CN113072648B

CN113072648B - 一种靶向afp的肝癌疫苗

Info

Publication number: CN113072648B
Application number: CN202110624831.6A
Authority: CN
Inventors: 孙忠杰; 齐海龙; 赵宏; 陈立功; 谢皇帆; 刘德芳; 王晓芳
Original assignee: Nuowei Technology Beijing Co ltd
Current assignee: Nuowei Technology Beijing Co ltd
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-08-24
Anticipated expiration: 2041-06-04
Also published as: CN113072648A

Abstract

本发明涉及癌症的免疫治疗和预防领域，特别涉及靶向甲胎蛋白（AFP)的肝癌疫苗。本发明通过将AFP蛋白与DC细胞配体XCL1融合表达，提高了AFP蛋白被DC细胞吞噬、加工、呈递的效率，改善了其抑制肿瘤生长的效果。经由实验验证，本发明的核酸疫苗表达的蛋白可以有效地诱导AFP特异性T细胞反应并在动物肿瘤模型中显著抑制高表达AFP的肿瘤的生长。

Description

一种靶向AFP的肝癌疫苗

技术领域

本发明涉及免疫治疗技术领域，特别涉及融合蛋白及靶向AFP的肝癌疫苗。

背景技术

肝癌是造成全球癌症相关死亡的第二大因素。肝细胞癌（hepatic cellcarcinoma，HCC）是最常见的肝癌，约占肝癌病例的90％。全球肝细胞癌（HCC）发病率正持续增加，其年发病率高达一百万例，据估计，到2025年，全球HCC将超过 100万例。HCC的主要诱因是乙型肝炎病毒和丙型肝炎病毒的感染，而在西方国家其常见诱因则是非酒精性脂肪性肝炎或糖尿病造成的代谢综合征。HCC具备复杂多步骤的发病过程，其中70-80％患者具有肝硬化的病史。肝硬化患者经过一系列临床病理事件的进展出现癌前肝硬化结节，最终可以转化为HCC。尽管有40–50％的肝癌患者可在发病早期确诊且得到外科手术治疗，但其5年复发率高达70%，10年生存率仅7-15%，且复发后无有效治疗手段。更严峻的问题是，几乎一半的患者确诊时已为肝癌晚期，几无治愈可能。目前晚期肝癌患者可依据病况接受6种系统性疗法（atezolizumab 联合贝伐单抗，索拉非尼，lenvatinib，regorafenib，cabozantinib和ramucirumab），另有3种疗法处于临床试验III期，但其疗效仍不乐观。最佳总生存期（overall survival）中位数仅有19.6个月，一般约为1年。肝癌治疗仍存在巨大挑战，新型有效的疗法亟待开发。

人甲胎蛋白（alpha-fetoprotein，AFP）是由单个含3-5％的碳水化合物的分子量为69 kD的多肽链构成的肿瘤相关蛋白，可在肝癌细胞中合成。据统计，AFP在约70％的HCC患者中高度表达，但健康人群中几乎不表达。因此，AFP可作为HCC免疫治疗的靶标。目前存在多种正在开发的针对AFP的治疗方法，包括重组质粒DNA、嵌合病毒样颗粒、表达AFP蛋白的病毒或细菌载体以及肿瘤特异性T细胞的过继转移等。尽管这些疗法可以改善动物模型或患者已有的抗肿瘤免疫力,产生针对AFP的特异性CD8+ 细胞毒性T细胞（CD8+ CTL），但其免疫应答仍然很弱，不足以提供可靠的抗肿瘤保护。

大多数免疫治疗方法对CTL的激活依赖于呈递肿瘤相关抗原的成熟树突状细胞（dendritic cell，DC）。其中，I型常规DC（conventional DC1，cDC1）可通过交叉呈递作用从死细胞中摄取抗原并经由MHC-I分子进行呈递，进而激活并扩增特异性的CTL。CD103+ DC1是一种非常重要的cDC1，具备迁移能力，可携带肿瘤抗原至引流淋巴结进行交叉呈递, 其表面表达XCL1受体(XCR1),可被XCL1分子吸引。因此，可通过XCL1融合AFP蛋白，增加CD103+DC与AFP抗原的接触，从而增强cDC1对自然杀伤细胞（natural killer cell，NK）和CD8+CTL的抗原提呈和免疫活化，增强免疫应答，提供更有效的抗肿瘤保护。

发明内容

有鉴于此，本发明提供了一种新的靶向AFP的抗原疫苗及其制备方法；具体地，本发明提供了一种靶向AFP的核酸和蛋白疫苗，通过将cDC1配体XCL1与AFP蛋白融合表达，提高了DC对AFP蛋白的吞噬和呈递的效率，增强了AFP特异性的CD8+ CTL反应，改善了肝癌肿瘤的抑制效果。

本发明提供了融合蛋白，包括：

甲胎蛋白（AFP）的能够诱发特异性CD8+T反应的或其MHCI类分子结合表位优化形式的去除分泌肽序列的氨基酸序列；接头；和特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白；

其中：

（Ⅰ）、所述AFP的能够诱发特异性CD8+T反应的或其MHCI类分子结合表位优化形式的去除分泌肽序列的氨基酸序列如SEQ ID No.1或SEQ ID No.2所示；

所述特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白的氨基酸序列如SEQ ID No.3或4所示；

或

（Ⅱ）、如（Ⅰ）所述的氨基酸序列经取代、缺失或添加一个或两个氨基酸残基获得的氨基酸序列，且与（Ⅰ）所示的氨基酸序列功能相同或相似的氨基酸序列；

或

（III）、与（Ⅰ）或（Ⅱ）所述序列具有至少90%序列一致性，且与（Ⅰ）所示的氨基酸序列功能相同或相似的氨基酸序列。

在本发明的一些具体实施方案中，所述融合蛋白包括如SEQ ID No.9或SEQ IDNo.10所示氨基酸序列。

基于上述研究，本发明还提供了编码所述的融合蛋白的核酸分子，所述核酸分子包括DNA和/或mRNA，所述核酸分子的序列：

（Ⅰ）、如SEQ ID No.15~30任意所示的核苷酸序列；

或

（Ⅱ）、如SEQ ID No.15~30任意所示的核苷酸序列的互补核苷酸序列所示；或

（Ⅲ）、如与（Ⅰ）或（Ⅱ）的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与（Ⅰ）或（Ⅱ）的核苷酸序列不同的核苷酸序列所示；

或

（Ⅳ）、与（Ⅰ）、（Ⅱ）或（Ⅲ）所示的核苷酸序列经取代、缺失或添加一个或两个核苷酸序列获得的核苷酸序列，且与（Ⅰ）、（Ⅱ）或（Ⅲ）所示的核苷酸序列功能相同或相似的核苷酸序列；

或

（V）、与（Ⅰ）、（Ⅱ）、（Ⅲ）或（Ⅳ）所述核苷酸序列具有至少90%序列一致性的核苷酸序列。

本发明还提供了重组表达载体，包括载体以及所述的融合蛋白或所述的核酸分子。

在本发明的一些具体实施方案中，所述载体包括pcDNA3.1(+)、pcDNA3.1(-)、pFastbac1-dual-MBP、pVAX1。

本发明还提供了包括所述的融合蛋白或所述的核酸分子的重组菌株或细胞。

本发明还提供了所述的融合蛋白、所述的核酸分子、所述的重组表达载体或所述的重组菌株或细胞在制备肝癌或AFP高表达的肿瘤的疫苗或制备预防和/或治疗肝癌或AFP高表达的肿瘤的药物中的应用。

本发明还提供了肝癌或AFP高表达的肿瘤的疫苗，包括所述的融合蛋白、所述的核酸分子、所述的重组表达载体或所述的重组菌株或细胞以及药学上可接受的运载体、赋形剂和/或佐剂。

本发明还提供了预防和/或治疗肝癌或AFP高表达的肿瘤的药物，包括所述的融合蛋白、所述的核酸分子、所述的重组表达载体或所述的重组菌株或细胞以及药学上可接受的辅料。

本发明提供了肝癌疫苗，该疫苗是由甲胎蛋白（AFP）的去除分泌信号肽的氨基酸序列和N端通过接头融合表达特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白组成，包含所述融合蛋白的核酸以及核酸所在的载体，包含本发明所述融合蛋白，核酸和载体在预防和治疗肝癌中的应用。本发明通过将AFP蛋白与DC细胞配体XCL1融合表达，提高了AFP蛋白被DC细胞吞噬、加工、呈递的效率，改善了其抑制肿瘤生长的效果。经由实验验证，本发明的核酸疫苗表达的蛋白可以有效地诱导AFP特异性T细胞反应并在肿瘤动物模型中显著抑制肝癌肿瘤的生长。

附图说明

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。

图1示编码融合蛋白核苷酸的载体图谱；其中图1A示CMV启动子之后接AFP野生型序列用于核酸疫苗编码序列；图1B示CMV启动子之后接XCL1序列之后连接AFP野生型去除分泌信号肽序列的用于核酸疫苗编码序列；

图2示融合蛋白的三维结构预测结果图；将编码融合蛋白的核苷酸序列输入http://raptorx.uchicago.edu/网址对融合蛋白的三维空间结构预测，图示XCL1-AFP融合蛋白空间结构图；

图3示检测编码融合蛋白的核酸在HEK293T细胞内的表达情况；将载由编码融合蛋白核酸的质粒载体转染HEK292T细胞48小时后，利用蛋白免疫印迹技术（Westernblot）检测C端带有Flag标签的融合蛋白编码核苷酸的表达情况；

图4示编码AFP或XCL1-AFP融合蛋白的融合基因免疫小鼠后，小鼠外周血中AFP 特异性tetramer+ CD8A+T细胞所占比例分析图；

图5示编码AFP或XCL1-AFP融合蛋白的融合基因免疫小鼠后，小鼠Hepa1-6的生长情况；图5A示融合基因免疫方法以及免疫过程图；图5B示接种Hepa1-6后，测量肿瘤的体积做出的肿瘤生长曲线图。

具体实施方式

本发明公开了靶向AFP的肝癌疫苗，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。

本发明采用了AFP的去除分泌信号肽的氨基酸序列作为免疫原，以方便可溶性蛋白疫苗的纯化，本发明的疫苗可在小鼠或人体内有效诱导出针对AFP的特异性T细胞免疫反应，打破了原肿瘤相关抗原AFP的免疫耐受性，因而可以有效的发挥直接清除表达AFP的肿瘤细胞，从而抑制肿瘤生长。

本发明一方面涉及一种肝癌的融合蛋白疫苗，所述疫苗由1）甲胎蛋白（AFP）的去除分泌信号肽的氨基酸序列，以及2）N端通过接头融合表达特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白组成。例如小鼠或人AFP，SEQ ID No.1和SEQ ID No.2所示的氨基酸序列构成，其N端通过接头融合表达特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1，由SEQ ID No.3和SEQ ID No.4的1-114位氨基酸所示序列构成。

在一些实施例中，所述肝癌疫苗包含SEQ ID No.9和SEQ ID No.10中的任意序列。

本发明另一方面涉及一种编码该融合蛋白疫苗的核酸，核酸疫苗由1）甲胎蛋白（AFP）的去除分泌信号肽的氨基酸序列，以及2）N端通过接头融合表达特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白的核苷酸序列组成。即编码SEQ ID No.1和SEQID No.2氨基酸序列和特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1序列，即SEQ ID No.3和SEQ ID No.4所示的氨基酸序列。

在一些实施例中，编码甲胎蛋白（AFP）的去除分泌信号肽的氨基酸序列和特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1的序列包含SEQ ID No.9和SEQ IDNo.10中的核苷酸序列。

本发明同时还提供了一种负载所述核酸疫苗的重组表达载体，其包含本发明所述融合蛋白疫苗以及对应核算疫苗的核苷酸序列以及载体。所述载体可以为哺乳动物细胞细胞表达载体或昆虫杆状细胞表达载体，具体的，所述载体可以为pcDNA3.1(+)、pcDNA3.1(-)、pFastbac1-dual-MBP、pVAX1。

本发明最终提供一种肝癌疫苗，包含所述的融合蛋白疫苗，核酸疫苗以及重组表达载体。

本发明的疫苗为肝癌提供一种预防复发或治疗的办法。包括给与其他适应症本发明的疫苗。

本发明的也提供了所述的疫苗，核酸疫苗以及载体在制备肝癌以及高表达AFP的肿瘤疫苗中的应用。

本发明也提供了所述的疫苗，核酸疫苗以及载体用于治疗肝癌。

本发明的疫苗或药物包括所述融合蛋白、所述核酸、所述重组表达载体以及任选存在的药物可接受的运载体、赋形剂和/或佐剂。

综上，本发明提供了肝癌疫苗，该疫苗是由甲胎蛋白（AFP）的去除分泌信号肽的氨基酸序列和N端通过接头融合表达特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白组成，包含所述融合蛋白的核酸以及核酸所在的载体，包含本发明所述融合蛋白，核酸和载体在预防和治疗肝癌中的应用。本发明通过将AFP蛋白与DC细胞配体XCL1融合表达，提高了AFP蛋白被DC细胞吞噬、加工、呈递的效率，改善了其抑制肿瘤生长的效果。经由实验验证，本发明的核酸疫苗表达的蛋白可以有效地诱导AFP特异性T细胞反应并在肿瘤动物模型中显著抑制肝癌肿瘤的生长。

本发明中涉及的原料及试剂均可由市场购得。

下面结合实施例，进一步阐述本发明：

实施例1 融合蛋白疫苗抗原的设计方案以及哺乳动物细胞表达质粒的构建与制备

（1）融合基因哺乳动物细胞表达载体的构建：

根据小鼠或人AFP (SEQ ID No.1和SEQ ID No.2)以及小鼠或人XCL1 (SEQ IDNo.3和SEQ ID No.4)蛋白的氨基酸序列构建融合蛋白XCL1-AFP，为了促进表达融合蛋白的核酸在体内翻译出的融合蛋白可以有效的分泌到细胞外并趋化MHC-II+CD11c+CD8A+抗原交叉呈递DC细胞，我们保留了XCL1蛋白的分泌信号肽(SEQ ID No.5和SEQ ID No.6)，同时将AFP的分泌信号肽去掉(SEQ ID No.7和SEQ ID No.8)。最终得到的融合蛋白的氨基酸序列(SEQ ID No.9和SEQ ID No.10)。对于单独AFP蛋白则保留自身信号肽的蛋白N端(SEQ IDNo.11和SEQ ID No.12)，以保证单独AFP表达载体表达出的AFP蛋白也能同样的被分泌到细胞外。我们将氨基酸序列对应的核苷酸序列进行哺乳动物细胞表达偏好的密码子优化，由擎科公司进行合成，并连接到pVAX1表达载体上（SEQ ID No.13和SEQ ID No.14）。

编码SEQ ID No.1所示氨基酸序列的核酸分子具有如SEQ ID No.15所示的核苷酸序列；

编码SEQ ID No.2所示氨基酸序列的核酸分子具有如SEQ ID No.16所示的核苷酸序列；

编码SEQ ID No.3所示氨基酸序列的核酸分子具有如SEQ ID No.17所示的核苷酸序列；

编码SEQ ID No.4所示氨基酸序列的核酸分子具有如SEQ ID No.18所示的核苷酸序列；

编码SEQ ID No.5所示氨基酸序列的核酸分子具有如SEQ ID No.19所示的核苷酸序列；

编码SEQ ID No.6所示氨基酸序列的核酸分子具有如SEQ ID No.20所示的核苷酸序列；

编码SEQ ID No.7所示氨基酸序列的核酸分子具有如SEQ ID No.21所示的核苷酸序列；

编码SEQ ID No.8所示氨基酸序列的核酸分子具有如SEQ ID No.22所示的核苷酸序列；

编码SEQ ID No.9所示氨基酸序列的核酸分子具有如SEQ ID No.23所示的核苷酸序列；

编码SEQ ID No.10所示氨基酸序列的核酸分子具有如SEQ ID No.24所示的核苷酸序列；

编码SEQ ID No.11所示氨基酸序列的核酸分子具有如SEQ ID No.25所示的核苷酸序列；

编码SEQ ID No.12所示氨基酸序列的核酸分子具有如SEQ ID No.26所示的核苷酸序列；

（2）融合基因哺乳动物细胞表达载体的扩增

细菌的转化，取-80℃冻存的感受态细菌置于冰上融化，待接近完全融化时加入100 ng 质粒，轻柔混匀，冰上放置 30 mins。将感受态置于 42℃水浴锅中热激 60 s，取出后立即置于冰上放置 2 mins。向管内加入 500 µL 不含抗生素的 LB 培养基， 37℃摇床内振荡培养 1 h。将细菌 4000 rpm 室温离心 2 mins，弃去部分上清（约 450 µL）后重悬细菌，取适量细菌悬液涂于含有相应抗生素的培养皿上。培养皿正面朝下放置， 37℃培养箱培养过夜。待克隆大小适宜后（约 16 h）用枪尖将克隆挑至加好抗生素的 LB 培养基中，37℃摇床中振荡培养至浑浊。取 15 mL 培养至合适浓度的细菌菌液， 4000 rpm 室温离心 5mins，弃掉上清。按照北京天根公司的《质粒小提试剂盒》货号（DP103)说明书提取细菌 DNA。首先加入 250 µL 含 RNA酶的细胞重悬液 P1 充分重悬细菌沉淀，并将沉淀转移至 1.5 mL EP 管中。加入 250 µL 碱性细胞裂解液 P2，轻柔颠倒混匀至液体澄清。加入350 µL 中和液 P3，颠倒混合均匀至出现絮状沉淀物。混悬液 12000 rpm室温离心 10mins。将 DNA 吸附柱放入回收管中，加入 500 µL 平衡液活化吸附膜， 12000 rpm 离心 1min 后弃掉液体。将步骤 4 中离心获得的上清转移至 DNA 吸附柱中， 12000 rpm 离心 1min 后弃去液体。向吸附柱内加入 600 µL 清洗液， 12000 rpm 离心 1min 后弃去液体，重复洗涤一次。 12000 rpm 空离回收管 2 mins。将收集管更换为新的 1.5 mL EP 管，室温放置 5 mins 晾干吸附柱。加入 70 µL 65℃预热的洗脱缓冲液或者高压过的纯净水，室温放置 5 mins 充分溶解 DNA， 12000 rpm 室温离心 3 mins，收集液体。弃掉吸附柱后对管内质粒进行浓度测定并标记质粒名称、浓度、提取日期。

编码SEQ ID No.9所示氨基酸序列的核酸分子具有如SEQ ID No.29所示的核苷酸序列；

编码SEQ ID No.10所示氨基酸序列的核酸分子具有如SEQ ID No.30所示的核苷酸序列。

实施例2 融合蛋白三维结构预测

根据融合蛋白的核苷酸序列，我们利用http://raptorx.uchicago.edu/ 网站对XCL1以及AFP融合蛋白的三维结构进行预测。XCL1属于趋化因子，其趋化功能的正常发挥需要保有完整的空间结构。为了确保XCL1与AFP融合后，XCL1仍然保留有自身的空间结构，我们对融合后的氨基酸序列在http://raptorx.uchicago.edu/网站上进行三维结构预测，结果如图2显示，XCL1与AFP融合后，二者仍然各自保有原来的空间结构，并不影响XCL1的趋化功能发挥。

实施例3 编码XCL1-AFP融合蛋白的哺乳动物细胞表达载体表达效果检测

转染前24小时，在6孔细胞培养板内接种1*10⁶个HEK293T细胞，待细胞密度长到70%-80%时开始转染试验。转染时提前在37°水浴锅中预热细胞培养基、无血清的 Opti-MEM培养基。转染时将 5 微克空载体（Vector），单独AFP表达载体，XCL1-AFP野生型融合基因质粒和20μL PEI 转染试剂先后加入到 200 μL无血清的 Opti-MEM 中，混合均匀后，室温下静置10分钟。将需要转染的细胞更换新鲜培养基，轻柔加入上述转染体系，轻轻摇匀。将细胞放回细胞培养箱中培养 6 小时后换液。转 48小时后收取细胞用Western Blot检测XCL1-AFP融合基因质粒HEK293T细胞中的表达效果。

为了方便检测融合基因的表达效果我们在融合蛋白的C端连接一个DDDDK5个氨基酸组成的Flag标签，以便利用Flag标签抗体检测融合蛋白的表达。收集细胞，加入60μL的含有 PMSF 或 Cocktail 蛋白酶抑制剂的 0.5% NP40 裂解缓冲液。充分重悬细胞， 4℃旋转裂解细胞 30 分钟。12000 rpm， 4℃离心裂解液 10 分钟，收集上清至新的 1.5 mL EP 管中，弃掉沉淀。根据样品实际体积加入 5×SDS-PAGE 蛋白上样缓冲液，混合均匀后将样品放在 100℃空气浴中加热 10分钟，立即进行 Western blot，利用Flag标签抗体(Sigma,F3165)进行检测,结果如图3显示空载体（Vector）无蛋白表达，单独AFP表达载体，XCL1-AFP野生型融合基因质粒均能有效表达并且表达量相当。

实施例4 探索融合基因免疫是否诱导出了更强的细胞特异性T细胞反应

我们提取单独AFP和XCL1-AFP野生型质粒，利用Wealtec公司的基因枪（GDS-80）对小鼠进行免疫质粒注射。14天后利用AFP特异的tetramer流式抗体检测小鼠外周血AFP特异性T细胞反应。

具体地，先取小鼠眦内外周血，加入肝素钠抗凝剂后混匀，200g离心10分钟后，弃上清，利用R&D公司红细胞裂解液（货号WL2000）对外周血进行裂红处理。具体为先将裂解液A用蒸馏水稀释10倍配成工作液，每个外周血样本加入2mL工作液重悬，室温放置10min，期间将中和液B用蒸馏水稀释10倍配成工作液。十分钟后将10mL中和液B加入裂解液中中和后200g离心10min。细胞团用含1%灭活FBS的PBS缓冲液洗涤一次并进行流式染色：AFP-tetramer antibody-FITC，CD8α-PE。2小时后作流式检测。结果如图4所示，经XCL1-AFP野生型质粒免疫后的小鼠外周血针对AFP的特异性T细胞与单独AFP免疫组相比显著增多，说明XCL1-AFP野生型质粒免疫确实诱导出了特异性反应的CD8A细胞毒性T淋巴细胞。

表1 图4的数据

实施例5 融合基因对hepa1-6肝癌细胞移植瘤发生的干预效果

我们提取单独AFP和XCL1-AFP野生型质粒，利用Wealtec公司的基因枪（GDS-80）对小鼠进行免疫质粒注射。并同种移植hepa1-6肝癌细胞后观察融合基因免疫对hepa1-6肝癌细胞生长的抑制情况。

我们按照图5A的时间轴标注的免疫策略对小鼠进行基因枪质粒注射。将C57B6（购自维通利华）6周龄雄性小鼠，分为对照组、注射单独AFP和XCL1-AFP野生型质粒的三组，每组五只，利用脱毛膏进行小鼠右侧靠近腹股沟淋巴结处脱毛处理。之后利用基因枪在脱毛处注射质粒，每只50μg，每周一次，共注射四次，在最后一次注射后一周接种之前摸索好成瘤条件的hepa1-6肝癌细胞，观察肿瘤形成时间并每两天测量肿瘤的长径a和短径b，按照a*b*b/2进行肿瘤体积计算，绘制肿瘤生长曲线。结果如图5A，5B所示，免疫注射XCL1-AFP野生型质粒均能有效地延迟肿瘤的发生时间以及生长速度。说明融合基因免疫的治疗效果显著有效。

表2 图5B数据

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

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Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys

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

305 310 315 320

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

325 330 335

Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile

340 345 350

Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln

355 360 365

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

370 375 380

Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu

385 390 395 400

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

405 410 415

Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile

420 425 430

Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu

435 440 445

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

450 455 460

His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly

465 470 475 480

Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser

485 490 495

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

500 505 510

Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala Leu Gln

515 520 525

Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln

530 535 540

Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser Gly Leu

545 550 555 560

Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu

565 570 575

Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly Val

580 585 590

<210> 9

<211> 712

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys

20 25 30

Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr

35 40 45

Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg

50 55 60

Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala

65 70 75 80

Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu

85 90 95

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

100 105 110

Thr Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Lys Ala Leu

115 120 125

His Glu Asn Glu Phe Gly Ile Ala Ser Thr Leu Asp Ser Ser Gln Cys

130 135 140

Val Thr Glu Lys Asn Val Leu Ser Ile Ala Thr Ile Thr Phe Thr Gln

145 150 155 160

Phe Val Pro Glu Ala Thr Glu Glu Glu Val Asn Lys Met Thr Ser Asp

165 170 175

Val Leu Ala Ala Met Lys Lys Asn Ser Gly Asp Gly Cys Leu Glu Ser

180 185 190

Gln Leu Ser Val Phe Leu Asp Glu Ile Cys His Glu Thr Glu Leu Ser

195 200 205

Asn Lys Tyr Gly Leu Ser Gly Cys Cys Ser Gln Ser Gly Val Glu Arg

210 215 220

His Gln Cys Leu Leu Ala Arg Lys Lys Thr Ala Pro Ala Ser Val Pro

225 230 235 240

Pro Phe Gln Phe Pro Glu Pro Ala Glu Ser Cys Lys Ala His Glu Glu

245 250 255

Asn Arg Ala Val Phe Met Asn Arg Phe Ile Tyr Glu Val Ser Arg Arg

260 265 270

Asn Pro Phe Met Tyr Ala Pro Ala Ile Leu Ser Leu Ala Ala Gln Tyr

275 280 285

Asp Lys Val Val Leu Ala Cys Cys Lys Ala Asp Asn Lys Glu Glu Cys

290 295 300

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

305 310 315 320

Met Leu Asn Glu His Val Cys Ser Val Ile Arg Lys Phe Gly Ser Arg

325 330 335

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

340 345 350

Ala Asn Phe Thr Glu Ile Gln Lys Leu Ala Leu Asp Val Ala His Ile

355 360 365

His Glu Glu Cys Cys Gln Gly Asn Ser Leu Glu Cys Leu Gln Asp Gly

370 375 380

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

385 390 395 400

Lys Ile Ala Glu Cys Cys Lys Leu Pro Met Ile Gln Leu Gly Phe Cys

405 410 415

Ile Ile His Ala Glu Asn Gly Val Lys Pro Glu Gly Leu Ser Leu Asn

420 425 430

Pro Ser Gln Phe Leu Gly Asp Arg Asn Phe Ala Gln Phe Ser Ser Glu

435 440 445

Glu Lys Ile Met Phe Met Ala Ser Phe Leu His Glu Tyr Ser Arg Thr

450 455 460

His Pro Asn Leu Pro Val Ser Val Ile Leu Arg Ile Ala Lys Thr Tyr

465 470 475 480

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

485 490 495

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

500 505 510

Ala Leu Ser Lys Gln Ser Cys Ala Leu Tyr Gln Thr Leu Gly Asp Tyr

515 520 525

Lys Leu Gln Asn Leu Phe Leu Ile Gly Tyr Thr Arg Lys Ala Pro Gln

530 535 540

Leu Thr Ser Ala Glu Leu Ile Asp Leu Thr Gly Lys Met Val Ser Ile

545 550 555 560

Ala Ser Thr Cys Cys Gln Leu Ser Glu Glu Lys Trp Ser Gly Cys Gly

565 570 575

Glu Gly Met Ala Asp Ile Phe Ile Gly His Leu Cys Ile Arg Asn Glu

580 585 590

Ala Ser Pro Val Asn Ser Gly Ile Ser His Cys Cys Asn Ser Ser Tyr

595 600 605

Ser Asn Arg Arg Leu Cys Ile Thr Ser Phe Leu Arg Asp Glu Thr Tyr

610 615 620

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

625 630 635 640

Cys Gln Ala Gln Gly Lys Ala Leu Gln Thr Met Lys Gln Glu Leu Leu

645 650 655

Ile Asn Leu Val Lys Gln Lys Pro Glu Leu Thr Glu Glu Gln Leu Ala

660 665 670

Ala Val Thr Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Lys Ala

675 680 685

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

690 695 700

Lys Thr Arg Asp Ala Leu Gly Val

705 710

<210> 10

<211> 715

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

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

100 105 110

Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Arg Thr Leu His

115 120 125

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

130 135 140

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

145 150 155 160

Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val Lys Asp Ala

165 170 175

Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser Ser Gly Cys

180 185 190

Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys His Glu Lys

195 200 205

Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser Gln Ser Glu

210 215 220

Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro Thr Pro Ala

225 230 235 240

Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser Cys Glu Ala

245 250 255

Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile Tyr Glu Ile

260 265 270

Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu Leu Trp Ala

275 280 285

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

290 295 300

Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys Glu Leu Arg

305 310 315 320

Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met Lys Asn Phe

325 330 335

Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu Ser Gln Lys

340 345 350

Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val Leu Asp Val

355 360 365

Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu Asp Cys Leu

370 375 380

Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr

385 390 395 400

Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg

405 410 415

Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu

420 425 430

Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe

435 440 445

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

450 455 460

Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala

465 470 475 480

Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro

485 490 495

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

500 505 510

Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu

515 520 525

Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu Val Ala Tyr Thr Lys Lys

530 535 540

Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met

545 550 555 560

Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu

565 570 575

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

580 585 590

Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln Cys Cys Thr

595 600 605

Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu Val Val Asp

610 615 620

Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe Ile Phe His

625 630 635 640

Lys Asp Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Thr Met Lys Gln

645 650 655

Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile Thr Glu Glu

660 665 670

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

675 680 685

Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys

690 695 700

Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly Val

705 710 715

<210> 11

<211> 18

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 11

Met Lys Trp Ile Thr Pro Ala Ser Leu Ile Leu Leu Leu His Phe Ala

1 5 10 15

Ala Ser

<210> 12

<211> 18

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 12

Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr

1 5 10 15

Glu Ser

<210> 13

<211> 5132

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccgaatt cgccaccatg agacttctcc tcctgacttt cctgggagtc 780

tgctgcctca ccccatgggt tgtggaaggt gtggggactg aagtcctaga agagagtagc 840

tgtgtgaact tacaaaccca gcggctgcca gttcaaaaaa tcaagaccta tatcatctgg 900

gagggggcca tgagagctgt aatttttgtc accaaacgag gactaaaaat ttgtgctgat 960

ccagaagcca aatgggtgaa agcagcgatc aagactgtgg atggcagggc cagtaccaga 1020

aagaacatgg ctgaaactgt tcccacagga gcccagaggt ccaccagcac agcgataacc 1080

ctgactgggg gcggaggcgg aggatcaggg ggagggggag gaaaagcatt gcacgaaaat 1140

gagtttggga tagcttccac gttagattcc tcccagtgcg tgacggagaa gaatgtgctt 1200

agcatagcta ccatcacctt tacccagttt gttccggaag ccaccgagga ggaagtgaac 1260

aaaatgacta gcgatgtgtt ggctgcaatg aagaaaaact ctggcgatgg gtgtttagaa 1320

agccagctat ctgtgtttct ggatgaaatt tgtcatgaga cggaactctc taacaagtat 1380

ggactctcag gctgctgcag ccaaagtgga gtggaaagac atcagtgtct gctggcacgc 1440

aagaagactg ctccggcctc tgtcccaccc ttccagtttc cagaacctgc cgagagttgc 1500

aaagcacatg aagaaaacag ggcagtgttc atgaacaggt tcatctatga agtgtcaagg 1560

aggaacccct tcatgtatgc cccagccatt ctgtccttgg ctgctcagta cgacaaggtc 1620

gttctggcat gctgcaaagc tgacaacaag gaggagtgct tccagacaaa gagagcatcc 1680

attgcaaagg aattaagaga aggaagcatg ttaaatgagc atgtatgttc agtgataaga 1740

aaatttggat cccgaaacct ccaggcaaca accattatta agctaagtca aaagttaact 1800

gaagcaaatt ttactgagat tcagaagctg gccctggatg tggctcacat ccacgaggag 1860

tgttgccaag gaaactcgct ggagtgtctg caggatgggg aaaaagtcat gacatatata 1920

tgttctcaac aaaatattct gtcaagcaaa atagcagagt gctgcaaatt acccatgatc 1980

caactaggct tctgcataat tcacgcagag aatggcgtca aacctgaagg cttatctcta 2040

aatccaagcc agtttttggg agacagaaat tttgcccaat tttcttcaga ggaaaaaatc 2100

atgttcatgg caagctttct tcatgaatac tcaagaactc accccaacct tcctgtctca 2160

gtcattctaa gaattgctaa aacgtaccag gaaatattgg agaagtgttc ccagtctgga 2220

aatctacctg gatgtcagga caatctggaa gaagaattgc agaaacacat cgaggagagc 2280

caggcactgt ccaagcaaag ctgcgctctc taccagacct taggagacta caaattacaa 2340

aatctgttcc ttattggtta cacgaggaaa gcccctcagc tgacctcagc agagctgatc 2400

gacctcaccg ggaagatggt gagcattgcc tccacgtgct gccagctcag cgaggagaaa 2460

tggtccggct gtggtgaggg aatggccgac attttcattg gacatttgtg tataaggaat 2520

gaagcaagcc ctgtgaactc tggtatcagc cactgctgca actcttcgta ttccaacagg 2580

aggctatgca tcaccagttt tctgagggat gaaacctatg cccctccccc attctctgag 2640

gataaattca tcttccacaa ggatctgtgc caagctcagg gcaaagccct acagaccatg 2700

aaacaagagc ttctcattaa cctggtgaag caaaagcctg aactgacaga ggagcagctg 2760

gcggctgtca ctgcagattt ctcgggcctt ttggagaagt gctgcaaagc ccaggaccag 2820

gaagtctgtt tcacagaaga gggtccaaag ttgatttcca aaactcgtga tgctttgggc 2880

gttgattaca aggatgacga cgataagtaa gcggccgcct cgagtctaga gggcccgttt 2940

aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 3000

cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 3060

aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 3120

aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 3180

ctatggcttc tactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc 3240

gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct cgccgccaag 3300

gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat 3360

gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 3420

ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 3480

gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 3540

agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 3600

cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 3660

tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 3720

gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 3780

cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 3840

gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 3900

cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 3960

ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4020

agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4080

cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4140

cgagttcttc tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca 4200

tctgtgcggt atttcacacc gcatacaggt ggcacttttc ggggaaatgt gcgcggaacc 4260

cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 4320

tgataaatgc ttcaataata gcacgtgcta aaacttcatt tttaatttaa aaggatctag 4380

gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 4440

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 4500

gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 4560

caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 4620

actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 4680

acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 4740

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 4800

gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 4860

cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 4920

gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 4980

tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 5040

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 5100

ggcttttgct ggccttttgc tcacatgttc tt 5132

<210> 14

<211> 5141

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccgaatt cgccaccatg agacttctca tcctggccct ccttggcatc 780

tgctctctca ctgcatacat tgtggaaggt gtagggagtg aagtctcaga taagaggacc 840

tgtgtgagcc tcactaccca gcgactgccg gttagcagaa tcaagaccta caccatcacg 900

gaaggctcct tgagagcagt aatttttatt accaaacgtg gcctaaaagt ctgtgctgat 960

ccacaagcca catgggtgag agacgtggtc aggagcatgg acaggaaatc caacaccaga 1020

aataacatga tccagaccaa gccaacagga acccagcaat cgaccaatac agctgtgact 1080

ctgactggcg gaggcggagg atcaggggga gggggaggaa gaacactgca tagaaatgaa 1140

tatggaatag cttccatatt ggattcttac caatgtactg cagagataag tttagctgac 1200

ctggctacca tattttttgc ccagtttgtt caagaagcca cttacaagga agtaagcaaa 1260

atggtgaaag atgcattgac tgcaattgag aaacccactg gagatgaaca gtcttcaggg 1320

tgtttagaaa accagctacc tgcctttctg gaagaacttt gccatgagaa agaaattttg 1380

gagaagtacg gacattcaga ctgctgcagc caaagtgaag agggaagaca taactgtttt 1440

cttgcacaca aaaagcccac tccagcatcg atcccacttt tccaagttcc agaacctgtc 1500

acaagctgtg aagcatatga agaagacagg gagacattca tgaacaaatt catttatgag 1560

atagcaagaa ggcatccctt cctgtatgca cctacaattc ttctttgggc tgctcgctat 1620

gacaaaataa ttccatcttg ctgcaaagct gaaaatgcag ttgaatgctt ccaaacaaag 1680

gcagcaacag ttacaaaaga attaagagaa agcagcttgt taaatcaaca tgcatgtgca 1740

gtaatgaaaa attttgggac ccgaactttc caagccataa ctgttactaa actgagtcag 1800

aagtttacca aagttaattt tactgaaatc cagaaactag tcctggatgt ggcccatgta 1860

catgagcact gttgcagagg agatgtgctg gattgtctgc aggatgggga aaaaatcatg 1920

tcctacatat gttctcaaca agacactctg tcaaacaaaa taacagaatg ctgcaaactg 1980

accacgctgg aacgtggtca atgtataatt catgcagaaa atgatgaaaa acctgaaggt 2040

ctatctccaa atctaaacag gtttttagga gatagagatt ttaaccaatt ttcttcaggg 2100

gaaaaaaata tcttcttggc aagttttgtt catgaatatt caagaagaca tcctcagctt 2160

gctgtctcag taattctaag agttgctaaa ggataccagg agttattgga gaagtgtttc 2220

cagactgaaa accctcttga atgccaagat aaaggagaag aagaattaca gaaatacatc 2280

caggagagcc aagcattggc aaagcgaagc tgcggcctct tccagaaact aggagaatat 2340

tacttacaaa atgcgtttct cgttgcttac acaaagaaag ccccccagct gacctcgtcg 2400

gagctgatgg ccatcaccag aaaaatggca gccacagcag ccacttgttg ccaactcagt 2460

gaggacaaac tattggcctg tggcgaggga gcggctgaca ttattatcgg acacttatgt 2520

atcagacatg aaatgactcc agtaaaccct ggtgttggcc agtgctgcac ttcttcatat 2580

gccaacagga ggccatgctt cagcagcttg gtggtggatg aaacatatgt ccctcctgca 2640

ttctctgatg acaagttcat tttccataag gatctgtgcc aagctcaggg tgtagcgctg 2700

caaacaatga agcaagagtt tctcattaac cttgtgaagc aaaagccaca aataacagag 2760

gaacaacttg aggctgtcat tgcagatttc tcaggcctgt tggagaaatg ctgccaaggc 2820

caggaacagg aagtctgctt tgctgaagag ggacaaaaac tgatttcaaa aactcgtgct 2880

gctttgggag ttgattacaa ggatgacgac gataagtaag cggccgcctc gagtctagag 2940

ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 3000

tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 3060

aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 3120

gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 3180

cggtgggctc tatggcttct actgggcggt tttatggaca gcaagcgaac cggaattgcc 3240

agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctc 3300

gccgccaagg atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc 3360

gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3420

gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3480

gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3540

tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3600

agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3660

ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3720

tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3780

acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3840

ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 3900

gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3960

ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4020

tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4080

ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 4140

ccttcttgac gagttcttct gaattattaa cgcttacaat ttcctgatgc ggtattttct 4200

ccttacgcat ctgtgcggta tttcacaccg catacaggtg gcacttttcg gggaaatgtg 4260

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 4320

caataaccct gataaatgct tcaataatag cacgtgctaa aacttcattt ttaatttaaa 4380

aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 4440

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 4500

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4560

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4620

ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4680

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4740

aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4800

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4860

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4920

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4980

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 5040

ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 5100

cggttcctgg gcttttgctg gccttttgct cacatgttct t 5141

<210> 15

<211> 1815

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

atgaagtgga tcacacccgc ttccctcatc ctcctgctac atttcgctgc gtccaaagca 60

ttgcacgaaa atgagtttgg gatagcttcc acgttagatt cctcccagtg cgtgacggag 120

aagaatgtgc ttagcatagc taccatcacc tttacccagt ttgttccgga agccaccgag 180

gaggaagtga acaaaatgac tagcgatgtg ttggctgcaa tgaagaaaaa ctctggcgat 240

gggtgtttag aaagccagct atctgtgttt ctggatgaaa tttgtcatga gacggaactc 300

tctaacaagt atggactctc aggctgctgc agccaaagtg gagtggaaag acatcagtgt 360

ctgctggcac gcaagaagac tgctccggcc tctgtcccac ccttccagtt tccagaacct 420

gccgagagtt gcaaagcaca tgaagaaaac agggcagtgt tcatgaacag gttcatctat 480

gaagtgtcaa ggaggaaccc cttcatgtat gccccagcca ttctgtcctt ggctgctcag 540

tacgacaagg tcgttctggc atgctgcaaa gctgacaaca aggaggagtg cttccagaca 600

aagagagcat ccattgcaaa ggaattaaga gaaggaagca tgttaaatga gcatgtatgt 660

tcagtgataa gaaaatttgg atcccgaaac ctccaggcaa caaccattat taagctaagt 720

caaaagttaa ctgaagcaaa ttttactgag attcagaagc tggccctgga tgtggctcac 780

atccacgagg agtgttgcca aggaaactcg ctggagtgtc tgcaggatgg ggaaaaagtc 840

atgacatata tatgttctca acaaaatatt ctgtcaagca aaatagcaga gtgctgcaaa 900

ttacccatga tccaactagg cttctgcata attcacgcag agaatggcgt caaacctgaa 960

ggcttatctc taaatccaag ccagtttttg ggagacagaa attttgccca attttcttca 1020

gaggaaaaaa tcatgttcat ggcaagcttt cttcatgaat actcaagaac tcaccccaac 1080

cttcctgtct cagtcattct aagaattgct aaaacgtacc aggaaatatt ggagaagtgt 1140

tcccagtctg gaaatctacc tggatgtcag gacaatctgg aagaagaatt gcagaaacac 1200

atcgaggaga gccaggcact gtccaagcaa agctgcgctc tctaccagac cttaggagac 1260

tacaaattac aaaatctgtt ccttattggt tacacgagga aagcccctca gctgacctca 1320

gcagagctga tcgacctcac cgggaagatg gtgagcattg cctccacgtg ctgccagctc 1380

agcgaggaga aatggtccgg ctgtggtgag ggaatggccg acattttcat tggacatttg 1440

tgtataagga atgaagcaag ccctgtgaac tctggtatca gccactgctg caactcttcg 1500

tattccaaca ggaggctatg catcaccagt tttctgaggg atgaaaccta tgcccctccc 1560

ccattctctg aggataaatt catcttccac aaggatctgt gccaagctca gggcaaagcc 1620

ctacagacca tgaaacaaga gcttctcatt aacctggtga agcaaaagcc tgaactgaca 1680

gaggagcagc tggcggctgt cactgcagat ttctcgggcc ttttggagaa gtgctgcaaa 1740

gcccaggacc aggaagtctg tttcacagaa gagggtccaa agttgatttc caaaactcgt 1800

gatgctttgg gcgtt 1815

<210> 16

<211> 1827

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

atgaagtggg tggaatcaat ttttttaatt ttcctactaa attttactga atccagaaca 60

ctgcatagaa atgaatatgg aatagcttcc atattggatt cttaccaatg tactgcagag 120

ataagtttag ctgacctggc taccatattt tttgcccagt ttgttcaaga agccacttac 180

aaggaagtaa gcaaaatggt gaaagatgca ttgactgcaa ttgagaaacc cactggagat 240

gaacagtctt cagggtgttt agaaaaccag ctacctgcct ttctggaaga actttgccat 300

gagaaagaaa ttttggagaa gtacggacat tcagactgct gcagccaaag tgaagaggga 360

agacataact gttttcttgc acacaaaaag cccactccag catcgatccc acttttccaa 420

gttccagaac ctgtcacaag ctgtgaagca tatgaagaag acagggagac attcatgaac 480

aaattcattt atgagatagc aagaaggcat cccttcctgt atgcacctac aattcttctt 540

tgggctgctc gctatgacaa aataattcca tcttgctgca aagctgaaaa tgcagttgaa 600

tgcttccaaa caaaggcagc aacagttaca aaagaattaa gagaaagcag cttgttaaat 660

caacatgcat gtgcagtaat gaaaaatttt gggacccgaa ctttccaagc cataactgtt 720

actaaactga gtcagaagtt taccaaagtt aattttactg aaatccagaa actagtcctg 780

gatgtggccc atgtacatga gcactgttgc agaggagatg tgctggattg tctgcaggat 840

ggggaaaaaa tcatgtccta catatgttct caacaagaca ctctgtcaaa caaaataaca 900

gaatgctgca aactgaccac gctggaacgt ggtcaatgta taattcatgc agaaaatgat 960

gaaaaacctg aaggtctatc tccaaatcta aacaggtttt taggagatag agattttaac 1020

caattttctt caggggaaaa aaatatcttc ttggcaagtt ttgttcatga atattcaaga 1080

agacatcctc agcttgctgt ctcagtaatt ctaagagttg ctaaaggata ccaggagtta 1140

ttggagaagt gtttccagac tgaaaaccct cttgaatgcc aagataaagg agaagaagaa 1200

ttacagaaat acatccagga gagccaagca ttggcaaagc gaagctgcgg cctcttccag 1260

aaactaggag aatattactt acaaaatgcg tttctcgttg cttacacaaa gaaagccccc 1320

cagctgacct cgtcggagct gatggccatc accagaaaaa tggcagccac agcagccact 1380

tgttgccaac tcagtgagga caaactattg gcctgtggcg agggagcggc tgacattatt 1440

atcggacact tatgtatcag acatgaaatg actccagtaa accctggtgt tggccagtgc 1500

tgcacttctt catatgccaa caggaggcca tgcttcagca gcttggtggt ggatgaaaca 1560

tatgtccctc ctgcattctc tgatgacaag ttcattttcc ataaggatct gtgccaagct 1620

cagggtgtag cgctgcaaac aatgaagcaa gagtttctca ttaaccttgt gaagcaaaag 1680

ccacaaataa cagaggaaca acttgaggct gtcattgcag atttctcagg cctgttggag 1740

aaatgctgcc aaggccagga acaggaagtc tgctttgctg aagagggaca aaaactgatt 1800

tcaaaaactc gtgctgcttt gggagtt 1827

<210> 17

<211> 345

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 17

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120

ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180

gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240

atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300

ggagcccaga ggtccaccag cacagcgata accctgactg ggggc 345

<210> 18

<211> 342

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 18

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120

ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg actctgactg gc 342

<210> 19

<211> 63

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 19

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggt 63

<210> 20

<211> 63

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggt 63

<210> 21

<211> 1761

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 21

aaagcattgc acgaaaatga gtttgggata gcttccacgt tagattcctc ccagtgcgtg 60

acggagaaga atgtgcttag catagctacc atcaccttta cccagtttgt tccggaagcc 120

accgaggagg aagtgaacaa aatgactagc gatgtgttgg ctgcaatgaa gaaaaactct 180

ggcgatgggt gtttagaaag ccagctatct gtgtttctgg atgaaatttg tcatgagacg 240

gaactctcta acaagtatgg actctcaggc tgctgcagcc aaagtggagt ggaaagacat 300

cagtgtctgc tggcacgcaa gaagactgct ccggcctctg tcccaccctt ccagtttcca 360

gaacctgccg agagttgcaa agcacatgaa gaaaacaggg cagtgttcat gaacaggttc 420

atctatgaag tgtcaaggag gaaccccttc atgtatgccc cagccattct gtccttggct 480

gctcagtacg acaaggtcgt tctggcatgc tgcaaagctg acaacaagga ggagtgcttc 540

cagacaaaga gagcatccat tgcaaaggaa ttaagagaag gaagcatgtt aaatgagcat 600

gtatgttcag tgataagaaa atttggatcc cgaaacctcc aggcaacaac cattattaag 660

ctaagtcaaa agttaactga agcaaatttt actgagattc agaagctggc cctggatgtg 720

gctcacatcc acgaggagtg ttgccaagga aactcgctgg agtgtctgca ggatggggaa 780

aaagtcatga catatatatg ttctcaacaa aatattctgt caagcaaaat agcagagtgc 840

tgcaaattac ccatgatcca actaggcttc tgcataattc acgcagagaa tggcgtcaaa 900

cctgaaggct tatctctaaa tccaagccag tttttgggag acagaaattt tgcccaattt 960

tcttcagagg aaaaaatcat gttcatggca agctttcttc atgaatactc aagaactcac 1020

cccaaccttc ctgtctcagt cattctaaga attgctaaaa cgtaccagga aatattggag 1080

aagtgttccc agtctggaaa tctacctgga tgtcaggaca atctggaaga agaattgcag 1140

aaacacatcg aggagagcca ggcactgtcc aagcaaagct gcgctctcta ccagacctta 1200

ggagactaca aattacaaaa tctgttcctt attggttaca cgaggaaagc ccctcagctg 1260

acctcagcag agctgatcga cctcaccggg aagatggtga gcattgcctc cacgtgctgc 1320

cagctcagcg aggagaaatg gtccggctgt ggtgagggaa tggccgacat tttcattgga 1380

catttgtgta taaggaatga agcaagccct gtgaactctg gtatcagcca ctgctgcaac 1440

tcttcgtatt ccaacaggag gctatgcatc accagttttc tgagggatga aacctatgcc 1500

cctcccccat tctctgagga taaattcatc ttccacaagg atctgtgcca agctcagggc 1560

aaagccctac agaccatgaa acaagagctt ctcattaacc tggtgaagca aaagcctgaa 1620

ctgacagagg agcagctggc ggctgtcact gcagatttct cgggcctttt ggagaagtgc 1680

tgcaaagccc aggaccagga agtctgtttc acagaagagg gtccaaagtt gatttccaaa 1740

actcgtgatg ctttgggcgt t 1761

<210> 22

<211> 1773

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 22

agaacactgc atagaaatga atatggaata gcttccatat tggattctta ccaatgtact 60

gcagagataa gtttagctga cctggctacc atattttttg cccagtttgt tcaagaagcc 120

acttacaagg aagtaagcaa aatggtgaaa gatgcattga ctgcaattga gaaacccact 180

ggagatgaac agtcttcagg gtgtttagaa aaccagctac ctgcctttct ggaagaactt 240

tgccatgaga aagaaatttt ggagaagtac ggacattcag actgctgcag ccaaagtgaa 300

gagggaagac ataactgttt tcttgcacac aaaaagccca ctccagcatc gatcccactt 360

ttccaagttc cagaacctgt cacaagctgt gaagcatatg aagaagacag ggagacattc 420

atgaacaaat tcatttatga gatagcaaga aggcatccct tcctgtatgc acctacaatt 480

cttctttggg ctgctcgcta tgacaaaata attccatctt gctgcaaagc tgaaaatgca 540

gttgaatgct tccaaacaaa ggcagcaaca gttacaaaag aattaagaga aagcagcttg 600

ttaaatcaac atgcatgtgc agtaatgaaa aattttggga cccgaacttt ccaagccata 660

actgttacta aactgagtca gaagtttacc aaagttaatt ttactgaaat ccagaaacta 720

gtcctggatg tggcccatgt acatgagcac tgttgcagag gagatgtgct ggattgtctg 780

caggatgggg aaaaaatcat gtcctacata tgttctcaac aagacactct gtcaaacaaa 840

ataacagaat gctgcaaact gaccacgctg gaacgtggtc aatgtataat tcatgcagaa 900

aatgatgaaa aacctgaagg tctatctcca aatctaaaca ggtttttagg agatagagat 960

tttaaccaat tttcttcagg ggaaaaaaat atcttcttgg caagttttgt tcatgaatat 1020

tcaagaagac atcctcagct tgctgtctca gtaattctaa gagttgctaa aggataccag 1080

gagttattgg agaagtgttt ccagactgaa aaccctcttg aatgccaaga taaaggagaa 1140

gaagaattac agaaatacat ccaggagagc caagcattgg caaagcgaag ctgcggcctc 1200

ttccagaaac taggagaata ttacttacaa aatgcgtttc tcgttgctta cacaaagaaa 1260

gccccccagc tgacctcgtc ggagctgatg gccatcacca gaaaaatggc agccacagca 1320

gccacttgtt gccaactcag tgaggacaaa ctattggcct gtggcgaggg agcggctgac 1380

attattatcg gacacttatg tatcagacat gaaatgactc cagtaaaccc tggtgttggc 1440

cagtgctgca cttcttcata tgccaacagg aggccatgct tcagcagctt ggtggtggat 1500

gaaacatatg tccctcctgc attctctgat gacaagttca ttttccataa ggatctgtgc 1560

caagctcagg gtgtagcgct gcaaacaatg aagcaagagt ttctcattaa ccttgtgaag 1620

caaaagccac aaataacaga ggaacaactt gaggctgtca ttgcagattt ctcaggcctg 1680

ttggagaaat gctgccaagg ccaggaacag gaagtctgct ttgctgaaga gggacaaaaa 1740

ctgatttcaa aaactcgtgc tgctttggga gtt 1773

<210> 23

<211> 2136

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 23

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120

ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180

gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240

atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300

ggagcccaga ggtccaccag cacagcgata accctgactg ggggcggagg cggaggatca 360

gggggagggg gaggaaaagc attgcacgaa aatgagtttg ggatagcttc cacgttagat 420

tcctcccagt gcgtgacgga gaagaatgtg cttagcatag ctaccatcac ctttacccag 480

tttgttccgg aagccaccga ggaggaagtg aacaaaatga ctagcgatgt gttggctgca 540

atgaagaaaa actctggcga tgggtgttta gaaagccagc tatctgtgtt tctggatgaa 600

atttgtcatg agacggaact ctctaacaag tatggactct caggctgctg cagccaaagt 660

ggagtggaaa gacatcagtg tctgctggca cgcaagaaga ctgctccggc ctctgtccca 720

cccttccagt ttccagaacc tgccgagagt tgcaaagcac atgaagaaaa cagggcagtg 780

ttcatgaaca ggttcatcta tgaagtgtca aggaggaacc ccttcatgta tgccccagcc 840

attctgtcct tggctgctca gtacgacaag gtcgttctgg catgctgcaa agctgacaac 900

aaggaggagt gcttccagac aaagagagca tccattgcaa aggaattaag agaaggaagc 960

atgttaaatg agcatgtatg ttcagtgata agaaaatttg gatcccgaaa cctccaggca 1020

acaaccatta ttaagctaag tcaaaagtta actgaagcaa attttactga gattcagaag 1080

ctggccctgg atgtggctca catccacgag gagtgttgcc aaggaaactc gctggagtgt 1140

ctgcaggatg gggaaaaagt catgacatat atatgttctc aacaaaatat tctgtcaagc 1200

aaaatagcag agtgctgcaa attacccatg atccaactag gcttctgcat aattcacgca 1260

gagaatggcg tcaaacctga aggcttatct ctaaatccaa gccagttttt gggagacaga 1320

aattttgccc aattttcttc agaggaaaaa atcatgttca tggcaagctt tcttcatgaa 1380

tactcaagaa ctcaccccaa ccttcctgtc tcagtcattc taagaattgc taaaacgtac 1440

caggaaatat tggagaagtg ttcccagtct ggaaatctac ctggatgtca ggacaatctg 1500

gaagaagaat tgcagaaaca catcgaggag agccaggcac tgtccaagca aagctgcgct 1560

ctctaccaga ccttaggaga ctacaaatta caaaatctgt tccttattgg ttacacgagg 1620

aaagcccctc agctgacctc agcagagctg atcgacctca ccgggaagat ggtgagcatt 1680

gcctccacgt gctgccagct cagcgaggag aaatggtccg gctgtggtga gggaatggcc 1740

gacattttca ttggacattt gtgtataagg aatgaagcaa gccctgtgaa ctctggtatc 1800

agccactgct gcaactcttc gtattccaac aggaggctat gcatcaccag ttttctgagg 1860

gatgaaacct atgcccctcc cccattctct gaggataaat tcatcttcca caaggatctg 1920

tgccaagctc agggcaaagc cctacagacc atgaaacaag agcttctcat taacctggtg 1980

aagcaaaagc ctgaactgac agaggagcag ctggcggctg tcactgcaga tttctcgggc 2040

cttttggaga agtgctgcaa agcccaggac caggaagtct gtttcacaga agagggtcca 2100

aagttgattt ccaaaactcg tgatgctttg ggcgtt 2136

<210> 24

<211> 2145

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 24

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120

ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg actctgactg gcggaggcgg aggatcaggg 360

ggagggggag gaagaacact gcatagaaat gaatatggaa tagcttccat attggattct 420

taccaatgta ctgcagagat aagtttagct gacctggcta ccatattttt tgcccagttt 480

gttcaagaag ccacttacaa ggaagtaagc aaaatggtga aagatgcatt gactgcaatt 540

gagaaaccca ctggagatga acagtcttca gggtgtttag aaaaccagct acctgccttt 600

ctggaagaac tttgccatga gaaagaaatt ttggagaagt acggacattc agactgctgc 660

agccaaagtg aagagggaag acataactgt tttcttgcac acaaaaagcc cactccagca 720

tcgatcccac ttttccaagt tccagaacct gtcacaagct gtgaagcata tgaagaagac 780

agggagacat tcatgaacaa attcatttat gagatagcaa gaaggcatcc cttcctgtat 840

gcacctacaa ttcttctttg ggctgctcgc tatgacaaaa taattccatc ttgctgcaaa 900

gctgaaaatg cagttgaatg cttccaaaca aaggcagcaa cagttacaaa agaattaaga 960

gaaagcagct tgttaaatca acatgcatgt gcagtaatga aaaattttgg gacccgaact 1020

ttccaagcca taactgttac taaactgagt cagaagttta ccaaagttaa ttttactgaa 1080

atccagaaac tagtcctgga tgtggcccat gtacatgagc actgttgcag aggagatgtg 1140

ctggattgtc tgcaggatgg ggaaaaaatc atgtcctaca tatgttctca acaagacact 1200

ctgtcaaaca aaataacaga atgctgcaaa ctgaccacgc tggaacgtgg tcaatgtata 1260

attcatgcag aaaatgatga aaaacctgaa ggtctatctc caaatctaaa caggttttta 1320

ggagatagag attttaacca attttcttca ggggaaaaaa atatcttctt ggcaagtttt 1380

gttcatgaat attcaagaag acatcctcag cttgctgtct cagtaattct aagagttgct 1440

aaaggatacc aggagttatt ggagaagtgt ttccagactg aaaaccctct tgaatgccaa 1500

gataaaggag aagaagaatt acagaaatac atccaggaga gccaagcatt ggcaaagcga 1560

agctgcggcc tcttccagaa actaggagaa tattacttac aaaatgcgtt tctcgttgct 1620

tacacaaaga aagcccccca gctgacctcg tcggagctga tggccatcac cagaaaaatg 1680

gcagccacag cagccacttg ttgccaactc agtgaggaca aactattggc ctgtggcgag 1740

ggagcggctg acattattat cggacactta tgtatcagac atgaaatgac tccagtaaac 1800

cctggtgttg gccagtgctg cacttcttca tatgccaaca ggaggccatg cttcagcagc 1860

ttggtggtgg atgaaacata tgtccctcct gcattctctg atgacaagtt cattttccat 1920

aaggatctgt gccaagctca gggtgtagcg ctgcaaacaa tgaagcaaga gtttctcatt 1980

aaccttgtga agcaaaagcc acaaataaca gaggaacaac ttgaggctgt cattgcagat 2040

ttctcaggcc tgttggagaa atgctgccaa ggccaggaac aggaagtctg ctttgctgaa 2100

gagggacaaa aactgatttc aaaaactcgt gctgctttgg gagtt 2145

<210> 25

<211> 53

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 25

tgaagtggat cacacccgct tccctcatcc tcctgctaca tttcgctgcg tcc 53

<210> 26

<211> 54

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 26

atgaagtggg tggaatcaat ttttttaatt ttcctactaa attttactga atcc 54

Claims

1.融合蛋白，其特征在于，包括：

甲胎蛋白的能够诱发特异性CD8+T反应的或其MHCI类分子结合表位优化形式的去除分泌肽序列的氨基酸序列；接头；和特异性结合具有抗原交叉呈递能力的DC细胞的人或鼠XCL1蛋白；

所述融合蛋白的氨基酸序列如SEQ ID No.9或SEQ ID No.10所示。

2.编码如权利要求1所述的融合蛋白的核酸分子。

3.重组表达载体，其特征在于，包括如权利要求2所述的核酸分子。

4.如权利要求3所述的重组表达载体，其特征在于，所述重组表达载体选自pcDNA3.1(+)、pcDNA3.1(-)、pFastbac1-dual-MBP或pVAX1。

5.包括如权利要求1所述的融合蛋白或如权利要求2所述的核酸分子的重组菌株或细胞。

6.如权利要求1所述的融合蛋白、如权利要求2所述的核酸分子、如权利要求3或4所述的重组表达载体或如权利要求5所述的重组菌株或细胞在制备肝癌或AFP高表达的肿瘤的疫苗或制备预防和/或治疗肝癌或AFP高表达的肿瘤的药物中的应用。

7.肝癌或AFP高表达的肿瘤的疫苗，其特征在于，包括如权利要求1所述的融合蛋白、如权利要求2所述的核酸分子或如权利要求3或4所述的重组表达载体以及药学上可接受的运载体、赋形剂和/或佐剂。

8.预防和/或治疗肝癌或AFP高表达的肿瘤的药物，其特征在于，包括如权利要求1所述的融合蛋白、如权利要求2所述的核酸分子或如权利要求3或4所述的重组表达载体以及药学上可接受的辅料。