CN106749678A - Her2 car重组慢病毒载体及其构建方法与应用 - Google Patents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及一种HER2 CAR重组慢病毒载体及其构建方法与应用，其中CAR结构基因的核苷酸序列如SEQ ID 1所示；通过将抗HER2 ScFV基因和CAR结构基因进行拼接，得到HER2 CAR基因，其核苷酸序列如SEQ ID 3所示；将Pre‑Lenti‑EF1‑MCS载体和HER2 CAR基因重组构建，得到HER2 CAR重组慢病毒载体，核苷酸序列如SEQ ID 4所示。本发明提供的HER2 CAR重组慢病毒载体，通过包装成HER2 CAR慢病毒并感染新鲜分离的T细胞，制备得到的HER2 CART可以有效杀死A431靶细胞，明显抑制肿瘤细胞的生长，可以用于制备抗肿瘤药物。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种HER2 CAR重组慢病毒载体及其构建方法与应用。
背景技术
肿瘤引起的死亡率在世界范围内排名第一。据国家肿瘤登记中心发布的《2013中国肿瘤登记年报》预测，2015年，中国肿瘤发病人数为428万人，约280万人死亡。目前肿瘤的治疗方式主要是手术、放疗、化疗、靶向治疗和最新的免疫治疗，手术、放化疗和靶向治疗作为临床治疗肿瘤的主要手段，有着不同的适应症和限制因素。免疫治疗和前述四种疗法的区别主要在于通过活化机体的免疫系统，杀死肿瘤细胞，即免疫治疗药物或技术没有针对肿瘤细胞，而是调动机体内处于免疫抑制状态或对肿瘤处于免疫逃逸状态的免疫细胞杀死肿瘤。免疫治疗提供了一种全新的治疗肿瘤的策略，能够开发出许多种药物和技术，为肿瘤治疗添加了新武器。免疫治疗目前最为可行的两个手段是免疫检查点抑制剂和嵌合抗原受体T细胞(Chimeric Antigen Receptor T cells，CART)。其中CART主要通过基因修饰T细胞赋予了T细胞识别免疫逃逸的肿瘤细胞的能力，识别的肿瘤细胞会被T细胞杀死，在治疗白血病和淋巴瘤时显示了惊人的疗效，约93％的儿童复发、难治性白血病被CART治愈。最为经典的CART识别分子为CD19，主要表达于急性B淋巴细胞性白血病细胞。CART细胞可以识别别的肿瘤细胞标志分子，比如，髓系白血病的CD33标志分子，多发性骨髓瘤的BCMA、CD138标志分子，实体瘤的EGFR、HER2等标志分子。鉴于CART治疗白血病的惊人疗效，一些研究机构正在尝试利用EGFR、HER2等肿瘤组织高表达的分子作为靶点，研究观察CART在实体瘤治疗方面的效果。
在CART的技术发展中，经过了不同代的演化，第一代CART只有CD3信号，没有协同刺激信号，在临床试验中，效果不好；宾西法尼亚大学的Carl June教授在2011年，利用二代CART技术治疗3例CLL(慢性淋巴细胞性白血病)取得了非常好的效果，2例完全缓解(CR)，1例部分缓解(PR)；二代CART根据选用的协同刺激分子不同，分为CD28协同刺激分子和4-1BB协同刺激分子，两类CART都在临床治疗ALL(急性淋巴细胞性白血病)时取得了成功；CD28协同刺激信号促使细胞快速扩增，4-1BB信号促使细胞扩增、持续存活时间更长。以HER2分子为靶点的CART，最早见于2009年的文章，报道了HER2 CART能够杀死HER2阳性的肿瘤细胞，在动物实验中显示了确切的效果，此CART结构为二代CART，包括CD28协同刺激信号和CD3信号。2015年，Ahmed,N.等利用HER2 CART细胞治疗19名HER2+肉瘤患者的临床试验中，使用细胞数量为1x108/m2，17名可评估患者中，3人在治疗后6个月，12个月与16个月持续缓解，Ahmed,N.等使用的HER2 CART结构也为CD28分子和CD3分子的二代CART。然而，现有2代HER2CART在治疗肿瘤的临床试验中，效果一般，只有大约17.6％的完全缓解率(3/17)，需要对二代CART再次进行改造，以取得更好的疗效。
发明内容
针对现有技术中的缺陷，本发明目的在于提供一种HER2 CAR重组慢病毒载体及其构建方法与应用。
本发明提供一种蛋白，是如下(a)或(b)：(a)由序列表中序列12所示的氨基酸序列组成的蛋白质；(b)将序列表中序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列12衍生的蛋白质。
本发明也保护编码上述蛋白的HER2 CAR基因。
本发明提供一种HER2 CAR基因，HER2 CAR基因是如下的任一种DNA分子：(a)核苷酸序列如SEQ ID 3所示的DNA分子；(b)在严格条件下与(a)限定的核苷酸序列杂交且编码具有相同功能的蛋白的DNA分子；(c)与(a)限定的核苷酸序列至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码具有相同功能的蛋白的DNA分子。
严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
含有上述HER2 CAR基因的重组载体、表达盒、转基因细胞系或重组菌属于本发明的保护范围；重组载体具体为将上述的HER2 CAR基因插入表达载体中，得到表达权利要求1所述蛋白的重组载体；当然，也保护扩增基因全长或其任意片段的引物对。
本发明提供一种HER2 CAR基因的合成方法，包括如下步骤：将抗HER2 ScFV基因和CAR结构基因采用重叠PCR进行拼接，得到HER2 CAR基因；其中，CAR结构基因的核苷酸序列如SEQ ID 1所示，抗HER2 ScFV基因的核苷酸序列如SEQ ID 2所示；得到的HER2 CAR基因的核苷酸序列如SEQ ID 3所示。其中，抗HER2 ScFV基因的序列来源于美国专利US5821337(1998年)，其编码的蛋白质为全长人源化抗体，商品名Herceptin，包括重链氨基酸密码子优化的核苷酸、linker(G4S)3和轻链氨基酸密码子优化的核苷酸序列。
本发明提供一种CAR结构基因，CAR结构基因是如下的任一种DNA分子：(a)核苷酸序列如SEQ ID 1所示的DNA分子；(b)在严格条件下与(a)限定的核苷酸序列杂交且编码具有相同功能的蛋白的DNA分子；(c)与(a)限定的核苷酸序列至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码具有相同功能的蛋白的DNA分子。严格条件同上面所述。
含有上述CAR结构基因的重组载体、表达盒、转基因细胞系或重组菌属于本发明的保护范围，也保护扩增基因全长或其任意片段的引物对。
由上述CAR结构基因编码得到的蛋白属于本发明的保护范围。CAR结构基因编码的蛋白包括CD8铰链区，CD28铰链区、跨膜区、胞浆区，4-1BB胞浆区和CD3ζ胞浆区。
本发明提供一种HER2 CAR重组慢病毒载体，HER2 CAR重组慢病毒载体为Pre-Lenti-EF1-HER2 CAR，其核苷酸序列如SEQ ID 4所示。
本发明提供一种HER2 CAR重组慢病毒载体的构建方法，包括如下步骤：采用重叠PCR合成HER2 CAR基因，两端设置XbaI、BamHI限制性内切酶位点；其中，HER2 CAR基因的核苷酸序列如SEQ ID 3所示；用限制性内切酶XbaI、BamHI双酶切Pre-Lenti-EF1-MCS载体和HER2 CAR基因，分别回收酶切后的目的产物；其中，Pre-Lenti-EF1-MCS载体的核苷酸序列如SEQ ID 5所示；连接回收获得的酶切后的目的产物，得到HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR。需要说明的是，将得到的HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR转化，挑取克隆，提取质粒，测序，得到测序正确的能够表达HER2 CAR的慢病毒载体Pre-Lenti-EF1-HER2 CAR。
在上述的构建过程，采用的引物序列包括：第一引物序列，其核苷酸序列如SEQ ID6所示；第二引物序列，其核苷酸序列如SEQ ID 7所示；第三引物序列，其核苷酸序列如SEQID 8所示；第四引物序列，其核苷酸序列如SEQ ID 9所示。在构建过程中，采用第一引物序列和第三引物序列扩增HER2 ScFV基因得到前面片段，采用第二引物序列和第四引物序列扩增3代CAR结构的基因得到后面片段，然后重叠，得到全长3代HER2 CAR基因，再克隆到Pre-Lenti-EF1-MCS载体。
本发明还保护下述任一项物质在制备抗肿瘤药物中的应用：1)含有上述的蛋白；2)含有CAR基因或HER2 CAR基因的重组载体、表达盒、转基因细胞系或重组菌；3)HER2 CAR重组慢病毒载体；4)含有HER2 CAR重组慢病毒载体的表达盒、转基因细胞系或重组菌。
本发明提供的技术方案，具有如下的有益效果：构建得到的HER2 CAR重组慢病毒载体Pre-Lenti-EF1-MCS-HER2 CAR，通过包装成HER2 CAR慢病毒并感染新鲜分离的T细胞，制备得到的HER2 CART可以有效杀死A431靶细胞，明显抑制肿瘤细胞的生长，为应用于制备抗肿瘤药物提供了可能。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1为本发明实施例中的CART载体设计的演化示意图；
图2为本发明实施例中的HER2 CAR慢病毒载体构建流程图；
图3为本发明实施例中的HER2 CART表面的单链抗体识别HER2蛋白实验的流式细胞分析阴性对照图；
图4为本发明实施例中的HER2 CART表面的单链抗体识别HER2蛋白实验的流式细胞分析图；
图5为本发明实施例中的HER2 CART有效杀死A431肿瘤细胞实验的显微镜图；
图6为本发明实施例中的HER2 CART有效杀死A431肿瘤细胞实验的LDH释放实验图；
图7为本发明实施例中的HER2 CART有效杀死A431肿瘤细胞实验检测得到的IFN-γ浓度情况图；
图8为本发明实施例中的HER2 CART抑制小鼠皮下肿瘤生长实验的动物实验设计图；
图9为本发明实施例中的HER2 CART抑制小鼠皮下肿瘤生长实验的肿瘤生长曲线图；
图10为本发明实施例中的HER2 CART抑制小鼠皮下肿瘤生长实验的小鼠皮下肿瘤大体观察图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只是作为示例，而不能以此来限制本发明的保护范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。
本发明中，HER2 ScFV基因、CAR结构基因和所有的引物均由中美泰和公司合成；重叠PCR反应、酶切反应、回收酶切产物和酶连反应使用的试剂盒、反应体系和反应程序都是采用常规使用的试剂盒和对应的方法。
图1为CART载体设计的演化示意图。一代载体胞外通过单链抗体(scFv)识别肿瘤标志分子，如CD19，胞内利用CD3分子ζ链传递信号；二代载体分为两类，一种为协同刺激分子CD28+CD3ζ，一种为协同刺激分子4-1BB+CD3ζ，CD28分子促进T细胞扩增，4-1BB分子不仅促进T细胞扩增，并且延长CART细胞体内存活时间；包括协同刺激分子CD28、4-1BB加上CD3ζ分子的三代载体，基本模拟了T细胞正常发挥作用时受到的刺激信号，和二代载体相比，具有更好的效果。
实施例一：HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR的构建
1.引物设计
使用软件pDRAW32分析HER2 CAR基因序列，限制性内切酶XbaI、BamHI适合用于基因克隆，能够和Pre-Lenti-EF1-MCS载体的多克隆位点中的XbaI、BamHI匹配，两端引物设计如下。
XbaI-up序列如SEQ ID 6所示：
GCTCTAGA GCCACCATGCCTCTGCTGCTGCTGCTG；
BamHI-down序列如SEQ ID 7所示：
CGGGATCCTTAGCGAGGGGGCAGGGCCTG；
Middle-R序列如SEQ ID 8所示：
GTCGTGGTGGGCTTCGCTGGCTTGATTTCCACTTTTGTGC；
Middle-F序列如SEQ ID 9所示：
GCACAAAAGTGGAAATCAAGCCAGCGAAGCCCACCACGAC。
2.PCR扩增、分子克隆
采用XbaI-up和Middle-R PCR扩增HER2 ScFV基因得到前面片段，Middle-F和BamHI-down PCR扩增3代CAR结构的基因得到后面片段，然后重叠，得到全长3代HER2 CAR基因，其核苷酸序列如SEQ ID 3所示。
将3代HER2 CAR基因克隆到Pre-Lenti-EF1-MCS载体上，具体如下步骤：
双酶切：用限制性内切酶XbaI、BamHI双酶切Pre-Lenti-EF1-MCS载体和HER2 CAR基因，分别回收酶切后的目的产物；其中，Pre-Lenti-EF1-MCS载体的核苷酸序列如SEQ ID5所示。
酶连反应：连接所述回收获得的酶切后的目的产物，得到HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR(核苷酸序列如SEQ ID 4所示)。
将得到的产物送测序，引物为：
Pre-up-Seq如SEQ ID10所示：GGAGCCTACCTAGACTCAGC
Pre-down-Seq如SEQ ID 11所示：CAACCAGGATTTATACAAGG。
测序正确，得到HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR。构建过程示意图如图2所示。
实施例二：细胞杀伤实验
(1)包装慢病毒
293T细胞培养于37℃，5％CO2孵箱内，培养基为DMEM(Hyclone)/10％FBS(兰州百灵)。包装前一天，消化293T细胞，7×106细胞/皿种植10cm培养皿。转染细胞时，除了Pre-Lenti-EF1-MCS-HER2 CAR质粒外，还要和包装质粒psPAX2、pMD2.0G(Addgene)共转染。其中Pre-Lenti-EF1-MCS-HER2 CAR 5μg，psPAX2质粒3.75μg，pMD2.0G质粒1.25μg。转染时，将三种质粒的混合物加入500μl opti-MEM培养基(Invitrogen)内，在另一个1.5ml微型离心管内将25μl Lipofectamine 2000试剂加入500μl opti-MEM培养基内，然后，将稀释的转染试剂加入稀释的质粒上部，混匀，瞬时离心，室温静置20分钟，最后将质粒和转染试剂混合物加入10cm培养皿的培养基内(室温孵育质粒期间，旧培养基换为5ml opti-MEM培养基，增加转染效率)，轻晃数下混匀，放入37℃，5％CO2孵箱，5小时后，换为10ml新鲜DMEM/10％FBS培养基。
细胞转染3天后，收获病毒，将10ml含病毒培养基转入15ml离心管内，4℃，1250rpm，离心5分钟，去除死亡的293T细胞，然后，将含病毒上清过滤，浓缩，分装，-80℃冻存，留存部分病毒测定滴度。
(2)外周血单核细胞分离
抽血：在无菌的条件下抽取人体的静脉血10ml。
分离外周血单核细胞(PBMC)：使用人淋巴细胞分离液(达科为生物工程有限公司)，依照说明书进行，获得PBMC，用含IL2、灭活血清的1640培养基重悬，计数。
(3)T细胞纯化
淋巴细胞计数，取1x107个淋巴细胞于1.5ml微型离心管中。250g，22℃，离心10分钟。吸弃上层液体，80μL磁珠分离缓冲液重悬细胞，加入20μl的CD3MicroBeads(美天旎)。4℃冰箱中放置1小时。1小时后，加入1mL的磁珠分离缓冲液，轻轻颠倒混匀，250g，5℃离心10分钟。将过滤柱安放在磁铁上，用500μl的缓冲液润洗。离心洗涤的细胞弃上清，500μl缓冲液重悬，加入过滤柱上方，细胞悬液流出后，柱子用500μl的缓冲液洗四次。将过滤柱从磁铁上卸掉，使用1mL缓冲液将磁性吸附的T细胞冲出，至1ml离心管。250g，5℃，10分钟。含IL2、含10％灭活血清的1640培养基重悬，计数。
(4)T细胞慢病毒感染
纯化得到的T细胞计数，2×106T细胞/孔(6孔板)种植，培养过夜后，加入MOI为2的对照病毒液(空载体包装，Control)和Pre-Lenti-EF1-MCS-HER2 CAR病毒液，1000g，22℃，离心30分钟，放入孵箱，感染过夜。
感染第一天，补加1ml新鲜培养基。
感染第三天，T细胞增殖旺盛，将T细胞收获、离心后转入25cm2培养瓶。
细胞扩增至一定数量后，利用流式细胞仪鉴定HER2 CART表面识别HER2的单链抗体的表达阳性率，具体结果如图3和图4所示。取一定数量的CART细胞，分为两管，一管使用HER2-Fc融合蛋白和HER2 CART孵育，然后加入PE二抗(HER2-Fc)(对应图4)，另一管只加入PE二抗，作为阴性对照(control)(对应图3)，流式细胞术分析结果图3和图4可见，约86％的CART细胞表达能识别HER2蛋白的单链抗体，即CART病毒感染阳性率为86％。
(5)杀伤实验
计数高表达HER2的A431细胞，1×104个A431细胞/孔(96孔板)；计数感染的T细胞(Control病毒液感染的Control T细胞和Pre-Lenti-EF1-MCS-HER2 CAR病毒液感染的HER2CART细胞)，按照效靶比10:1,5:1,2.5:1的比例加入A431细胞上层，实验设计如表1所示。
表1杀伤实验设计表(靶细胞：A431)
(6)杀伤测量
通过测量死亡细胞释放的LDH，确定CART细胞杀死A431靶细胞的效果。
操作按照Promega LDH释放试剂盒说明书(CytoTox 96非放射性细胞毒性检测，货号：G1780)。
步骤(5)中杀伤实验过夜，约18h后，最大释放孔加入10×细胞裂解液20ul。
2小时后，每孔取50μl上清，然后加入50μl LDH酶底物，室温静置反应20分钟，酶标仪测量492nm处的吸光度OD492。同时，取50ul上清，进行ELISA实验，观察IFN-γ分泌水平。
杀伤率计算公式为：
实验孔为不同效靶比得到的OD492吸光度值，效应细胞自发为Control T细胞或HER2 CART细胞的OD492值，靶细胞自发为A431最小释放，靶细胞最大为A431最大释放(加入裂解液孔)。
计算得到的杀伤率见图5、图6和图7所示：从图5的细胞实验后显微镜图片，可见下面的图中没有肿瘤细胞，已被杀死，CART细胞活化后聚集成团；从图6可以看到LDH释放实验检测杀伤效果，从图7可以看到杀伤实验后，ELISA检测培养液内IFN-γ浓度，杀伤效果明显的HER2 CART分泌高浓度IFN-γ，以上结果表明Pre-Lenti-EF1-MCS-HER2 CART的杀伤功能较好。
实施例三：动物实验
小鼠品系：为NSG免疫缺陷小鼠(缺乏T细胞、B细胞和NK细胞功能)。
肿瘤接种：采用A431肿瘤细胞系，3x106细胞/100ul PBS皮下注射(10只小鼠)。
CART注射：A431肿瘤细胞皮下接种8天后，尾静脉注射Control或HER2 CART细胞(1x107细胞/200ul PBS，一组5只小鼠，隔日注射，每只小鼠共注射3次)，动物实验设计如图8所示。
每3天观察，测量小鼠皮下肿瘤体积，直至CART细胞注射18天后，HER2 CART组皮下肿瘤消失，小鼠麻醉后照相。
实验结果：肿瘤生长曲线如图9所示，尾静脉注射CART细胞后，每3天测量肿瘤体积，绘制肿瘤生长曲线，可见HER2 CART明显抑制肿瘤生长；小鼠皮下肿瘤大体观察如图10所示(HER2 CART应用18天后)。从图9和图10的结果可知，经过HER2 CART处理，可以显著抑制肿瘤的生长。
本发明提供的HER2 CAR重组慢病毒载体及其构建方法与应用，构建得到的HER2CAR重组慢病毒载体Pre-Lenti-EF1-MCS-HER2 CAR，通过包装成HER2 CAR慢病毒并感染新鲜分离的T细胞，制备得到的HER2 CART可以有效杀死A431靶细胞，明显抑制肿瘤细胞的生长，为应用于制备抗肿瘤药物提供了可能。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对步骤、数字表达式和数值并不限制本发明的范围。在这里示出和描述的所有示例中，除非另有规定，任何具体值应被解释为仅仅是示例性的，而不是作为限制，因此，示例性实施例的其他示例可以具有不同的值。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。
SEQUENCE LISTING
<110> 北京普瑞金科技有限公司、深圳咖荻生物科技有限公司
<120> HER2 CAR重组慢病毒载体及其构建方法与应用
<130> 2017
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 939
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<213> 人工合成
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ccagcgaagc ccaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 60
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 120
acgagggggc tggacttcgc cccacgcaaa attgaagtta tgtatcctcc tccttaccta 180
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 240
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg tggagtcctg 300
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 360
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 420
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 480
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 540
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 600
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 660
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 720
gaccctgaga tggggggaaa gccgcagaga aggaagaacc ctcaggaagg cctgtacaat 780
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 840
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 900
tacgacgccc ttcacatgca ggccctgccc cctcgctaa 939
<210> 2
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atgcctctgc tgctgctgct gccactgctg tgggcaggag cactggctga ggtgcagctg 60
gtcgaaagcg gaggaggact ggtgcagcct ggagggagtc tgaggctgtc atgcgccgct 120
agcggcttta acatcaaaga cacctacatt cactgggtgc gacaggctcc aggaaaggga 180
ctggagtggg tcgcacgaat ctatcctact aatggctaca cccgatatgc cgactccgtg 240
aaagggcggt ttacaatttc cgccgataca tctaagaaca ctgcttacct gcagatgaat 300
agtctgagag ctgaagacac tgcagtctac tattgcagca gatggggagg cgacggcttc 360
tacgccatgg attattgggg ccagggaacc ctggtgacag tcagctccgg aggaggaggg 420
agcggcggag gaggcagcgg aggaggaggc tctgatatcc agatgaccca gtccccctct 480
agtctgtccg cctctgtggg cgacagggtc actattacct gtcgcgcttc tcaggatgtg 540
aacacagcag tcgcctggta ccagcagaag ccaggaaaag cacccaagct gctgatctac 600
agtgcctcat tcctgtatag tggcgtgcca tcacggttta gcgggtccag atctggaact 660
gacttcacac tgactatttc aagcctgcag cccgaggatt ttgccaccta ctattgtcag 720
cagcattata ccacaccccc taccttcgga cagggcacaa aagtggaaat caag 774
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atgcctctgc tgctgctgct gccactgctg tgggcaggag cactggctga ggtgcagctg 60
gtcgaaagcg gaggaggact ggtgcagcct ggagggagtc tgaggctgtc atgcgccgct 120
agcggcttta acatcaaaga cacctacatt cactgggtgc gacaggctcc aggaaaggga 180
ctggagtggg tcgcacgaat ctatcctact aatggctaca cccgatatgc cgactccgtg 240
aaagggcggt ttacaatttc cgccgataca tctaagaaca ctgcttacct gcagatgaat 300
agtctgagag ctgaagacac tgcagtctac tattgcagca gatggggagg cgacggcttc 360
tacgccatgg attattgggg ccagggaacc ctggtgacag tcagctccgg aggaggaggg 420
agcggcggag gaggcagcgg aggaggaggc tctgatatcc agatgaccca gtccccctct 480
agtctgtccg cctctgtggg cgacagggtc actattacct gtcgcgcttc tcaggatgtg 540
aacacagcag tcgcctggta ccagcagaag ccaggaaaag cacccaagct gctgatctac 600
agtgcctcat tcctgtatag tggcgtgcca tcacggttta gcgggtccag atctggaact 660
gacttcacac tgactatttc aagcctgcag cccgaggatt ttgccaccta ctattgtcag 720
cagcattata ccacaccccc taccttcgga cagggcacaa aagtggaaat caagccagcg 780
aagcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900
gggctggact tcgccccacg caaaattgaa gttatgtatc ctcctcctta cctagacaat 960
gagaagagca atggaaccat tatccatgtg aaagggaaac acctttgtcc aagtccccta 1020
tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttggtggagt cctggcttgc 1080
tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg 1140
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1200
taccagccct atgccccacc acgcgacttc gcagcctatc gctccaaacg gggcagaaag 1260
aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1320
gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1380
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1440
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gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1560
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1620
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1680
gcccttcaca tgcaggccct gccccctcgc taa 1713
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tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
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tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataaggat ctgcgatcgc tccggtgccc gtcagtgggc 2220
agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg 2280
tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 2340
ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 2400
tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc 2460
acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc 2520
tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc 2580
gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac 2640
gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg 2700
ttacagatcc aagctgtgac cggcgcctac gctagacgaa ttcctcgaga ctagttctag 2760
aatgccacca tgcctctgct gctgctgctg ccactgctgt gggcaggagc actggctgag 2820
gtgcagctgg tcgaaagcgg aggaggactg gtgcagcctg gagggagtct gaggctgtca 2880
tgcgccgcta gcggctttaa catcaaagac acctacattc actgggtgcg acaggctcca 2940
ggaaagggac tggagtgggt cgcacgaatc tatcctacta atggctacac ccgatatgcc 3000
gactccgtga aagggcggtt tacaatttcc gccgatacat ctaagaacac tgcttacctg 3060
cagatgaata gtctgagagc tgaagacact gcagtctact attgcagcag atggggaggc 3120
gacggcttct acgccatgga ttattggggc cagggaaccc tggtgacagt cagctccgga 3180
ggaggaggga gcggcggagg aggcagcgga ggaggaggct ctgatatcca gatgacccag 3240
tccccctcta gtctgtccgc ctctgtgggc gacagggtca ctattacctg tcgcgcttct 3300
caggatgtga acacagcagt cgcctggtac cagcagaagc caggaaaagc acccaagctg 3360
ctgatctaca gtgcctcatt cctgtatagt ggcgtgccat cacggtttag cgggtccaga 3420
tctggaactg acttcacact gactatttca agcctgcagc ccgaggattt tgccacctac 3480
tattgtcagc agcattatac cacaccccct accttcggac agggcacaaa agtggaaatc 3540
aagccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 3600
gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 3660
cacacgaggg ggctggactt cgccccacgc aaaattgaag ttatgtatcc tcctccttac 3720
ctagacaatg agaagagcaa tggaaccatt atccatgtga aagggaaaca cctttgtcca 3780
agtcccctat ttcccggacc ttctaagccc ttttgggtgc tggtggtggt tggtggagtc 3840
ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 3900
aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 3960
cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccaaacgg 4020
ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 4080
caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 4140
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 4200
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 4260
cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 4320
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 4380
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 4440
acctacgacg cccttcacat gcaggccctg ccccctcgct aaggatccac gcgtctggaa 4500
caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 4560
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 4620
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 4680
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 4740
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 4800
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 4860
gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct 4920
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 4980
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 5040
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattaa 5100
ttctgcagtc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc 5160
aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca 5220
cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta 5280
aaagaaaaga ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat 5340
ctgtggatct accacacaca aggctacttc cctgattagc agaactacac accagggcca 5400
ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat 5460
aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat 5520
gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt 5580
catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc 5640
tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag 5700
tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg 5760
gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 5820
tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 5880
taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc 5940
atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag 6000
gccttgacat tgctagcgtt taccgtcgac ctctagctag agcttggcgt aatcatggtc 6060
atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 6120
aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 6180
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 6240
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 6300
ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 6360
acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 6420
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 6480
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 6540
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 6600
gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 6660
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 6720
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 6780
ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 6840
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6900
aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6960
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 7020
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 7080
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 7140
cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 7200
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 7260
tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 7320
gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 7380
agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 7440
tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 7500
agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 7560
gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 7620
catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 7680
ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 7740
atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 7800
tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 7860
cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7920
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7980
atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 8040
aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 8100
ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 8160
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg 8220
atcgggagat caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 8280
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 8340
tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa aatcatccca 8400
aacttcccac cccataccct attaccactg ccaattacct gtggtttcat ttactctaaa 8460
cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta 8520
caaacttagt agtttttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt 8580
<210> 5
<211> 6867
<212> DNA
<213> 人工合成
<400> 5
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataaggat ctgcgatcgc tccggtgccc gtcagtgggc 2220
agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg 2280
tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 2340
ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 2400
tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc 2460
acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc 2520
tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc 2580
gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac 2640
gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg 2700
ttacagatcc aagctgtgac cggcgcctac gctagacgaa ttcctcgaga ctagttctag 2760
agcggccgcg gatccacgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag 2820
attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 2880
gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 2940
ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 3000
cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 3060
ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 3120
tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 3180
gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 3240
gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 3300
gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 3360
ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa acatggagca 3420
atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag 3480
gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag 3540
gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg gctaattcac 3600
tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct 3660
gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc 3720
tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc 3780
agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag 3840
tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac 3900
ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg 3960
aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct 4020
gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 4080
ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 4140
gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 4200
gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc 4260
aaagaaatga atatcagaga gtgagaggcc ttgacattgc tagcgtttac cgtcgacctc 4320
tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 4380
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4440
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4500
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4560
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4620
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4680
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4740
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4800
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4860
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4920
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4980
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5040
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5100
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5160
gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5220
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5280
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5340
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5400
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5460
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5520
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5580
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5640
accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5700
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5760
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5820
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5880
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5940
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 6000
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 6060
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 6120
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 6180
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 6240
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 6300
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6360
actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6420
cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6480
ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata 6540
atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 6600
attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga 6660
taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca 6720
attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa 6780
gaaattgtat ttgttaaata tgtactacaa acttagtagt ttttaaagaa attgtatttg 6840
ttaaatatgt actacaaact tagtagt 6867
<210> 6
<211> 35
<212> DNA
<213> 人工合成
<400> 6
gctctagagc caccatgcct ctgctgctgc tgctg 35
<210> 7
<211> 29
<212> DNA
<213> 人工合成
<400> 7
cgggatcctt agcgaggggg cagggcctg 29
<210> 8
<211> 40
<212> DNA
<213> 人工合成
<400> 8
gtcgtggtgg gcttcgctgg cttgatttcc acttttgtgc 40
<210> 9
<211> 40
<212> DNA
<213> 人工合成
<400> 9
gcacaaaagt ggaaatcaag ccagcgaagc ccaccacgac 40
<210> 10
<211> 20
<212> DNA
<213> 人工合成
<400> 10
ggagcctacc tagactcagc 20
<210> 11
<211> 20
<212> DNA
<213> 人工合成
<400> 11
caaccaggat ttatacaagg 20
<210> 12
<211> 570
<212> PRT
<213> 人工合成
<400> 12
Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala
1 5 10 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
20 25 30
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
35 40 45
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
50 55 60
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
65 70 75 80
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
85 90 95
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
100 105 110
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
145 150 155 160
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
165 170 175
Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
225 230 235 240
Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
245 250 255
Ile Lys Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Pro Arg Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn
305 310 315 320
Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys
325 330 335
Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val
340 345 350
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
355 360 365
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
370 375 380
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
385 390 395 400
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys
405 410 415
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
420 425 430
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
435 440 445
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
450 455 460
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
465 470 475 480
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
485 490 495
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro
500 505 510
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
515 520 525
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
530 535 540
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
545 550 555 560
Ala Leu His Met Gln Ala Leu Pro Pro Arg
565 570
Claims (10)
1.一种蛋白，其特征在于，是如下(a)或(b)：
(a)由序列表中序列12所示的氨基酸序列组成的蛋白质；
(b)将序列表中序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列12衍生的蛋白质。
2.编码权利要求1所述蛋白的HER2 CAR基因。
3.如权利要求2所述的HER2 CAR基因，其特征在于，所述基因是如下的任意一种DNA分子：
(a)核苷酸序列如SEQ ID 3所示的DNA分子；
(b)在严格条件下与(a)限定的核苷酸序列杂交且编码具有相同功能的蛋白的DNA分子；
(c)与(a)限定的核苷酸序列至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码具有相同功能的蛋白的DNA分子。
4.含有权利要求2或3所述的HER2 CAR基因的重组载体、表达盒、转基因细胞系或重组菌；
所述重组载体具体为将权利要求2或3所述的HER2 CAR基因插入表达载体中，得到表达权利要求1所述蛋白的重组载体。
5.一种HER2 CAR基因的合成方法，其特征在于，包括如下步骤：
将抗HER2 ScFV基因和CAR结构基因采用重叠PCR进行拼接，得到HER2 CAR基因；
其中，所述CAR结构基因的核苷酸序列如SEQ ID 1所示，所述HER2 ScFV基因的核苷酸序列如SEQ ID 2所示；得到的HER2 CAR基因的核苷酸序列如SEQ ID 3所示。
6.一种HER2 CAR重组慢病毒载体，其特征在于：
所述HER2 CAR重组慢病毒载体为Pre-Lenti-EF1-HER2 CAR，其核苷酸序列如SEQ ID 4所示。
7.含有权利要求6所述的HER2 CAR重组慢病毒载体的表达盒、转基因细胞系或重组菌。
8.如权利要求6所述的HER2 CAR重组慢病毒载体的构建方法，其特征在于，包括如下步骤：
采用重叠PCR合成HER2 CAR基因，两端设置XbaI、BamHI限制性内切酶位点；其中，所述HER2 CAR基因的核苷酸序列如SEQ ID 3所示；
用限制性内切酶XbaI、BamHI双酶切Pre-Lenti-EF1-MCS载体和所述HER2 CAR基因，分别回收酶切后的目的产物；其中，所述Pre-Lenti-EF1-MCS载体的核苷酸序列如SEQ ID 5所示；
连接所述回收获得的酶切后的目的产物，得到HER2 CAR重组慢病毒载体Pre-Lenti-EF1-HER2 CAR。
9.如权利要求8所述的HER2 CAR重组慢病毒载体的构建方法，其特征在于，采用的引物序列包括：
第一引物序列，其核苷酸序列如SEQ ID 6所示；
第二引物序列，其核苷酸序列如SEQ ID 7所示；
第三引物序列，其核苷酸序列如SEQ ID 8所示；
第四引物序列，其核苷酸序列如SEQ ID 9所示。
10.下述任一项物质在制备抗肿瘤药物中的应用：1)含有权利要求1所述的蛋白；2)含有权利要求2或3所述的HER2 CAR基因的重组载体、表达盒、转基因细胞系或重组菌；3)权利要求6所述的HER2 CAR重组慢病毒载体；4)含有权利要求6所述的HER2 CAR重组慢病毒载体的表达盒、转基因细胞系或重组菌。
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