Gly Thr Pro Ala Arg Ser Leu Ser Asn
195 200 205
Cys Val Ala His His Gly Thr Gln Glu Ser Gly Pro Val Lys Pro Asp
210 215 220
Val Ser Ser Ser Asp Leu Thr Lys Glu Glu Pro Asp Ala Ala Asn Gly
225 230 235 240
Ile Ala Ser Gly Ser Phe Arg Val Val Asp Val Tyr Arg Thr Gly Ala
245 250 255
Lys Cys Val Pro Gly Glu Thr Gly Asp Leu Arg Glu Pro Gly Ala Ala
260 265 270
Tyr His Gln Val Gly Leu Leu Arg Val Lys Pro Gly Arg Gly Asp Arg
275 280 285
Thr Cys Ser Met Ser Cys Ser Asp Lys Met Ala Arg Trp Asn Val Leu
290 295 300
Gly Cys Gln Gly Ala Leu Leu Met His Phe Leu Glu Lys Pro Ile Tyr
305 310 315 320
Leu Ser Ala Val Val Ile Gly Lys Cys Pro Tyr Ser Gln Glu Ala Met
325 330 335
Arg Arg Ala Leu Thr Gly Arg Cys Glu Glu Thr Leu Val Leu Pro Arg
340 345 350
Gly Phe Gly Val Gln Glu Leu Glu Ile Gln Gln Ser Gly Leu Leu Phe
355 360 365
Glu Gln Ser Arg Cys Ala Val His Arg Lys Arg Gly Asp Ser Pro Gly
370 375 380
Arg Leu Val Pro Cys Gly Ala Ala Ile Ser Trp Ser Ala Val Pro Gln
385 390 395 400
Gln Pro Leu Asp Val Thr Ala Asn Gly Phe Pro Gln Gly Thr Thr Lys
405 410 415
Lys Glu Ile Gly Ser Pro Arg Ala Arg Ser Arg Ile Ser Lys Val Glu
420 425 430
Leu Phe Arg Ser Phe Gln Lys Leu Leu Ser Ser Ile Ala Asp Asp Glu
435 440 445
Gln Pro Asp Ser Ile Arg Val Thr Lys Lys Leu Asp Thr Tyr Gln Glu
450 455 460
Tyr Lys Asp Ala Ala Ser Ala Tyr Gln Glu Ala Trp Gly Ala Leu Arg
465 470 475 480
Arg Ile Gln Pro Phe Ala Ser Trp Ile Arg Asn Pro Pro Asp Tyr His
485 490 495
Gln Phe Lys
<210> 28
<211> 502
<212> PRT
<213> 人类
<400> 28
Met Trp Thr Ala Asp Glu Ile Ala Gln Leu Cys Tyr Glu His Tyr Gly
1 5 10 15
Ile Arg Leu Pro Lys Lys Gly Lys Pro Glu Pro Asn His Glu Trp Thr
20 25 30
Leu Leu Ala Ala Val Val Lys Ile Gln Ser Pro Ala Asp Lys Ala Cys
35 40 45
Asp Thr Pro Asp Lys Pro Val Gln Val Thr Lys Glu Val Val Ser Met
50 55 60
Gly Thr Gly Thr Lys Cys Ile Gly Gln Ser Lys Met Arg Lys Asn Gly
65 70 75 80
Asp Ile Leu Asn Asp Ser His Ala Glu Val Ile Ala Arg Arg Ser Phe
85 90 95
Gln Arg Tyr Leu Leu His Gln Leu Gln Leu Ala Ala Thr Leu Lys Glu
100 105 110
Asp Ser Ile Phe Val Pro Gly Thr Gln Lys Gly Val Trp Lys Leu Arg
115 120 125
Arg Asp Leu Ile Phe Val Phe Phe Ser Ser His Thr Pro Cys Gly Asp
130 135 140
Ala Ser Ile Ile Pro Met Leu Glu Phe Glu Asp Gln Pro Cys Cys Pro
145 150 155 160
Val Phe Arg Asn Trp Ala His Asn Ser Ser Val Glu Ala Ser Ser Asn
165 170 175
Leu Glu Ala Pro Gly Asn Glu Arg Lys Cys Glu Asp Pro Asp Ser Pro
180 185 190
Val Thr Lys Lys Met Arg Leu Glu Pro Gly Thr Ala Ala Arg Glu Val
195 200 205
Thr Asn Gly Ala Ala His His Gln Ser Phe Gly Lys Gln Lys Ser Gly
210 215 220
Pro Ile Ser Pro Gly Ile His Ser Cys Asp Leu Thr Val Glu Gly Leu
225 230 235 240
Ala Thr Val Thr Arg Ile Ala Pro Gly Ser Ala Lys Val Ile Asp Val
245 250 255
Tyr Arg Thr Gly Ala Lys Cys Val Pro Gly Glu Ala Gly Asp Ser Gly
260 265 270
Lys Pro Gly Ala Ala Phe His Gln Val Gly Leu Leu Arg Val Lys Pro
275 280 285
Gly Arg Gly Asp Arg Thr Arg Ser Met Ser Cys Ser Asp Lys Met Ala
290 295 300
Arg Trp Asn Val Leu Gly Cys Gln Gly Ala Leu Leu Met His Leu Leu
305 310 315 320
Glu Glu Pro Ile Tyr Leu Ser Ala Val Val Ile Gly Lys Cys Pro Tyr
325 330 335
Ser Gln Glu Ala Met Gln Arg Ala Leu Ile Gly Arg Cys Gln Asn Val
340 345 350
Ser Ala Leu Pro Lys Gly Phe Gly Val Gln Glu Leu Lys Ile Leu Gln
355 360 365
Ser Asp Leu Leu Phe Glu Gln Ser Arg Ser Ala Val Gln Ala Lys Arg
370 375 380
Ala Asp Ser Pro Gly Arg Leu Val Pro Cys Gly Ala Ala Ile Ser Trp
385 390 395 400
Ser Ala Val Pro Glu Gln Pro Leu Asp Val Thr Ala Asn Gly Phe Pro
405 410 415
Gln Gly Thr Thr Lys Lys Thr Ile Gly Ser Leu Gln Ala Arg Ser Gln
420 425 430
Ile Ser Lys Val Glu Leu Phe Arg Ser Phe Gln Lys Leu Leu Ser Arg
435 440 445
Ile Ala Arg Asp Lys Trp Pro His Ser Leu Arg Val Gln Lys Leu Asp
450 455 460
Thr Tyr Gln Glu Tyr Lys Glu Ala Ala Ser Ser Tyr Gln Glu Ala Trp
465 470 475 480
Ser Thr Leu Arg Lys Gln Val Phe Gly Ser Trp Ile Arg Asn Pro Pro
485 490 495
Asp Tyr His Gln Phe Lys
500
<210> 29
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 29
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser
1 5 10
<210> 30
<211> 1580
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 30
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Asp Lys
20 25 30
Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala
35 40 45
Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu
50 55 60
Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu
65 70 75 80
Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr
85 90 95
Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln
100 105 110
Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His
115 120 125
Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg
130 135 140
His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys
145 150 155 160
Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp
165 170 175
Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys
180 185 190
Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser
195 200 205
Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
210 215 220
Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile
225 230 235 240
Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
245 250 255
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala
260 265 270
Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
275 280 285
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu
290 295 300
Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
305 310 315 320
Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg
325 330 335
Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys
340 345 350
Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val
355 360 365
Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser
370 375 380
Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
385 390 395 400
Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu
405 410 415
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg
420 425 430
Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu
435 440 445
His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp
450 455 460
Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
465 470 475 480
Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg
485 490 495
Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp
500 505 510
Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
515 520 525
Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr
530 535 540
Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
545 550 555 560
Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala
565 570 575
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln
580 585 590
Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu
595 600 605
Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His
610 615 620
Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu
625 630 635 640
Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
645 650 655
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe
660 665 670
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp
675 680 685
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
690 695 700
Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
705 710 715 720
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp
725 730 735
Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His
740 745 750
Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
755 760 765
Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
770 775 780
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln
785 790 795 800
Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
805 810 815
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn
820 825 830
Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly
835 840 845
Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
850 855 860
Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
865 870 875 880
Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
885 890 895
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp
900 905 910
Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
915 920 925
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly
930 935 940
Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
945 950 955 960
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
965 970 975
Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val
980 985 990
Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn
995 1000 1005
Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly
1010 1015 1020
Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
1025 1030 1035
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1040 1045 1050
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1055 1060 1065
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1070 1075 1080
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1085 1090 1095
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1100 1105 1110
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1115 1120 1125
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1130 1135 1140
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1145 1150 1155
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1160 1165 1170
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1175 1180 1185
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1190 1195 1200
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1205 1210 1215
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1220 1225 1230
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1235 1240 1245
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1250 1255 1260
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1265 1270 1275
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1280 1285 1290
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1295 1300 1305
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1310 1315 1320
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1325 1330 1335
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1340 1345 1350
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1355 1360 1365
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1370 1375 1380
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
1385 1390 1395
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr
1400 1405 1410
Val Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe
1415 1420 1425
Gly Tyr Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe
1430 1435 1440
Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr
1445 1450 1455
Arg Val Thr Trp Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala
1460 1465 1470
Arg His Val Ala Asp Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu
1475 1480 1485
Arg Ile Phe Thr Ala Arg Leu Tyr Phe Cys Glu Asp Arg Lys Ala
1490 1495 1500
Glu Pro Glu Gly Leu Arg Arg Leu His Arg Ala Gly Val Gln Ile
1505 1510 1515
Ala Ile Met Thr Phe Lys Asp Tyr Phe Tyr Cys Trp Asn Thr Phe
1520 1525 1530
Val Glu Asn His Glu Arg Thr Phe Lys Ala Trp Glu Gly Leu His
1535 1540 1545
Glu Asn Ser Val Arg Leu Ser Arg Gln Leu Arg Arg Ile Leu Leu
1550 1555 1560
Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala Phe Arg Thr Leu
1565 1570 1575
Gly Leu
1580
<210> 31
<211> 1564
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 31
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
180 185 190
Gly Gly Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
195 200 205
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
210 215 220
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
225 230 235 240
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
245 250 255
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
260 265 270
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
275 280 285
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
290 295 300
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
305 310 315 320
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
325 330 335
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
340 345 350
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
355 360 365
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
370 375 380
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
385 390 395 400
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
405 410 415
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
420 425 430
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
435 440 445
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
450 455 460
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
465 470 475 480
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
485 490 495
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
500 505 510
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
515 520 525
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
530 535 540
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
545 550 555 560
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
565 570 575
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
580 585 590
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
595 600 605
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
610 615 620
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
625 630 635 640
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
645 650 655
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
660 665 670
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
675 680 685
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
690 695 700
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
705 710 715 720
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
725 730 735
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
740 745 750
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
755 760 765
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
770 775 780
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
785 790 795 800
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
805 810 815
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
820 825 830
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
835 840 845
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn
850 855 860
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
865 870 875 880
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp
885 890 895
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
900 905 910
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
915 920 925
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
930 935 940
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
945 950 955 960
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg
965 970 975
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu
980 985 990
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr
995 1000 1005
Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu
1010 1015 1020
Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile Val
1025 1030 1035
Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu
1040 1045 1050
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
1055 1060 1065
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu
1070 1075 1080
Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys
1085 1090 1095
Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile
1100 1105 1110
Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala
1115 1120 1125
Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
1130 1135 1140
Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu
1145 1150 1155
Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
1160 1165 1170
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
1175 1180 1185
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
1190 1195 1200
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
1205 1210 1215
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1220 1225 1230
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu
1235 1240 1245
Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
1250 1255 1260
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr
1265 1270 1275
Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1280 1285 1290
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
1295 1300 1305
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp
1310 1315 1320
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser
1325 1330 1335
Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
1340 1345 1350
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser
1355 1360 1365
Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1370 1375 1380
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1385 1390 1395
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1400 1405 1410
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1415 1420 1425
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1430 1435 1440
Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His
1445 1450 1455
Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
1460 1465 1470
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser
1475 1480 1485
Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu
1490 1495 1500
Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala
1505 1510 1515
Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr
1520 1525 1530
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1535 1540 1545
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly
1550 1555 1560
Asp
<210> 32
<211> 1580
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 32
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Asp Lys
20 25 30
Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala
35 40 45
Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu
50 55 60
Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu
65 70 75 80
Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr
85 90 95
Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln
100 105 110
Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His
115 120 125
Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg
130 135 140
His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys
145 150 155 160
Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp
165 170 175
Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys
180 185 190
Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser
195 200 205
Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
210 215 220
Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile
225 230 235 240
Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
245 250 255
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala
260 265 270
Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
275 280 285
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu
290 295 300
Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
305 310 315 320
Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg
325 330 335
Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys
340 345 350
Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val
355 360 365
Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser
370 375 380
Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
385 390 395 400
Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu
405 410 415
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg
420 425 430
Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu
435 440 445
His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp
450 455 460
Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
465 470 475 480
Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg
485 490 495
Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp
500 505 510
Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
515 520 525
Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr
530 535 540
Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
545 550 555 560
Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala
565 570 575
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln
580 585 590
Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu
595 600 605
Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His
610 615 620
Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu
625 630 635 640
Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
645 650 655
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe
660 665 670
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp
675 680 685
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
690 695 700
Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
705 710 715 720
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp
725 730 735
Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His
740 745 750
Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
755 760 765
Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
770 775 780
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln
785 790 795 800
Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
805 810 815
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn
820 825 830
Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly
835 840 845
Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
850 855 860
Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
865 870 875 880
Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
885 890 895
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp
900 905 910
Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
915 920 925
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly
930 935 940
Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
945 950 955 960
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
965 970 975
Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val
980 985 990
Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn
995 1000 1005
Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly
1010 1015 1020
Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
1025 1030 1035
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1040 1045 1050
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1055 1060 1065
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1070 1075 1080
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1085 1090 1095
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1100 1105 1110
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1115 1120 1125
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1130 1135 1140
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1145 1150 1155
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1160 1165 1170
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1175 1180 1185
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1190 1195 1200
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1205 1210 1215
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1220 1225 1230
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1235 1240 1245
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1250 1255 1260
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1265 1270 1275
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1280 1285 1290
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1295 1300 1305
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1310 1315 1320
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1325 1330 1335
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1340 1345 1350
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1355 1360 1365
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1370 1375 1380
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
1385 1390 1395
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr
1400 1405 1410
Val Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe
1415 1420 1425
Gly His Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe
1430 1435 1440
Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr
1445 1450 1455
Arg Val Thr Trp Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala
1460 1465 1470
Arg His Val Ala Glu Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu
1475 1480 1485
Arg Ile Phe Thr Ala Arg Leu Tyr Phe Cys Glu Asp Arg Lys Ala
1490 1495 1500
Glu Pro Glu Gly Leu Arg Arg Leu His Arg Ala Gly Val Gln Ile
1505 1510 1515
Gly Ile Met Thr Phe Lys Asp Tyr Phe Tyr Cys Trp Asn Thr Phe
1520 1525 1530
Val Glu Asn Arg Glu Arg Thr Phe Lys Ala Trp Glu Gly Leu His
1535 1540 1545
Glu Asn Ser Val Arg Leu Thr Arg Gln Leu Arg Arg Ile Leu Leu
1550 1555 1560
Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala Phe Arg Met Leu
1565 1570 1575
Gly Phe
1580
<210> 33
<211> 1724
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 33
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Met Glu Leu Lys Tyr
1 5 10 15
His Pro Glu Met Arg Phe Phe His Trp Phe Ser Lys Trp Arg Lys Leu
20 25 30
His Arg Asp Gln Glu Tyr Glu Val Thr Trp Tyr Ile Ser Trp Ser Pro
35 40 45
Cys Thr Lys Cys Thr Arg Asp Met Ala Thr Phe Leu Ala Glu Asp Pro
50 55 60
Lys Val Thr Leu Thr Ile Phe Val Ala Arg Leu Tyr Tyr Phe Trp Asp
65 70 75 80
Pro Asp Tyr Gln Glu Ala Leu Arg Ser Leu Cys Gln Lys Arg Asp Gly
85 90 95
Pro Arg Ala Thr Met Lys Ile Met Asn Tyr Asp Glu Phe Gln His Cys
100 105 110
Trp Ser Lys Phe Val Tyr Ser Gln Arg Glu Leu Phe Glu Pro Trp Asn
115 120 125
Asn Leu Pro Lys Tyr Tyr Ile Leu Leu His Ile Met Leu Gly Glu Ile
130 135 140
Leu Arg His Ser Met Asp Pro Pro Thr Phe Thr Phe Asn Phe Asn Asn
145 150 155 160
Glu Pro Trp Val Arg Gly Arg His Glu Thr Tyr Leu Cys Tyr Glu Val
165 170 175
Glu Arg Met His Asn Asp Thr Trp Val Leu Leu Asn Gln Arg Arg Gly
180 185 190
Phe Leu Cys Asn Gln Ala Pro His Lys His Gly Phe Leu Glu Gly Arg
195 200 205
His Ala Glu Leu Cys Phe Leu Asp Val Ile Pro Phe Trp Lys Leu Asp
210 215 220
Leu Asp Gln Asp Tyr Arg Val Thr Cys Phe Thr Ser Trp Ser Pro Cys
225 230 235 240
Phe Ser Cys Ala Gln Glu Met Ala Lys Phe Ile Ser Lys Asn Lys His
245 250 255
Val Ser Leu Cys Ile Phe Thr Ala Arg Ile Tyr Asp Asp Gln Gly Arg
260 265 270
Cys Gln Glu Gly Leu Arg Thr Leu Ala Glu Ala Gly Ala Lys Ile Ser
275 280 285
Ile Met Thr Tyr Ser Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp
290 295 300
His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser
305 310 315 320
Gln Asp Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Glu Asn
325 330 335
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Ser Pro Lys Lys Lys
340 345 350
Arg Lys Val Glu Ala Ser Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
355 360 365
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
370 375 380
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
385 390 395 400
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
405 410 415
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
420 425 430
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
435 440 445
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
450 455 460
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
465 470 475 480
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
485 490 495
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
500 505 510
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
515 520 525
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
530 535 540
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
545 550 555 560
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
565 570 575
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
580 585 590
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
595 600 605
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
610 615 620
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
625 630 635 640
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
645 650 655
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
660 665 670
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
675 680 685
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
690 695 700
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
705 710 715 720
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
725 730 735
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
740 745 750
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
755 760 765
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
770 775 780
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
785 790 795 800
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
805 810 815
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
820 825 830
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
835 840 845
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
850 855 860
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
865 870 875 880
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
885 890 895
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
900 905 910
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
915 920 925
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
930 935 940
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
945 950 955 960
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
965 970 975
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
980 985 990
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
995 1000 1005
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile
1010 1015 1020
Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
1025 1030 1035
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
1040 1045 1050
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
1055 1060 1065
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu
1070 1075 1080
Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys
1085 1090 1095
Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu
1100 1105 1110
Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys
1115 1120 1125
Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
1130 1135 1140
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu
1145 1150 1155
Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
1160 1165 1170
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
1175 1180 1185
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu
1190 1195 1200
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys
1205 1210 1215
Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1220 1225 1230
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile
1235 1240 1245
Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly
1250 1255 1260
Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val
1265 1270 1275
Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
1280 1285 1290
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1295 1300 1305
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg
1310 1315 1320
Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His
1325 1330 1335
His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu
1340 1345 1350
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp
1355 1360 1365
Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln
1370 1375 1380
Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
1385 1390 1395
Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1400 1405 1410
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1415 1420 1425
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu
1430 1435 1440
Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr
1445 1450 1455
Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1460 1465 1470
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly
1475 1480 1485
Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala
1490 1495 1500
Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu
1505 1510 1515
Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
1520 1525 1530
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1535 1540 1545
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu
1550 1555 1560
Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys
1565 1570 1575
Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr
1580 1585 1590
Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
1595 1600 1605
Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp
1610 1615 1620
Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu
1625 1630 1635
Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1640 1645 1650
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1655 1660 1665
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
1670 1675 1680
Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val
1685 1690 1695
Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1700 1705 1710
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1715 1720
<210> 34
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 34
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 35
<211> 1851
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 35
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Ser Met
20 25 30
Gly Thr Gly Thr Lys Cys Ile Gly Gln Ser Lys Met Arg Lys Asn Gly
35 40 45
Asp Ile Leu Asn Asp Ser His Ala Glu Val Ile Ala Arg Arg Ser Phe
50 55 60
Gln Arg Tyr Leu Leu His Gln Leu Gln Leu Ala Ala Thr Leu Lys Glu
65 70 75 80
Asp Ser Ile Phe Val Pro Gly Thr Gln Lys Gly Val Trp Lys Leu Arg
85 90 95
Arg Asp Leu Ile Phe Val Phe Phe Ser Ser His Thr Pro Cys Gly Asp
100 105 110
Ala Ser Ile Ile Pro Met Leu Glu Phe Glu Asp Gln Pro Cys Cys Pro
115 120 125
Val Phe Arg Asn Trp Ala His Asn Ser Ser Val Glu Ala Ser Ser Asn
130 135 140
Leu Glu Ala Pro Gly Asn Glu Arg Lys Cys Glu Asp Pro Asp Ser Pro
145 150 155 160
Val Thr Lys Lys Met Arg Leu Glu Pro Gly Thr Ala Ala Arg Glu Val
165 170 175
Thr Asn Gly Ala Ala His His Gln Ser Phe Gly Lys Gln Lys Ser Gly
180 185 190
Pro Ile Ser Pro Gly Ile His Ser Cys Asp Leu Thr Val Glu Gly Leu
195 200 205
Ala Thr Val Thr Arg Ile Ala Pro Gly Ser Ala Lys Val Ile Asp Val
210 215 220
Tyr Arg Thr Gly Ala Lys Cys Val Pro Gly Glu Ala Gly Asp Ser Gly
225 230 235 240
Lys Pro Gly Ala Ala Phe His Gln Val Gly Leu Leu Arg Val Lys Pro
245 250 255
Gly Arg Gly Asp Arg Thr Arg Ser Met Ser Cys Ser Asp Lys Met Ala
260 265 270
Arg Trp Asn Val Leu Gly Cys Gln Gly Ala Leu Leu Met His Leu Leu
275 280 285
Glu Glu Pro Ile Tyr Leu Ser Ala Val Val Ile Gly Lys Cys Pro Tyr
290 295 300
Ser Gln Glu Ala Met Gln Arg Ala Leu Ile Gly Arg Cys Gln Asn Val
305 310 315 320
Ser Ala Leu Pro Lys Gly Phe Gly Val Gln Glu Leu Lys Ile Leu Gln
325 330 335
Ser Asp Leu Leu Phe Glu Gln Ser Arg Ser Ala Val Gln Ala Lys Arg
340 345 350
Ala Asp Ser Pro Gly Arg Leu Val Pro Cys Gly Ala Ala Ile Ser Trp
355 360 365
Ser Ala Val Pro Glu Gln Pro Leu Asp Val Thr Ala Asn Gly Phe Pro
370 375 380
Gln Gly Thr Thr Lys Lys Thr Ile Gly Ser Leu Gln Ala Arg Ser Gln
385 390 395 400
Ile Ser Lys Val Glu Leu Phe Arg Ser Phe Gln Lys Leu Leu Ser Arg
405 410 415
Ile Ala Arg Asp Lys Trp Pro His Ser Leu Arg Val Gln Lys Leu Asp
420 425 430
Thr Tyr Gln Glu Tyr Lys Glu Ala Ala Ser Ser Tyr Gln Glu Ala Trp
435 440 445
Ser Thr Leu Arg Lys Gln Val Phe Gly Ser Trp Ile Arg Asn Pro Pro
450 455 460
Asp Tyr His Gln Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
465 470 475 480
Gly Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr
485 490 495
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
500 505 510
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
515 520 525
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
530 535 540
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
545 550 555 560
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
565 570 575
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
580 585 590
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
595 600 605
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
610 615 620
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
625 630 635 640
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
645 650 655
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
660 665 670
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
675 680 685
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
690 695 700
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
705 710 715 720
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
725 730 735
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
740 745 750
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
755 760 765
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
770 775 780
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
785 790 795 800
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
805 810 815
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
820 825 830
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
835 840 845
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
850 855 860
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
865 870 875 880
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
885 890 895
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
900 905 910
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
915 920 925
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
930 935 940
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
945 950 955 960
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
965 970 975
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
980 985 990
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
995 1000 1005
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
1010 1015 1020
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
1025 1030 1035
Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
1040 1045 1050
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
1055 1060 1065
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
1070 1075 1080
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile
1085 1090 1095
Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu
1100 1105 1110
Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp
1115 1120 1125
Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly
1130 1135 1140
Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
1145 1150 1155
Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn
1160 1165 1170
Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
1175 1180 1185
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
1190 1195 1200
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
1205 1210 1215
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val
1220 1225 1230
Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
1235 1240 1245
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg
1250 1255 1260
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile
1265 1270 1275
Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys
1280 1285 1290
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
1295 1300 1305
Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala
1310 1315 1320
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys
1325 1330 1335
Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val
1340 1345 1350
Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln
1355 1360 1365
Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu
1370 1375 1380
Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly
1385 1390 1395
Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His
1400 1405 1410
Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
1415 1420 1425
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
1430 1435 1440
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val
1445 1450 1455
Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn
1460 1465 1470
Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1475 1480 1485
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys
1490 1495 1500
Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys
1505 1510 1515
Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile
1520 1525 1530
Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
1535 1540 1545
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1550 1555 1560
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val
1565 1570 1575
Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile
1580 1585 1590
Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp
1595 1600 1605
Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala
1610 1615 1620
Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys
1625 1630 1635
Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu
1640 1645 1650
Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1655 1660 1665
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1670 1675 1680
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala
1685 1690 1695
Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser
1700 1705 1710
Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1715 1720 1725
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu
1730 1735 1740
Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu
1745 1750 1755
Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val
1760 1765 1770
Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
1775 1780 1785
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala
1790 1795 1800
Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg
1805 1810 1815
Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln
1820 1825 1830
Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu
1835 1840 1845
Gly Gly Asp
1850
<210> 36
<211> 1846
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 36
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Asp Lys
20 25 30
Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala
35 40 45
Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu
50 55 60
Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu
65 70 75 80
Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr
85 90 95
Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln
100 105 110
Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His
115 120 125
Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg
130 135 140
His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys
145 150 155 160
Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp
165 170 175
Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys
180 185 190
Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser
195 200 205
Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
210 215 220
Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile
225 230 235 240
Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
245 250 255
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala
260 265 270
Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
275 280 285
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu
290 295 300
Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
305 310 315 320
Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg
325 330 335
Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys
340 345 350
Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val
355 360 365
Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser
370 375 380
Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
385 390 395 400
Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu
405 410 415
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg
420 425 430
Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu
435 440 445
His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp
450 455 460
Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
465 470 475 480
Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg
485 490 495
Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp
500 505 510
Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
515 520 525
Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr
530 535 540
Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
545 550 555 560
Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala
565 570 575
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln
580 585 590
Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu
595 600 605
Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His
610 615 620
Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu
625 630 635 640
Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
645 650 655
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe
660 665 670
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp
675 680 685
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
690 695 700
Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
705 710 715 720
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp
725 730 735
Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His
740 745 750
Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
755 760 765
Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
770 775 780
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln
785 790 795 800
Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
805 810 815
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn
820 825 830
Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly
835 840 845
Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
850 855 860
Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
865 870 875 880
Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
885 890 895
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp
900 905 910
Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
915 920 925
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly
930 935 940
Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
945 950 955 960
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
965 970 975
Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val
980 985 990
Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn
995 1000 1005
Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly
1010 1015 1020
Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
1025 1030 1035
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1040 1045 1050
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1055 1060 1065
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1070 1075 1080
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1085 1090 1095
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1100 1105 1110
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1115 1120 1125
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1130 1135 1140
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1145 1150 1155
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1160 1165 1170
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1175 1180 1185
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1190 1195 1200
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1205 1210 1215
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1220 1225 1230
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1235 1240 1245
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1250 1255 1260
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1265 1270 1275
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1280 1285 1290
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1295 1300 1305
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1310 1315 1320
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1325 1330 1335
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1340 1345 1350
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1355 1360 1365
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1370 1375 1380
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
1385 1390 1395
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Met Gly Thr Gly Thr
1400 1405 1410
Lys Cys Ile Gly Gln Ser Lys Met Arg Lys Asn Gly Asp Ile Leu
1415 1420 1425
Asn Asp Ser His Ala Glu Val Ile Ala Arg Arg Ser Phe Gln Arg
1430 1435 1440
Tyr Leu Leu His Gln Leu Gln Leu Ala Ala Thr Leu Lys Glu Asp
1445 1450 1455
Ser Ile Phe Val Pro Gly Thr Gln Lys Gly Val Trp Lys Leu Arg
1460 1465 1470
Arg Asp Leu Ile Phe Val Phe Phe Ser Ser His Thr Pro Cys Gly
1475 1480 1485
Asp Ala Ser Ile Ile Pro Met Leu Glu Phe Glu Asp Gln Pro Cys
1490 1495 1500
Cys Pro Val Phe Arg Asn Trp Ala His Asn Ser Ser Val Glu Ala
1505 1510 1515
Ser Ser Asn Leu Glu Ala Pro Gly Asn Glu Arg Lys Cys Glu Asp
1520 1525 1530
Pro Asp Ser Pro Val Thr Lys Lys Met Arg Leu Glu Pro Gly Thr
1535 1540 1545
Ala Ala Arg Glu Val Thr Asn Gly Ala Ala His His Gln Ser Phe
1550 1555 1560
Gly Lys Gln Lys Ser Gly Pro Ile Ser Pro Gly Ile His Ser Cys
1565 1570 1575
Asp Leu Thr Val Glu Gly Leu Ala Thr Val Thr Arg Ile Ala Pro
1580 1585 1590
Gly Ser Ala Lys Val Ile Asp Val Tyr Arg Thr Gly Ala Lys Cys
1595 1600 1605
Val Pro Gly Glu Ala Gly Asp Ser Gly Lys Pro Gly Ala Ala Phe
1610 1615 1620
His Gln Val Gly Leu Leu Arg Val Lys Pro Gly Arg Gly Asp Arg
1625 1630 1635
Thr Arg Ser Met Ser Cys Ser Asp Lys Met Ala Arg Trp Asn Val
1640 1645 1650
Leu Gly Cys Gln Gly Ala Leu Leu Met His Leu Leu Glu Glu Pro
1655 1660 1665
Ile Tyr Leu Ser Ala Val Val Ile Gly Lys Cys Pro Tyr Ser Gln
1670 1675 1680
Glu Ala Met Gln Arg Ala Leu Ile Gly Arg Cys Gln Asn Val Ser
1685 1690 1695
Ala Leu Pro Lys Gly Phe Gly Val Gln Glu Leu Lys Ile Leu Gln
1700 1705 1710
Ser Asp Leu Leu Phe Glu Gln Ser Arg Ser Ala Val Gln Ala Lys
1715 1720 1725
Arg Ala Asp Ser Pro Gly Arg Leu Val Pro Cys Gly Ala Ala Ile
1730 1735 1740
Ser Trp Ser Ala Val Pro Glu Gln Pro Leu Asp Val Thr Ala Asn
1745 1750 1755
Gly Phe Pro Gln Gly Thr Thr Lys Lys Thr Ile Gly Ser Leu Gln
1760 1765 1770
Ala Arg Ser Gln Ile Ser Lys Val Glu Leu Phe Arg Ser Phe Gln
1775 1780 1785
Lys Leu Leu Ser Arg Ile Ala Arg Asp Lys Trp Pro His Ser Leu
1790 1795 1800
Arg Val Gln Lys Leu Asp Thr Tyr Gln Glu Tyr Lys Glu Ala Ala
1805 1810 1815
Ser Ser Tyr Gln Glu Ala Trp Ser Thr Leu Arg Lys Gln Val Phe
1820 1825 1830
Gly Ser Trp Ile Arg Asn Pro Pro Asp Tyr His Gln Phe
1835 1840 1845
<210> 37
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 37
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 38
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 38
guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu ccguuaucaa cuugaaaaag 60
uggcaccgag ucggugcuuu uu 82
<210> 39
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 39
gatgacattg catacattcg aaagacccta gccttagata aaactgagca agaggctttg 60
gagtatttca tgaaacaaat gaatgatgca cgtcatggtg gctggacaac aaaaatggat 120
tggatcttcc acacaattaa acagcatgca ttgaactgaa agataactga gaaaatgaaa 180
<210> 40
<211> 59
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 40
Asp Asp Ile Ala Tyr Ile Arg Lys Thr Leu Ala Leu Asp Lys Thr Glu
1 5 10 15
Gln Glu Ala Leu Glu Tyr Phe Met Lys Gln Met Asn Asp Ala Arg His
20 25 30
Gly Gly Trp Thr Thr Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln
35 40 45
His Ala Leu Asn Lys Ile Thr Glu Lys Met Lys
50 55
<210> 41
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 41
aucggaauct auuuugacuc 20
<210> 42
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 42
ucggaaucua uuuugacucg 20
<210> 43
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 43
cuuagauaaa acugagcaag 20
<210> 44
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 44
aucuauuuug acucguucuc 20
<210> 45
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 45
uaaaacugag caagaggcuu 20
<210> 46
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 46
ugguggcugg acaacaaaaa 20
<210> 47
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 47
gcuggacaac aaaaauggau 20
<210> 48
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 48
guguuaauuu gucguacgua 20
<210> 49
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 49
aatcacattt ttccacttct tgaaaagtac tgtggcttcc atgaagataa cattccccag 60
ctggaagacg tttctcaatt cctgcagact tgcactggtc tccgcctccg acctgtggct 120
ggcctgcttt cctctcggga tttcttgggt ggcctggcct tccgagtctt ccactgcaca 180
<210> 50
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 50
Asn His Ile Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp
1 5 10 15
Asn Ile Pro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr
20 25 30
Gly Ser Arg Leu Arg Pro Val Ala Gly Leu Leu Ser Ser Arg Asp Phe
35 40 45
Leu Gly Gly Leu Ala Phe Arg Val Phe His Cys Thr
50 55 60
<210> 51
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 51
atgcctgcct ggggagccct gttcctgctc tgggccacag cagaggccac caaggactgc 60
cccagcccac gtacctgccg cgccctggaa accatggggc tgtgggtgga ctgcaggggc 120
cacggactca cggccctgcc tgccctgccg gcccgcaccc gccaccttct gctggccaac 180
<210> 52
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 52
Met Pro Ala Trp Gly Ala Leu Phe Leu Leu Trp Ala Thr Ala Glu Ala
1 5 10 15
Thr Lys Asp Cys Pro Ser Pro Arg Thr Cys Arg Ala Leu Glu Thr Met
20 25 30
Gly Leu Trp Val Asp Cys Arg Gly His Gly Leu Thr Ala Leu Pro Ala
35 40 45
Leu Pro Ala Arg Thr Arg His Leu Leu Leu Ala Asn
50 55 60
<210> 53
<211> 120
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 53
ggttatggtc ctgtctgccc tcctggtggc atacaagaag tcactatcaa ccagagccct 60
cttcagcccc tcaatgtgga gattgaccct gagatccaaa aggtgaagtc tcgagaaagg 120
<210> 54
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 54
Gly Tyr Gly Pro Val Cys Pro Pro Gly Gly Ile Gln Glu Val Thr Ile
1 5 10 15
Asn Gln Ser Pro Leu Gln Pro Leu Asn Val Glu Ile Asp Pro Glu Ile
20 25 30
Gln Lys Val Lys Ser Arg Glu Arg
35 40
<210> 55
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 55
gtctccctgg ctgaggatcc ccagggagat gctgcccaga agacagatac atcccaccat 60
gatcaggatc acccaacctt caacaagatc acccccaacc cggctgagtt cgccttcagc 120
ctataccgcc agctggcaca ccagtccaac agcaccaata tcttcttctc cccagtgagc 180
<210> 56
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 56
Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp
1 5 10 15
Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro
20 25 30
Asn Pro Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln
35 40 45
Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser
50 55 60
<210> 57
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 57
ggccactgcc tcattatcaa caatgtgaac ttctgccgtg agtccgggct ccgcacccgc 60
actggctcca acatcgactg tgagaagttg cggcgtcgct tctcctcgcc gcatttcatg 120
gtggaggtga agggcgacct gactgccaag aaaatggtgc tggctttgct ggagctggcg 180
<210> 58
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 58
Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly
1 5 10 15
Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg
20 25 30
Arg Phe Ser Ser Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr
35 40 45
Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala
50 55 60
<210> 59
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 59
actagagcta gatactttct agttgggagc aataatgcag aaacgaaata tcgtgtcttg 60
aagactgata gaacagaacc aaaagatttg gtcataattg atgacaggca tgtctatact 120
caacaagaag taagggaact tcttggccgc ttggatcttg gaaatagaac aaagatggga 180
<210> 60
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 60
Thr Arg Ala Arg Tyr Phe Leu Val Gly Ser Asn Asn Ala Glu Thr Lys
1 5 10 15
Tyr Arg Val Leu Lys Thr Asp Arg Thr Glu Pro Lys Asp Leu Val Ile
20 25 30
Ile Asp Asp Arg His Val Tyr Thr Gln Gln Glu Val Arg Glu Leu Leu
35 40 45
Gly Arg Leu Asp Leu Gly Asn Arg Thr Lys Met Gly
50 55 60
<210> 61
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 61
acagatgccc cggtgagccc caccactctg tatgtggagg acatctcgga accgccgttg 60
cacgatttct accgcagcag gctactggac ctggtcttcc tgctggatgg ctcctccagg 120
ctgtccgagg ctgagtttga agtgctgaag gcctttgtgg tggacatgat ggagcggctg 180
<210> 62
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 62
Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser
1 5 10 15
Glu Pro Pro Leu His Asp Phe Tyr Arg Ser Arg Leu Leu Asp Leu Val
20 25 30
Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val
35 40 45
Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu
50 55 60
<210> 63
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 63
atctgtgctg ctgtcctcag caaattcatg tctgtgttct gcggggtata tgagcagcca 60
tactactact ctgatatcct gacggtgggc tgtgctgtgg gagtcggccg ttgttttggg 120
acaccacttg gaggagtgct atttagcatc gaggtcacct ccacctactt tgctgttcgg 180
<210> 64
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 64
Ile Cys Ala Ala Val Leu Ser Lys Phe Met Ser Val Phe Cys Gly Val
1 5 10 15
Tyr Glu Gln Pro Tyr Tyr Tyr Ser Asp Ile Leu Thr Val Gly Cys Ala
20 25 30
Val Gly Val Gly Arg Cys Phe Gly Thr Pro Leu Gly Gly Val Leu Phe
35 40 45
Ser Ile Glu Val Thr Ser Thr Tyr Phe Ala Val Arg
50 55 60
<210> 65
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 65
tactttgaaa agtcaaagga gcagctgaca cccctgatca agaaggctgg aacggaactg 60
gttaacttct tgagctattt cgtggaactt ggaacacagc ctgccaccca gcgaagtgtc 120
cagcaccatt gtcttccaac cccagctggc ctctagaaca cccactggcc agtcctagag 180
<210> 66
<211> 59
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 66
Tyr Phe Glu Lys Ser Lys Glu Gln Leu Thr Pro Leu Ile Lys Lys Ala
1 5 10 15
Gly Thr Glu Leu Val Asn Phe Leu Ser Tyr Phe Val Glu Leu Gly Thr
20 25 30
Gln Pro Ala Thr Gln Arg Ser Val Gln His His Cys Leu Pro Thr Pro
35 40 45
Ala Gly Leu Asn Thr His Trp Pro Val Leu Glu
50 55
<210> 67
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 67
ccgcacaagc gcctcacgct cagcggcatc tgcgccttca ttagtgaccg cttcccctac 60
taccgccgca agttccccgc ccggcagaac agcatccgcc acaacctctc gctgaacgac 120
tgcttcgtca agatcccccg cgagccgggc cgcccaggca agggcaacta ctggagcctg 180
<210> 68
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 68
Pro His Lys Arg Leu Thr Leu Ser Gly Ile Cys Ala Phe Ile Ser Asp
1 5 10 15
Arg Phe Pro Tyr Tyr Arg Arg Lys Phe Pro Ala Arg Gln Asn Ser Ile
20 25 30
Arg His Asn Leu Ser Leu Asn Asp Cys Phe Val Lys Ile Pro Arg Glu
35 40 45
Pro Gly Arg Pro Gly Lys Gly Asn Tyr Trp Ser Leu
50 55 60
<210> 69
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 69
gctgaggacc tgtggctgag cccgctgacc atggaagatc ttgtctgcta cagcttccag 60
gtggccagag ggatggagtt cctggcttcc cgaaagtgca tccgcagaga cctggctgct 120
cggaacattc tgctgtcgga aagcgacgtg gtgaagatct gtgactttgg ccttgcccgg 180
<210> 70
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 70
Ala Glu Asp Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys
1 5 10 15
Tyr Ser Phe Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys
20 25 30
Cys Ile Arg Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser
35 40 45
Asp Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg
50 55 60
<210> 71
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 71
gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcatgatc 60
agtgtcgttg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 120
gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 180
<210> 72
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 72
Asp Thr Glu Thr Val Gly Gln Arg Ala Leu His Ser Ile Leu Asn Ala
1 5 10 15
Ala Ile Met Ile Ser Val Val Val Val Met Thr Ile Leu Leu Val Val
20 25 30
Leu Tyr Lys Tyr Arg Cys Tyr Lys Val Ile His Ala Trp Leu Ile Ile
35 40 45
Ser Ser Leu Leu Leu Leu Phe Phe Phe Ser Phe Ile
50 55 60
<210> 73
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 73
aagccgagta agccaaaaac caacatgaag cacatggctg gtgctgcagc agctggggca 60
gtggtggggg gccttggcgg ctacgtgctg ggaagtgcca tgagcaggcc catcatacat 120
ttcggcagtg actatgagga ccgttactat cgtgaaaaca tgcaccgtta ccccaaccaa 180
<210> 74
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 74
Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met Ala Gly Ala Ala
1 5 10 15
Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Val Leu Gly Ser
20 25 30
Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp Tyr Glu Asp Arg
35 40 45
Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln
50 55 60
<210> 75
<211> 120
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 75
cttcccagcc gagacgtgac agtccttctg gaaaactatg gcaaattcga aaaggggtgt 60
ttgatttttg ttgtacgttt cctctttggc ctggtaaacc aggagaggac ctcctacttg 120
<210> 76
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 76
Leu Pro Ser Arg Asp Val Thr Val Leu Leu Glu Asn Tyr Gly Lys Phe
1 5 10 15
Glu Lys Gly Cys Leu Ile Phe Val Val Arg Phe Leu Phe Gly Leu Val
20 25 30
Asn Gln Glu Arg Thr Ser Tyr Leu
35 40
<210> 77
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 77
gtgaagcact tctccccaga ggaactcaaa gttaaggtgt tgggagatgt gattgaggtg 60
catggaaaac atgaagagcg ccaggatgaa catggtttca tctccaggga gttccacggg 120
aaataccgga tcccagctga tgtagaccct ctcaccatta cttcatccct gtcatctgat 180
<210> 78
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 78
Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp
1 5 10 15
Val Ile Glu Val His Gly Lys His Glu Glu Arg Gln Asp Glu His Gly
20 25 30
Phe Ile Ser Arg Glu Phe His Gly Lys Tyr Arg Ile Pro Ala Asp Val
35 40 45
Asp Pro Leu Thr Ile Thr Ser Ser Leu Ser Ser Asp
50 55 60
<210> 79
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 79
gagctgcact gtgacaagct gcacgtggat cctgagaact tcaggctcct gggcaacgtg 60
ctggtctgtg tgccggccca tcactttggc aaagaattca ccccaccagt gcaggctgcc 120
tatcagaaag tggtggctgg tgtggctaat gccctggccc acaagtatca ctaagctcgc 180
<210> 80
<211> 59
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 80
Glu Leu His Cys Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg Leu
1 5 10 15
Leu Gly Asn Val Leu Val Cys Val Pro Ala His His Phe Gly Lys Glu
20 25 30
Phe Thr Pro Pro Val Gln Ala Ala Tyr Gln Lys Val Val Ala Gly Val
35 40 45
Ala Asn Ala Leu Ala His Lys Tyr His Ala Arg
50 55
<210> 81
<211> 102
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 81
aucggaauct auuuugacuc guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 82
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 82
ucggaaucua uuuugacucg guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 83
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 83
cuuagauaaa acugagcaag guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 84
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 84
aucuauuuug acucguucuc guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 85
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 85
uaaaacugag caagaggcuu guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 86
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 86
ugguggcugg acaacaaaaa guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 87
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 87
gcuggacaac aaaaauggau guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 88
<211> 102
<212> RNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 88
guguuaauuu gucguacgua guuuuagagc uagaaauagc aaguuaaaau aaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uu 102
<210> 89
<211> 180
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 89
gcctccgcca acgtggactt cgctttcagc ctgtacaagc agttagtcct gaaggcccct 60
gataagaatg tcatcttctc cccaccgagc atctccaccg ccttggcctt cctgtctctg 120
ggggcccata ataccaccct gacagagatt ctcaaaggcc tcaagttcta cctcacggag 180
<210> 90
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 90
Ala Ser Ala Asn Val Asp Phe Ala Phe Ser Leu Tyr Lys Gln Leu Val
1 5 10 15
Leu Lys Ala Pro Asp Lys Asn Val Ile Phe Ser Pro Pro Ser Ile Ser
20 25 30
Thr Ala Leu Ala Phe Leu Ser Leu Gly Ala His Asn Thr Thr Leu Thr
35 40 45
Glu Ile Leu Lys Gly Leu Lys Phe Tyr Leu Thr Glu
50 55 60
<210> 91
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 91
Gly Gly Gly Gly Ser
1 5
<210> 92
<211> 1636
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 92
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Met Thr Ser Glu Lys
1 5 10 15
Gly Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro Trp
20 25 30
Glu Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu Arg Lys Glu Ala Cys
35 40 45
Leu Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg Lys Ile Trp Arg Ser
50 55 60
Ser Gly Lys Asn Thr Thr Asn His Val Glu Val Asn Phe Ile Lys Lys
65 70 75 80
Phe Thr Ser Glu Arg Asp Phe His Pro Ser Met Ser Cys Ser Ile Thr
85 90 95
Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser Gln Ala Ile Arg
100 105 110
Glu Phe Leu Ser Arg His Pro Gly Val Thr Leu Val Ile Tyr Val Ala
115 120 125
Arg Leu Phe Trp His Met Asp Gln Gln Asn Arg Gln Gly Leu Arg Asp
130 135 140
Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Arg Ala Ser Glu Tyr
145 150 155 160
Tyr His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro Gly Asp Glu Ala
165 170 175
His Trp Pro Gln Tyr Pro Pro Leu Trp Met Met Leu Tyr Ala Leu Glu
180 185 190
Leu His Cys Ile Ile Leu Ser Leu Pro Pro Cys Leu Lys Ile Ser Arg
195 200 205
Arg Trp Gln Asn His Leu Thr Phe Phe Arg Leu His Leu Gln Asn Cys
210 215 220
His Tyr Gln Thr Ile Pro Pro His Ile Leu Leu Ala Thr Gly Leu Ile
225 230 235 240
His Pro Ser Val Ala Trp Arg Ser Pro Lys Lys Lys Arg Lys Val Glu
245 250 255
Ala Ser Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Asp Lys Lys
260 265 270
Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val
275 280 285
Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly
290 295 300
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
305 310 315 320
Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala
325 330 335
Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu
340 345 350
Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg
355 360 365
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His
370 375 380
Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
385 390 395 400
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys
405 410 415
Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe
420 425 430
Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp
435 440 445
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe
450 455 460
Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
465 470 475 480
Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln
485 490 495
Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu
500 505 510
Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu
515 520 525
Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp
530 535 540
Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
545 550 555 560
Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
565 570 575
Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
580 585 590
Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg
595 600 605
Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
610 615 620
Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
625 630 635 640
Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu
645 650 655
Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr
660 665 670
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His
675 680 685
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
690 695 700
Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val
705 710 715 720
Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys
725 730 735
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
740 745 750
Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys
755 760 765
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
770 775 780
Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu
785 790 795 800
Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile
805 810 815
Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu
820 825 830
Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile
835 840 845
Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
850 855 860
Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
865 870 875 880
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp
885 890 895
Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp
900 905 910
Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly
915 920 925
Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly
930 935 940
Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn
945 950 955 960
Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile
965 970 975
Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile
980 985 990
Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr
995 1000 1005
Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
1010 1015 1020
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1025 1030 1035
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1040 1045 1050
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1055 1060 1065
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1070 1075 1080
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1085 1090 1095
Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser
1100 1105 1110
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1115 1120 1125
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1130 1135 1140
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1145 1150 1155
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1160 1165 1170
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1175 1180 1185
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1190 1195 1200
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1205 1210 1215
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1220 1225 1230
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1235 1240 1245
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1250 1255 1260
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1265 1270 1275
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1280 1285 1290
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1295 1300 1305
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1310 1315 1320
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1325 1330 1335
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1340 1345 1350
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1355 1360 1365
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1370 1375 1380
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1385 1390 1395
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1400 1405 1410
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1415 1420 1425
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1430 1435 1440
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1445 1450 1455
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1460 1465 1470
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1475 1480 1485
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1490 1495 1500
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1505 1510 1515
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1520 1525 1530
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1535 1540 1545
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1550 1555 1560
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1565 1570 1575
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1580 1585 1590
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1595 1600 1605
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1610 1615 1620
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1625 1630 1635
<210> 93
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 93
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
1 5 10 15
<210> 94
<211> 1600
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 94
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Gly Gly Ser Met Asp Lys Lys Tyr Ser Ile Gly
225 230 235 240
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
245 250 255
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
260 265 270
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
275 280 285
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
290 295 300
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
305 310 315 320
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
325 330 335
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
340 345 350
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
355 360 365
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
370 375 380
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
385 390 395 400
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
405 410 415
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
420 425 430
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
435 440 445
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
450 455 460
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
465 470 475 480
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
485 490 495
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
500 505 510
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
515 520 525
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
530 535 540
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
545 550 555 560
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
565 570 575
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
580 585 590
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
595 600 605
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
610 615 620
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
625 630 635 640
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
645 650 655
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
660 665 670
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
675 680 685
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
690 695 700
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
705 710 715 720
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
725 730 735
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
740 745 750
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
755 760 765
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
770 775 780
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
785 790 795 800
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
805 810 815
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
820 825 830
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
835 840 845
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
850 855 860
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
865 870 875 880
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
885 890 895
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
900 905 910
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
915 920 925
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
930 935 940
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
945 950 955 960
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
965 970 975
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
980 985 990
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
995 1000 1005
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu
1010 1015 1020
Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu
1025 1030 1035
Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp
1040 1045 1050
Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr
1055 1060 1065
Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
1070 1075 1080
Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys
1085 1090 1095
Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
1100 1105 1110
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
1115 1120 1125
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu
1130 1135 1140
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln
1145 1150 1155
Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
1160 1165 1170
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile
1175 1180 1185
Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln
1190 1195 1200
Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp
1205 1210 1215
Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
1220 1225 1230
Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
1235 1240 1245
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys
1250 1255 1260
Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe
1265 1270 1275
Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro
1280 1285 1290
Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys
1295 1300 1305
Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln
1310 1315 1320
Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser
1325 1330 1335
Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1340 1345 1350
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1355 1360 1365
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys
1370 1375 1380
Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile
1385 1390 1395
Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
1400 1405 1410
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile
1415 1420 1425
Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys
1430 1435 1440
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu
1445 1450 1455
Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
1460 1465 1470
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1475 1480 1485
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu
1490 1495 1500
Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn
1505 1510 1515
Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro
1520 1525 1530
Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr
1535 1540 1545
Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile
1550 1555 1560
Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr
1565 1570 1575
Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
1580 1585 1590
Leu Ser Gln Leu Gly Gly Asp
1595 1600
<210> 95
<211> 1606
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<400> 95
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Asp
225 230 235 240
Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp
245 250 255
Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val
260 265 270
Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala
275 280 285
Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg
290 295 300
Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu
305 310 315 320
Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe
325 330 335
His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu
340 345 350
Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
355 360 365
Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr
370 375 380
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile
385 390 395 400
Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn
405 410 415
Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln
420 425 430
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala
435 440 445
Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile
450 455 460
Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile
465 470 475 480
Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
485 490 495
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp
500 505 510
Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe
515 520 525
Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu
530 535 540
Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile
545 550 555 560
Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu
565 570 575
Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln
580 585 590
Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu
595 600 605
Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr
610 615 620
Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln
625 630 635 640
Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu
645 650 655
Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys
660 665 670
Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr
675 680 685
Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr
690 695 700
Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val
705 710 715 720
Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe
725 730 735
Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu
740 745 750
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val
755 760 765
Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys
770 775 780
Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys
785 790 795 800
Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val
805 810 815
Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr
820 825 830
His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu
835 840 845
Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe
850 855 860
Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
865 870 875 880
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly
885 890 895
Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln
900 905 910
Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn
915 920 925
Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu
930 935 940
Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu
945 950 955 960
His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu
965 970 975
Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His
980 985 990
Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
995 1000 1005
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
1010 1015 1020
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
1025 1030 1035
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
1040 1045 1050
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
1055 1060 1065
Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser
1070 1075 1080
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser
1085 1090 1095
Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
1100 1105 1110
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys
1115 1120 1125
Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
1130 1135 1140
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
1145 1150 1155
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
1160 1165 1170
Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
1175 1180 1185
Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
1190 1195 1200
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
1205 1210 1215
Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
1220 1225 1230
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1235 1240 1245
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1250 1255 1260
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1265 1270 1275
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1280 1285 1290
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
1295 1300 1305
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1310 1315 1320
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val
1325 1330 1335
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
1340 1345 1350
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys
1355 1360 1365
Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1370 1375 1380
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1385 1390 1395
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
1400 1405 1410
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
1415 1420 1425
Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
1430 1435 1440
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
1445 1450 1455
Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1460 1465 1470
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1475 1480 1485
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1490 1495 1500
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1505 1510 1515
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1520 1525 1530
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
1535 1540 1545
His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1550 1555 1560
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
1565 1570 1575
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1580 1585 1590
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1595 1600 1605
<210> 96
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 96
attattatta ttccgcggat ttatttattt atttatttat tt 42
<210> 97
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (8)..(9)
<223> n为a, c, g, 或t
<220>
<221> misc_feature
<222> (10)..(12)
<223> 核苷酸可以重复多次
<220>
<221> misc_feature
<222> (16)..(23)
<223> n为a, c, g, 或t
<220>
<221> misc_feature
<222> (30)..(35)
<223> n为a, c, g, 或t
<400> 97
atcttccnnn nncgtnnnnn nnncctcctn nnnnn 35
<210> 98
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<220>
<221> misc_feature
<222> (1)..(6)
<223> n为a, c, g, 或t
<220>
<221> misc_feature
<222> (13)..(20)
<223> n为a, c, g, 或t
<220>
<221> misc_feature
<222> (24)..(26)
<223> n为a或t
<220>
<221> misc_feature
<222> (24)..(26)
<223> 核苷酸可以重复多次
<220>
<221> misc_feature
<222> (27)..(28)
<223> n为a, c, g, 或t
<400> 98
nnnnnnagga ggnnnnnnnn acgnnnnngg aagat 35
<210> 99
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 99
attattatta ttccgcggat ttatttattt atttatttat tt 42
<210> 100
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 100
attattatta ttcugcggat ttatttattt atttatttat tt 42
<210> 101
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 101
attattatta ttcgcggatt tatttattta tttatttatt t 41
<210> 102
<211> 13
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 102
attattatta ttc 13
<210> 103
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 103
gcggatttat ttatttattt atttattt 28
<210> 104
<211> 13
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 104
attattatta ttc 13
<210> 105
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 105
gcggatttat ttatttattt atttattt 28
<210> 106
<211> 36
<212> PRT
<213> 人工序列
<220>
<223> 合成多肽
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa 可以是任意天然发生的氨基酸
<220>
<221> MISC_FEATURE
<222> (4)..(7)
<223> 这些氨基酸可以不存在
<220>
<221> misc_feature
<222> (8)..(29)
<223> Xaa 可以是任意天然发生的氨基酸
<220>
<221> misc_feature
<222> (32)..(32)
<223> Xaa 可以是任意天然发生的氨基酸
<220>
<221> MISC_FEATURE
<222> (33)..(34)
<223> 这些氨基酸可以不存在
<220>
<221> misc_feature
<222> (35)..(35)
<223> Xaa 可以是任意天然发生的氨基酸
<400> 106
His Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Cys Xaa
20 25 30
Xaa Xaa Xaa Cys
35

Claims (62)

1.一种融合蛋白，该融合蛋白包括
(i)核酸酶非活性Cas9结构域；和
(ii)核酸编辑结构域。
2.如权利要求1所述的融合蛋白，其中所述核酸编辑结构域是DNA编辑结构域。
3.如权利要求1所述的融合蛋白，其中所述核酸编辑结构域是脱氨酶结构域。
4.如权利要求1所述的融合蛋白，其中所述脱氨酶是胞苷脱氨酶。
5.如权利要求1所述的融合蛋白，其中所述脱氨酶是载脂蛋白B mRNA编辑复合体(APOBEC)家族脱氨酶。
6.如权利要求5所述的融合蛋白，其中所述脱氨酶是APOBEC1家族脱氨酶。
7.如权利要求5所述的融合蛋白，其中所述脱氨酶是激活诱导的胞苷脱氨酶(AID)。
8.如权利要求1所述的融合蛋白，其中所述脱氨酶是ACF1/ASE脱氨酶。
9.如权利要求1所述的融合蛋白，其中所述脱氨酶是腺苷脱氨酶。
10.如权利要求9所述的融合蛋白，其中所述脱氨酶是ADAT家族脱氨酶。
11.如权利要求1所述的融合蛋白，其中所述核酸编辑结构域被融合至Cas9结构域的N末端。
12.如权利要求1所述的融合蛋白，其中所述核酸编辑结构域被融合至Cas9结构域的C末端。
13.如权利要求1所述的融合蛋白，其中所述Cas9结构域与所述核酸编辑结构域是通过连接体融合的。
14.如权利要求1所述的融合蛋白，其中所述连接体包含(GGGGS)n(SEQ ID NO:91)、(G)n、(EAAAK)n(SEQ ID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:93)、或(XP)n基序，或这些中任意的组合，其中n独立地是1至30之间的整数。
15.一种DNA编辑的方法，该方法包括使DNA分子接触
(a)包括核酸酶非活性的Cas9结构域和核酸编辑结构域的融合蛋白；和
(b)将(a)的融合蛋白靶向DNA分子的靶DNA序列的sgRNA；
其中所述DNA分子与处于有效量的和在适合于该DNA分子的核苷酸碱基的脱氨基作用的条件下的所述融合蛋白和所述sgRNA接触。
16.如权利要求15所述的方法，其中所述核酸编辑结构域是脱氨酶结构域。
17.如权利要求16所述的方法，其中所述脱氨酶是胞苷脱氨酶。
18.如权利要求16或17中任一项所述的方法，其中所述脱氨酶是载脂蛋白B mRNA编辑复合体(APOBEC)家族脱氨酶。
19.如权利要求16所述的方法，其中所述脱氨酶是APOBEC1家族脱氨酶。
20.如权利要求16所述的方法，其中所述脱氨酶是激活诱导的胞苷脱氨酶(AID)。
21.如权利要求16所述的方法，其中所述脱氨酶是ACF1/ASE脱氨酶。
22.如权利要求16所述的方法，其中所述脱氨酶是腺苷脱氨酶。
23.如权利要求16所述的方法，其中所述脱氨酶是ADAT家族脱氨酶。
24.如权利要求15-23中任一项所述的方法，其中所述核酸编辑结构域被融合至Cas9结构域的N末端。
25.如权利要求15-23中任一项所述的方法，其中所述核酸编辑结构域被融合至Cas9结构域的C末端。
26.如权利要求15-25中任一项所述的方法，其中所述Cas9结构域和所述核酸编辑结构域是通过连接体融合的。
27.如权利要求26所述的方法，其中所述连接体包含(GGGGS)n(SEQ ID NO:91)、(G)n、(EAAAK)n(SEQ ID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:93)、或(XP)n基序，或这些中任意的组合，其中n独立地是1至30之间的整数。
28.如权利要求15-27中任一项所述的方法，其中所述靶DNA序列包括与疾病或失调相关的序列，并且其中所述核苷酸碱基的脱氨基作用产生与疾病或失调无关的序列。
29.如权利要求28所述的方法，其中所述靶DNA序列包括与疾病或失调相关的点突变，并且其中所述脱氨基作用修正该点突变。
30.如权利要求15-28中任一项所述的方法，其中所述靶DNA序列包括与疾病或失调相关的T→C或A→G点突变，并且其中所述突变的C或G碱基的脱氨基作用产生与疾病或失调无关的序列。
31.如权利要求28-30中任一项所述的方法，其中所述与疾病或失调相关的序列编码蛋白，并且其中所述脱氨基作用在所述与疾病或失调相关的序列中引入终止密码子，导致所编码蛋白的截短。
32.如权利要求28-31中任一项所述的方法，其中所述接触是在具有或被诊断出具有疾病或失调的受试者的体内进行的。
33.如权利要求28-32中任一项所述的方法，其中所述疾病或失调是囊胞性纤维症、苯丙酮酸尿症、表皮松解性角化过度(EHK)、进行性神经性腓骨肌萎缩症4J型、神经母细胞瘤(NB)、血管性血友病(vWD)、先天性肌强直、遗传性肾淀粉样变性、扩张性心肌病(DCM)、遗传性淋巴水肿、家族性阿尔茨海默病、朊病毒疾病、慢性小儿神经皮肤关节综合征(CINCA)、结蛋白相关肌病(DRM)、或与突变的PI3KCA蛋白相关的肿瘤性疾病。
34.如权利要求15-33中任一项所述的方法，其中所述靶DNA序列包括与野生型PI3K蛋白相比导致PI3KCA蛋白的氨基酸序列突变的T→C和/或A→G点突变，并且其中所述方法导致突变的C或G碱基的脱氨基作用。
35.如权利要求34所述的方法，其中所述点突变是导致H1047R置换的A3140G突变。
36.如权利要求15-33中任一项所述的方法，其中所述靶DNA序列包括与野生型PSEN1蛋白相比导致PSEN1蛋白的氨基酸序列突变的T→C和/或A→G点突变，并且其中所述方法导致突变的C或G碱基的脱氨基作用。
37.如权利要求36所述的方法，其中所述PSEN1蛋白包括由PSEN1基因的密码子143中A-→G点突变导致的I143V置换。
38.如权利要求36或37所述的方法，其中所述PSEN1点突变是与阿尔茨海默病相关的。
39.如权利要求36-38中任一项所述的方法，其中所述接触导致所述PSEN1基因的密码子143内突变的胞嘧啶残基脱去氨基，从而修正A→G点突变。
40.如权利要求15-33中任一项所述的方法，其中所述靶DNA序列包括与野生型α-抗胰蛋白酶蛋白相比导致α-抗胰蛋白酶蛋白的氨基酸序列突变的T→C和/或A→G点突变，并且其中所述方法导致突变的C或G碱基的脱氨基作用。
41.如权利要求40所述的方法，其中所述α-抗胰蛋白酶蛋白包括L55P置换该置换是由α-抗胰蛋白酶基因的密码子55中的T→C点突变导致的。
42.如权利要求40或41所述的方法，其中所述α-抗胰蛋白酶点突变是与慢性阻塞性肺病(COPD)相关的。
43.如权利要求40-42中任一项所述的方法，其中所述接触导致所述α-抗胰蛋白酶基因的密码子55内突变的胞嘧啶残基脱去氨基，从而修正T→C点突变。
44.如权利要求15-33中任一项所述的方法，其中所述靶DNA序列包括与野生型vWF蛋白相比导致vWF蛋白的氨基酸序列突变的T→C和/或A→G点突变，并且其中所述方法导致突变的C或G碱基的脱氨基作用。
45.如权利要求44所述的方法，其中所述vWF蛋白包括由vWF基因的密码子509中T→C点突变导致的C509A置换。
46.如权利要求45所述的方法，其中所述vWF点突变是与血管性血友病相关的。
47.如权利要求44或45所述的方法，其中所述接触导致所述vWF基因的密码子509内突变的胞嘧啶残基脱去氨基，从而修正T→C点突变。
48.如权利要求15-33中任一项所述的方法，其中所述靶DNA序列包括与野生型半胱天冬酶-9蛋白相比导致半胱天冬酶-9蛋白的氨基酸序列突变的T→C和/或A→G点突变，并且其中所述方法导致突变的C或G碱基的脱氨基作用。
49.如权利要求48所述的方法，其中所述半胱天冬酶-9蛋白包括由半胱天冬酶-9基因的密码子197中T→C点突变导致的L197P置换。
50.如权利要求48或49所述的方法，其中所述半胱天冬酶-9点突变是与神经母细胞瘤相关的。
51.如权利要求48-50中任一项所述的方法，其中所述接触导致所述半胱天冬酶-9基因的密码子197内突变的胞嘧啶残基脱去氨基，从而修正T→C点突变。
52.如权利要求15-51中任一项所述的方法，其中所述方法进一步包括检测核苷酸碱基的脱氨基作用。
53.如权利要求52所述的方法，其中所述检测是通过PCR进行的。
54.如权利要求15-53中任一项所述的方法，其中所述融合蛋白是如权利要求1-14中任一项所述的融合蛋白。
55.一种用于检测Cas9 DNA编辑蛋白的DNA编辑活性的报告构建体，该构建体包括
(a)包含用于Cas9 DNA编辑蛋白的靶位点的报告基因，其中靶向的DNA编辑导致所述报告基因表达的增加；以及
(b)控制所述报告基因表达的启动子序列。
56.如权利要求55所述的报告构建体，其中该构建体进一步包括
(c)编码将Cas9 DNA编辑蛋白靶向所述报告基因的靶位点的sgRNA的序列，其中所述sgRNA的表达与所述报告基因的表达是独立的。
57.如权利要求55或56所述的报告构建体，其中所述报告基因的靶位点包括未成熟的终止密码子，并且其中通过Cas9 DNA编辑蛋白对靶向的DNA的模板链的编辑导致未成熟的终止密码子转变为编码氨基酸残基的密码子。
58.如权利要求55-57中任一项所述的报告构建体，其中所述报告基因编码荧光素酶、荧光蛋白或抗生素抗性标记。
59.一种试剂盒，包括
核酸构建体，该构建体包括
编码核酸酶非活性Cas9序列的序列；
包含克隆位点的序列，该克隆位点被定位以允许编码与Cas9编码序列同框的核酸编辑酶或酶结构域的序列的克隆；
任选地，编码连接体的序列，该连接体被定位于Cas9编码序列和克隆位点之间；以及
合适的试剂、缓冲液、和/或说明书，用于将编码核酸编辑酶或酶结构域的序列框架克隆进入核酸构建体以产生Cas9核酸编辑融合蛋白。
60.如权利要求59所述的试剂盒，其中所述包括克隆位点的序列是Cas9序列的N末端。
61.如权利要求59所述的试剂盒，其中所述包括克隆位点的序列是Cas9序列的C末端。
62.如权利要求59-61中任一项所述的试剂盒，其中所编码的连接体包括(GGGGS)n(SEQID NO:91)、(G)n、(EAAAK)n(SEQ ID NO:5)、(GGS)n、SGSETPGTSESATPES(SEQ ID NO:93)、或(XP)n基序，或这些中任意的组合，其中n独立地是1至30之间的整数。
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