一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用的制作方法

文档序号：12284572阅读：342来源：国知局

本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用，具体为以肿瘤特异靶点pl-CS为基础的嵌合抗原受体T(CAR-T)细胞技术的构建方法及其在抗肿瘤治疗中的应用。

背景技术：

嵌合抗原受体T细胞(CAR-T)技术是目前在进行大规模临床试验的新型细胞疗法。在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。其基本技术是通过基因工程手段，将T细胞改造成不依赖HLA的方式识别肿瘤抗原，使得CAR-T细胞相较于天然T细胞具有更强的识别和杀伤肿瘤细胞的能力。CAR-T的核心部件是CAR，CAR一般包括一个肿瘤相关抗原结合区，通常来源于肿瘤相关抗原的单克隆抗体scFV段；一个胞外铰链区；一个跨膜区和一个胞内刺激信号区。

CAR-T技术成功的关键是肿瘤抗原即靶点的选择。理想的抗原是在肿瘤细胞表面高表达，在正常细胞不表达的抗原。只有选择这样的抗原，才会使CAR-T细胞专一的结合并杀伤肿瘤细胞，而对正常细胞不结合，不会对正常组织造成伤害。但这样理想的抗原并不多，大多数抗原是在肿瘤组织中高表达或者过度表达，在正常组织中低表达。目前，CAR-T在实体肿瘤的治疗远不及白血病的治疗效果，其原因之一就是各实体瘤的中均没有找到非常理想的靶标。

CAR-T技术用于治疗实体瘤的效果不好的另一个原因是肿瘤免疫抑制微环境的存在。肿瘤免疫抑制微环境是肿瘤发生发展过程中形成的内环境，由肿瘤细胞，间质细胞，免疫调节细胞，微血管，组织液，细胞因子，细胞外基质等组成。由于免疫微环境的存在，免疫杀伤细胞无法有效到达肿瘤细胞部位或者到达目标部位后无法对肿瘤细胞进行有效的杀伤。CAR-T细胞要对实体瘤进行有效的治疗，必需突破肿瘤免疫微环境限制，进入实体瘤实现对肿瘤细胞的杀伤。

CN 104788573 A公开了一种嵌合抗原受体hCD19scFv-CD8α-CD28-CD3ζ及其用途，该嵌合抗原受体由抗人CD19单克隆抗体HI19a轻链和重链可变区(hCD19scFv)、人CD8α铰链区、人CD28跨膜区和胞内区、以及人CD3ζ胞内区结构串联构成。该嵌合抗原受体用于修饰T淋巴细胞，修饰后的T细胞(CAR-T细胞)能用于表面CD19阳性的肿瘤的治疗。该专利的嵌合抗原受体只针对于表面CD19阳性的肿瘤，且识别效果不好，因此，筛选合适的肿瘤靶标，同时还能突破肿瘤微环境，是CAR-T技术治疗实体瘤的关键。

硫酸软骨素(CS)是共价连接在蛋白质上形成蛋白聚糖的一类糖胺聚糖。硫酸软骨素广泛分布于动物组织的细胞外基质和细胞表面，糖链由交替的葡萄糖醛酸和N-乙酰半乳糖胺(又称N-乙酰氨基半乳糖)二糖单位组成，通过一个似糖链接区连接到核心蛋白的丝氨酸残基上。硫酸软骨素存在于从线虫到人除植物外的所有生物中，发挥着许多重要的生理功能。虽然多糖的主链结构并不复杂，但就硫酸化程度、硫酸基和两种差异向异构糖醛酸在链内的分布来说，呈现高度的不均一性。硫酸软骨素的精细结构决定着功能的特异性和与多种蛋白质分子的相互作用。

胎盘中存在着一种特殊的硫酸软骨素，其糖基化模式与常规的硫酸软骨素不同，疟原虫中存在一种名为VAR2CSA蛋白，可以特异的结合胎盘中的硫酸软骨素，而在肿瘤细胞的表面，存在着胎盘样硫酸软骨素(pl-CS)，因此，pl-CS是CAR-T技术治疗肿瘤的理想靶点。

技术实现要素：

针对目前CAR-T技术治疗肿瘤中靶点选择不十分理想，以及肿瘤微环境影响CAR-T技术治疗效果的情况，本发明提供一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用，所述嵌合抗原受体，可以更特异的识别肿瘤细胞，对肿瘤微环境具有更好的靶向性。

为达此目的，本发明采用以下技术方案：

一方面，本发明提供一种抗胎盘样硫酸软骨素的嵌合抗原受体，所述嵌合抗原受体主要包括抗pl-CS的抗原识别区、铰链区、跨膜区和胞内区串联构成；

其中，所述抗pl-CS的抗原识别区为疟原虫蛋白VAR2CSA、疟原虫蛋白VAR2CSA的部分肽段或pl-CS抗体中的任意一种；

所述疟原虫蛋白VAR2CSA的部分肽段为疟原虫蛋白VAR2CSA中的氨基酸数量大于500的肽段。

本发明中，所述疟原虫蛋白VAR2CSA的部分肽段指的是疟原虫蛋白VAR2CSA中的氨基酸数量大于500的肽段，可以是疟原虫蛋白VAR2CSA中的任意500个氨基酸或是500个氨基酸以上的肽段都是可行的。本发明中，疟原虫蛋白VAR2CSA、疟原虫蛋白VAR2CSA的部分肽段ID1-ID2a和pl-CS抗体均可以特异性结合pl-CS，pl-CS几乎存在于所有类型的肿瘤细胞表面，而正常细胞的表面不存在pl-CS；选择pl-CS为肿瘤的特异性靶点，以VAR2CSA作为CAR的识别结构域来识别肿瘤的pl-CS，通过VAR2CSA识别pl-CS的方式，可以使CAR-T细胞特异的识别肿瘤细胞，更容易靶向到肿瘤微环境，进入到肿瘤组织内部，而不会对正常的细胞和组织造成伤害；此外，pl-CS还是肿瘤细胞外基质的组成部分，是形成肿瘤微环境的物质基础，选择pl-CS为靶点，有利于CAR-T细胞突破肿瘤免疫抑制微环境。

优选地，所述疟原虫蛋白VAR2CSA为疟原虫pf 3D7的VAR2CSA蛋白和/或疟原虫pf FCR3的VAR2CSA蛋白。

优选地，所述疟原虫pf 3D7的VAR2CSA蛋白的氨基酸序列为SEQ ID NO.3，核酸序列为SEQ ID NO.4。

优选地，所述疟原虫pf FCR3的VAR2CSA蛋白的氨基酸序列为SEQ ID NO.5，核酸序列为SEQ ID NO.6。

优选地，所述疟原虫蛋白VAR2CSA的部分肽段为ID1-ID2a，所述ID1-ID2a的氨基酸序列为SEQ ID NO.7，核酸序列为SEQ ID NO.8。

本发明中，所述ID1-ID2a是这个部分肽段中，长度比较小，是效果最好的一段。

优选地，所述铰链区为人CD8α铰链区。

优选地，所述人CD8α铰链区的氨基酸序列为SEQ ID NO.9，核酸序列为SEQ ID NO.10。

优选地，所述跨膜区为人CD28跨膜区。

优选地，所述人CD28跨膜区的氨基酸序列为SEQ ID NO.11，核酸序列为SEQ ID NO.12。

作为优选技术方案，所述胞内区为人CD3ζ胞内区、人CD28胞内区或人4-1BB胞内区中的任意一种或至少两种的组合，例如可以是CD3ζ胞内区、人CD28胞内区、人4-1BB胞内区、人CD28胞内区与人CD3ζ胞内区的串联、人4-1BB胞内区与人CD3ζ胞内区的串联、人CD3ζ胞内区与人4-1BB胞内区的串联或人CD28胞内区、人4-1BB胞内区与人CD3ζ胞内区的串联，优选为CD3ζ胞内区、人CD28胞内区与人CD3ζ胞内区的串联、人4-1BB胞内区与人CD3ζ胞内区的串联或人CD28胞内区、人4-1BB胞内区与人CD3ζ胞内区的串联。

优选地，所述人CD3ζ胞内区的氨基酸序列为SEQ ID NO.13，核酸序列为SEQ ID NO.14。

优选地，所述人CD28胞内区与人CD3ζ胞内区的串联的氨基酸序列为SEQ ID NO.15，核酸序列为SEQ ID NO.16。

优选地，所述人4-1BB胞内区与人CD3ζ胞内区的串联的氨基酸序列为SEQ ID NO.17，核酸序列为SEQ ID NO.18。

优选地，所述人CD28胞内区、人4-1BB胞内区与人CD3ζ胞内区的串联的氨基酸序列为SEQ ID NO.19，核酸序列为SEQ ID NO.20。

优选地，所述嵌合抗原受体的氨基末端含有一个人CD8α信号肽。

优选地，所述人CD8α信号肽的氨基酸序列为SEQ ID NO.21，核酸序列为SEQ ID NO.22。

作为优选技术方案，所述嵌合抗原受体由人CD8α信号肽、VAR2CSA、人CD8α铰链区、人CD28跨膜区和胞内区、人4-1BB胞内区以及人CD3ζ胞内区串联构成。

优选地，所述嵌合抗原受体的氨基酸序列为SEQ ID NO.1，核酸序列为SEQ ID NO.2。

优选地，所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。

优选地，所述转染的方式为病毒载体、真核表达质粒或mRNA序列中的任意一种或至少两种的组合。

优选地，所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体中的任意一种或至少两种的组合。

第二方面，本发明提供一种组合物，所述组合物包括第一方面所述的嵌合抗原受体。

第三方面，本发明提供如第一方面所述的嵌合抗原受体和/或如第二方面所述的组合物在制备嵌合抗原受体T细胞及其在肿瘤治疗药物中的应用。

与现有技术相比，本发明具有如下有益效果：

(1)本发明的嵌合抗原受体，通过VAR2CSA识别pl-CS的方式，可以使CAR-T细胞特异的识别肿瘤细胞，更容易靶向到肿瘤微环境，进入到肿瘤组织内部，可以更特异的识别肿瘤细胞，对肿瘤微环境具有更好的靶向性；

(2)本发明表达此嵌合抗原受体的T细胞能最大程度的杀伤肿瘤细胞，对正常组织的伤害程度很小，能突破肿瘤免疫抑制微环境，从而对实体瘤具有更好的疗效，且几乎能治疗各种类型的肿瘤。

附图说明

图1为靶向pl-CS的CAR慢病毒表达载体示意图；

图2(A)为荧光显微镜下观察慢病毒感染96h后T细胞的绿色荧光(GFP)表达情况，图2(B)为流式细胞仪显示慢病毒感染96h后T细胞的GFP阳性率；

图3(A)为实验组、阴性对照组、阳性对照组和野生型T细胞组上清中IL-2浓度比较，图3(B)为实验组、阴性对照组、阳性对照组和野生型T细胞组上清中IFN-γ浓度比较；

图4为在不同效靶比例情况下，实验组、阴性对照组和野生型T细胞组对B细胞淋巴瘤细胞系Raji的细胞毒性比较，其中，实验组、阴性对照组合野生组的柱状图从左到右分别为效靶比8:1、4:1和1:1。

具体实施方式

为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。

实施例1：靶向pl-CS的CAR基因序列的确定

从GenBank数据库以及文献中搜索到疟原虫VAR2CSA基因、人CD8α信号肽基因、人CD8α铰链区基因、人CD28跨膜区和胞内区基因、人4-1BB胞内区基因以及人CD3ζ胞内区基因序列。将其进行密码子优化，保证在编码氨基酸序列不变的情况下更适合人类细胞表达。

各基因序列信息见SEQUENCE LISTING(序列表SEQ ID NO.1-22)。

实施例2：慢病毒表达载体pLHlentiCAR004的构建

将上述基因序列依次按人CD8α信号肽基因、VAR2CSA、人CD8α铰链区基因、人CD28跨膜区和胞内区基因、人4-1BB胞内区基因以及人CD3ζ胞内区基因序列进行连接，组成嵌合抗原受体，所述抗原受体的核酸序列如SEQ ID NO.2所示，引入合适的酶切位点，进行人工全基因合成，克隆到慢病毒表达载体中得到pLHlentiCAR004质粒。质粒示意图见图1。

将重组质粒送上海英骏生物技术有限公司进行测序，测序结果与拟合成的序列比对以证实序列完全正确。

实施例3：慢病毒表达载体和包装载体的大量提取

将pLHlentiCAR004质粒以及psPAX2和pMD2.G包装质粒的stbl3大肠杆菌在LB培养液中大量培养，以碱裂解法大量提取质粒，以备转染，具体过程为：

1)分别将带有pLHlentiCAR004、psPAX2、pMD2.G的stbl3大肠杆菌种接种于含有500mL LB/抗生素培养液的培养瓶中，37℃摇床培养12-16小时；

2)3000*g离心10min，收集菌体，倒掉培养基并反扣于吸水纸轻轻拍打以吸尽残液。收集的菌体采用广州美基生物科技有限公司MaxPure Plasmid Maxi Kit大提质粒，提取质粒的具体步骤为：

(1)加入20mL BufferP1/RNase A至菌体中，涡旋重悬细菌(确保细菌完全重悬，重悬后的溶液没有细胞团块)；向重悬液中加入20mL Buffer P2，颠倒混匀10-15次，静置1-2min(一定要轻轻混匀，注意操作时间不能超过4分钟)；

(2)向裂解液中加入20mL Buffer E3，立即颠倒混匀15-20次，混匀过程要轻柔而充分，中和充分的标准是：溶液变得不粘稠，沉淀物分散显白色；3000*g离心10min，然后转移上清液至新的离心管中，加入1/3倍体积Buffer E4至上清液中，涡旋混匀；

(3)将MaxPure Micro Column套在50mL离心管中，转移15mL混合液至柱子中，3000*g离心10min；倒掉流出液，把柱子套回收集管中，继续转移剩下的溶液至柱子中，3000*g离心10分钟；倒掉流出液，把柱子套回收集管中，加入20mL Buffer E5至柱子，3000*g离心10分钟；倒掉滤液把柱子套回收集管中；

(4)加入20mL Buffer PW2(已用无水乙醇稀释)至柱子，3000*g离心10分钟；倒掉滤液把珠子套回收集管中，加入20mL Buffer PW2(已用无水乙醇稀释)至柱子，3000*g离心10分钟；倒弃滤液把柱子套回收集管中，4000*g离心10分钟；

(5)把柱子套在灭菌的50mL离心管中，加入30-200mL灭菌水至柱子的膜中央，静置2分钟，10000*g离心5min，收集分离液；

(6)进一步抽提去除内毒素，用灭菌水调整质粒浓度在0.1g-0.6mg/mL之间；

(7)加入0.1倍体积Buffer ER1和0.1倍体积Buffer ER2至质粒溶液中，颠倒混匀；冰上放置20min，期间颠倒混匀多次；42℃水浴5min后室温在10000*g速度下离心3min；

(8)轻轻取出样品，发现底部形成了一层红色的溶液，小心把上清液转移到离心管仲，加入0.7倍体积的异丙醇至上清液中，颠倒混匀多次；室温下，在10000*g速度下离心10min，倒掉上清液，加入1倍体积的70％乙醇至沉淀中。涡旋混匀15-30s；室温下，在10000*g速度下离心3min，小心倒掉上清液；室温下，在10000*g速度下离心1min收集管壁上的液滴，不要吸到沉淀，在空气中干燥10min；

(9)加入适量无菌水到质粒中，涡旋10-20s混匀，室温静置30min让质粒充分溶解，期间颠倒混匀多次；

在Nanodrop紫外分光光度计上测量质粒浓度，然后将质粒保存在-20℃中。

实施例4：慢病毒的包装、浓缩和滴定

1)慢病毒的包装

(1)细胞预板：在转染前24h，取长满的293T细胞进行预板，以第二天能达到70％汇合度为宜(75mm培养皿内预板500万)。

(2)第二天：转染

(a)观察预板细胞的生长情况，以细胞的汇合度在90％左右为宜，吸去培养基，加入新鲜预热的完全培养基(75mm培养皿内15mL)；

(b)转染前将转染所需的PEI、质粒、Opti-MEM I Reduced Serum Medium等放置室温平衡；

(c)制备质粒的混合液：本次包装共10皿293T细胞，每一皿细胞准备混合液500μL，故需准备混合液总体积为5mL；先根据各种质粒用量加入三种质粒，共420μg(pLHlentiCAR004:psPAX2:pMD2.G＝3:2:1)，加入Opti-MEM I Reduced Serum Medium调整体积到2.5mL，吹打混匀后室温放置10min；再取PEI和Opti-MEM I Reduced Serum Medium各1.25mL，混匀后室温放置10min，最后将以上两种溶液混合，混匀后室温放置10min，即为转染用的混合液；

(d)混合液逐滴加入培养皿中，每皿500μL；

(3)转染6h以后，需把每皿培养基换成17mL UltraCLUTURE无血清培养基；

(4)第三至五天，病毒的收集：转染48h-72h，分别收集培养上清，收集的病毒上清暂存于4℃。

2)慢病毒浓缩

将收集的培养上清用0.45um的膜过滤后用SW-32Ti的转子25000rpm(对应离心力为106750g)，4℃离心2h，去上清后，用200uL的PBS重悬，保存于-80℃。

3)慢病毒的滴定

(1)6孔板中预铺板293T细胞，3×10⁵cells/well，37℃5％CO₂培养箱中培养过夜(大约18-20h)；

(2)配制polybrene母液：灭菌纯水，8mg/mL，0.22μm滤头过滤，分装，保存于-20℃备用；

(3)配制含polybrene的DMEM培养基：10％FBS，1％双抗，8μg/mL polybrene(培养基体积的千分之一)；

(4)融化病毒：从-80℃取出病毒冻存液，在室温融化，融化后置于冰上拿进细胞间备用；

(5)稀释病毒：用配好的含polybrene的5％FBS DMEM完全培养基将病毒原液进行10倍稀释，得到10^-1-10^-4的病毒稀释液，注意每次吸取病毒液进行下一步稀释前要混匀；

(6)弃掉旧培养基，第一孔中加入1mL不含病毒的培养基作为阴性对照。其余每孔中加入1mL对应的病毒稀释液，37℃培养18-20h；

(7)第二天早上去掉含病毒的培养基，替换以2mL不含聚凝胺的5％FBS DMEM培养基；显微镜下观察细胞有表达GFP；

(8)吸去培养基，然后用胰酶消化细胞；将细胞吹成单个细胞，加入适量完全培养基终止胰酶反应，300g离心3min收集细胞；

(9)倒去上清，用适量预冷PBS重悬后，300g离心3min，收集细胞，再重复一次；

(10)最后用1mL预冷PBS重悬，置于冰上，用于流式分析。

(11)选择GFP阳性率在1％-20％的孔，各孔分别计算病毒滴度，最后取平均值；

计算公式：titer＝{(F×Cn)/V}×DF，单位为TU/mL；

其中，F：GFP阳性细胞率；Cn：每孔铺板的细胞数量；V：所加入病毒稀释液体积；DF：病毒稀释倍数

计算得到病毒滴度为：1.6×10⁸-1×10¹⁰之间。

实施例5：T细胞的分离，培养和感染

1)PBMC细胞的分离

(1)采血50mL，800g离心10min(加速设置为3，降速设置为4)；

(2)取白细胞层，用2％FBS稀释至8mL；

(3)吸取4mL LymphoPrep加入15mL离心管中，再将稀释血液小心沿管壁加至分层液上，保持两者界面清晰；

(4)800g离心20min(加速设置为6，降速设置为1)；

(5)用巴氏吸管轻轻吸出灰白色的单个核细胞，加入另一支已含有10mL RF-10的离心管中，混匀；

(6)以500g离心5min，弃上清；

(7)加10mL RF-10重悬细胞，trypan blue计数，350g离心10min，弃上清。

2)磁珠分选CD4+T(CD8+T)细胞

(1)涡旋混匀磁珠，取25μL磁珠于试管中，加入1mL Buffer 1混匀，将试管放在磁力架上1min，去上清。加25μL Bffer1重悬磁珠备用；

(2)PBMC用Buffer 1重悬至密度为107个/mL；

(3)向1mL的PBMC中加入25μL洗涤的磁珠，2-8℃孵育20min，放在摇床上倾斜旋转；

(4)将试管放在磁铁上2min，收集上清；

(5)移开试管，加1mL Buffer 1，吹打混匀，放在磁珠上2min，收集上清，重复一次；

(6)加100μL Buffer 2重悬细胞，加10μL DETCHaBEAD，室温下孵育45min使细胞从磁珠上释放；

(7)将试管放在磁力架上1min，含有细胞的上清转移到新的试管中，加500μL Buffer 2洗涤磁珠2-3次，收集上清；

(8)加4mL Buffer 2，350g离心5min，去上清，用Buffer 2重悬细胞；

(9)分选CD4+T细胞过程中收集的上清按试剂盒操作收集CD8+T细胞。

3)T细胞的培养

(1)磁珠分选后的CD4+T(CD8+T)细胞350g离心10min；

(2)RF-10重悬计数；

(3)按照1：1的比例加CD4+T和CD8+T细胞培养板中培养，细胞密度为5×10⁵个/mL；

(4)在T细胞专用培养基加入CD3/CD28抗体磁珠，加入磁珠的量按与细胞的比例1:1加入；

(5)加rhIL-2，使终浓度为10ng/mL；

(6)每周计数2-3次，记录细胞的增殖情况。

4)T细胞的慢病毒转染。

(1)磁珠分选后的CD4+T(CD8+T)细胞350g离心10min，加完全培养基重悬计数；

(2)按照1：1的比例加CD4+T和CD8+T细胞于96孔板中培养，细胞密度为5×10⁵个/mL，每孔细胞数为1×10⁵个；

(3)洗涤磁珠，向培养皿中加入与细胞1:1比例的磁珠；

(4)加rhIL-2，使终浓度为10μg/L；

(5)刺激24h后，感染细胞，加polybrene，使终浓度为6μg/mL，混匀；

(6)16-24h后换液；

(7)3天后进行荧光显微镜拍照。

荧光显微镜的结果图如图2(A)，流式检测感染效率如图2(B)所示，可见，流式检测的感染效率为50％左右，可以高效制得CAR-T细胞。

实施例6：CAR-T细胞体外细胞因子释放及检测

(1)48孔板中，每孔CAR-T细胞1.5×10⁵个与Huh7细胞每孔1.5×10⁵个共培养24h，加500μL RPMI-1640培养基(不含血清和rhIL-2)；

(2)样品收集和储存：离心收集上清，直接开始实验或分装保存于-20℃(100μL每管)；

(3)试剂准备：使用前将所有试剂拿出恢复至室温，用去离子水将Wash Buffer浓缩液稀释至500mL；底物显色液，将A与B等体积混合，15min内用完，每孔200μL混合液；细胞因子标准品，去500μL储存液，梯度浓度稀释6组；

(4)将样品以及浓度梯度稀释的标准品加入已包被抗体的ELISA孔板中，100μL每孔，除去气泡室温孵育2h(样品每组设三个复孔)；

(5)移去孔内液体，每孔加300μLWB洗涤，洗涤三次，最后一次吸干WB；

(6)加200μL对应的检测抗体，室温孵育2h；

(7)移去孔内液体，每孔加300μLWB洗涤，洗涤三次，最后一次吸干WB；

(8)加200μL底物液，室温孵育20min(避光)；

(9)加50μL终止液，溶液由蓝色变为黄色，如果颜色为绿色或者没有改变颜色，轻拍使其混匀；

(10)30min内检测450nm处的吸光度；

(11)根据标准曲线计数各细胞因子浓度，

结果如图3(A)和3(B)所示，可见，CAR-T细胞与肿瘤细胞共培养不会释放细胞因子，表明CAR-T细胞不会具有强烈的细胞因子风暴有害作用，也就不会引起炎症反应的副作用。

实施例7：CAR-T细胞体外杀伤肿瘤细胞

1)确定最佳的细胞铺板浓度

(1)收集各肿瘤细胞，用分析培养基洗涤，调整细胞浓度为1×10⁵个/mL；

(2)在96孔板中加100μL分析用培养基，铺满；

(3)加100μL细胞悬液于96孔板中，依次稀释，最后一个浓度梯度吸出100μL细胞悬液。设置六个复孔，三孔为High control组，三孔为Low control组，并设置三孔无细胞的分析培养基作为Background control(量程，500-50000)；

(4)培养箱孵育18h；

(5)HC组每孔加入5μL细胞溶解液，孵育15min，震动加速细胞溶解；

(6)每孔加入100μL现配的反应液，15℃-25℃孵育30min(注意96孔板避光)；

(7)每孔加入50μL终止液，震动10s；

(8)490nm测吸光度；

(9)分析数据，确定最佳细胞浓度(HC与LC的最大差值)。

2)细胞介导的毒性检测

(1)收集CAR-T细胞，用分析培养基洗涤细胞；

(2)根据不同的效靶比和1)中结果的最佳浓度来确定铺于96孔板中的CAR-T细胞数；

(3)在96孔板中铺的相应细胞悬液每孔体积调为50μL；

(4)收集各肿瘤细胞，调整细胞浓度为最佳浓度的2倍，加50μL细胞悬液于T细胞中；

(5)设置不同对照组；

(6)37度培养箱孵育18h；

(7)每孔加入5μL细胞溶解液，孵育15min，震动加速细胞溶解；

(8)每孔加入100μL现配的反应液，15℃-25℃孵育30min(注意96孔板避光)；

(9)每孔加入50μL终止液，震动10s；

(10)490nm测吸光度；

(11)计算每个样品细胞毒性的百分比。

结果如图4所示，不同效靶比的CAR-T细胞具有对肿瘤细胞的毒性，能高效的杀伤肿瘤细胞，释放LDH，作为对照组的野生型T细胞，不能杀伤肿瘤细胞。

综上所述，本发明表达此嵌合抗原受体的T细胞能最大程度的杀伤肿瘤细胞，不释放细胞因子，对正常组织的伤害很小，能突破肿瘤免疫抑制微环境，从而对实体瘤具有更好的疗效，且几乎能治疗各种类型的肿瘤。

申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

序列表

<110> 广州中科蓝华生物科技有限公司

<120> 一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用

<130> 2015

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 900

<212> PRT

<213> 人工合成序列

<400> 1

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

1 5 10 15

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

20 25 30

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

35 40 45

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

50 55 60

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

65 70 75 80

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

85 90 95

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

100 105 110

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

115 120 125

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

130 135 140

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

145 150 155 160

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

165 170 175

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

180 185 190

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

195 200 205

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

210 215 220

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

225 230 235 240

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

245 250 255

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

260 265 270

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

275 280 285

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

290 295 300

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

305 310 315 320

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

325 330 335

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

340 345 350

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

355 360 365

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

370 375 380

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

385 390 395 400

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

405 410 415

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

420 425 430

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

435 440 445

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

450 455 460

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

465 470 475 480

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

485 490 495

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

500 505 510

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

515 520 525

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

530 535 540

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

545 550 555 560

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

565 570 575

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

580 585 590

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

595 600 605

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

610 615 620

Arg Ser Asn Ser Ser Lys Leu Asp Thr Thr Thr Pro Ala Pro Arg Pro

625 630 635 640

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

645 650 655

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

660 665 670

Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu

675 680 685

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

690 695 700

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

705 710 715 720

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

725 730 735

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu

740 745 750

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

755 760 765

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

770 775 780

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

785 790 795 800

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

805 810 815

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

820 825 830

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

835 840 845

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

850 855 860

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

865 870 875 880

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

885 890 895

Leu Pro Pro Arg

900

<210> 2

<211> 2700

<212> DNA

<213> 人工合成序列

<400> 2

aactacatca agggcgaccc ctacttcgcc gagtacgcca ccaagctgag cttcatcctg 60

aacagcagcg acgccaacaa ccccagcgag aagatccaga agaacaacga cgaggtgtgc 120

aactgcaacg agagcggcat cgccagcgtg gagcaggagc agatcagcga ccccagcagc 180

aacaagacct gcatcaccca cagcagcatc aaggccaaca agaagaaggt gtgcaagcac 240

gtgaagctgg gcgtgcgcga gaacgacaag gacctgcgcg tgtgcgtgat cgagcacacc 300

agcctgagcg gcgtggagaa ctgctgctgc caggacttcc tgcgcatcct gcaggagaac 360

tgcagcgaca acaagagcgg cagcagcagc aacggcagct gcaacaacaa gaaccaggag 420

gcctgcgaga agaacctgga gaaggtgctg gccagcctga ccaactgcta caagtgcgac 480

aagtgcaaga gcgagcagag caagaagaac aacaagaact ggatctggaa gaagagcagc 540

ggcaaggagg gcggcctgca gaaggagtac gccaacacca tcggcctgcc cccccgcacc 600

cagagcctgt gcctggtggt gtgcctggac gagaagggca agaagaccca ggagctgaag 660

aacatccgca ccaacagcga gctgctgaag gagtggatca tcgccgcctt ccacgagggc 720

aagaacctga agcccagcca cgagaagaag aacgacgaca acggcaagaa gctgtgcaag 780

gccctggagt acagcttcgc cgactacggc gacctgatca agggcaccag catctgggac 840

aacgagtaca ccaaggacct ggagctgaac ctgcagaaga tcttcggcaa gctgttccgc 900

aagtacatca agaagaacaa caccgccgag caggacacca gctacagcag cctggacgag 960

ctgcgcgaga gctggtggaa caccaacaag aagtacatct ggctggccat gaagcacggc 1020

gccggcatga acagcaccac ctgctgcggc gacggcagcg tgaccggcag cggcagcagc 1080

tgcgacgaca tccccaccat cgacctgatc ccccagtacc tgcgcttcct gcaggagtgg 1140

gtggagcact tctgcaagca gcgccaggag aaggtgaagc ccgtgatcga gaactgcaag 1200

agctgcaagg agagcggcgg cacctgcaac ggcgagtgca agaccgagtg caagaacaag 1260

tgcgaggtgt acaagaagtt catcgaggac tgcaagggcg gcgacggcac cgccggcagc 1320

agctgggtga agcgctggga ccagatctac aagcgctaca gcaagtacat cgaggacgcc 1380

aagcgcaacc gcaaggccgg caccaagaac tgcggcccca gcagcaccac caacgccgcc 1440

gagaacaagt gcgtgcagag cgacatcgac agcttcttca agcacctgat cgacatcggc 1500

ctgaccaccc ccagcagcta cctgagcatc gtgctggacg acaacatctg cggcgccgac 1560

aaggccccct ggaccaccta caccacctac accaccaccg agaagtgcaa caaggagacc 1620

gacaagagca agctgcagca gtgcaacacc gccgtggtgg tgaacgtgcc cagccccctg 1680

ggcaacaccc cccacggcta caagtacgcc tgccagtgca agatccccac caacgaggag 1740

acctgcgacg accgcaagga gtacatgaac cagtggagct gcggcagcgc ccgcaccatg 1800

aagcgcggct acaagaacga caactacgag ctgtgcaagt acaacggcgt ggacgtgaag 1860

cccaccaccg tgcgcagcaa cagcagcaag ctggacacca ccacccccgc cccccgcccc 1920

cccacccccg cccccaccat cgccagccag cccctgagcc tgcgccccga ggcctgccgc 1980

cccgccgccg gcggcgccgt gcacacccgc ggcctggact tcgcctgcga cttctgggtg 2040

ctggtggtgg tgggcggcgt gctggcctgc tacagcctgc tggtgaccgt ggccttcatc 2100

atcttctggg tgcgcagcaa gcgcagccgc ctgctgcaca gcgactacat gaacatgacc 2160

ccccgccgcc ccggccccac ccgcaagcac taccagccct acgccccccc ccgcgacttc 2220

gccgcctacc gcagcaagcg cggccgcaag aagctgctgt acatcttcaa gcagcccttc 2280

atgcgccccg tgcagaccac ccaggaggag gacggctgca gctgccgctt ccccgaggag 2340

gaggagggcg gctgcgagct gcgcgtgaag ttcagccgca gcgccgacgc ccccgcctac 2400

cagcagggcc agaaccagct gtacaacgag ctgaacctgg gccgccgcga ggagtacgac 2460

gtgctggaca agcgccgcgg ccgcgacccc gagatgggcg gcaagcccca gcgccgcaag 2520

aacccccagg agggcctgta caacgagctg cagaaggaca agatggccga ggcctacagc 2580

gagatcggca tgaagggcga gcgccgccgc ggcaagggcc acgacggcct gtaccagggc 2640

ctgagcaccg ccaccaagga cacctacgac gccctgcaca tgcaggccct gcccccccgc 2700

<210> 3

<211> 2646

<212> PRT

<213> 人工合成序列

<400> 3

Met Asp Lys Ser Ser Ile Ala Asn Lys Ile Glu Ala Tyr Leu Gly Ala

1 5 10 15

Lys Ser Asp Asp Ser Lys Ile Asp Gln Ser Leu Lys Ala Asp Pro Ser

20 25 30

Glu Val Gln Tyr Tyr Gly Ser Gly Gly Asp Gly Tyr Tyr Leu Arg Lys

35 40 45

Asn Ile Cys Lys Ile Thr Val Asn His Ser Asp Ser Gly Thr Asn Asp

50 55 60

Pro Cys Asp Arg Ile Pro Pro Pro Tyr Gly Asp Asn Asp Gln Trp Lys

65 70 75 80

Cys Ala Ile Ile Leu Ser Lys Val Ser Glu Lys Pro Glu Asn Val Phe

85 90 95

Val Pro Pro Arg Arg Gln Arg Met Cys Ile Asn Asn Leu Glu Lys Leu

100 105 110

Asn Val Asp Lys Ile Arg Asp Lys His Ala Phe Leu Ala Asp Val Leu

115 120 125

Leu Thr Ala Arg Asn Glu Gly Glu Arg Ile Val Gln Asn His Pro Asp

130 135 140

Thr Asn Ser Ser Asn Val Cys Asn Ala Leu Glu Arg Ser Phe Ala Asp

145 150 155 160

Ile Ala Asp Ile Ile Arg Gly Thr Asp Leu Trp Lys Gly Thr Asn Ser

165 170 175

Asn Leu Glu Gln Asn Leu Lys Gln Met Phe Ala Lys Ile Arg Glu Asn

180 185 190

Asp Lys Val Leu Gln Asp Lys Tyr Pro Lys Asp Gln Asn Tyr Arg Lys

195 200 205

Leu Arg Glu Asp Trp Trp Asn Ala Asn Arg Gln Lys Val Trp Glu Val

210 215 220

Ile Thr Cys Gly Ala Arg Ser Asn Asp Leu Leu Ile Lys Arg Gly Trp

225 230 235 240

Arg Thr Ser Gly Lys Ser Asn Gly Asp Asn Lys Leu Glu Leu Cys Arg

245 250 255

Lys Cys Gly His Tyr Glu Glu Lys Val Pro Thr Lys Leu Asp Tyr Val

260 265 270

Pro Gln Phe Leu Arg Trp Leu Thr Glu Trp Ile Glu Asp Phe Tyr Arg

275 280 285

Glu Lys Gln Asn Leu Ile Asp Asp Met Glu Arg His Arg Glu Glu Cys

290 295 300

Thr Ser Glu Asp His Lys Ser Lys Glu Gly Thr Ser Tyr Cys Ser Thr

305 310 315 320

Cys Lys Asp Lys Cys Lys Lys Tyr Cys Glu Cys Val Lys Lys Trp Lys

325 330 335

Ser Glu Trp Glu Asn Gln Lys Asn Lys Tyr Thr Glu Leu Tyr Gln Gln

340 345 350

Asn Lys Asn Glu Thr Ser Gln Lys Asn Thr Ser Arg Tyr Asp Asp Tyr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

420 425 430

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

435 440 445

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

450 455 460

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

465 470 475 480

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

485 490 495

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

500 505 510

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

515 520 525

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

530 535 540

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

545 550 555 560

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

565 570 575

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

580 585 590

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

595 600 605

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

610 615 620

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

625 630 635 640

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

645 650 655

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

660 665 670

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

675 680 685

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

690 695 700

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

705 710 715 720

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

725 730 735

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

740 745 750

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

755 760 765

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

770 775 780

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

785 790 795 800

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

805 810 815

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

820 825 830

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

835 840 845

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

850 855 860

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

865 870 875 880

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

885 890 895

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

900 905 910

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

915 920 925

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

930 935 940

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

945 950 955 960

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

965 970 975

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

980 985 990

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

995 1000 1005

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

1010 1015 1020

Asn Leu Phe Glu Gln Trp Asn Lys Glu Ile Gln Tyr Gln Ile Glu

1025 1030 1035

Gln Tyr Met Thr Asn Thr Lys Ile Ser Cys Asn Asn Glu Lys Asn

1040 1045 1050

Val Leu Ser Arg Val Ser Asp Glu Ala Ala Gln Pro Lys Phe Ser

1055 1060 1065

Asp Asn Glu Arg Asp Arg Asn Ser Ile Thr His Glu Asp Lys Asn

1070 1075 1080

Cys Lys Glu Lys Cys Lys Cys Tyr Ser Leu Trp Ile Glu Lys Ile

1085 1090 1095

Asn Asp Gln Trp Asp Lys Gln Lys Asp Asn Tyr Asn Lys Phe Gln

1100 1105 1110

Arg Lys Gln Ile Tyr Asp Ala Asn Lys Gly Ser Gln Asn Lys Lys

1115 1120 1125

Val Val Ser Leu Ser Asn Phe Leu Phe Phe Ser Cys Trp Glu Glu

1130 1135 1140

Tyr Ile Gln Lys Tyr Phe Asn Gly Asp Trp Ser Lys Ile Lys Asn

1145 1150 1155

Ile Gly Ser Asp Thr Phe Glu Phe Leu Ile Lys Lys Cys Gly Asn

1160 1165 1170

Asp Ser Gly Asp Gly Glu Thr Ile Phe Ser Glu Lys Leu Asn Asn

1175 1180 1185

Ala Glu Lys Lys Cys Lys Glu Asn Glu Ser Thr Asn Asn Lys Met

1190 1195 1200

Lys Ser Ser Glu Thr Ser Cys Asp Cys Ser Glu Pro Ile Tyr Ile

1205 1210 1215

Arg Gly Cys Gln Pro Lys Ile Tyr Asp Gly Lys Ile Phe Pro Gly

1220 1225 1230

Lys Gly Gly Glu Lys Gln Trp Ile Cys Lys Asp Thr Ile Ile His

1235 1240 1245

Gly Asp Thr Asn Gly Ala Cys Ile Pro Pro Arg Thr Gln Asn Leu

1250 1255 1260

Cys Val Gly Glu Leu Trp Asp Lys Arg Tyr Gly Gly Arg Ser Asn

1265 1270 1275

Ile Lys Asn Asp Thr Lys Glu Ser Leu Lys Gln Lys Ile Lys Asn

1280 1285 1290

Ala Ile Gln Lys Glu Thr Glu Leu Leu Tyr Glu Tyr His Asp Lys

1295 1300 1305

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

1310 1315 1320

Lys Glu Glu Lys Asn Asn Asp Ser Asn Gly Leu Pro Lys Gly Phe

1325 1330 1335

Cys His Ala Val Gln Arg Ser Phe Ile Asp Tyr Lys Asn Met Ile

1340 1345 1350

Leu Gly Thr Ser Val Asn Ile Tyr Glu Tyr Ile Gly Lys Leu Gln

1355 1360 1365

Glu Asp Ile Lys Lys Ile Ile Glu Lys Gly Thr Thr Lys Gln Asn

1370 1375 1380

Gly Lys Thr Val Gly Ser Gly Ala Glu Asn Val Asn Ala Trp Trp

1385 1390 1395

Lys Gly Ile Glu Gly Glu Met Trp Asp Ala Val Arg Cys Ala Ile

1400 1405 1410

Thr Lys Ile Asn Lys Lys Gln Lys Lys Asn Gly Thr Phe Ser Ile

1415 1420 1425

Asp Glu Cys Gly Ile Phe Pro Pro Thr Gly Asn Asp Glu Asp Gln

1430 1435 1440

Ser Val Ser Trp Phe Lys Glu Trp Ser Glu Gln Phe Cys Ile Glu

1445 1450 1455

Arg Leu Gln Tyr Glu Lys Asn Ile Arg Asp Ala Cys Thr Asn Asn

1460 1465 1470

Gly Gln Gly Asp Lys Ile Gln Gly Asp Cys Lys Arg Lys Cys Glu

1475 1480 1485

Glu Tyr Lys Lys Tyr Ile Ser Glu Lys Lys Gln Glu Trp Asp Lys

1490 1495 1500

Gln Lys Thr Lys Tyr Glu Asn Lys Tyr Val Gly Lys Ser Ala Ser

1505 1510 1515

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

1520 1525 1530

Asp Phe Ile Phe Asn Asp Asn Ile Glu Tyr Lys Thr Tyr Tyr Pro

1535 1540 1545

Tyr Gly Asp Tyr Ser Ser Ile Cys Ser Cys Glu Gln Val Lys Tyr

1550 1555 1560

Tyr Glu Tyr Asn Asn Ala Glu Lys Lys Asn Asn Lys Ser Leu Cys

1565 1570 1575

His Glu Lys Gly Asn Asp Arg Thr Trp Ser Lys Lys Tyr Ile Lys

1580 1585 1590

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

1595 1600 1605

Arg Arg Gln Gln Leu Cys Leu Tyr Glu Leu Phe Pro Ile Ile Ile

1610 1615 1620

Lys Asn Lys Asn Asp Ile Thr Asn Ala Lys Lys Glu Leu Leu Glu

1625 1630 1635

Thr Leu Gln Ile Val Ala Glu Arg Glu Ala Tyr Tyr Leu Trp Lys

1640 1645 1650

Gln Tyr His Ala His Asn Asp Thr Thr Tyr Leu Ala His Lys Lys

1655 1660 1665

Ala Cys Cys Ala Ile Arg Gly Ser Phe Tyr Asp Leu Glu Asp Ile

1670 1675 1680

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

1685 1690 1695

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

1700 1705 1710

Ile Glu Thr Lys Arg Ala Arg Thr Asp Trp Trp Glu Asn Glu Ala

1715 1720 1725

Ile Ala Val Pro Asn Ile Thr Gly Ala Asn Lys Ser Asp Pro Lys

1730 1735 1740

Thr Ile Arg Gln Leu Val Trp Asp Ala Met Gln Ser Gly Val Arg

1745 1750 1755

Lys Ala Ile Asp Glu Glu Lys Glu Lys Lys Lys Pro Asn Glu Asn

1760 1765 1770

Phe Pro Pro Cys Met Gly Val Gln His Ile Gly Ile Ala Lys Pro

1775 1780 1785

Gln Phe Ile Arg Trp Leu Glu Glu Trp Thr Asn Glu Phe Cys Glu

1790 1795 1800

Lys Tyr Thr Lys Tyr Phe Glu Asp Met Lys Ser Asn Cys Asn Leu

1805 1810 1815

Arg Lys Gly Ala Asp Asp Cys Asp Asp Asn Ser Asn Ile Glu Cys

1820 1825 1830

Lys Lys Ala Cys Ala Asn Tyr Thr Asn Trp Leu Asn Pro Lys Arg

1835 1840 1845

Ile Glu Trp Asn Gly Met Ser Asn Tyr Tyr Asn Lys Ile Tyr Arg

1850 1855 1860

Lys Ser Asn Lys Glu Ser Glu Asp Gly Lys Asp Tyr Ser Met Ile

1865 1870 1875

Met Glu Pro Thr Val Ile Asp Tyr Leu Asn Lys Arg Cys Asn Gly

1880 1885 1890

Glu Ile Asn Gly Asn Tyr Ile Cys Cys Ser Cys Lys Asn Ile Gly

1895 1900 1905

Glu Asn Ser Thr Ser Gly Thr Val Asn Lys Lys Leu Gln Lys Lys

1910 1915 1920

Glu Thr Gln Cys Glu Asp Asn Lys Gly Pro Leu Asp Leu Met Asn

1925 1930 1935

Lys Val Leu Asn Lys Met Asp Pro Lys Tyr Ser Glu His Lys Met

1940 1945 1950

Lys Cys Thr Glu Val Tyr Leu Glu His Val Glu Glu Gln Leu Lys

1955 1960 1965

Glu Ile Asp Asn Ala Ile Lys Asp Tyr Lys Leu Tyr Pro Leu Asp

1970 1975 1980

Arg Cys Phe Asp Asp Lys Ser Lys Met Lys Val Cys Asp Leu Ile

1985 1990 1995

Gly Asp Ala Ile Gly Cys Lys His Lys Thr Lys Leu Asp Glu Leu

2000 2005 2010

Asp Glu Trp Asn Asp Val Asp Met Arg Asp Pro Tyr Asn Lys Tyr

2015 2020 2025

Lys Gly Val Leu Ile Pro Pro Arg Arg Arg Gln Leu Cys Phe Ser

2030 2035 2040

Arg Ile Val Arg Gly Pro Ala Asn Leu Arg Asn Leu Lys Glu Phe

2045 2050 2055

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

2060 2065 2070

Gly Asn Tyr Tyr Asn Glu Asp Lys Asp Lys Glu Lys Ala Leu Glu

2075 2080 2085

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

2090 2095 2100

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

2105 2110 2115

Leu Asp Arg Leu Leu Glu Lys Glu Thr Asn Asn Thr Glu Lys Val

2120 2125 2130

Asp Asp Trp Trp Glu Thr Asn Lys Lys Ser Ile Trp Asn Ala Met

2135 2140 2145

Leu Cys Gly Tyr Lys Lys Ser Gly Asn Lys Ile Ile Asp Pro Ser

2150 2155 2160

Trp Cys Thr Ile Pro Thr Thr Glu Thr Pro Pro Gln Phe Leu Arg

2165 2170 2175

Trp Ile Lys Glu Trp Gly Thr Asn Val Cys Ile Gln Lys Glu Glu

2180 2185 2190

His Lys Glu Tyr Val Lys Ser Lys Cys Ser Asn Val Thr Asn Leu

2195 2200 2205

Gly Ala Gln Glu Ser Glu Ser Lys Asn Cys Thr Ser Glu Ile Lys

2210 2215 2220

Lys Tyr Gln Glu Trp Ser Arg Lys Arg Ser Ile Gln Trp Glu Ala

2225 2230 2235

Ile Ser Glu Gly Tyr Lys Lys Tyr Lys Gly Met Asp Glu Phe Lys

2240 2245 2250

Asn Thr Phe Lys Asn Ile Lys Glu Pro Asp Ala Asn Glu Pro Asn

2255 2260 2265

Ala Asn Glu Tyr Leu Lys Lys His Cys Ser Lys Cys Pro Cys Gly

2270 2275 2280

Phe Asn Asp Met Gln Glu Ile Thr Lys Tyr Thr Asn Ile Gly Asn

2285 2290 2295

Glu Ala Phe Lys Gln Ile Lys Glu Gln Val Asp Ile Pro Ala Glu

2300 2305 2310

Leu Glu Asp Val Ile Tyr Arg Leu Lys His His Glu Tyr Asp Lys

2315 2320 2325

Gly Asn Asp Tyr Ile Cys Asn Lys Tyr Lys Asn Ile Asn Val Asn

2330 2335 2340

Met Lys Lys Asn Asn Asp Asp Thr Trp Thr Asp Leu Val Lys Asn

2345 2350 2355

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

2360 2365 2370

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

2375 2380 2385

Arg Asp Pro Lys Leu Phe Lys Asp Phe Ile Tyr Ser Ser Ala Ile

2390 2395 2400

Ser Glu Val Glu Arg Leu Lys Lys Val Tyr Gly Glu Ala Lys Thr

2405 2410 2415

Lys Val Val His Ala Met Lys Tyr Ser Phe Ala Asp Ile Gly Ser

2420 2425 2430

Ile Ile Lys Gly Asp Asp Met Met Glu Asn Asn Ser Ser Asp Lys

2435 2440 2445

Ile Gly Lys Ile Leu Gly Asp Gly Val Gly Gln Asn Glu Lys Arg

2450 2455 2460

Lys Lys Trp Trp Asp Met Asn Lys Tyr His Ile Trp Glu Ser Met

2465 2470 2475

Leu Cys Gly Tyr Lys His Ala Tyr Gly Asn Ile Ser Glu Asn Asp

2480 2485 2490

Arg Lys Met Leu Asp Ile Pro Asn Asn Asp Asp Glu His Gln Phe

2495 2500 2505

Leu Arg Trp Phe Gln Glu Trp Thr Glu Asn Phe Cys Thr Lys Arg

2510 2515 2520

Asn Glu Leu Tyr Glu Asn Met Val Thr Ala Cys Asn Ser Ala Lys

2525 2530 2535

Cys Asn Thr Ser Asn Gly Ser Val Asp Lys Lys Glu Cys Thr Glu

2540 2545 2550

Ala Cys Lys Asn Tyr Ser Asn Phe Ile Leu Ile Lys Lys Lys Glu

2555 2560 2565

Tyr Gln Ser Leu Asn Ser Gln Tyr Asp Met Asn Tyr Lys Glu Thr

2570 2575 2580

Lys Ala Glu Lys Lys Glu Ser Pro Glu Tyr Phe Lys Asp Lys Cys

2585 2590 2595

Asn Gly Glu Cys Ser Cys Leu Ser Glu Tyr Phe Lys Asp Glu Thr

2600 2605 2610

Arg Trp Lys Asn Pro Tyr Glu Thr Leu Asp Asp Thr Glu Val Lys

2615 2620 2625

Asn Asn Cys Met Cys Lys Pro Pro Pro Pro Ala Ser Asn Ser Gly

2630 2635 2640

Arg Glu Leu

2645

<210> 4

<211> 7938

<212> DNA

<213> 人工合成序列

<400> 4

atggacaagt cctctatcgc caacaagatc gaagcctacc tgggagccaa gtctgacgac 60

tctaagatcg accagtctct gaaggccgac ccctctgaag tccagtacta cggatctggt 120

ggcgacggtt actacctgag gaagaacatc tgcaagatca ccgtcaacca ctctgactct 180

ggcaccaacg acccctgcga caggatcccc cctccctacg gcgacaacga ccagtggaag 240

tgcgccatca tcctgtctaa ggtgtccgaa aagcccgaaa acgtgttcgt cccccctagg 300

cgccagagga tgtgcatcaa caacctggaa aagctgaacg tcgacaagat cagggacaag 360

cacgccttcc tcgccgacgt cctgctgacc gctaggaacg aaggcgaaag gatcgtccag 420

aaccaccccg acaccaactc ttctaacgtc tgcaacgccc tggaacgctc tttcgccgac 480

atcgctgaca tcatccgcgg aaccgacctg tggaagggca ccaactctaa cctggaacag 540

aacctgaagc agatgttcgc caagatccgc gaaaacgaca aggtcctgca ggataagtac 600

cccaaggacc agaactaccg caagctgcgc gaggactggt ggaacgccaa caggcagaag 660

gtctgggaag tcatcacttg cggagccagg tctaacgacc tgctgatcaa gaggggatgg 720

cgcacctctg gaaagtctaa cggcgacaac aagctggaac tgtgccgcaa gtgcggacac 780

tacgaagaaa aggtccccac caagctggac tacgtccccc agttcctgcg ctggctgacc 840

gaatggatcg aggacttcta cagggaaaag cagaacctga tcgacgacat ggaaaggcac 900

agggaagaat gcacctctga ggaccacaag tctaaggaag gcacctctta ctgctctacc 960

tgcaaggaca agtgcaagaa gtactgcgag tgcgtcaaga agtggaagtc tgaatgggag 1020

aaccagaaga acaagtacac cgaactgtac cagcagaaca agaacgaaac ctctcagaag 1080

aacacctctc gctacgacga ctacgtcaag gacttcttca agaagctcga ggccaactac 1140

tcctccctgg aaaactacat caagggcgac ccctacttcg ccgaatacgc taccaagctg 1200

tccttcatcc tgaactcttc cgacgccaac aacccctctg agaagatcca aaagaacaac 1260

gacgaagtct gcaactgcaa cgaatctgga atcgcctctg tcgaacaaga gcagatctct 1320

gacccctcta gcaacaagac ctgcatcacc cactcttcta tcaaggccaa caagaagaag 1380

gtctgcaagc acgtcaagct gggagtcaga gagaacgaca aggacctccg cgtctgcgtc 1440

atcgaacaca cctccctgtc tggtgtcgaa aactgctgct gccaggactt cctgaggatc 1500

ctgcaagaaa actgctccga caacaagtcc ggatcttctt ctaacggctc ctgcaacaac 1560

aagaaccagg aagcctgcga gaagaacctc gaaaaggtcc tggcctctct gaccaactgc 1620

tacaagtgcg acaagtgcaa gtctgagcag agcaagaaga acaacaagaa ctggatctgg 1680

aagaagtcct ccggaaagga aggtggactg cagaaggaat acgccaacac catcggactg 1740

ccccccagga cccagtctct gtgcctggtc gtctgcctgg acgaaaaggg aaagaagacc 1800

caagagctga agaacatccg caccaactcc gaactgctga aggaatggat catcgccgcc 1860

ttccacgagg gaaagaacct caagccctct cacgaaaaga agaacgacga caacggcaag 1920

aagctgtgca aggccctcga gtactccttc gccgactacg gcgacctgat caagggaacc 1980

tctatctggg acaacgagta caccaaggac ttggaactga acctgcaaaa gatcttcggc 2040

aagctgttcc gcaagtacat caagaagaac aacaccgccg agcaggacac ctcctactct 2100

agcctggacg agctgaggga atcttggtgg aacactaaca agaagtacat ctggctggcc 2160

atgaagcacg gtgccggaat gaactctacc acttgctgcg gcgacggatc tgtgaccgga 2220

tctggatctt cttgcgacga catccccacc atcgacctga tccctcagta cctgcgcttc 2280

ctgcaagagt gggtcgaaca cttctgcaag cagaggcaag agaaggtcaa gcccgtcatc 2340

gagaactgca agtcttgcaa ggaaagcgga ggaacctgca acggcgaatg caagaccgag 2400

tgcaagaaca agtgtgaagt ctacaagaag ttcatcgagg actgcaaggg tggcgacgga 2460

accgccggat cttcctgggt caagcgctgg gaccagatct acaagaggta ctctaagtac 2520

atcgaggacg ccaagaggaa ccgcaaggcc ggcaccaaga actgcggacc ctcttctacc 2580

accaacgccg ccgaaaacaa gtgcgtccag tctgacatcg actcattctt caagcacctc 2640

atcgacatcg gactgaccac cccctcctct tacctgtcta tcgtcctgga cgacaacatc 2700

tgcggagccg acaaggcccc ctggaccacc tacaccactt acaccaccac cgaaaagtgc 2760

aacaaggaaa ccgacaagtc caagctccag cagtgcaaca ccgctgtcgt cgtcaacgtc 2820

ccctctcccc tgggaaacac cccccacgga tacaagtacg cctgccagtg caagatcccc 2880

accaacgaag aaacctgcga cgaccgcaag gagtacatga accagtggtc ttgcggatct 2940

gccaggacca tgaagagggg atacaagaac gacaactacg aactgtgcaa gtacaacggt 3000

gtcgacgtca agcccaccac cgtcaggtct aactcctcta agctggacga caaggacgtc 3060

accttcttca acctgttcga gcagtggaac aaggaaatcc agtaccagat cgagcagtac 3120

atgaccaaca ccaagatcag ctgcaacaac gaaaagaacg tcctgtctcg cgtcagcgac 3180

gaagccgccc agcccaagtt ctctgacaac gaaagggacc gcaactctat cacccacgag 3240

gacaagaact gcaaggaaaa gtgcaagtgc tactccctgt ggatcgagaa gatcaacgac 3300

caatgggaca agcagaagga caactacaac aagttccagc gcaagcagat ctacgacgct 3360

aacaagggat cccaaaacaa gaaggtcgtc agcctgtcca acttcctgtt cttcagctgc 3420

tgggaagagt acatccagaa gtacttcaac ggcgactggt ccaagatcaa gaacatcgga 3480

tctgacacct tcgagttcct catcaagaag tgcggaaacg actctggcga cggcgaaacc 3540

atcttctctg agaagctgaa caacgccgag aagaagtgca aggaaaacga gtccaccaac 3600

aacaagatga agtcctctga aacctcctgc gactgctctg aacccatcta catcagggga 3660

tgccagccca agatctacga cggaaagatc ttccccggaa agggcggaga aaagcagtgg 3720

atctgcaagg acaccatcat ccacggcgac accaacggtg cctgcatccc tcctaggacc 3780

cagaacctgt gcgtcggaga actgtgggac aagcgctacg gtggaaggtc taacatcaag 3840

aacgacacca aggaatccct gaagcagaag atcaagaacg ccatccagaa ggaaaccgaa 3900

ctgctgtacg agtaccacga caagggaacc gccatcatct ctaggaaccc catgaaggga 3960

cagaaggaaa aggaagagaa gaacaacgac tccaacggac tgcccaaggg attctgccac 4020

gccgtccagc gcagcttcat cgactacaag aacatgatcc tgggcacctc cgtcaacatc 4080

tacgagtaca tcggaaagct gcaagaggac atcaagaaga tcatcgagaa gggaaccacc 4140

aagcagaacg gaaagaccgt cggaagcgga gccgaaaacg tcaacgcctg gtggaaggga 4200

atcgaaggcg agatgtggga cgccgtccgc tgcgccatca ctaagatcaa caagaagcag 4260

aagaagaacg gcaccttctc tatcgacgag tgcggaatct tcccccccac cggaaacgac 4320

gaggaccagt ctgtctcctg gttcaaggaa tggtccgaac agttctgcat cgaaaggctg 4380

cagtacgaga agaacatcag ggacgcctgc accaacaacg gacagggcga caagatccaa 4440

ggcgactgca agcgcaagtg cgaagagtac aagaagtaca tcagcgagaa gaagcaagag 4500

tgggacaagc aaaagactaa gtacgagaac aagtacgtcg gaaagtctgc ctctgacctg 4560

ctcaaggaaa actaccccga atgcatctct gccaacttcg acttcatctt caacgacaac 4620

atcgagtaca agacctacta cccttacggc gactactctt ccatctgctc ttgcgaacaa 4680

gtgaagtact acgagtacaa caacgctgaa aagaagaaca acaagtccct gtgccacgag 4740

aagggaaacg accgcacctg gtctaagaag tacatcaaga agctcgagaa cggaaggacc 4800

ctggaaggcg tctacgtccc accccgcagg cagcagctgt gcctgtacga actgttcccc 4860

atcatcatca agaacaagaa cgacatcacc aacgccaaga aggaattgct ggaaaccctg 4920

cagatcgtcg ccgaaaggga agcctactac ctctggaagc agtaccacgc ccacaacgac 4980

accacctacc tggcccacaa gaaggcctgc tgcgctatca ggggatcttt ctacgacctc 5040

gaggatatca tcaagggcaa cgacctggtc cacgacgagt acaccaagta catcgactct 5100

aagctgaacg agatcttcga ctcctccaac aagaacgaca tcgaaactaa gagggccagg 5160

accgactggt gggaaaacga agctatcgcc gtccccaaca tcaccggtgc caacaagtct 5220

gaccccaaga ccatcaggca gctcgtctgg gacgccatgc agtctggtgt ccgcaaggcc 5280

atcgacgaag agaaggaaaa gaagaagccc aacgaaaact tcccaccctg catgggagtc 5340

cagcacatcg gaatcgccaa gccccagttc atccgttggc tggaagaatg gaccaacgag 5400

ttctgcgaga agtacactaa gtacttcgag gacatgaagt ccaactgcaa cctgcgcaag 5460

ggcgccgacg actgcgacga caactctaac atcgaatgca agaaggcttg cgccaactac 5520

accaactggc tgaaccccaa gaggatcgaa tggaacggaa tgtccaacta ctacaacaag 5580

atctaccgca agtccaacaa ggaatctgag gacggaaagg actactcaat gatcatggaa 5640

cccaccgtga tcgactacct gaacaagagg tgtaacggcg agatcaacgg caactacatc 5700

tgctgctctt gcaagaacat cggcgagaac tctacctctg gcaccgtgaa caagaagctg 5760

caaaagaagg aaactcagtg cgaggacaac aagggacccc tggacctgat gaacaaggtc 5820

ctcaacaaga tggaccccaa gtactctgag cacaagatga agtgcaccga ggtctacctg 5880

gaacacgtcg aagaacagtt gaaggaaatc gacaacgcca tcaaggacta caagctgtac 5940

cctctggacc gctgcttcga cgacaagagc aagatgaagg tttgcgacct catcggcgac 6000

gccatcggat gcaagcacaa gaccaagctc gacgaactgg acgagtggaa cgacgtcgac 6060

atgagggacc cttacaacaa gtacaagggc gtcctgatcc ccccaaggcg caggcagctg 6120

tgcttctcta ggatcgtcag gggacccgcc aacctgagga acctgaagga gttcaaggaa 6180

gagatcctga agggcgccca gtctgaggga aagttcctgg gaaactacta caacgaggac 6240

aaggacaagg aaaaggccct ggaagccatg aagaacagct tctacgacta cgagtacatc 6300

atcaagggct ctgacatgct gaccaacatc cagttcaagg acatcaagcg caagctggac 6360

aggctgctgg aaaaggaaac taacaacacc gaaaaggtgg acgactggtg ggagactaac 6420

aagaagagta tctggaacgc catgttgtgc ggttacaaga agtccggcaa caagatcatc 6480

gacccctctt ggtgcaccat ccctaccacc gaaacccccc ctcagttctt gcgttggatc 6540

aaggaatggg gcaccaacgt ctgcatccaa aaggaagaac acaaggaata cgtcaagagc 6600

aagtgctcca acgtcaccaa cctcggagcc caagaatctg aatctaagaa ctgcacctcc 6660

gagatcaaga agtaccaaga gtggtcccgc aagcgctcta tccagtggga agccatctct 6720

gagggctaca agaagtacaa gggtatggac gagttcaaga acaccttcaa gaacatcaag 6780

gaacccgacg ccaacgagcc caacgccaac gaatacctga agaagcactg ctctaagtgc 6840

ccctgcggat tcaacgacat gcaagaaatc actaagtaca ccaacatcgg caacgaggcc 6900

ttcaagcaga tcaaggaaca ggtcgacatc cccgccgaat tggaggacgt catctacagg 6960

ctgaagcacc acgaatacga caagggaaac gactacatct gcaacaagta caagaacatc 7020

aacgtcaaca tgaagaagaa caacgacgac acctggaccg acctggtcaa gaactcttct 7080

gacatcaaca agggtgtcct gctgccccct cgccgcaaga acctgttcct gaagatcgac 7140

gaatccgaca tctgcaagta caagagggac cctaagctgt tcaaggactt catctactct 7200

tccgccatct ccgaagtcga acgcctcaag aaggtctacg gcgaggccaa gaccaaggtc 7260

gtccacgcta tgaagtactc attcgcagac atcggctcca tcatcaaggg cgacgacatg 7320

atggaaaaca acagctctga caagatcggc aagatcctgg gcgacggtgt cggccagaac 7380

gaaaagcgca agaagtggtg ggacatgaac aagtaccaca tctgggagtc tatgctgtgc 7440

ggctacaagc acgcttacgg aaacatctct gagaacgacc gaaagatgct ggacatcccc 7500

aacaacgacg acgaacacca gttcctccgc tggttccaag aatggactga aaacttctgc 7560

accaagcgca acgagctgta cgaaaacatg gtcaccgcct gcaactctgc caagtgcaac 7620

acttctaacg gatccgtgga caagaaggaa tgcaccgaag cctgcaagaa ctactccaac 7680

ttcatcctga tcaagaagaa ggaataccag tccctgaact cccagtacga catgaactac 7740

aaggaaacta aggccgaaaa gaaggaatcc cccgagtact tcaaggacaa gtgtaacggc 7800

gaatgctctt gcctgagcga atacttcaag gacgaaacca ggtggaagaa cccctacgaa 7860

accctcgacg acaccgaagt caagaacaac tgcatgtgca agcccccacc ccctgcctct 7920

aacagcggcc gcgagctc 7938

<210> 5

<211> 2649

<212> PRT

<213> 人工合成序列

<400> 5

Met Asp Ser Thr Ser Thr Ile Ala Asn Lys Ile Glu Glu Tyr Leu Gly

1 5 10 15

Ala Lys Ser Asp Asp Ser Lys Ile Asp Glu Leu Leu Lys Ala Asp Pro

20 25 30

Ser Glu Val Glu Tyr Tyr Arg Ser Gly Gly Asp Gly Asp Tyr Leu Lys

35 40 45

Asn Asn Ile Cys Lys Ile Thr Val Asn His Ser Asp Ser Gly Lys Tyr

50 55 60

Asp Pro Cys Glu Lys Lys Leu Pro Pro Tyr Asp Asp Asn Asp Gln Trp

65 70 75 80

Lys Cys Gln Gln Asn Ser Ser Asp Gly Ser Gly Lys Pro Glu Asn Ile

85 90 95

Cys Val Pro Pro Arg Arg Glu Arg Leu Cys Thr Tyr Asn Leu Glu Asn

100 105 110

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

115 120 125

Leu Leu Thr Ala Arg Asn Glu Gly Glu Lys Ile Val Gln Asn His Pro

130 135 140

Asp Thr Asn Ser Ser Asn Val Cys Asn Ala Leu Glu Arg Ser Phe Ala

145 150 155 160

Asp Leu Ala Asp Ile Ile Arg Gly Thr Asp Gln Trp Lys Gly Thr Asn

165 170 175

Ser Asn Leu Glu Lys Asn Leu Lys Gln Met Phe Ala Lys Ile Arg Glu

180 185 190

Asn Asp Lys Val Leu Gln Asp Lys Tyr Pro Lys Asp Gln Lys Tyr Thr

195 200 205

Lys Leu Arg Glu Ala Trp Trp Asn Ala Asn Arg Gln Lys Val Trp Glu

210 215 220

Val Ile Thr Cys Gly Ala Arg Ser Asn Asp Leu Leu Ile Lys Arg Gly

225 230 235 240

Trp Arg Thr Ser Gly Lys Ser Asp Arg Lys Lys Asn Phe Glu Leu Cys

245 250 255

Arg Lys Cys Gly His Tyr Glu Lys Glu Val Pro Thr Lys Leu Asp Tyr

260 265 270

Val Pro Gln Phe Leu Arg Trp Leu Thr Glu Trp Ile Glu Asp Phe Tyr

275 280 285

Arg Glu Lys Gln Asn Leu Ile Asp Asp Met Glu Arg His Arg Glu Glu

290 295 300

Cys Thr Arg Glu Asp His Lys Ser Lys Glu Gly Thr Ser Tyr Cys Ser

305 310 315 320

Thr Cys Lys Asp Lys Cys Lys Lys Tyr Cys Glu Cys Val Lys Lys Trp

325 330 335

Lys Thr Glu Trp Glu Asn Gln Glu Asn Lys Tyr Lys Asp Leu Tyr Glu

340 345 350

Gln Asn Lys Asn Lys Thr Ser Gln Lys Asn Thr Ser Arg Tyr Asp Asp

355 360 365

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

370 375 380

Glu Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr Lys

385 390 395 400

Leu Ser Phe Ile Leu Asn Pro Ser Asp Ala Asn Asn Pro Ser Gly Glu

405 410 415

Thr Ala Asn His Asn Asp Glu Ala Cys Asn Cys Asn Glu Ser Gly Ile

420 425 430

Ser Ser Val Gly Gln Ala Gln Thr Ser Gly Pro Ser Ser Asn Lys Thr

435 440 445

Cys Ile Thr His Ser Ser Ile Lys Thr Asn Lys Lys Lys Glu Cys Lys

450 455 460

Asp Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Lys Ile Cys

465 470 475 480

Val Ile Glu Asp Thr Ser Leu Ser Gly Val Asp Asn Cys Cys Cys Gln

485 490 495

Asp Leu Leu Gly Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Arg Gly

500 505 510

Ser Ser Ser Asn Asp Ser Cys Asp Asn Lys Asn Gln Asp Glu Cys Gln

515 520 525

Lys Lys Leu Glu Lys Val Phe Ala Ser Leu Thr Asn Gly Tyr Lys Cys

530 535 540

Asp Lys Cys Lys Ser Gly Thr Ser Arg Ser Lys Lys Lys Trp Ile Trp

545 550 555 560

Lys Lys Ser Ser Gly Asn Glu Glu Gly Leu Gln Glu Glu Tyr Ala Asn

565 570 575

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Tyr Leu Gly Asn Leu

580 585 590

Pro Lys Leu Glu Asn Val Cys Glu Asp Val Lys Asp Ile Asn Phe Asp

595 600 605

Thr Lys Glu Lys Phe Leu Ala Gly Cys Leu Ile Val Ser Phe His Glu

610 615 620

Gly Lys Asn Leu Lys Lys Arg Tyr Pro Gln Asn Lys Asn Ser Gly Asn

625 630 635 640

Lys Glu Asn Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly

645 650 655

Asp Leu Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp

660 665 670

Leu Glu Leu Asn Leu Gln Asn Asn Phe Gly Lys Leu Phe Gly Lys Tyr

675 680 685

Ile Lys Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu

690 695 700

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

705 710 715 720

Thr Ala Met Lys His Gly Ala Glu Met Asn Ile Thr Thr Cys Asn Ala

725 730 735

Asp Gly Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr

740 745 750

Ile Asp Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu

755 760 765

Asn Phe Cys Glu Gln Arg Gln Ala Lys Val Lys Asp Val Ile Thr Asn

770 775 780

Cys Lys Ser Cys Lys Glu Ser Gly Asn Lys Cys Lys Thr Glu Cys Lys

785 790 795 800

Thr Lys Cys Lys Asp Glu Cys Glu Lys Tyr Lys Lys Phe Ile Glu Ala

805 810 815

Cys Gly Thr Ala Gly Gly Gly Ile Gly Thr Ala Gly Ser Pro Trp Ser

820 825 830

Lys Arg Trp Asp Gln Ile Tyr Lys Arg Tyr Ser Lys His Ile Glu Asp

835 840 845

Ala Lys Arg Asn Arg Lys Ala Gly Thr Lys Asn Cys Gly Thr Ser Ser

850 855 860

Thr Thr Asn Ala Ala Ala Ser Thr Asp Glu Asn Lys Cys Val Gln Ser

865 870 875 880

Asp Ile Asp Ser Phe Phe Lys His Leu Ile Asp Ile Gly Leu Thr Thr

885 890 895

Pro Ser Ser Tyr Leu Ser Asn Val Leu Asp Asp Asn Ile Cys Gly Ala

900 905 910

Asp Lys Ala Pro Trp Thr Thr Tyr Thr Thr Tyr Thr Thr Thr Glu Lys

915 920 925

Cys Asn Lys Glu Arg Asp Lys Ser Lys Ser Gln Ser Ser Asp Thr Leu

930 935 940

Val Val Val Asn Val Pro Ser Pro Leu Gly Asn Thr Pro Tyr Arg Tyr

945 950 955 960

Lys Tyr Ala Cys Gln Cys Lys Ile Pro Thr Asn Glu Glu Thr Cys Asp

965 970 975

Asp Arg Lys Glu Tyr Met Asn Gln Trp Ser Cys Gly Ser Ala Arg Thr

980 985 990

Met Lys Arg Gly Tyr Lys Asn Asp Asn Tyr Glu Leu Cys Lys Tyr Asn

995 1000 1005

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

1010 1015 1020

Leu Asp Gly Asn Asp Val Thr Phe Phe Asn Leu Phe Glu Gln Trp

1025 1030 1035

Asn Lys Glu Ile Gln Tyr Gln Ile Glu Gln Tyr Met Thr Asn Ala

1040 1045 1050

Asn Ile Ser Cys Ile Asp Glu Lys Glu Val Leu Asp Ser Val Ser

1055 1060 1065

Asp Glu Gly Thr Pro Lys Val Arg Gly Gly Tyr Glu Asp Gly Arg

1070 1075 1080

Asn Asn Asn Thr Asp Gln Gly Thr Asn Cys Lys Glu Lys Cys Lys

1085 1090 1095

Cys Tyr Lys Leu Trp Ile Glu Lys Ile Asn Asp Gln Trp Gly Lys

1100 1105 1110

Gln Lys Asp Asn Tyr Asn Lys Phe Arg Ser Lys Gln Ile Tyr Asp

1115 1120 1125

Ala Asn Lys Gly Ser Gln Asn Lys Lys Val Val Ser Leu Ser Asn

1130 1135 1140

Phe Leu Phe Phe Ser Cys Trp Glu Glu Tyr Ile Gln Lys Tyr Phe

1145 1150 1155

Asn Gly Asp Trp Ser Lys Ile Lys Asn Ile Gly Ser Asp Thr Phe

1160 1165 1170

Glu Phe Leu Ile Lys Lys Cys Gly Asn Asn Ser Ala His Gly Glu

1175 1180 1185

Glu Ile Phe Ser Glu Lys Leu Lys Asn Ala Glu Lys Lys Cys Lys

1190 1195 1200

Glu Asn Glu Ser Thr Asp Thr Asn Ile Asn Lys Ser Glu Thr Ser

1205 1210 1215

Cys Asp Leu Asn Ala Thr Asn Tyr Ile Arg Gly Cys Gln Ser Lys

1220 1225 1230

Thr Tyr Asp Gly Lys Ile Phe Pro Gly Lys Gly Gly Glu Lys Gln

1235 1240 1245

Trp Ile Cys Lys Asp Thr Ile Ile His Gly Asp Thr Asn Gly Ala

1250 1255 1260

Cys Ile Pro Pro Arg Thr Gln Asn Leu Cys Val Gly Glu Leu Trp

1265 1270 1275

Asp Lys Ser Tyr Gly Gly Arg Ser Asn Ile Lys Asn Asp Thr Lys

1280 1285 1290

Glu Leu Leu Lys Glu Lys Ile Lys Asn Ala Ile His Lys Glu Thr

1295 1300 1305

Glu Leu Leu Tyr Glu Tyr His Asp Thr Gly Thr Ala Ile Ile Ser

1310 1315 1320

Lys Asn Asp Lys Lys Gly Gln Lys Gly Lys Asn Asp Pro Asn Gly

1325 1330 1335

Leu Pro Lys Gly Phe Cys His Ala Val Gln Arg Ser Phe Ile Asp

1340 1345 1350

Tyr Lys Asn Met Ile Leu Gly Thr Ser Val Asn Ile Tyr Glu His

1355 1360 1365

Ile Gly Lys Leu Gln Glu Asp Ile Lys Lys Ile Ile Glu Lys Gly

1370 1375 1380

Thr Pro Gln Gln Lys Asp Lys Ile Gly Gly Val Gly Ser Ser Thr

1385 1390 1395

Glu Asn Val Asn Ala Trp Trp Lys Gly Ile Glu Arg Glu Met Trp

1400 1405 1410

Asp Ala Val Arg Cys Ala Ile Thr Lys Ile Asn Lys Lys Asn Asn

1415 1420 1425

Asn Ser Ile Phe Asn Gly Asp Glu Cys Gly Val Ser Pro Pro Thr

1430 1435 1440

Gly Asn Asp Glu Asp Gln Ser Val Ser Trp Phe Lys Glu Trp Gly

1445 1450 1455

Glu Gln Phe Cys Ile Glu Arg Leu Arg Tyr Glu Gln Asn Ile Arg

1460 1465 1470

Glu Ala Cys Thr Ile Asn Gly Lys Asn Glu Lys Lys Cys Ile Asn

1475 1480 1485

Ser Lys Ser Gly Gln Gly Asp Lys Ile Gln Gly Ala Cys Lys Arg

1490 1495 1500

Lys Cys Glu Lys Tyr Lys Lys Tyr Ile Ser Glu Lys Lys Gln Glu

1505 1510 1515

Trp Asp Lys Gln Lys Thr Lys Tyr Glu Asn Lys Tyr Val Gly Lys

1520 1525 1530

Ser Ala Ser Asp Leu Leu Lys Glu Asn Tyr Pro Glu Cys Ile Ser

1535 1540 1545

Ala Asn Phe Asp Phe Ile Phe Asn Asp Asn Ile Glu Tyr Lys Thr

1550 1555 1560

Tyr Tyr Pro Tyr Gly Asp Tyr Ser Ser Ile Cys Ser Cys Glu Gln

1565 1570 1575

Val Lys Tyr Tyr Lys Tyr Asn Asn Ala Glu Lys Lys Asn Asn Lys

1580 1585 1590

Ser Leu Cys Tyr Glu Lys Asp Asn Asp Met Thr Trp Ser Lys Lys

1595 1600 1605

Tyr Ile Lys Lys Leu Glu Asn Gly Arg Ser Leu Glu Gly Val Tyr

1610 1615 1620

Val Pro Pro Arg Arg Gln Gln Leu Cys Leu Tyr Glu Leu Phe Pro

1625 1630 1635

Ile Ile Ile Lys Asn Glu Glu Gly Met Glu Lys Ala Lys Glu Glu

1640 1645 1650

Leu Leu Glu Thr Leu Gln Ile Val Ala Glu Arg Glu Ala Tyr Tyr

1655 1660 1665

Leu Trp Lys Gln Tyr Asn Pro Thr Gly Lys Gly Ile Asp Asp Ala

1670 1675 1680

Asn Lys Lys Ala Cys Cys Ala Ile Arg Gly Ser Phe Tyr Asp Leu

1685 1690 1695

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

1700 1705 1710

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

1715 1720 1725

Thr Asn Asp Ile Asp Thr Lys Arg Ala Arg Thr Asp Trp Trp Glu

1730 1735 1740

Asn Glu Thr Ile Thr Asn Gly Thr Asp Arg Lys Thr Ile Arg Gln

1745 1750 1755

Leu Val Trp Asp Ala Met Gln Ser Gly Val Arg Tyr Ala Val Glu

1760 1765 1770

Glu Lys Asn Glu Asn Phe Pro Leu Cys Met Gly Val Glu His Ile

1775 1780 1785

Gly Ile Ala Lys Pro Gln Phe Ile Arg Trp Leu Glu Glu Trp Thr

1790 1795 1800

Asn Glu Phe Cys Glu Lys Tyr Thr Lys Tyr Phe Glu Asp Met Lys

1805 1810 1815

Ser Lys Cys Asp Pro Pro Lys Arg Ala Asp Thr Cys Gly Asp Asn

1820 1825 1830

Ser Asn Ile Glu Cys Lys Lys Ala Cys Ala Asn Tyr Thr Asn Trp

1835 1840 1845

Leu Asn Pro Lys Arg Ile Glu Trp Asn Gly Met Ser Asn Tyr Tyr

1850 1855 1860

Asn Lys Ile Tyr Arg Lys Ser Asn Lys Glu Ser Glu Asp Gly Lys

1865 1870 1875

Asp Tyr Ser Met Ile Met Ala Pro Thr Val Ile Asp Tyr Leu Asn

1880 1885 1890

Lys Arg Cys His Gly Glu Ile Asn Gly Asn Tyr Ile Cys Cys Ser

1895 1900 1905

Cys Lys Asn Ile Gly Ala Tyr Asn Thr Thr Ser Gly Thr Val Asn

1910 1915 1920

Lys Lys Leu Gln Lys Lys Glu Thr Glu Cys Glu Glu Glu Lys Gly

1925 1930 1935

Pro Leu Asp Leu Met Asn Glu Val Leu Asn Lys Met Asp Lys Lys

1940 1945 1950

Tyr Ser Ala His Lys Met Lys Cys Thr Glu Val Tyr Leu Glu His

1955 1960 1965

Val Glu Glu Gln Leu Asn Glu Ile Asp Asn Ala Ile Lys Asp Tyr

1970 1975 1980

Lys Leu Tyr Pro Leu Asp Arg Cys Phe Asp Asp Gln Thr Lys Met

1985 1990 1995

Lys Val Cys Asp Leu Ile Ala Asp Ala Ile Gly Cys Lys Asp Lys

2000 2005 2010

Thr Lys Leu Asp Glu Leu Asp Glu Trp Asn Asp Met Asp Leu Arg

2015 2020 2025

Gly Thr Tyr Asn Lys His Lys Gly Val Leu Ile Pro Pro Arg Arg

2030 2035 2040

Arg Gln Leu Cys Phe Ser Arg Ile Val Arg Gly Pro Ala Asn Leu

2045 2050 2055

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

2060 2065 2070

Ser Glu Gly Lys Phe Leu Gly Asn Tyr Tyr Lys Glu His Lys Asp

2075 2080 2085

Lys Glu Lys Ala Leu Glu Ala Met Lys Asn Ser Phe Tyr Asp Tyr

2090 2095 2100

Glu Asp Ile Ile Lys Gly Thr Asp Met Leu Thr Asn Ile Glu Phe

2105 2110 2115

Lys Asp Ile Lys Ile Lys Leu Asp Arg Leu Leu Glu Lys Glu Thr

2120 2125 2130

Asn Asn Thr Lys Lys Ala Glu Asp Trp Trp Lys Thr Asn Lys Lys

2135 2140 2145

Ser Ile Trp Asn Ala Met Leu Cys Gly Tyr Lys Lys Ser Gly Asn

2150 2155 2160

Lys Ile Ile Asp Pro Ser Trp Cys Thr Ile Pro Thr Thr Glu Thr

2165 2170 2175

Pro Pro Gln Phe Leu Arg Trp Ile Lys Glu Trp Gly Thr Asn Val

2180 2185 2190

Cys Ile Gln Lys Gln Glu His Lys Glu Tyr Val Lys Ser Lys Cys

2195 2200 2205

Ser Asn Val Thr Asn Leu Gly Ala Gln Ala Ser Glu Ser Asn Asn

2210 2215 2220

Cys Thr Ser Glu Ile Lys Lys Tyr Gln Glu Trp Ser Arg Lys Arg

2225 2230 2235

Ser Ile Gln Trp Glu Thr Ile Ser Lys Arg Tyr Lys Lys Tyr Lys

2240 2245 2250

Arg Met Asp Ile Leu Lys Asp Val Lys Glu Pro Asp Ala Asn Thr

2255 2260 2265

Tyr Leu Arg Glu His Cys Ser Lys Cys Pro Cys Gly Phe Asn Asp

2270 2275 2280

Met Glu Glu Met Asn Asn Asn Glu Asp Asn Glu Lys Glu Ala Phe

2285 2290 2295

Lys Gln Ile Lys Glu Gln Val Lys Ile Pro Ala Glu Leu Glu Asp

2300 2305 2310

Val Ile Tyr Arg Ile Lys His His Glu Tyr Asp Lys Gly Asn Asp

2315 2320 2325

Tyr Ile Cys Asn Lys Tyr Lys Asn Ile His Asp Arg Met Lys Lys

2330 2335 2340

Asn Asn Gly Asn Phe Val Thr Asp Asn Phe Val Lys Lys Ser Trp

2345 2350 2355

Glu Ile Ser Asn Gly Val Leu Ile Pro Pro Arg Arg Lys Asn Leu

2360 2365 2370

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

2375 2380 2385

Pro Lys Leu Phe Lys Asp Phe Ile Tyr Trp Ser Ala Phe Thr Glu

2390 2395 2400

Val Glu Arg Leu Lys Lys Ala Tyr Gly Gly Ala Arg Ala Lys Val

2405 2410 2415

Val His Ala Met Lys Tyr Ser Phe Thr Asp Ile Gly Ser Ile Ile

2420 2425 2430

Lys Gly Asp Asp Met Met Glu Lys Asn Ser Ser Asp Lys Ile Gly

2435 2440 2445

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

2450 2455 2460

Trp Trp Asp Met Asn Lys Tyr His Ile Trp Glu Ser Met Leu Cys

2465 2470 2475

Gly Tyr Arg Glu Ala Glu Gly Asp Thr Glu Thr Asn Glu Asn Cys

2480 2485 2490

Arg Phe Pro Asp Ile Glu Ser Val Pro Gln Phe Leu Arg Trp Phe

2495 2500 2505

Gln Glu Trp Ser Glu Asn Phe Cys Asp Arg Arg Gln Lys Leu Tyr

2510 2515 2520

Asp Lys Leu Asn Ser Glu Cys Ile Ser Ala Glu Cys Thr Asn Gly

2525 2530 2535

Ser Val Asp Asn Ser Lys Cys Thr His Ala Cys Val Asn Tyr Lys

2540 2545 2550

Asn Tyr Ile Leu Thr Lys Lys Thr Glu Tyr Glu Ile Gln Thr Asn

2555 2560 2565

Lys Tyr Asp Asn Glu Phe Lys Asn Lys Asn Ser Asn Asp Lys Asp

2570 2575 2580

Ala Pro Asp Tyr Leu Lys Glu Lys Cys Asn Asp Asn Lys Cys Glu

2585 2590 2595

Cys Leu Asn Lys His Ile Asp Asp Lys Asn Lys Thr Trp Lys Asn

2600 2605 2610

Pro Tyr Glu Thr Leu Glu Asp Thr Phe Lys Ser Lys Cys Asp Cys

2615 2620 2625

Pro Lys Pro Leu Pro Ser Pro Ile Lys Pro Asp Asp Leu Pro Pro

2630 2635 2640

Gln Ala Asp Glu Pro Phe

2645

<210> 6

<211> 7947

<212> DNA

<213> 人工合成序列

<400> 6

atggacagca ccagcacaat cgccaacaag atcgaggaat atctgggagc taagtccgat 60

gacagcaaga tcgacgaact gctgaaggcc gatcctagcg aagtggagta ctacagaagc 120

ggaggcgacg gcgactacct gaagaacaac atctgcaaga tcaccgtgaa ccacagcgat 180

agcggcaagt atgacccctg cgagaagaag ctgcccccct acgacgacaa cgaccagtgg 240

aagtgccagc agaacagcag cgacggcagc ggcaagcccg agaacatctg cgtgcccccc 300

agacgggagc ggctgtgcac ctacaacctg gaaaacctga agttcgacaa gatccgggac 360

aacaacgcct tcctggccga cgtgctgctg accgcccgga acgagggcga gaagatcgtg 420

cagaaccacc ccgacaccaa cagcagcaac gtgtgcaacg ccctggaacg gtccttcgct 480

gacctggctg acatcatccg gggcaccgat cagtggaagg gcaccaactc caatctggaa 540

aagaacctga agcagatgtt cgccaagatc agagaaaacg acaaggtgct gcaggacaag 600

taccccaagg accagaagta caccaagctg cgggaggcct ggtggaacgc caaccggcag 660

aaagtgtggg aagtgatcac ctgtggcgcc agaagcaacg atctgctgat caagcggggc 720

tggcggacca gcggcaagag cgaccggaag aaaaacttcg agctgtgccg gaagtgcggc 780

cactacgaga aagaggtgcc caccaagctg gactacgtgc cccagttcct gcggtggctg 840

accgagtgga tcgaggactt ctaccgggag aagcagaacc tgatcgacga catggaacgg 900

caccgggagg aatgcaccag agaggaccac aagagcaaag agggcaccag ctactgcagc 960

acatgcaagg acaagtgcaa gaaatactgc gagtgcgtga agaaatggaa aaccgagtgg 1020

gagaaccagg aaaacaagta caaggacctg tacgagcaga acaagaacaa gaccagccag 1080

aagaacacca gcagatacga cgactacgtg aaggacttct tcgagaagct ggaagccaac 1140

tacagcagcc tggaaaacta catcaagggc gacccctatt tcgctgagta cgctacaaaa 1200

ctgagcttca tcctgaaccc cagcgacgcc aacaacccca gcggcgagac agccaaccac 1260

aacgacgagg cctgcaactg caacgagagc ggcatcagca gcgtgggcca ggctcagaca 1320

tccggcccta gcagcaacaa gacctgtatc acccacagct ccatcaagac caacaagaaa 1380

aaagaatgca aggacgtgaa gctgggcgtg cgggagaacg acaaggatct gaagatctgc 1440

gtgatcgagg acaccagcct gagcggcgtg gacaactgct gctgccagga tctgctgggc 1500

atcctgcagg aaaactgcag cgacaacaag cggggcagca gctccaacga cagctgcgac 1560

aataagaacc aggacgagtg ccagaaaaag ctggaaaagg tgttcgccag cctgaccaac 1620

ggctacaagt gcgataagtg caagagcggc acctcccggt ccaagaagaa gtggatctgg 1680

aagaagtcca gcggcaacga ggaaggcctg caggaagagt acgccaacac catcggcctg 1740

ccccccagga cccagagcct gtacctgggc aatctgccca aactggaaaa cgtgtgcgag 1800

gatgtgaagg acatcaactt cgacaccaaa gagaagtttc tggccggctg cctgatcgtg 1860

tccttccacg agggcaagaa tctgaagaag cgctaccccc agaataagaa cagcggcaac 1920

aaagaaaacc tgtgcaaggc tctggaatac agcttcgccg actacggcga cctgatcaag 1980

ggcacctcca tctgggacaa cgagtacaca aaggacctgg aactgaatct gcagaacaac 2040

ttcggcaagc tgttcggcaa gtacatcaag aagaacaata ccgccgagca ggacacctcc 2100

tacagctccc tggacgagct gcgcgagtct tggtggaata ccaataagaa gtacatctgg 2160

accgccatga agcacggcgc cgagatgaac atcaccacct gtaacgccga cggctccgtg 2220

accggcagcg gctccagctg cgacgacatc cccaccatcg acctgatccc ccagtacctg 2280

agatttctgc aggaatgggt cgagaacttc tgcgagcagc ggcaggccaa agtgaaggac 2340

gtgatcacca actgcaagag ctgcaaagaa tccggcaaca aatgcaagac cgagtgcaaa 2400

accaagtgca aggatgagtg cgagaagtac aagaagttca tcgaggcctg cggcacagcc 2460

ggcggaggca tcggaacagc cggcagcccc tggtccaaga gatgggacca gatctacaag 2520

cggtacagca agcacatcga ggacgccaag cggaaccgga aggccggcac caagaactgc 2580

ggcaccagct ccaccaccaa cgccgctgcc agcaccgacg agaataagtg cgtgcagagc 2640

gacatcgaca gctttttcaa gcacctgatc gatatcggcc tgaccacccc cagcagctac 2700

ctgagcaacg tgctggacga caacatctgt ggcgccgaca aggccccctg gacaacctat 2760

acaacataca ctacaaccga gaagtgcaac aaagagcggg acaagagcaa gagccagagc 2820

agcgacaccc tggtggtggt gaacgtgccc agccccctgg gcaacacacc ctaccggtac 2880

aagtacgcct gccagtgcaa gatccccacc aacgaggaaa catgcgacga ccggaaagaa 2940

tacatgaacc agtggtcctg cgggagcgct cggaccatga agagagggta taagaacgat 3000

aactacgaac tgtgcaagta caacggcgtg gatgtgaagc ccaccaccgt gcggagcaac 3060

tccagcaagc tggacggcaa cgacgtgacc ttcttcaacc tgttcgagca gtggaacaaa 3120

gaaatccagt accagatcga gcagtacatg accaacgcca acatcagctg catcgatgag 3180

aaagaagtgc tggacagcgt ctccgacgag ggcaccccca aagtgcgggg aggctacgag 3240

gacggccgga acaacaacac agatcagggc acaaattgca aagaaaagtg caagtgctac 3300

aagctgtgga tcgagaagat caacgatcag tggggcaagc agaaggacaa ctacaacaag 3360

ttccggtcca agcagatcta cgacgccaat aagggcagcc agaacaaaaa ggtggtgtcc 3420

ctgagcaact tcctgttctt cagctgctgg gaggaataca tccagaagta cttcaacggc 3480

gattggagca agatcaagaa catcggctcc gacaccttcg agtttctgat caagaagtgc 3540

ggcaacaaca gcgcccacgg cgaggaaatc ttcagcgaga agctgaagaa cgccgagaag 3600

aagtgcaaag agaacgagag caccgacacc aatatcaaca agagcgagac atcctgcgac 3660

ctgaacgcca ccaactacat ccggggctgc cagagcaaga cctacgacgg caagatcttc 3720

cccggcaagg gcggcgagaa gcagtggatt tgcaaggaca ccatcatcca cggcgacacc 3780

aacggcgcct gcatcccccc tcgcacccag aacctgtgcg tgggcgagct gtgggacaag 3840

tcctacggcg gcagatccaa catcaagaac gataccaaag aactgctgaa agagaagatt 3900

aagaacgcca tccacaaaga gacagagctg ctgtacgagt accacgacac cggcaccgcc 3960

atcatcagca agaatgacaa gaagggccag aagggcaaga acgaccctaa cggcctgccc 4020

aaaggcttct gtcatgctgt gcagcggagc ttcatcgact acaagaacat gatcctgggc 4080

accagcgtga acatctacga gcacatcggc aagctgcagg aagatatcaa gaagatcatc 4140

gaaaagggca cccctcagca gaaggataag atcggcggcg tgggcagcag caccgagaac 4200

gtgaacgctt ggtggaaggg aatcgagcgg gagatgtggg acgccgtgag atgcgccatc 4260

accaagatca ataagaagaa taacaacagc atcttcaatg gcgacgagtg cggcgtgagc 4320

ccccccaccg gcaacgatga ggaccagagc gtgtcctggt tcaaagagtg gggcgagcag 4380

ttctgcatcg agcggctgag atacgaacag aacatcagag aggcctgcac catcaacggc 4440

aaaaacgaga agaaatgcat caacagcaag tccggccagg gcgataagat ccagggcgcc 4500

tgcaagcgga agtgtgaaaa gtataagaag tatatcagcg agaaaaaaca ggaatgggac 4560

aagcagaaaa ccaagtacga gaacaaatac gtgggcaaga gcgccagcga tctgctgaaa 4620

gaaaactacc ccgagtgcat cagcgccaac ttcgacttca tcttcaacga caatatcgag 4680

tacaagacct actaccccta cggcgattac agcagcatct gcagctgcga acaggtgaag 4740

tactataagt acaacaatgc cgagaaaaag aacaacaaga gcctgtgcta cgagaaggac 4800

aatgacatga cctggtctaa gaaatatatc aagaagctgg aaaacggccg gtccctggaa 4860

ggcgtgtacg tgccccctcg gcggcagcag ctgtgtctgt acgagctgtt ccctatcatc 4920

atcaaaaacg aagagggcat ggaaaaggcc aaagaggaac tgctggaaac cctgcagatc 4980

gtggccgagc gcgaggccta ctacctgtgg aagcagtaca accccacagg caaagggatt 5040

gatgatgcca acaagaaggc ctgctgcgcc atcagaggca gcttctacga cctggaagat 5100

attatcaagg gcaacgacct ggtgcacgac gagtacacca aatacatcga ctccaagctg 5160

aacgagatct tcggctccag caacaccaac gacatcgata ccaagcgggc cagaaccgat 5220

tggtgggaga acgagacaat caccaacggc accgacagaa agaccatccg gcagctggtg 5280

tgggatgcca tgcagagcgg agtgcgatat gctgtggagg aaaagaacga gaacttcccc 5340

ctgtgcatgg gcgtggagca cattggcatt gccaagcccc agttcatcag atggctggaa 5400

gagtggacca acgagttctg cgagaaatac accaagtact tcgaggacat gaagtccaag 5460

tgcgaccccc ccaagagggc cgacacatgc ggcgacaact ccaacatcga gtgcaagaag 5520

gcttgcgcca actacaccaa ctggctgaac cccaagcgga tcgagtggaa cggcatgagc 5580

aactactaca ataagatcta ccggaagtct aacaaagaga gcgaggacgg caaggactac 5640

agcatgatca tggcccccac cgtgatcgat tacctgaaca agcggtgcca cggcgagatc 5700

aatggcaact acatctgctg cagctgcaag aatatcggcg cctacaacac cacctccggc 5760

accgtgaaca agaagctgca gaaaaaagaa accgagtgcg aggaagagaa gggccctctg 5820

gacctgatga acgaggtgct gaacaagatg gacaagaagt actccgccca caagatgaag 5880

tgcaccgagg tgtacctgga acacgtggag gaacagctga atgagatcga caacgccatc 5940

aaggattaca agctgtaccc cctggacaga tgcttcgacg accagaccaa gatgaaagtg 6000

tgcgatctga tcgccgacgc catcggctgc aaggataaga caaagctgga tgagctggac 6060

gaatggaacg acatggacct gagaggcacc tacaataagc acaagggcgt gctgatcccc 6120

cccaggcgga gacagctgtg cttcagccgg atcgtgcggg gacccgccaa cctgagaagc 6180

ctgaacgagt tcaaagagga aatcctgaag ggcgctcagt ccgagggcaa gttcctgggc 6240

aattactaca aagagcacaa ggacaaagag aaggccctgg aagccatgaa gaacagcttt 6300

tacgactacg aggacatcat caagggaacc gacatgctga ccaacatcga attcaaggat 6360

atcaagatca agctggaccg gctgctggaa aaagagacaa acaacaccaa gaaagccgag 6420

gactggtgga aaaccaacaa aaagtctatt tggaacgcca tgctgtgcgg ctacaaaaag 6480

agcggcaata agatcattga ccccagctgg tgcaccatcc ctaccaccga gacacccccc 6540

cagtttctga gatggatcaa agaatggggc accaatgtgt gcatccagaa gcaggaacac 6600

aaagaatacg tcaagtctaa gtgctccaac gtgaccaatc tgggagcaca ggccagcgag 6660

agcaacaact gcaccagcga gatcaagaaa taccaggaat ggtcccggaa gcggagcatc 6720

cagtgggaga caatcagcaa gcgctacaag aaatacaagc ggatggacat cctgaaggat 6780

gtgaaagagc ctgacgccaa tacctacctg agagaacact gcagcaagtg cccctgcggc 6840

ttcaacgata tggaagagat gaacaacaac gaggacaatg agaaagaggc cttcaagcag 6900

atcaaagaac aggtgaaaat ccccgccgag ctggaagatg tgatctacag aatcaagcac 6960

cacgagtacg acaagggcaa tgactacatc tgtaacaaat acaagaatat ccacgaccgg 7020

atgaagaaaa acaacggcaa cttcgtgacc gacaatttcg tgaagaagtc ctgggagatt 7080

tccaatggag tgctgatccc tcctcggaga aagaacctgt tcctgtacat cgaccccagc 7140

aagatctgcg aatacaagaa ggaccccaag ctgttcaagg acttcatcta ttggagcgcc 7200

ttcaccgagg tggagcggct gaagaaggcc tacggcggag ccagagccaa ggtggtgcac 7260

gccatgaagt atagcttcac cgacatcggc agcatcatta agggcgacga catgatggaa 7320

aagaatagca gcgacaagat cggcaagatc ctgggggaca ccgacggcca gaacgagaag 7380

cgcaaaaagt ggtgggacat gaacaagtac cacatctggg agagcatgct gtgtggatac 7440

cgggaggccg agggagatac agagactaac gaaaactgcc ggttccccga catcgagagc 7500

gtgcctcagt ttctgcgctg gtttcaggaa tggtcagaga atttttgcga tagaaggcag 7560

aagctgtacg acaagctgaa cagcgagtgc atctccgccg agtgcaccaa tggctccgtg 7620

gacaatagca agtgcaccca cgcctgcgtg aactacaaga attacatcct gaccaaaaag 7680

accgagtacg agatccagac caataagtac gacaacgagt ttaagaacaa gaatagcaac 7740

gataaggacg cccccgacta tctgaaagag aaatgcaacg acaacaagtg cgagtgcctg 7800

aataagcaca ttgacgacaa gaacaaaacc tggaagaacc cctacgagac actggaagat 7860

accttcaaga gcaagtgcga ctgccctaag cccctgccct cccccatcaa gcccgacgat 7920

ctgccacccc aggccgacga gcccttc 7947

<210> 7

<211> 632

<212> PRT

<213> 人工合成序列

<400> 7

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

1 5 10 15

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

20 25 30

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

35 40 45

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

50 55 60

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

65 70 75 80

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

85 90 95

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

100 105 110

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

115 120 125

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

130 135 140

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

145 150 155 160

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

165 170 175

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

180 185 190

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

195 200 205

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

210 215 220

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

225 230 235 240

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

245 250 255

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

260 265 270

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

275 280 285

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

290 295 300

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

305 310 315 320

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

325 330 335

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

340 345 350

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

355 360 365

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

370 375 380

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

385 390 395 400

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

405 410 415

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

420 425 430

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

435 440 445

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

450 455 460

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

465 470 475 480

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

485 490 495

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

500 505 510

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

515 520 525

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

530 535 540

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

545 550 555 560

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

565 570 575

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

580 585 590

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

595 600 605

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

610 615 620

Arg Ser Asn Ser Ser Lys Leu Asp

625 630

<210> 8

<211> 1896

<212> DNA

<213> 人工合成序列

<400> 8