CN110272881B

CN110272881B - 核酸内切酶SpCas9高特异性截短变异体TSpCas9-V1/V2及其应用

Info

Publication number: CN110272881B
Application number: CN201910581265.8A
Authority: CN
Inventors: 黄强; 薛冬梅; 汤洪海; 朱海霞; 杜文豪
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-06-29
Filing date: 2019-06-29
Publication date: 2021-04-30
Anticipated expiration: 2039-06-29
Also published as: CN110272881A

Abstract

本发明属于蛋白质工程技术领域，具体为一种来源于酿脓链球菌的CRISPR核酸酶SpCas9的截短型高特异性变异体及其应用。本发明中的CRISPR‑Cas9（TSpCas9‑V1/V2）核酸酶属于CRISPR‑Cas9系统，TSpCas9‑V1核酸酶将截短型CRISPR‑Cas9（TSpCas9）核酸酶的第863位的氨基酸H突变成N，TSpCas9‑V2核酸酶将截短型CRISPR‑Cas9（TSpCas9）核酸酶的第862位的氨基酸H突变成A，将第863位的氨基酸H突变成N；该截短型高特异性变异体具有与野生型CRISPR‑Cas9核酸酶相当的基因编辑活性，能够降低基因编辑中的脱靶，因而能用于对基因组DNA片段特定位置的精准编辑。

Description

核酸内切酶SpCas9高特异性截短变异体TSpCas9-V1/V2及其应用

技术领域

本发明属于蛋白质工程技术领域，具体涉及来源于酿脓链球菌的CRISPR-Cas9核酸酶SpCas9的高特异性截短变异体TSpCas9-V1/V2及其在基因编辑领域中的应用。

背景技术

CRISPR/Cas系统可方便地对基因组特定基因进行的切割编辑，是最新发现的一种革命性的基因编辑技术，近年来，发展尤为迅速。来源于酿脓链球菌(Streptococcuspyogenes)的CRISPR-Cas9核酸酶SpCas9是目前最为广泛使用的CRISPR核酸酶^[1]，因其具有易用、高效、强特异性和多功能性等优点，已被应用到多个领域，包括医学研究以及生物技术等，比如细胞或动物模型的快速构建、功能基因的便捷筛选以及部分遗传疾病的治疗^[2-6]。

虽然，CRISPR-Cas9系统的优势促使其能够广泛发展，但是，其仍然存在着许多有待解决的问题。比如SpCas9体积过于庞大，难以通过病毒载体将其高效靶向地运载到体内或细胞中，从而限制了其在临床医学治疗上的发展^[7]；SpCas9由sgRNA引导至特定靶序列进行切割编辑时，sgRNA可能会与其他类似靶序列的基因位点发生局部匹配，进一步激活Cas9切割非靶DNA，因而难以精准的靶向特异性疾病基因位点，导致产生脱靶现象，进而限制了该技术在精准医疗方面的应用^[8-10]。

基于上述问题，寻找一个体积较小，特异性较高的Cas9核酸酶，从而有效地实现体内DNA片段的精准遗传编辑是十分必要的。

发明内容

本发明的目的是提供一种来源于酿脓链球菌的CRISPR-Cas9核酸酶SpCas9的截短型高特异性变异体及其用途。

本发明提供的来源于酿脓链球菌的CRISPR-Cas9核酸酶SpCas9的截短型高特异性变异体有两种，一种是将截短型CRISPR-Cas9(TSpCas9)核酸酶的第863位的氨基酸H突变成N，记为TSpCas9-V1核酸酶；一种是将截短型CRISPR-Cas9(TSpCas9)核酸酶的第862位的氨基酸H突变成A，第863位的氨基酸H突变成N，记为TSpCas9-V2核酸酶；两种截短型高特异性变异体记为TSpCas9-V1/V2；它们具有与野生型CRISPR-Cas9核酸酶相当的基因编辑活性，但比野生型特异性更高，能实现精准编辑。

所述野生型SpCas9核酸酶的核苷酸序列和氨基酸序列分别为SEQIDNO.1和SEQ IDNO.2所示。

所述截短型TSpCas9核酸酶的核苷酸序列和氨基酸序列分别为SEQ ID NO.3和SEQID NO.4所示。

所述截短型高特异性TSpCas9-V1核酸酶的核苷酸序列和氨基酸序列分别为SEQID NO.5和SEQ ID NO.6所示，与野生型SpCas9的相似度达90％以上。

所述截短型高特异性TSpCas9-V2核酸酶的核苷酸序列和氨基酸序列分别为SEQID NO.7和SEQ ID NO.8所示，与野生型SpCas9的相似度达90％以上。

本发明还提供一种多核苷酸序列，可以转录和翻译成所述的CRISPR-Cas9核酸酶(TSpCas9-V1/V2)。

本发明还提供一种表达载体，其含有上述多核苷酸序列。

本发明还提供一种宿主细胞，可以用于转化上述表达载体。

本发明还提供所述CRISPR-Cas9核酸酶(TSpCas9-V1/V2)的制备方法，具体步骤包括：首先，构建所述CRISPR-Cas9核酸酶的多核苷酸序列表达载体；然后，将所述表达载体转化至宿主细胞，筛选并挑出单克隆；最后，将所述单克隆诱导表达，并通过亲和层析、离子交换等方法从表达产物中分离出所述的CRISPR-Cas9核酸酶。

本发明提供的上述CRISPR-Cas9核酸酶、多核苷酸序列以及表达载体均可作为编辑基因组DNA的编辑工具，用于基因组DNA片段的相关编辑。

本发明中，所述的基因编辑可以是单点编辑，也可以是编辑位点大于等于两个的多点编辑。

所述编辑的手段包括删除、突变、插入、倒位、移位、重复或易位。

本发明中，所述CRISPR-Cas9编辑工具包括与靶标DNA片段匹配的引导sgRNA。

所述的CRISPR-Cas9核酸酶与能够介导它的sgRNA组合，从而对目的基因进行编辑。

本发明中，所述含有编码CRISPR-Cas9核酸酶多核苷酸序列的载体和与之匹配的引导sgRNA一同转入宿主细胞，对基因进行编辑。

本发明中，所述单位点或多位点基因编辑，包括利用所述CRISPR-Cas9核酸酶对双链DNA进行剪切，并通过宿主细胞的修复系统对断裂的缺口进行修复。

本发明中，所述的单位点或多位点的基因编辑，是改变单位点或多位点编辑时的碱基突变特征。

与现有技术相比，本发明的(TSpCas9-V1/V2)，属于CRISPR-Cas9免疫系统，分别含有SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7、SEQ ID NO.8的核苷酸序列和氨基酸序列，能够提高基因编辑特异性，实现对基因组DNA片段的特定位置的精准编辑，具有潜在的精准生物医学应用价值。

附图说明

图1为Pet21-6His-TEV-TSpCas9-V1/V2质粒构建图示。

图2为Pet21-6His-TEV-TSpCas9-V1/V2-V1/V2质粒筛选与培养。

图3为含有TSpCas9-V1/V2的质粒测序。

图4为TSpCas9-V1/V2-V1/V2目标蛋白的纯化方法。

图5为TSpCas9-V1/V2-V1/V2目标蛋白纯化获取的过程。

图6为CRISPR-Cas9目标蛋白纯化的电泳鉴定。

图7为sgRNA的在靶和脱靶序列。

图8为野生型SpCas9、TSpCas9及TSpCas9-V1/V2体外剪切活性的检测。

图9为野生型SpCas9体外在靶和脱靶效应的检测。

图10为截短型TSpCas9体外在靶和脱靶效应的检测。

图11为截短变异体TSpCas9-V1体外在靶和脱靶效应的检测。

图12为截短变异体TSpCas9-V2体外在靶和脱靶效应的检测。

具体实施方式

下面通过具体实施例进一步描述本发明。

下述实施例中所用的实验方法，如无特定说明，均为常规方法。

下述实施例中所用的材料、试剂等，如无特定说明，均为从商业途径获得。

一、CRISPR-Cas9核酸酶

本发明的TSpCas9-V1核酸酶将截短型CRISPR-Cas9(TSpCas9)核酸酶的第863位的氨基酸H突变成N，TSpCas9-V2核酸酶将截短型CRISPR-Cas9(TSpCas9)核酸酶的第862位的氨基酸H突变成A、第863位的氨基酸H突变成N，属于CRISPR-Cas9系统，具有与野生型CRISPR-Cas9核酸酶相当的基因编辑活性，但比野生型更能特异性靶向基因编辑位点，实现精准编辑。

二、编码CRISPR-Cas9核酸酶的多核苷酸序列

转录和翻译所述CRISPR-Cas9(TSpCas9-V1/V2)的多核苷酸序列，包括DNA或RNA。DNA还可以细分为质粒DNA、基因组DNA或人工合成的DNA。

编码所述CRISPR-Cas9(TSpCas9-V1/V2)的多核苷酸序列，可以利用该领域科研或技术人员所熟悉的相关分子生物学技术来制备，其不局限于重组DNA技术和化学合成方法。

三、表达载体

所述表达载体含有编码所述CRISPR-Cas9核酸酶(TSpCas9-V1/V2)的多核苷酸序列。该表达载体可以通过科研或技术人员所熟悉的分子生物学方法来构建，包括DNA重组技术和DNA合成技术等，主要将CRISPR-Cas9核酸酶(TSpCas9-V1/V2)的DNA有效连接到载体上的克隆位点中，然后通过转录翻译等过程表达目的蛋白TSpCas9-V1/V2。

四、宿主细胞

所述宿主细胞是用来转化表达CRISPR-Cas9核酸酶的重组质粒。主要包括原核细胞(如细菌)，低等真核细胞(如酵母)，高等真核细胞(如哺乳动物细胞)等。常用的宿主细胞如大肠杆菌DH5α、毕赤酵母、HEK293、CHO、Hela细胞等。

五、CRISPR-Cas9核酸酶(TSpCas9-V1/V2)及其编码该酶的核苷酸序列和所述表达载体的用途

本发明的CRISPR-Cas9核酸酶(TSpCas9-V1/V2)及其编码该酶的多核苷酸序列和所述的表达载体能够用于基因组DNA片段的编辑或用于制备基因编辑工具。CRISPR-Cas9核酸酶(TSpCas9-V1/V2)编辑包括单位点和多位点编辑，其编辑手段包括删除、突变、插入、倒位、移位、重复或易位等。

六、基因编辑工具及其方法

本发明的基因编辑工具属于CRISPR-Cas9系统，CRISPR-Cas9(TSpCas9-V1/V2)在特定的sgRNA的引导下可以在目的基因DNA片段PAM(NGG)位点上游3到4位碱基间剪切底物DNA。该编辑过程可以在体内或体内进行。当sgRNA是单个的时候可以进行单点编辑，当sgRNA是两个或两个以上时可以进行多位点编辑。

如本发明的一些实施方式中所列举的，CRISPR-Cas9核酸酶(TSpCas9-V1/V2)在sgRNA的引导下，可以在体外对底物DNA(920bp)进行剪切，其产物长度分别为760bp和260bp。

在本发明中，Cas9可作为CRISPR-Cas9核酸酶的简称使用，其含义与CRISPR-Cas9核酸酶相同。本发明中的截短型高特异性TSpCas9-V1核酸酶是将截短型CRISPR-Cas9(TSpCas9)核酸酶的第863位的氨基酸H突变成N后表达得到的蛋白，TSpCas9-V2核酸酶是将截短型CRISPR-Cas9(TSpCas9)核酸酶的第862位的氨基酸H突变成A、第863位的氨基酸H突变成N后表达得到的蛋白。

在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围并不局限为下述特定的具体实施方案，还应理解为，本发明实施例中的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下例实施例中未注明具体条件的试验方法，通常按照常规条件操作，或者按照各生产厂商所建议的条件操作。

在实施例给出的数值范围中，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。

除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、重组DNA技术及相关领域的常规技术。

实施例1，构建CRISPR-Cas9核酸酶(TSpCas9-V1/V2)的质粒

1.变异体的设计

以pet21-6His-TEV-TSpCas9质粒，即SEQ ID NO.3(3744bp)为模板，将2587位的碱基C和2584-2587位的碱基CACC分别突变为A和GCCA，其余位置的碱基保持不变，将其分别命名为TSpCas9-V1和TSpCas9-V2。其改造设计思路如图1所示，其详细步骤简述如下：

利用引物F2587和R2587，F2584和R2584分别对质粒Pet21-6His-TEV-TSpCas9(相当于SEQ ID NO.3)进行点突变，然后质粒模板消化，即可获得目的产物TSpCas9-V1/V2。

(1)采购点突变试剂盒

所用点突变试剂盒Fast Site-Directed Mutagenesis Kit从天根生化科技(北京)有限公司订购。

(2)采购引物

所用引物均从上海生工生物工程有限公司订购。它们的序列如下：

2587位碱基C突变为A的正反引物：

F2587：GATCAACAATTACCACAATGCGCATGATGCC(SEQ ID NO.9)

R2587：TGTGGTAATTGTTGATCTCTCTCACCTTATA(SEQ ID NO.10)

2584-2587位的碱基CACC突变为GCCA的正反引物：

F2584：GATCAACAATTACGCCAATGCGCATGATGCCTAC(SEQ ID NO.11)

R2584：GTAGGCATCATGCGCATTGGCGTAATTGTTGATC(SEQ ID NO.12)。

点突变体系如下：

PCR反应条件：

点突变体系如下：

PCR反应条件：

该反应体系在37℃孵育1小时后，转化大肠杆菌DH5α(购于TIANGEN)37℃过夜培养筛选单克隆TSpCas9-V1/V2，如图2所示，其核苷酸序列和氨基酸分别为序列为SEQ IDNO.5、SEQ ID NO.6和SEQ ID NO.7、SEQ ID NO.8，培养细菌。

(3)采购质粒抽提试剂盒

所用质粒小提试剂盒TIANprep Mini Plasmid Kit从天根生化科技(北京)有限公司订购。

抽提质粒，抽提方法见天根质粒小提试剂盒使用说明。

(4)样品测序

在上海杰李生物技术有限公司通过一代测序对8个样品质粒进行测序，发现样品质粒1和6构建正确，其结果如图3所示。

实施例2，制备CRISPR-Cas9(TSpCas9-V1/V2)核酸酶

2.蛋白表达与纯化

2.1蛋白表达

(1)打开超净台，用含75％酒精的棉球擦拭桌面以及各种器具耗材，开紫外灯照射20min，启动风机备用；

(2)移液枪吸取10μl表达Pet21-6His-TEV-TSpCas9-V1/V2的Rosetta(DE3)(购于TIANGEN)菌液转至6ml含有双抗(Amp与Cm)的LB液体培养基中，37℃，200r/min振荡培养过夜；

(3)将过夜培养的菌液按照体积比为1：100转至500ml含双抗的LB(购于生工)液体培养基中，37℃，200r/min振荡培养。在培养过程中，随时检测菌液的OD值；

(4)当菌液的OD值接近0.4～0.8时，加入蛋白诱导剂IPTG，使其终浓度为0.1mM，然后16℃，200r/min振荡培养20h；

(5)收集菌液，5000r/min离心5min使菌体沉淀，弃上清，并称重Pet21-6His-TEV-TSpCas9-V1/V2菌体。

2.2蛋白纯化

所述蛋白纯化主要通过镍柱亲和层析技术，如图4所示；其纯化过程包括菌体破碎、蛋白样品离心收集、蛋白样品与镍柱介质共孵育以及目的蛋白的洗脱等，如图5所示。其详细步骤如下：

(1)向菌体中加入预先冰浴且PMSF终浓度为0.1mM的裂解液(20mM HEPES，500mMKCl，pH7.5；1g菌体加入5ml)，涡旋仪重悬使菌块分散混匀，细胞超声破碎仪破碎细胞，超声3sec停3sec，一次10min，超声两次，超声过程均在冰浴中进行；

(2)向破碎的菌液中加入终浓度为10μg/ml Rnase(生工)，5μg/ml DNase I(生工)，冰浴处理30min后，4℃10000r/min离心45～60min，收集上清；

(3)将上清与预先用平衡液(20mM HEPES，500mM KCl，1％蔗糖，pH7.5)处理的Qiagen Ni-NTA介质孵育，此过程在冰浴上进行，并加以振荡(150r/min)，1.5h后静置，待Qiagen Ni-NTA沉淀；

(4)将Qiagen Ni-NTA装载到重力柱中，BioLogic LP系统的监测下，分别以流速为2ml/min的平衡液和洗脱液(20mM HEPES，500mM KCl，500mM咪唑，1％蔗糖，pH7.5)，20、30、40、50、100、250、500Mm洗脱液冲洗Qiagen Ni-NTA，并收集蛋白；

(5)将不同咪唑浓度下的蛋白溶液跑SDS-PAGE(购于EpiZyme Scientific)电泳，考马斯亮蓝染色，脱色剂脱色，观察目的蛋白的表达和挂柱效果。

所述蛋白TSpCas9-V1/V2的纯化结果如图6所示，其显示该目的蛋白TSpCas9-V1/V2的纯化情况，杂条带较少，蛋白较为纯净。

实施例3，检验CRISPR-Cas9(TSpCas9-V1/V2)核酸酶剪切活性

3.变异体活性检测

所用底物DNA(SEQ ID NO.13)，主要利用引物QG-F和QG-R通过常规PCR扩增，然后割胶回收获取。

(1)采购扩增试剂盒

所用扩增试剂盒Fast HiFidelity PCR Kit从天根生化科技(北京)有限公司订购。

(2)采购引物

QG-F：TAGTCCTGTCGGGTTTCG(SEQ ID NO.14)

QG-R：TTCCATTCGCCATTCAGG(SEQ ID NO.15)。

其反应体系和扩增条件如下：

扩增体系如下：

PCR反应条件：

(3)采购割胶回收试剂盒

所用割胶回收试剂盒AxyPrep^TM DNA Gel Extraction Kit从Axygen公司订购，割胶回收操作均按其说明书进行，可以获得较纯的底物DNA(SEQ ID NO.13)。

Cas9与sgRNA以等摩尔混合，而根据实验需要，底物DNA可调为Cas9摩尔质量的0.2～1倍。其反应体系如下

将反应体系置37℃孵育，1h后70℃加热10min，最后通过琼脂糖凝胶电泳检测目的蛋白TSpCas9-V1/V2的体外切割活性。其结果如图8所示，从图8中可以发现，与野生型SpCas9和截短型TSpCas9相比，TSpCas9-V1和TSpCas9-V2在体外均能够剪切底物DNA (SEQID NO.13)(泳道5和6)，生成产物1和产物2。虽然TSpCas9-V1/V2在体外的DNA剪切活性并不比野生型SpCas9的强，但是其体积在小型化上却比野生型SpCas9有优势，即我们的TSpCas9-V1/V2体积比野生型SpCas9小，对方便腺病毒AAV运输而言，比野生型SpCas9有较大优势。

实施例4，CRISPR-Cas9(TSpCas9-V1/V2)体外脱靶检测的评价方法

4.脱靶效应检测

利用不同的sgRNA，如图7中1到8号所示检测TSpCas9-V1/V2的体外切割活性，从而评价TSpCas9-V1/V2的脱靶效应，其反应体系如下：

首先，评价野生型SpCas9在体外的脱靶效应，如图9所示，与0号sgRNA引导的SpCas9剪切活性相比(泳道3)，1到8号sgRNA引导的SpCas9均能够在体外剪切底物DNA(SEQID NO.13)(泳道4到11)，生成产物1和产物2。尽管只有部分sgRNA的引导活性强，即1到4号sgRNA引导的SpCas9体外剪切活性比较强(泳道4到7)，5到8号sgRNA引导的SpCas9体外剪切活性相对较弱(8号到11号)，但是，该结果依然反映野生型SpCas9在体外的脱靶效应比较严重。由此说明，野生型SpCas9有较强的脱靶效应，尤其在1到4号sgRNA的引导下更为突出。

其次，评价截短型TSpCas9在体外的脱靶效应，如图10所示，与0号sgRNA引导的TSpCas9剪切活性相比(泳道3)，1到8号sgRNA引导的截短型TSpCas9均能够在体外剪切底物DNA(SEQ ID NO.13)(泳道4到11)，生成产物1和产物2。尤其是1到4号sgRNA引导的截短型TSpCas9的体外剪切活性相对较强，由此说明截短型TSpCas9依然存在较强的脱靶效应。

再次，评价截短型TSpCas9-V1在体外的脱靶效应，如图11所示，与0号sgRNA引导的TSpCas9-V1剪切活性相比(泳道3)，1、2和4号sgRNA引导的截短型TSpCas9-V1体外剪切底物DNA(SEQ ID NO.13)的能力较为明显(泳道4、5、7)，生成产物1和产物2。其余sgRNA引导的TSpCas9-V1的在体外的切割活性非常弱，尤其是5到8号sgRNA引导的TSpCas9-V1几乎没有发挥切割作用，由此说明TSpCas9-V1能降低脱靶效应。

最后，评价截短型TSpCas9-V2在体外的脱靶效应，如图12所示，与0号sgRNA引导的TSpCas9-V2剪切活性相比(泳道3)，1和4号sgRNA引导的截短型TSpCas9-V2体外剪切底物DNA(SEQ ID NO.13)的能力明显减弱(泳道4和7)，生成产物1和产物2。而其余sgRNA引导的TSpCas9-V2的在体外几乎没有发挥切割作用，由此说明TSpCas9-V2能降低脱靶效应。

在sgRNA与底物DNA完全互补的情况下，TSpCas9-V1/V2保留了野生型SpCas9核酸酶的切割活性；同时，在sgRNA与底物DNA存在两个碱基错配的情况下，和野生型SpCas9和截短型TSpCas9相比较，TSpCas9-V1/V2对于对底物DNA的容错率更低，体外剪切特异性更高。即TSpCas9-V1/V2不仅具有野生型CRISPR-Cas9核酸酶基因编辑功能，而且与野生型核酸酶相比，更能特异性靶向基因编辑位点，实现精准编辑，同时体积更小，方便腺病毒AAV运输，比野生型SpCas9有更大优势，这对将来CRISPR-Cas9系统方便应用到临床医学上提供了潜在的价值。

参考文选

[1].Doudna,J.A.and E.Charpentier,Genome editing.The new frontier ofgenome engineering with CRISPR-Cas9.Science,2014.346(6213):p.1258096.

[2].Hsu,P.D.,E.S.Lander and F.Zhang,Development and applications ofCRISPR-Cas9 for genome engineering.Cell,2014.157(6):p.1262-78.

[3].Suzuki,K.,et al.,In vivo genome editing via CRISPR/Cas9 mediatedhomology-independent targeted integration.Nature,2016.540(7631):p.144-149.

[4].Jinek,M.,et al.,RNA-programmed genome editing in humancells.Elife,2013.2:p.e00471.

[5].Hille,F.,et al.,The Biology of CRISPR-Cas:Backward andForward.Cell,2018.172(6):p.1239-1259.

[6].Karginov,F.V.and G.J.Hannon,The CRISPR system:small RNA-guideddefense in bacteria and archaea.Mol Cell,2010.37(1):p.7-19.

[7].Niewoehner,J.,et al.,Increased brain penetration and potency of atherapeutic antibody using a monovalent molecular shuttle.Neuron,2014.81(1):p.49-60.

[8].Kleinstiver,B.P.,et al.,High-fidelity CRISPR-Cas9 nucleases withno detectable genome-wide off-target effects.Nature,2016.529(7587):p.490-5.

[9].Pattanayak,V.,et al.,High-throughput profiling of off-target DNAcleavage reveals RNA-programmed Cas9 nuclease specificity.Nat Biotechnol,2013.31(9):p.839-43.

[10].Jamal,M.,et al.,Keeping CRISPR/Cas on-Target.Curr Issues MolBiol,2016.20:p.1-12。

序列表

<110> 复旦大学

<120> 核酸内切酶SpCas9高特异性截短变异体TSpCas9-V1/V2及其应用

<130> 2019.06.28

<160> 16

<170> SIPOSequenceListing 1.0

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tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

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cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780

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atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320

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agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440

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tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700

actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2760

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080

gacctctctc agctcggtgg agac 4104

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His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

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Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

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Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

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Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

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Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

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Asp Leu Ser Gln Leu Gly Gly Asp

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<213> Streptococcus pyogenes serotype M1

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ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

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ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 600

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 660

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 720

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 780

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 840

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 900

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 960

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1020

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1080

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1140

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1200

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1260

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1320

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1380

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1440

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1500

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1560

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1620

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 1680

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 1740

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 1800

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 1860

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 1920

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 1980

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2040

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2100

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2160

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2220

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2280

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2340

actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2400

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2460

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2520

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2580

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 2640

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 2700

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 2760

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 2820

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 2880

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 2940

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3000

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3060

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3120

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3180

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3240

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3300

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3360

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3420

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3480

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3540

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3600

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 3660

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 3720

gacctctctc agctcggtgg agac 3744

<210> 4

<211> 1248

<212> PRT

<213> Streptococcus pyogenes serotype M1

<400> 4

Gly 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 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 Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile

180 185 190

Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His

195 200 205

His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro

210 215 220

Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala

225 230 235 240

Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile

245 250 255

Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys

260 265 270

Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly

275 280 285

Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg

290 295 300

Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile

305 310 315 320

Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala

325 330 335

Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr

340 345 350

Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala

355 360 365

Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn

370 375 380

Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val

385 390 395 400

Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys

405 410 415

Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu

420 425 430

Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr

435 440 445

Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu

450 455 460

Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile

465 470 475 480

Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu

485 490 495

Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile

500 505 510

Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met

515 520 525

Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg

530 535 540

Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu

545 550 555 560

Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu

565 570 575

Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln

580 585 590

Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala

595 600 605

Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val

610 615 620

Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val

625 630 635 640

Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn

645 650 655

Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly

660 665 670

Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn

675 680 685

Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val

690 695 700

Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His

705 710 715 720

Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val

725 730 735

Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser

740 745 750

Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn

755 760 765

Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu

770 775 780

Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln

785 790 795 800

Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp

805 810 815

Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu

820 825 830

Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys

835 840 845

Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala

850 855 860

His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys

865 870 875 880

Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

885 890 895

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

900 905 910

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

915 920 925

Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu

930 935 940

Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe

945 950 955 960

Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys

965 970 975

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

980 985 990

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

995 1000 1005

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1010 1015 1020

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val

1025 1030 1035 1040

Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys

1045 1050 1055

Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1060 1065 1070

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn

1075 1080 1085

Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn

1090 1095 1100

Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser

1105 1110 1115 1120

His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1125 1130 1135

Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln

1140 1145 1150

Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp

1155 1160 1165

Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu

1170 1175 1180

Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala

1185 1190 1195 1200

Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1205 1210 1215

Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1220 1225 1230

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1235 1240 1245

<210> 5

<211> 3744

<212> DNA

<213> Streptococcus pyogenes serotype M1

<400> 5

ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcatt 540

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 600

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 660

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 720

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 780

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 840

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 900

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 960

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1020

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1080

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1140

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1200

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1260

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1320

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1380

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1440

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1500

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1560

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1620

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 1680

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 1740

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 1800

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 1860

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 1920

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 1980

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2040

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2100

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2160

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2220

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2280

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2340

actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2400

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2460

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2520

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2580

taccacaatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 2640

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 2700

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 2760

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 2820

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 2880

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 2940

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3000

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3060

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3120

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3180

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3240

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3300

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3360

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3420

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3480

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3540

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3600

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 3660

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 3720

gacctctctc agctcggtgg agac 3744

<210> 6

<211> 1248

<212> PRT

<213> Streptococcus pyogenes serotype M1

<400> 6

Gly 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 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 Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile

180 185 190

Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His

195 200 205

His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro

210 215 220

Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala

225 230 235 240

Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile

245 250 255

Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys

260 265 270

Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly

275 280 285

Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg

290 295 300

Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile

305 310 315 320

Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala

325 330 335

Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr

340 345 350

Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala

355 360 365

Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn

370 375 380

Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val

385 390 395 400

Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys

405 410 415

Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu

420 425 430

Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr

435 440 445

Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu

450 455 460

Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile

465 470 475 480

Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu

485 490 495

Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile

500 505 510

Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met

515 520 525

Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg

530 535 540

Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu

545 550 555 560

Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu

565 570 575

Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln

580 585 590

Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala

595 600 605

Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val

610 615 620

Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val

625 630 635 640

Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn

645 650 655

Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly

660 665 670

Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn

675 680 685

Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val

690 695 700

Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His

705 710 715 720

Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val

725 730 735

Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser

740 745 750

Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn

755 760 765

Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu

770 775 780

Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln

785 790 795 800

Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp

805 810 815

Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu

820 825 830

Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys

835 840 845

Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His Asn Ala

850 855 860

His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys

865 870 875 880

Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

885 890 895

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

900 905 910

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

915 920 925

Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu

930 935 940

Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe

945 950 955 960

Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys

965 970 975

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

980 985 990

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

995 1000 1005

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1010 1015 1020

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val

1025 1030 1035 1040

Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys

1045 1050 1055

Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1060 1065 1070

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn

1075 1080 1085

Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn

1090 1095 1100

Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser

1105 1110 1115 1120

His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1125 1130 1135

Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln

1140 1145 1150

Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp

1155 1160 1165

Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu

1170 1175 1180

Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala

1185 1190 1195 1200

Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1205 1210 1215

Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1220 1225 1230

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1235 1240 1245

<210> 7

<211> 3744

<212> DNA

<213> Streptococcus pyogenes serotype M1

<400> 7

ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcatt 540

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 600

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 660

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 720

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 780

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 840

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 900

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 960

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1020

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1080

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1140

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1200

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1260

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1320

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1380

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1440

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1500

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1560

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1620

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 1680

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 1740

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 1800

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 1860

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 1920

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 1980

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2040

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2100

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2160

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2220

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2280

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2340

actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2400

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2460

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2520

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2580

tacgccaatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 2640

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 2700

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 2760

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 2820

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 2880

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 2940

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3000

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3060

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3120

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3180

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3240

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3300

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3360

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3420

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3480

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3540

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3600

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 3660

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 3720

gacctctctc agctcggtgg agac 3744

<210> 8

<211> 1248

<212> PRT

<213> Streptococcus pyogenes serotype M1

<400> 8

Gly 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 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 Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile

180 185 190

Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His

195 200 205

His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro

210 215 220

Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala

225 230 235 240

Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile

245 250 255

Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys

260 265 270

Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly

275 280 285

Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg

290 295 300

Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile

305 310 315 320

Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala

325 330 335

Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr

340 345 350

Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala

355 360 365

Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn

370 375 380

Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val

385 390 395 400

Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys

405 410 415

Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu

420 425 430

Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr

435 440 445

Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu

450 455 460

Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile

465 470 475 480

Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu

485 490 495

Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile

500 505 510

Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met

515 520 525

Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg

530 535 540

Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu

545 550 555 560

Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu

565 570 575

Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln

580 585 590

Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala

595 600 605

Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val

610 615 620

Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val

625 630 635 640

Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn

645 650 655

Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly

660 665 670

Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn

675 680 685

Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val

690 695 700

Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His

705 710 715 720

Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val

725 730 735

Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser

740 745 750

Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn

755 760 765

Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu

770 775 780

Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln

785 790 795 800

Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp

805 810 815

Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu

820 825 830

Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys

835 840 845

Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr Ala Asn Ala

850 855 860

His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys

865 870 875 880

Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

885 890 895

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

900 905 910

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

915 920 925

Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu

930 935 940

Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe

945 950 955 960

Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys

965 970 975

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

980 985 990

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

995 1000 1005

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1010 1015 1020

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val

1025 1030 1035 1040

Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys

1045 1050 1055

Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1060 1065 1070

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn

1075 1080 1085

Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn

1090 1095 1100

Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser

1105 1110 1115 1120

His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1125 1130 1135

Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln

1140 1145 1150

Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp

1155 1160 1165

Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu

1170 1175 1180

Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala

1185 1190 1195 1200

Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1205 1210 1215

Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1220 1225 1230

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1235 1240 1245

<210> 9

<211> 31

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

gatcaacaat taccacaatg cgcatgatgc c 31

<210> 10

<211> 31

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

tgtggtaatt gttgatctct ctcaccttat a 31

<210> 11

<211> 34

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 11

gatcaacaat tacgccaatg cgcatgatgc ctac 34

<210> 12

<211> 34

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

gtaggcatca tgcgcattgg cgtaattgtt gatc 34

<210> 13

<211> 920

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 60

ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 120

ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 180

taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 240

agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 300

gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 360

cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 420

ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 480

ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc tggagctcca 540

ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatatcaa 600

gcttatcgat taccgctcca gtcgttcatg aggttagagc tagaaatagc aagttaaaat 660

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctctc gagggggggc 720

ccggtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 780

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 840

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 900

agcctgaatg gcgaatggaa 920

<210> 14

<211> 18

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

tagtcctgtc gggtttcg 18

<210> 15

<211> 18

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

ttccattcgc cattcagg 18

<210> 16

<211> 3046

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 60

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 120

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 180

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 240

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 300

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 360

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 420

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 480

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 540

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 600

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 660

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 720

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 780

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 840

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 900

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 960

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1020

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1080

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1140

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1200

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1260

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1320

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1380

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1440

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1500

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1560

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1620

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1680

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1740

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1800

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1860

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1920

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1980

gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 2040

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 2100

gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 2160

agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc 2220

gctctagaac tagtggatcc cccgggctgc aggaattcga tatcaagctt atcgattacc 2280

gctccagtcg ttcatgaggt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2340

atcaacttga aaaagtggca ccgagtcggt gctctcgagg gggggcccgg tacccaattc 2400

gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga 2460

aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 2520

taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 2580

atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 2640

tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 2700

gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 2760

tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 2820

ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 2880

agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 2940

aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 3000

accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtg 3046

Claims

1.来源于酿脓链球菌的CRISPR-Cas9核酸酶SpCas9的截短型高特异性变异体，其特征在于，有两种，一种是将截短型CRISPR-Cas9（TSpCas9）核酸酶的第863位的氨基酸H突变成N，记为TSpCas9-V1核酸酶；一种是将截短型CRISPR-Cas9（TSpCas9）核酸酶的第862位的氨基酸H突变成A，第863位的氨基酸H突变成N，记为TSpCas9-V2核酸酶；两种截短型高特异性变异体记为TSpCas9-V1/V2；它们具有与野生型CRISPR-Cas9核酸酶相当的基因编辑活性，但比野生型特异性更高，能实现精准编辑；

所述野生型SpCas9核酸酶的核苷酸序列和氨基酸序列分别为SEQIDNO.1和SEQ IDNO.2所示；

所述截短型TSpCas9核酸酶的核苷酸序列和氨基酸序列分别为SEQ ID NO.3和SEQ IDNO.4所示；

所述截短型高特异性TSpCas9-V1核酸酶的核苷酸序列和氨基酸序列分别为SEQ IDNO.5和SEQ ID NO.6所示；

所述截短型高特异性TSpCas9-V2核酸酶的核苷酸序列和氨基酸序列分别为SEQ IDNO.7和SEQ ID NO.8所示。

2.一种多核苷酸序列，可以转录和翻译成如权利要求1所述的TSpCas9-V1或者转录和翻译成如权利要求1所述的TSpCas9-V2。

3.一种表达载体，其含有权利要求2所述的多核苷酸序列。

4.一种如权利要求1所述的截短型高特异性变异体的制备方法，其特征在于，具体步骤为：首先，构建所述CRISPR-Cas9核酸酶的多核苷酸序列表达载体；然后，将所述表达载体转化至宿主细胞，筛选并挑出单克隆；最后，将所述单克隆诱导表达，并通过亲和层析、离子交换方法从表达产物中分离出所述的CRISPR-Cas9核酸酶。

5.如权利要求1所述的截短型高特异性变异体、如权利要求2所述的多核苷酸序列、如权利要求3所述的表达载体作为编辑基因组DNA的编辑工具在基因组DNA片段的相关编辑中的应用。

6.根据权利要求5所述的应用，所述的编辑基因组DNA是单点编辑，或者是编辑位点大于等于两个的多点编辑；编辑的手段包括删除、突变、插入、倒位、移位、重复或易位。

7.根据权利要求6所述的应用，所述CRISPR-Cas9编辑工具包括与靶标DNA片段匹配的引导sgRNA；所述的CRISPR-Cas9核酸酶与能够介导它的sgRNA组合，对目的基因进行编辑。

8.根据权利要求5所述的应用，其特性在于，将权利要求3所述的表达载体和与之匹配的引导sgRNA一同转入宿主细胞，对基因进行编辑。

9.根据权利要求7所述的应用，其特性在于，所述单位点或多位点基因编辑，是利用权利要求1中所述TSpCas9-V1或TSpCas9-V2核酸酶对双链DNA进行剪切，并通过宿主细胞的修复系统对断裂的缺口进行修复。