CN113249342B - 一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用 - Google Patents
一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用 Download PDFInfo
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Abstract
本发明涉及一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用。所述溶瘤腺病毒具有三重肿瘤靶向调控机制、三个病毒结构基因改造、三种血清型腺病毒嵌合、三类抗癌免疫基因装载的特点,能激活多种病毒结构蛋白的内在抗癌活性,提高感染肿瘤细胞能力的同时又能保证病毒本身逃避机体预存中和抗体的拦截以及肝细胞的粘附和摄取,武装三类抗癌免疫调节基因提高增强杀伤癌细胞的作用。关键技术是在基因组内巧妙地预置了多重机制相互协同的设计理念,能打破肿瘤微环境的免疫抑制壁垒,对免疫检查点抑制剂治疗和免疫细胞治疗具有明显增效作用。本发明属于全新类型的溶瘤腺病毒,实现了真正意义的多重机制协同增效,靶向性好,抗癌活性强,具有广谱性,可单独或辅助用于癌症特别是实体瘤的治疗。
Description
技术领域
本发明属生命科学、生物医学领域,具体涉及应用于肿瘤治疗的一款多重机制协同和增效免疫治疗的广谱嵌合型溶瘤腺病毒及其在肿瘤治疗中的应用。
背景技术
溶瘤病毒介导的基因治疗对肿瘤细胞杀伤效率高、特异性好、安全性高、副作用小、成本低廉,使其成为继三大常规治疗方法(手术、放疗和化疗)以及免疫治疗之后的又一重要新兴肿瘤治疗模式。溶瘤病毒疗法最大的挑战,在于如何提高产品的安全性和有效性。通过一些安全的调控机制,能够保障溶瘤病毒很大程度上在肿瘤细胞内复制,而在正常细胞内不复制,肿瘤的靶向特异性提高,其安全性也就得到了保障。实现腺病毒靶向肿瘤细胞增殖杀伤的机制是丰富多样的,如早期第一代的溶瘤腺病毒(OAV)通过删除E1b-55kD蛋白基因实现靶向P53基因缺陷肿瘤细胞的特异性复制,之后第二代OAV采用各种肿瘤特异性启动子调控腺病毒增殖基因E1a或/和E1b的表达,发展至第三代OAV则是多种调控元件的联合应用。经过数代、多样化的改造,OAV针对肿瘤的特异性和有效性有所提高。从理论上讲,OAV作为载体携带抗癌基因,只要有肿瘤细胞存在,就能启动病毒增殖,抗癌基因随之高拷贝复制且高效率表达,继而产生病毒增殖溶瘤和基因表达抑癌的双重肿瘤杀伤效应,而且对正常细胞不产生影响。但在实际应用中,包括OAV在内的溶瘤病毒,其肿瘤靶向特异性以及破坏肿瘤细胞的效应,还没有达到人们的期望值,临床上单独应用溶瘤病毒***的效果依然不理想。此外,由于受调控元件差异和抗癌基因选择的限制,OAV个性化程度较高,难以在异质性大的不同肿瘤中均产生较好的治疗效果。
发明内容
本发明的目的是针对现有肿瘤免疫治疗对实体瘤效果不佳的难题,提供一款具有多重机制协同抗癌、能作为免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗增效剂的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用。
第一方面,本发明提供了一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,所述溶瘤腺病毒同时表达IL-12、IFN-γ和CCL5三个因子;所述溶瘤腺病毒其外壳蛋白Hexon由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成,其纤毛蛋白Fiber由Ad5和Ad11两个血清型病毒的Fiber序列嵌合而成;所述溶瘤腺病毒同时采用肿瘤特异性强势启动子和氧依赖元件开关,并删除Elb-55kD基因;所述溶瘤腺病毒同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列。
作为本发明的一种优选实施方式,在所述溶瘤腺病毒的基因组中,IFN-γ和CCL5基因cDNA序列共有一个表达框,以人巨细胞病毒(hCMV)启动子驱动,IL-12基因cDNA序列表达框,以鼠巨细胞病毒(mCMV)启动子驱动,两个表达框呈“脚对脚”排列。
更优选地,同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
作为本发明的另一种优选实施方式,所述溶瘤腺病毒其外壳蛋白Hexon的编码序列如SEQ ID NO:3的第18327-21170bp所示。
作为本发明的另一种优选实施方式,所述溶瘤腺病毒其纤毛蛋白Fiber的编码序列如SEQ ID NO:3的第33373-34356bp所示。
作为本发明的另一种优选实施方式,所述溶瘤腺病毒的全基因组序列如SEQ IDNO:3所示。
第二方面,本发明提供了一种同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒,所述溶瘤腺病毒的基因组中,IFN-γ和CCL5基因cDNA序列共有一个表达框,以人巨细胞病毒(hCMV)启动子驱动,IL-12基因cDNA序列表达框,以鼠巨细胞病毒(mCMV)启动子驱动,两个表达框呈“脚对脚”排列。
作为本发明的一种优选实施方式,同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
第三方面,本发明提供了如上所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒或如上所述的同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒在制备治疗和/或预防和/或辅助治疗癌症或者抗肿瘤的药物中的用途。
作为本发明的一种优选实施方式,所述癌症或者肿瘤为乳腺癌、肝癌、胆囊癌、胃癌、结肠癌、肺癌、***癌、淋巴瘤、大肠癌、卵巢癌、***、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、膀胱癌或头颈癌。
本发明提供***基于4T技术(4triple-construction techniques)的OAV设计方案,尽最大可能将OAV抗癌疗效和生物安全性提高到一个新高度。主要特点如下:
1)三重装载(Triple Transgene):本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,同时在腺病毒E3区装载3个抗癌基因,其中包括2个增强免疫的细胞因子基因(IL-12和IFN-γ)和1个免疫细胞趋化因子基因(CCL5),建立OAV联合免疫治疗协同增效的直接的、主动的分子机制。以OAV为载体装载抗癌基因,有助于抗癌基因在癌细胞内特异性高拷贝复制和高效率表达。但选择装载的抗癌基因,不仅仅满足于抗癌疗效叠加的要求,更优的方案是建立能与OAV本身、肿瘤微环境、机体免疫***互作的协同机制。肿瘤微环境的抑制状态是实体瘤免疫治疗的主要障碍,有研究证实,溶瘤病毒感染的肿瘤细胞能释放大量细胞因子,裂解的肿瘤细胞还能释放多种肿瘤相关抗原,这些细胞因子和肿瘤抗原不但具有免疫激活作用,也能改变肿瘤微环境,不仅能增强放化疗作用,同样能提高免疫检查点分子的抗体治疗和免疫细胞治疗的效果(Oncoimmunology 2015,4:e988098)。但现有溶瘤病毒提高免疫治疗的作用是有限的、缺乏直接的协同增效机制。本发明所提供的OAV,表达IL-12、IFN-γ和CCL5三个因子,具有主动的、直接的增效免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗疗效的协同机制。首先,OAV装载2个增强免疫的细胞因子IL-12和IFN-γ,由于PD-1抗体、PD-L1抗体治疗时需要激活T细胞才能发挥抗癌作用,这个过程先由树突状细胞(DC)合成分泌IFN-γ和IL-12,这两个细胞因子与T细胞进行信息交流,才能激活T细胞的杀伤活性(Immunity.2018,49:1148-1161.e7)。但肿瘤微环境中常常缺乏DC,IFN-γ和IL-12分泌量不足。当OAV装载增强免疫的细胞因子IL-12和IFN-γ,在OAV与PD-1抗体联用时无需DC参与,病毒表达的IL-12和IFN-γ可直接激活微环境杀伤性T细胞而快速发挥抗癌疗效。在与CAR-T/CAR-NK连用时,OAV表达的IL-12能延长CAR-T/CAR-NK细胞存活时间,IFN-γ能增强CAR-T/CAR-NK杀伤活性。当然,IL-12和IFN-γ本身也具有很强的抗癌活性,IFN-γ是老牌的抗癌免疫因子,能诱导Interlukins、TNF等多种细胞因子表达。IL-12是肿瘤免疫治疗的新宠,能提高机体保护性免疫应答水平,促进刺激T细胞及NK细胞增殖和激活,诱导分泌IFN-γ等多种细胞因子。其次,OAV装载免疫细胞趋化因子CCL5,可趋化NK、T细胞(包括CAR-T/CAR-NK)、DC等多种免疫细胞迁移到肿瘤内积聚(Cell 2018,172:1022-1037)。在OAV与PD-1抗体联用时,CCL5可增加肿瘤微环境中NK、T细胞、DC等细胞数量,协同增强杀伤癌细胞的作用。在与CAR-T/CAR-NK连用时,CCL5既可趋化更多CAR-T/CAR-NK细胞进入肿瘤区,也能促进趋化来的NK、T细胞、DC等细胞协同增强CAR-T/CAR-NK功能。实验证实,三个因子在溶瘤病毒基因组中的排列连接顺序对其抗肿瘤活性有明显的影响,通过比较三个因子多重组合排列顺序的溶瘤病毒抑制肿瘤细胞的活性,最终确定抗癌活性最佳的VirRon中三因子排列顺序,即:VirRon装载的IFN-γ和CCL5基因cDNA序列共有一个表达框,以人巨细胞病毒(hCMV)启动子驱动,IFN-γ和CCL5基因cDNA序列间由T2A连接。VirRon装载的IL-12基因cDNA序列表达框,以鼠巨细胞病毒(mCMV)启动子驱动。两个表达框呈“脚对脚”排列。两个表达框分别选择人和鼠两个不同的巨细胞病毒(CMV)启动子驱动,可避免相同序列重复造成的不可预见的高级结构影响病毒的包装成功率和抗癌活性。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的IFN-γ基因cDNA编码序列(不包含终止密码子)的反向互补序列如SEQ ID NO:3的第32263-32760bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的IL-12基因cDNA编码序列如SEQ ID NO:3的第29973-31583bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的CCL5基因cDNA编码序列的反向互补序列如SEQ ID NO:3的第31924-32199bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的IFN-γ和CCL5基因cDNA序列共有一个表达框,IFN-γ和CCL5基因cDNA序列间由T2A连接,其T2A序列的反向互补序列如SEQ ID NO:3的第32200-32262bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的IFN-γ和CCL5基因cDNA序列共有一个表达框,以人巨细胞病毒(hCMV)启动子驱动。其hCMV启动子编码序列的反向互补序列如SEQ ID NO:3的第32767-33108bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的IL-12基因cDNA序列表达框,以鼠巨细胞病毒(mCMV)启动子驱动。其mCMV启动子序列如SEQ ID NO:3的第29422-29944bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其装载的抗癌基因并不限于IL-12、IFN-γ和CCL5,还包括其他具有抗癌活性的细胞因子、免疫因子、抑癌基因、***基因、抗体基因等及其突变体改构序列;其多基因连接方式不限于T2A序列,还包括F2A、P2A、IRES等序列及其突变体;其采用的启动子不限于mCMV和hCMV,还包括其他具有基因驱动功能的启动子、增强子及其突变体序列。
2)三重嵌合(Triple Chimerism):本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,同时应用B亚属Ad11血清型的腺病毒Fiber编码序列和D亚属Ad48血清型的腺病毒Hexon编码序列,与C亚属Ad5型腺病毒骨架相应序列嵌合,构建出三个血清型嵌合的OAV,实现了在一个治疗***上同时提高病毒对肿瘤细胞感染效率、避免病毒被机体预存中和抗体拦截、降低机体肝细胞对病毒的粘附摄取三大优势。人类腺病毒家族有52个血清型,分6个亚属(A至F)。除了B组以外,各组腺病毒都用柯萨奇病毒腺病毒共用受体(Coxsackievirus-adenovirus receptor,CAR)作为其主要吸附受体,对缺乏CAR的骨髓造血细胞、造血干细胞、树突状细胞、部分肿瘤细胞尤其是肿瘤干细胞等感染效率很低。B组腺病毒(Ad3、Ad7、Ad11、Ad14、Ad16、Ad21、Ad34-35、Ad50、Ad55等)则主要识别一种广泛表达的补体调节蛋白CD46。采用B组腺病毒的纤毛蛋白(fiber knob)替代Ad5纤毛蛋白,构建嵌合病毒,使之增加识别CD46受体的能力,则有利于提高病毒对肿瘤细胞感染效率,特别是感染肿瘤干细胞的能力,有可能更彻底地根除肿瘤复发的根源;Ad5在自然界存在较广,大多数人已被感染并产生了中和抗体,能够拦截相同血清型的病毒。况且Ad5具有嗜肝细胞性,能够被肝细胞吸附。腺病毒外壳蛋白Hexon的高变区(HVR)因位置暴露于腺病毒表面,是导致不同血清型腺病毒之间对肝脏感染能力和免疫原性差异的关键部位。将Ad5的Hexon分子内7个HVR(L1:HVR1-6;L2:HVR7)选择性地与稀有血清型如D亚群(Ad8-10、Ad25-30、Ad36-39、Ad42-49、Ad51等)病毒的Hexon相应区域进行嵌合,是帮助Ad5逃避预存免疫和肝脏摄取的有效方法。然而,Hexon作为腺病毒的主要结构蛋白,对其进行改造往往会导致腺病毒载体结构不稳定,从而无法有效地包装病毒,因此这是一项很具有挑战性的研究。我们前期通过深入研究,设计多个Ad5和Ad48血清型病毒的Hexon编码序列片段进行组合,进行病毒重组包装和扩增测试,终于攻克了这项技术难关,构建成功多个Hexon替代的嵌合型腺病毒载体骨架,能有效包装目的病毒颗粒。Ad48腺病毒Hexon的嵌合则能保证OAV逃避机体预存中和抗体的拦截以及肝细胞对腺病毒的粘附和摄取。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其B亚属Ad11血清型腺病毒纤毛蛋白(Fiber)编码序列与C亚属Ad5型腺病毒Fiber相应序列嵌合的嵌合型纤毛蛋白(Ad5F11b)的序列如SEQ ID NO:3的第33373-34356bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其D亚属Ad48血清型腺病毒六邻体(Hexon)编码序列与C亚属Ad5型腺病毒Hexon相应序列嵌合的嵌合型Hexon(Ad5H48)的序列如SEQ ID NO:3的第18327-21170bp所示。
本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,其血清型嵌合并不限于C亚属Ad5与B亚属Ad11、D亚属Ad48的嵌合,还包括其他亚属其他血清型的相互嵌合改造。
3)三重调控(Triple Regulation):本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,同时采用肿瘤特异性强势启动子和氧依赖元件开关,并删除Elb-55kD基因,构建在转录和翻译水平上具有双重安全调控机制的OAV产品。实现OAV对肿瘤细胞特异性增殖复制的机制之一,是以肿瘤特异性启动子作为顺式元件控制腺病毒增殖必需基因的表达。通过研究腺病毒E1a、E1b与癌细胞分子互作的机制,在启动子、增强子调控元件中优选调控模块组合,由肿瘤特异性广谱启动子在转录水平上控制腺病毒E1a基因只在肿瘤细胞内表达;E1a末端融合氧依赖元件开关,E1a蛋白在肿瘤缺氧环境中被保护,而在正常组织常氧状态下则被蛋白酶体降解,在翻译水平上有效防止E1a蛋白在正常细胞中表达的渗漏;Elb转录单元编码Elb-55kD和Elb-19kD,Elb-55kD是腺病毒在正常细胞增殖复制必需的而在肿瘤细胞中非必需的蛋白,Elb-55kD编码基因的选择性缺失可以使腺病毒在肿瘤细胞内保持增殖复制的能力,而在正常细胞中失去复制能力。Elb-55kD蛋白能够灭活并降解P53蛋白,Elb-55kD缺失有利于细胞保持P53的抗肿瘤活性,同时提高病毒载体的靶向性。因此在上述调控机制的三重控制下,VirRon的安全性和有效性得到提高。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其肿瘤特异性广谱启动子核心序列截取于BIRC5基因5’-UTR区域转录起始位点“-857~+4bp”片段,全长序列如SEQ ID NO:3的第467-1388bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon及其应用,其氧依赖元件ON/OFF开关序列如SEQ ID NO:3的第2376-2423bp所示。
我们前期已克隆、研究、保存了20多种肿瘤特异性的基因启动子、增强子及其突变体序列,本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,其调控元件包括Ki67、hTERT、CEA、AFP、EGFR、DF3/MUC1、VEGFR、E2F、GFAP、Survivin等因子的启动子、增强子及其突变体序列,还有HIF-1缺氧反应元件(HRE)、Egr-l辐射敏感元件(CArG)、hTERT内部E-box、氧依赖开关ODD等调控序列,以及上述序列的组合应用。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,删除其Elb转录单元中Elb-19kD全部编码序列以及Elb-55kD编码序列第1个密码子到437个密码子,Elb-55kD剩余编码序列如SEQ ID NO:3的第2818-2997bp所示。
4)三重改造(Triple Modification):本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列。OAV构建以致病力最弱的人类腺病毒C亚属中的5型血清型(Ad5)为基础,保留E1a蛋白表达并使其受肿瘤特异性启动子调控,实现针对肿瘤的特异性复制。E1a有3个功能区,即CR1、CR2、CR3,CR1区通过与转录调节因子P300/CBP结合能抑制Her-2/neu基因的表达,CR2区与Rb蛋白家族结合,CR3区是转录活化区。因此E1a蛋白本身就具有抗肿瘤作用,既可以通过抑制Her-2/neu基因的转录、阻断NF-κB的活性、提高p53的表达,又可以抑制Ⅳ型胶原酶、纤溶酶原激活剂等蛋白酶基因表达;E1a还能引起非特异性免疫反应,提高CTL细胞、NK细胞、巨噬细胞的杀伤效应等多种途径,诱导肿瘤细胞凋亡,抑制肿瘤侵袭转移,提高肿瘤细胞对化疗、放疗的敏感性。对E1a的CR2区引入12个碱基的缺失突变,使其不能结合Rb蛋白,保证去磷酸化的Rb蛋白与转录因子E2F形成复合物,阻断E2F的转录活性,可以释放E1a内在抗癌机制,增强产品抗癌活性;Elb-19kDa基因与凋亡抑制基因Bcl-2同源,Elb-19kD蛋白能够结合Bax或/和Bak启动下游的凋亡抑制程序,保护感染的细胞免受TNF-α介导的杀伤作用。Elb-19kD缺失,因而该病毒突变体在肿瘤细胞内增殖的特异性得以提高,而在正常细胞内的增殖活性被削弱。Elb-19kD缺失能够促进癌细胞凋亡通路的恢复,且有利于病毒在正常细胞内的快速清除以及在肿瘤细胞内的快速释放和播散,使OAV特异性更好,效能更强;E3转录单位有9个开放读框,编码蛋白保护感染细胞免受宿主免疫反应的杀伤,E3-gpl9k可减弱CTL介导的感染细胞杀伤效应;RID可阻断“死亡”配体包括TNF、Fas配体和TRAIL介导的细胞凋亡;RID还可抑制IL-1和TNF介导的细胞存活所必须的NF-κB的激活;E3-6.7k除了使TRAIL受体下调之外还可单独抑制外部和内部信号通路诱导的细胞凋亡;E3-14.7k是TNF介导细胞凋亡的广泛抑制剂,也可结合并抑制Caspases-8从而阻止Fas信号通路启动的细胞凋亡;E3-14.7k与受感染的细胞内FIP蛋白(for 14.7K-interacting protein,FIP-1、-2、-3)作用,可使E3-14.7k蛋白在细胞凋亡和存活、炎症反应、维持膜稳定、核浆转运等信号转导通路中处于重要地位,这一多功能的E3蛋白的分子机制仍需深入研究。腺病毒死亡蛋白(ADP)可促进细胞溶解以及病毒释放,然而分子机制不明。由此可见,在OAV构建过程中删除E3区同时保留ADP基因,既可以扩大载体容量,又能够促进受感染的癌细胞的凋亡,并有利于促进OAV溶解癌细胞以及子代病毒释放。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,敲除其E1a-CR2中12个碱基“cacgaggctggc”,并且突变型E1a(mE1a)序列终止密码子前***氧依赖元件开关,其序列如SEQ ID NO:3的第1405-2426bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,其病毒结构蛋白的改造不限于E1a-CR2、E1b-19kD、E3区部分蛋白编码序列的敲除,还包括E1a、E1b、E3以及其他蛋白的缺失、***、突变等改造。
综上所述,本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,是全新一代OAV产品,实现了真正意义的多重机制协同增效的目的,特异性好,抗癌活性强,对多种实体瘤具有明显的治疗效果,克服了以往溶瘤腺病毒单独使用疗效不佳和抗瘤谱窄的问题,既具有单独应用的良好抗癌效果,也能作为免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗增效剂,协同免疫治疗大幅提高实体瘤的疗效。其创新性意义如下:
1、多重机制精准调控OAV增殖提高其安全性和有效性:以肿瘤特异性广谱启动子控制腺病毒增殖基因E1a表达,在转录水平增强OAV针对肿瘤细胞的特异性复制活力;在E1a蛋白内部融合氧依赖元件开关,保证E1a在正常组织常氧状态下被降解,在翻译水平限制病毒在正常细胞内复制;选择性缺失Elb-55kD可使腺病毒在肿瘤细胞内保持增殖复制的能力,而在正常细胞中失去复制能力;Elb-19kD缺失能够促进癌细胞凋亡通路的恢复,有利于病毒在正常细胞内的快速清除,在肿瘤细胞内的快速释放;E3区的部分编码蛋白敲除和ADP基因保留,达到保护腺病毒长期生存并介导抗癌基因高效表达的目的。多重机制协同,共同提高OAV安全性和有效性。
2、激活病毒内在抗肿瘤机制协同提高OAV溶瘤作用和抗癌疗效:腺病毒E1a蛋白本身即具有抗肿瘤作用,既可以通过抑制Her-2/neu基因的转录、阻断NF-κB的活性、提高p53的表达,又可以抑制Ⅳ型胶原酶、纤溶酶原激活剂等蛋白酶基因表达;E1a还能引起非特异性免疫反应,提高CTL、NK、巨噬细胞的杀伤效应等多种途径,诱导肿瘤细胞凋亡,抑制肿瘤侵袭转移,提高肿瘤细胞对化疗、放疗的敏感性。在此基础上,对E1a的CR2区引入缺失突变,使其不能结合Rb蛋白,保证去磷酸化的Rb蛋白与转录因子E2F形成复合物,阻断E2F的转录活性,可以增强抗癌活性。特别是OAV感染癌细胞释放大量细胞因子,裂解癌细胞释放大量肿瘤相关抗原,发挥进一步的免疫激活作用。局限于癌细胞表达的病毒蛋白,类似于肿瘤特异抗原,还能发挥抗癌疫苗的功能。
3、三个血清型腺病毒嵌合形成的OAV提高其感染力并能有效逃避免疫拦截和肝脏摄取:采用B组腺病毒纤毛蛋白(fiber knob)与Ad5纤毛蛋白嵌合,有利于提高病毒对癌细胞特别是对肿瘤干细胞的感染效率。将Ad5的Hexon分子内HVR区选择性地与D亚群病毒的Hexon相应区域嵌合,有助于病毒逃避人体中和抗体拦截和肝细胞摄取。
4、建立免疫调节肿瘤微环境的多机制协同增效的治疗策略并在研究中获得明确疗效:以OAV为载体装载抗癌基因,有助于抗癌基因随病毒增殖在癌细胞内特异性高拷贝复制和高效率表达。通过表达能激活肿瘤微环境免疫的抗癌基因或免疫调节基因,研制多机制协同抗癌的高效OAV新产品,能够与免疫检查点抑制剂治疗和免疫细胞治疗等多种治疗手段具有协同增效,在提高病毒安全性和有效性方面实现多重重大突破,达到广谱、特异、安全、高效的抗癌效果。
附图说明
图1.腺病毒右臂骨架质粒pPE3F11bH48-RC(+)结构图。
图2.腺病毒左臂穿梭质粒pAdSVP-mE1aODD结构图。
图3.外源基因穿梭质粒pENTR12-C3结构图。
图4.溶瘤腺病毒VirRon基因组结构图。
图5.溶瘤病毒VirRon在肝癌、乳腺癌、胆囊癌、肺癌细胞系增殖活性高,增殖倍数在感染后96h介于104和106之间,在正常细胞系中增殖力很弱。
图6.溶瘤病毒VirRon对细胞杀伤作用的曲线图,显示VirRon在较低MOI值时即对多种实体瘤细胞均有明显的杀伤抑制作用,而对正常成纤维细胞BJ无杀伤活性。
图7.溶瘤病毒VirRon对细胞杀伤作用的IC50值比较,显示VirRon对多种实体瘤细胞杀伤抑制作用的IC50值较低,而对正常成纤维细胞BJ杀伤的IC50值高达1090。
图8.溶瘤病毒VirRon表达“IL-12+IFN-γ+CCL5”三个抗癌基因,随时间延长表达量逐渐增高。
图9.溶瘤腺病毒VirRon中三个抗癌基因排列连接顺序对其包装效率和抗癌活性的影响。
图10.溶瘤腺病毒VirRon中嵌合型Hexon(Ad5H48)片段组合对其包装和扩增效率的影响。
图11.溶瘤病毒VirRon抗肝癌HCCLM3裸鼠移植瘤的实验。
图12.溶瘤病毒VirRon抗乳腺癌MDA-MB-231裸鼠移植瘤的实验。
图13.溶瘤病毒VirRon抗胆囊癌SGC996裸鼠移植瘤的实验。
图14.VirRon联合PD-1抗体治疗结肠癌移植瘤的人源化小鼠模型实验。
图15.VirRon联合CAR-T细胞治疗***癌移植瘤的NCG小鼠模型实验。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的研制
本发明所述的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,由腺病毒右臂质粒、腺病毒左臂质粒和外源基因穿梭质粒重组而成。现介绍这3个质粒及重组过程,具体如下:
(1)腺病毒右臂骨架质粒
腺病毒右臂骨架质粒pPE3F11bH48-RC(+)由加拿大Microbix Biosystems公司提供的pBHGlox(delta)E13Cre质粒(Catalog no.PD-01-40)和pBHGE3质粒(Catalog no.PD-01-12)改建而来。将pBHGE3含有部分E3区序列的SpeI+NotI酶切片段以及合成的ADP基因序列(如SEQ ID NO:3的第30901-31182bp所示),***到pBHGlox(delta)E13Cre的SpeI+PacI位点间,构建pPE3质粒。将合成的attR1+ccdB+attR2序列(949bp,SEQ ID NO:1)***到pPE3的PacI位点,将合成的Ad5F11b片段替换Ad5-Fiber序列,将合成的Ad5H48片段替换Ad5-Hexon序列,最终构建pPE3F11bH48-RC(+)质粒(图1)。pPE3F11bH48-RC(+)全长36591bp,其纤毛蛋白(Ad5F11b)由Ad5和Ad11血清型腺病毒相应序列嵌合而成(如SEQ ID NO:3的第33373-34356bp所示),其六邻体蛋白(Ad5H48)由Ad5和Ad48血清型腺病毒相应序列嵌合而成(如SEQ ID NO:3的第18327-21170bp所示)。
(2)腺病毒左臂穿梭质粒
腺病毒左臂穿梭质粒pAdSVP-mE1aODD由加拿大Microbix Biosystems公司提供的pXC1质粒(Catalog no.PD-01-03)改建而来。在pXC1的E1a起始密码子上游12bp处引入BglII酶切位点,构建pXC2。合成BIRC5基因5’-UTR区域转录起始位点“-857~+4bp”片段、突变型E1a及氧依赖元件开关序列(mE1a-ODD),共2355bp(如SEQ ID NO:3的第467-2821bp所示),***到pXC2的2个BglII位点之间,替换pXC2中的E1a和E1b序列,构建成pAdSVP-mE1aODD,全长9402bp(图2)。
(3)外源基因穿梭质粒
穿梭质粒pENTR12由美国Invitrogen公司提供的pENTR11质粒(Catalogno.11819-018)改建而来。在pENTR11的NcoI和EcoRV酶切位点之间引入“mCMV启动子+SpeI/EcoRI/HindIII/BglII/NheI/BamHI/SalI+Poly(A)”序列(SEQ ID NO:2),替换原pENTR11质粒中的多克隆位点和ccdB基因序列,构建成新的质粒pENTR12。在pENTR12多克隆位点EcoRI和EcoRV之间***IL-12表达框和IFN-γ+CCL5表达框,双表达框呈“脚对脚”排列,构建成外源基因穿梭质粒pENTR12-C3,全长5985bp(图3)。最终由pENTR12-C3转移到VirRon基因组中包含IL-12表达框和IFN-γ+CCL5表达框的NcoI和EcoRV位点之间的完整序列(含位点序列)如SEQ ID NO:3的第29401-33114bp所示。
(4)VirRon病毒的重组包装程序
VirRon病毒的重组包装以腺病毒右臂质粒pPE3F11bH48-RC(+)为骨架,经两轮特异性重组包装而成。首先用外源基因穿梭质粒pENTR12-C3与腺病毒右臂骨架质粒pPE3F11bH48-RC(+)在DB3.1大肠杆菌感受态细菌内发生第一轮attL/attR位点特异性重组,将pENTR12-C3中的外源基因表达框转移到pPE3F11bH48-RC(+)的E3区;然后再用腺病毒左臂穿梭质粒pAdSVP-mE1aODD与第一轮重组的产物在293T细胞内发生第二轮序列特异性重组,将pAdSVP-mE1aODD中的肿瘤特异性启动子控制的mE1aODD表达框转移到腺病毒右臂骨架质粒的E1区。经过两轮特异性重组,准确快速地包装出理想的溶瘤腺病毒VirRon。溶瘤腺病毒VirRon结构图如图4所示。本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,其完整序列如SEQ ID NO:3所示。
实施例2本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的细胞学实验
本发明所述的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon,能够特异性地在肿瘤细胞内增殖复制,介导抗癌基因高效表达,最后破坏或抑制肿瘤细胞。
(1)溶瘤腺病毒VirRon的特异性增殖复制
收集对数生长期的实体瘤细胞系以及正常细胞系,铺96孔板,1×104/孔,细胞贴壁后,换用无血清培养液;以MOI=5pfu/cell加入VirRon进行细胞感染。病毒感染2h后换5%血清培养液,继续培养0h、48h、96h后,收集0h、48h、96h时间点的细胞及上清液,TCID50方法检测病毒滴度,以0h为基准,计算病毒增殖倍数。结果发现,VirRon在包括肝癌、乳腺癌、胆囊癌、肺癌等多数实体瘤细胞系中呈高拷贝增殖,增殖倍数最高可达316978.64倍;而在正常细胞系中增殖活性较低,在GES-1细胞中完全不增殖,在肝细胞L02中有较弱增殖活性,增殖倍数仅为399.05倍(图5)。
(2)溶瘤腺病毒VirRon对肿瘤细胞的特异性杀伤抑制
通过CCK8实验检测溶瘤腺病毒VirRon对肿瘤细胞和正常细胞的特异性杀伤抑制。收集对数生长期的肿瘤细胞系以及正常细胞系,铺96孔板,1×104/孔,细胞贴壁后,换用无血清培养液;以梯度MOI加入VirRon进行细胞感染,对应于每个MOI值设8个复孔,孵箱内培养2h;换血清培养液100μl/孔,培养48h后,弃培养液,加入CCK8溶液,置孵箱内4h,酶标仪测定490nm波长光吸收值,计算细胞存活率,绘制细胞生存曲线。结果发现,VirRon对多种实体瘤细胞均有明显的杀伤抑制作用,并且与病毒感染强度密切相关;同一类型肿瘤的不同细胞系对VirRon的敏感性有所不同,VirRon对肺癌H460细胞的杀伤IC50值仅有6.656pfu/cell。VirRon对正常成纤维细胞BJ无明显杀伤活性,其IC50值高达1091pfu/cell(图6-7,表1)。
表1.溶瘤腺病毒VirRon对培养细胞的杀伤作用(IC50对比)
细胞系 | IC50(pfu/cell) | |
乳腺癌 | MDA-MB-231 | 82.04 |
MDA-MB-453 | 10.52 | |
MCF-7 | 16.72 | |
肝癌 | Hep3B | 28.94 |
MHCC97L | 46.43 | |
MHCC97H | 82.09 | |
HCCLM3 | 9.85 | |
胆囊癌 | NOZ | 16.01 |
GBC-SD | 79.41 | |
胃癌 | MKN45 | 189.6 |
BGC823 | 11.40 | |
SNU-1 | 47.23 | |
结肠癌 | SW480 | 21.22 |
SW620 | 113.4 | |
Caco2 | 104.7 | |
肺癌 | H1299 | 12.65 |
H460 | 6.66 | |
A549 | 16.66 | |
正常细胞 | BJ | 1091 |
(3)溶瘤腺病毒VirRon介导抗癌基因的表达
收集对数生长期的实体瘤细胞系以及正常细胞系,铺6孔板,1×106/孔,24h细胞贴壁后,换用无血清培养液;以MOI=5pfu/cell的感染强度加入VirRon进行细胞感染。继续培养24h、48h、72h后,收集上清,通过ELISA方法检测IL-12、IFN-γ、CCL5基因蛋白表达情况。结果发现,VirRon能够介导IFN-γ、IL-12、CCL5蛋白高效表达,并且随时间延长表达量逐渐增高(图8)。
(4)溶瘤腺病毒中抗癌基因排列连接顺序对其包装效率和抗癌活性的影响
溶瘤腺病毒VirRon装载三个抗癌免疫因子IL-12、IFN-γ、CCL5,三个因子的排列连接顺序对病毒包装成功率和抗癌活性都有明显影响。通过设计构建三个因子不同排列顺序的穿梭质粒(V1~V4),与腺病毒骨架质粒重组,在HEK293细胞中包装溶瘤腺病毒(图9A)。结果发现,V1观察至第13天,细胞未出现噬斑病变,病毒包装不成功;V2和V3在第11天、V4在第7天分别出现噬斑,病毒重组成功(图9B)。在噬斑出现后48h收集细胞上清,ELISA检测三个因子的表达,V2、V3、V4表达IL-12和CCL5水平较高,但V2表达IFN-γ表达量极低,显著低于V3和V4(图9C)。培养肺癌A549细胞,以梯度(MOI=0.01~1000pfu/cell)感染强度感染V2、V3、V4病毒,采用CCK-8试剂盒检测72h病毒对细胞增殖活性的影响,证实V4对A549细胞的抑制效应最明显(图9D)。最终确定V4(即VirRon)中三个因子的排列连接顺序既有利于病毒的重组包装和基因表达,也有益于提高病毒的抗癌活性。
(5)溶瘤腺病毒中嵌合型Hexon(Ad5H48)片段组合对其包装和扩增效率的影响
溶瘤腺病毒VirRon的外壳蛋白Hexon(Ad5H48)由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成,目的是避免体内预存Ad5型病毒中和抗体的拦截和肝脏对病毒的吸附。Ad5H48编码cDNA全长2844bp,我们设计了3种不同片段组合的嵌合方式,在HEK293细胞中进行病毒重组包装和扩增(图10A)。实验发现,Ad5H48-1(即VirRon)在第7天出现细胞噬斑,Ad5H48-2观察至第14天未出现噬斑病变,Ad5H48-3在第12天出现噬斑,证实Ad5H48-1(即VirRon)出毒最快(图10B)。Ad5H48-1和Ad5H48-3以MOI=10pfu/cell感染HEK293细胞,96h后分别收集细胞和上清,TCID50法检测病毒滴度,无论是细胞内还是上清中Ad5H48-1滴度都高于Ad5H48-3(图10C)。最终证实Ad5H48-1(即VirRon)包装和扩增效率最佳。
实施例3本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的动物学实验
(1)VirRon抗肝癌的动物模型实验
健康纯种BALB/C裸鼠30只,4周龄,雄性,上海诚勤生物科技有限公司提供,合格证号SCXK(沪)2012-0002。取对数生长期肝癌HCCLM3细胞悬液注射于裸鼠右腋皮下,5×106细胞数/100μl/只。接种后12天,成瘤率100%,移植瘤最大最小直径平均约5.0mm。随机分为3组(VirRon组、AdSVP-H48-DsRed组、空白对照组),每组10只。每组首先剔除瘤体最大和最小的小鼠各一只,其余8只进入实验治疗性观察。2个病毒治疗组给予相应腺病毒瘤内多点注射,每次每只剂量3×108pfu/100μl,隔天一次,共5次;空白对照组同步给予病毒保存液,每次每只100μl。治疗后,定期测量瘤体大小,以“最大径×最小径2×0.5”公式计算瘤体体积,绘制生长曲线。实验过程中,任意一组瘤体体积超过动物实验伦理委员会允许的肿瘤体积上限2000mm3时,即终止实验观察。结果发现,实验观察至治疗后28天,VirRon治疗组抑瘤率为65.51%,与对照组有明显差异(P=0.0001);对照病毒为未携带治疗基因但***红色荧光报告基因的AdSVP-H48-DsRed,其也产生明显的溶瘤作用,抑瘤率45.91%,与对照组相比差异有统计学意义(P=0.01)(图11)。
(2)VirRon抗乳腺癌的动物模型实验
健康纯种BALB/C裸鼠30只,4周龄,雌性,上海诚勤生物科技有限公司提供,合格证号SCXK(沪)2012-0002。取对数生长期乳腺癌MDA-MB-231细胞悬液注射于裸鼠右腋皮下,5×106细胞数/100μl/只。接种后10天,成瘤率100%,移植瘤最大最小直径平均约3.0mm。剔除瘤体最大的3只、瘤体最小的2只,其余25只随机分为5组(VirRon低剂量组、VirRon中剂量组、VirRon高剂量组、AdSVP-H48组、空白对照组),每组5只。病毒治疗组给予相应腺病毒瘤内多点注射,高剂量组每次每只剂量3×108pfu/100μl,中剂量组每次每只剂量2×108pfu/100μl,低剂量组每次每只剂量1×108pfu/100μl,隔天一次,共5次;AdSVP-H48组为中剂量注射,空白对照组同步给予病毒保存液,每次每只100μl。治疗后,定期测量瘤体大小,以“最大径×最小径2×0.5”公式计算瘤体体积,绘制生长曲线。实验过程中,任意一组瘤体体积超过动物实验伦理委员会允许的肿瘤体积上限2000mm3时,即终止实验观察。结果发现,实验观察至治疗后30天,VirRon高、中、低剂量组组抑瘤率分别为74.15%、52.80%、40.66%,未携带治疗基因的对照病毒AdSVP-H48抑瘤率仅33.38%(图12)。
(3)VirRon抗胆囊癌的动物模型实验
胆囊癌恶性度高、进展速度快,除手术外临床无治疗措施。通过建立SGC-996快速增长的移植瘤模型,观察VirRon治疗的疗效及协同机制。健康纯种BALB/C裸鼠40只,4周龄,雄性,上海诚勤生物科技有限公司提供,合格证号SCXK(沪)2012-0002。取对数生长期胆囊癌SGC-996细胞悬液注射于裸鼠右腋皮下,5×106细胞数/100μl/只。接种后14天,成瘤率100%,移植瘤最大最小直径平均约5.0mm。随机分为4组(VirRon组、AdSVP-H48空载病毒组、Ad5-C3非增殖病毒组、空白对照组),每组10只。每组首先剔除瘤体最大的小鼠各一只,其余9只进入实验治疗性观察。VirRon组、AdSVP-H48组和Ad5-C3组给予相应病毒瘤内多点注射,每次每只剂量2×108pfu/100μl,隔天一次,共5次;空白对照组同步给予病毒保存液,每次每只100μl。治疗后,定期测量瘤体大小,以“最大径×最小径2×0.5”公式计算瘤体体积,绘制生长曲线。结果发现,实验观察至治疗后44天,VirRon治疗组抑瘤率为49.47%,与对照组比较有明显差异(P=0.0241);未携带治疗基因的空载对照溶瘤腺病毒AdSVP-H48和表达三个细胞因子IL-12、IFN-γ、CCL5的非增殖型对照腺病毒Ad5-C3的抑瘤率仅为24.03%和15.26%,统计显示药物协同作用的Q值为1.39,证实VirRon发挥出了AdSVP-H48病毒的溶瘤作用和细胞因子的抗癌作用的协同增效作用(图13)。
(4)VirRon联合PD-1抗体治疗结肠癌移植瘤的人源化小鼠模型实验
健康C57-hPD1人源化小鼠,雌性,5-6周龄,46只,购自北京百奥赛图基因生物技术有限公司,右侧背部皮下接种小鼠结肠癌细胞系MC38细胞1×106细胞数/只,肿瘤平均长到50mm3-80mm3时,剔除瘤体最大的3只,瘤体最小的3只,其余40只随机分成4组(空白对照、VirRon单药、Keytruda单药,联合用药组),开始给药治疗。VirRon瘤内多点注射,每次每只剂量3×108pfu/100μl,隔天一次,共5次;Keytruda腹腔注射给药,每次每只2.5mg/kg,每周2次,共6次。空白对照组同步给予病毒保存液,每次每只100μl。治疗后,定期测量瘤体大小,以“最大径×最小径2×0.5”公式计算瘤体体积,绘制生长曲线。至首次治疗后18天,VirRon单药组抑瘤率18.71%、Keytruda单药组抑瘤率69.05%,联合用药组抑瘤率88.86%(图14)。VirRon+Keytruda联合用药组与VirRon单药组、Keytruda单药组、对照组相比均有显著差异(P=0.0013、P=0.0110、P=0.0004),统计显示药物协同作用的Q值为1.19,证实VirRon与Keytruda联合用药具有协同增效的作用。
(5)VirRon联合CAR-T细胞治疗***癌移植瘤的NCG小鼠模型实验
CAR-T细胞由徐州医科大学肿瘤研究所制备,靶点为B7-H3。健康NCG小鼠,雄性,4-5周龄,19只,购自江苏集萃药康生物科技股份有限公司,右侧背部皮下接种***癌细胞系Du145细胞3×106细胞数/只,肿瘤平均长到50mm3左右时,随机分成4组(空白对照、VirRon单药、CAR-T单药,联合用药组),每组5只(CAR-T单药组4只),开始给药治疗。VirRon每只小鼠总剂量1×109pfu,分3次瘤内注射,隔天一次,共5次;完成病毒注射后第二天,给予CAR-T尾静脉注射,每只2×106细胞数。空白对照组同步给予PBS缓冲液。治疗后,定期测量瘤体大小,以“最大径×最小径2×0.5”公式计算瘤体体积,绘制生长曲线。至首次治疗后43天,VirRon单药组抑瘤率80.19%、CAR-T单药组抑瘤率48.83%,联合用药组抑瘤率高达97.56%。VirRon+CAR-T联合用药组与VirRon单药组、CAR-T单药组、对照组相比均有显著差异(P=0.0008、P=0.0000、P=0.0000)(图15左),证实VirRon与CAR-T联合用药能提高疗效。观察期结束,抽取小鼠血液,流式细胞术检测CAR-T细胞含量,结果发现,VirRon与CAR-T联合时,VirRon能明显延长CAR-T存活时间,提高血液中CAR-T细胞数量(图15右)。
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,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 江苏万戎生物医药科技有限公司
<120> 一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤
治疗中的应用
<130> /
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 949
<212> DNA
<213> 人工序列
<400> 1
taaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata 60
ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag 120
tcactatggc ggccgatctg gatccggctt actaaaagcc agataacagt atgcgtattt 180
gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa 240
aagaggtgtg ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg 300
ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag 360
cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg 420
cccggtttat tgaaatgaac ggctcttttg ctgacgagaa cagggactgg tgaaatgcag 480
tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt 540
gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg 600
tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc 660
atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat 720
ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa 780
atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact ggatatgttg 840
tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt 900
tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggttaat 949
<210> 2
<211> 769
<212> DNA
<213> 人工序列
<400> 2
ccatggctct agagatatac tgagtcatta gggactttcc aatgggtttt gcccagtaca 60
taaggtcaat aggggtgaat caacaggaaa gtcccattgg agccaagtac actgagtcaa 120
tagggacttt ccattgggtt ttgcccagta caaaaggtca atagggggtg agtcaatggg 180
tttttcccat tattggcacg tacataaggt caataggggt gagtcattgg gtttttccag 240
ccatttaatt aaaacgccat gtactttccc accattgacg tcaatgggct attgaaacta 300
atgcaacgtg acctttaaac ggtactttcc catagctgat taatgggaaa gtaccgttct 360
cgagccaata cacgtcaatg ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt 420
ttcccctgga aattccatat tggcactcat tctattggct gagctgcgtt ctacgtgggt 480
ataagaggcg cgaccagcgt cggtaccgtc gcagtcttcg gtctgaccac cgtagaacgc 540
agatcgaatt actagtcagg gaattcaagc ttagatctgc tagcaaggat ccagcttgtc 600
gacttcgagc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 660
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 720
caatgtatct tatcatgtct ggatcgtcta gcatcgaaga tccgatatc 769
<210> 3
<211> 37515
<212> DNA
<213> 人工序列
<400> 3
ttattttgga ttgaagccaa tatgataatg agggggtgga gtttgtgacg tggcgcgggg 60
cgtgggaacg gggcgggtga cgtagtagtg tggcggaagt gtgatgttgc aagtgtggcg 120
gaacacatgt aagcgacgga tgtggcaaaa gtgacgtttt tggtgtgcgc cggtgtacac 180
aggaagtgac aattttcgcg cggttttagg cggatgttgt agtaaatttg ggcgtaaccg 240
agtaagattt ggccattttc gcgggaaaac tgaataagag gaagtgaaat ctgaataatt 300
ttgtgttact catagcgcgt aatatttgtc tagggccgcg gggactttga ccgtttacgt 360
ggagactcgc ccaggtgttt ttctcaggtg ttttccgcgt tccgggtcaa agttggcgtt 420
ttattattat agtcagctga cgtgtaaccg gcgtacgttc ggccgcagat ctggtcgact 480
agtccatata aggtataaat ccatataagg tataaatcca tataaggtaa taggctgcag 540
gacttactgt tggtgggacg ccctgctttg cgaagggaaa ggaggagttt gccctgagca 600
caggccccca ccctccactg ggctttcccc agctcccttg tcttcttatc acggtagtgg 660
cccagtccct ggcccctgac tccagaaggt ggccctcctg gaaacccagg tcgtgcagtc 720
aacgatgtac tcgccgggac agcgatgtct gctgcactcc atccctcccc tgttcatttg 780
tccttcatgc ccgtctggag tagatgcttt ttgcagaggt ggcaccctgt aaagctctcc 840
tgtctgactt tttttttttt tttagactga gttttgctct tgttgcctag gctggagtgc 900
aatggcacaa tctcagctca ctgcaccctc tgcctcccgg gttcaagcga ttctcctgcc 960
tcagcctccc gagtagttgg gattacaggc atgcaccacc acgcccagct aatttttgta 1020
tttttagtag agacaaggtt tcaccgtgat ggccaggctg gtcttgaact ccaggactca 1080
agtgatgctc ctgcctaggc ctctcaaagt gttgggatta caggcgtgag ccactgcacc 1140
cggcctgcac gcgttctttg aaagcagtcg agggggcgct aggtgtgggc agggacgagc 1200
tggcgcggcg tcgctgggtg caccgcgacc acgggcagag ccacgcggcg ggaggactac 1260
aactcccggc acaccccgcg ccgccccgcc tctactccca gaaggccgcg gggggtggac 1320
cgcctaagag ggcgtgcgct cccgacatgc cccgcggcgc gccattaacc gccagatttg 1380
aatcgcggga caccgggact gaaaatgaga catattatct gccacggagg tgttattacc 1440
gaagaaatgg ccgccagtct tttggaccag ctgatcgaag aggtactggc tgataatctt 1500
ccacctccta gccattttga accacctacc cttcacgaac tgtatgattt agacgtgacg 1560
gcccccgaag atcccaacga ggaggcggtt tcgcagattt ttcccgactc tgtaatgttg 1620
gcggtgcagg aagggattga cttactcact tttccgccgg cgcccggttc tccggagccg 1680
cctcaccttt cccggcagcc cgagcagccg gagcagagag ccttgggtcc ggtttctatg 1740
ccaaaccttg taccggaggt gatcgatctt acctgctttc cacccagtga cgacgaggat 1800
gaagagggtg aggagtttgt gttagattat gtggagcacc ccgggcacgg ttgcaggtct 1860
tgtcattatc accggaggaa tacgggggac ccagatatta tgtgttcgct ttgctatatg 1920
aggacctgtg gcatgtttgt ctacagtaag tgaaaattat gggcagtggg tgatagagtg 1980
gtgggtttgg tgtggtaatt ttttttttaa tttttacagt tttgtggttt aaagaatttt 2040
gtattgtgat ttttttaaaa ggtcctgtgt ctgaacctga gcctgagccc gagccagaac 2100
cggagcctgc aagacctacc cgccgtccta aaatggcgcc tgctatcctg agacgcccga 2160
catcacctgt gtctagagaa tgcaatagta gtacggatag ctgtgactcc ggtccttcta 2220
acacacctcc tgagatacac ccggtggtcc cgctgtgccc cattaaacca gttgccgtga 2280
gagttggtgg gcgtcgccag gctgtggaat gtatcgagga cttgcttaac gagcctgggc 2340
aacctttgga cttgagctgt aaacgcccca ggccagactt ggagatgtta gctccctata 2400
tcccaatgga tgatgacttc cagtaacttg catggcgtgt taaatggggc ggggcttaaa 2460
gggtatataa tgcgccgtgg gctaatcttg gttacatctg acctcccggc gggtccggcg 2520
ggtcaccatg gagctcctcg actgataagc agatttgcac attagcatta acagatgtag 2580
ctaagcactt tgcttcagtt ctcatcagca ctttatagtg acttccagat gtagctaagc 2640
actttgcttc agttctcaat tagcattaat cagcactttc tagtgacttc gagctcctcg 2700
actgataagc agatttgcac tcagcacttt gtagtgactt ccagatgtag ctaagcactt 2760
tggagctcct cgactgataa gcagatttgc accttcagtt ctcaattagc attaaagatc 2820
tggaaggtgc tgaggtacga tgagacccgc accaggtgca gaccctgcga gtgtggcggt 2880
aaacatatta ggaaccagcc tgtgatgctg gatgtgaccg aggagctgag gcccgatcac 2940
ttggtgctgg cctgcacccg cgctgagttt ggctctagcg atgaagatac agattgaggt 3000
actgaaatgt gtgggcgtgg cttaagggtg ggaaagaata tataaggtgg gggtcttatg 3060
tagttttgta tctgttttgc agcagccgcc gccgccatga gcaccaactc gtttgatgga 3120
agcattgtga gctcatattt gacaacgcgc atgcccccat gggccggggt gcgtcagaat 3180
gtgatgggct ccagcattga tggtcgcccc gtcctgcccg caaactctac taccttgacc 3240
tacgagaccg tgtctggaac gccgttggag actgcagcct ccgccgccgc ttcagccgct 3300
gcagccaccg cccgcgggat tgtgactgac tttgctttcc tgagcccgct tgcaagcagt 3360
gcagcttccc gttcatccgc ccgcgatgac aagttgacgg ctcttttggc acaattggat 3420
tctttgaccc gggaacttaa tgtcgtttct cagcagctgt tggatctgcg ccagcaggtt 3480
tctgccctga aggcttcctc ccctcccaat gcggtttaaa acataaataa aaaaccagac 3540
tctgtttgga tttggatcaa gcaagtgtct tgctgtcttt atttaggggt tttgcgcgcg 3600
cggtaggccc gggaccagcg gtctcggtcg ttgagggtcc tgtgtatttt ttccaggacg 3660
tggtaaaggt gactctggat gttcagatac atgggcataa gcccgtctct ggggtggagg 3720
tagcaccact gcagagcttc atgctgcggg gtggtgttgt agatgatcca gtcgtagcag 3780
gagcgctggg cgtggtgcct aaaaatgtct ttcagtagca agctgattgc caggggcagg 3840
cccttggtgt aagtgtttac aaagcggtta agctgggatg ggtgcatacg tggggatatg 3900
agatgcatct tggactgtat ttttaggttg gctatgttcc cagccatatc cctccgggga 3960
ttcatgttgt gcagaaccac cagcacagtg tatccggtgc acttgggaaa tttgtcatgt 4020
agcttagaag gaaatgcgtg gaagaacttg gagacgccct tgtgacctcc aagattttcc 4080
atgcattcgt ccataatgat ggcaatgggc ccacgggcgg cggcctgggc gaagatattt 4140
ctgggatcac taacgtcata gttgtgttcc aggatgagat cgtcataggc catttttaca 4200
aagcgcgggc ggagggtgcc agactgcggt ataatggttc catccggccc aggggcgtag 4260
ttaccctcac agatttgcat ttcccacgct ttgagttcag atggggggat catgtctacc 4320
tgcggggcga tgaagaaaac ggtttccggg gtaggggaga tcagctggga agaaagcagg 4380
ttcctgagca gctgcgactt accgcagccg gtgggcccgt aaatcacacc tattaccggg 4440
tgcaactggt agttaagaga gctgcagctg ccgtcatccc tgagcagggg ggccacttcg 4500
ttaagcatgt ccctgactcg catgttttcc ctgaccaaat ccgccagaag gcgctcgccg 4560
cccagcgata gcagttcttg caaggaagca aagtttttca acggtttgag accgtccgcc 4620
gtaggcatgc ttttgagcgt ttgaccaagc agttccaggc ggtcccacag ctcggtcacc 4680
tgctctacgg catctcgatc cagcatatct cctcgtttcg cgggttgggg cggctttcgc 4740
tgtacggcag tagtcggtgc tcgtccagac gggccagggt catgtctttc cacgggcgca 4800
gggtcctcgt cagcgtagtc tgggtcacgg tgaaggggtg cgctccgggc tgcgcgctgg 4860
ccagggtgcg cttgaggctg gtcctgctgg tgctgaagcg ctgccggtct tcgccctgcg 4920
cgtcggccag gtagcatttg accatggtgt catagtccag cccctccgcg gcgtggccct 4980
tggcgcgcag cttgcccttg gaggaggcgc cgcacgaggg gcagtgcaga cttttgaggg 5040
cgtagagctt gggcgcgaga aataccgatt ccggggagta ggcatccgcg ccgcaggccc 5100
cgcagacggt ctcgcattcc acgagccagg tgagctctgg ccgttcgggg tcaaaaacca 5160
ggtttccccc atgctttttg atgcgtttct tacctctggt ttccatgagc cggtgtccac 5220
gctcggtgac gaaaaggctg tccgtgtccc cgtatacaga cttgagaggc ctgtcctcga 5280
gcggtgttcc gcggtcctcc tcgtatagaa actcggacca ctctgagaca aaggctcgcg 5340
tccaggccag cacgaaggag gctaagtggg aggggtagcg gtcgttgtcc actagggggt 5400
ccactcgctc cagggtgtga agacacatgt cgccctcttc ggcatcaagg aaggtgattg 5460
gtttgtaggt gtaggccacg tgaccgggtg ttcctgaagg ggggctataa aagggggtgg 5520
gggcgcgttc gtcctcactc tcttccgcat cgctgtctgc gagggccagc tgttggggtg 5580
agtactccct ctgaaaagcg ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg 5640
aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc gcatccatct 5700
ggtcagaaaa gacaatcttt ttgttgtcaa gcttggtggc aaacgacccg tagagggcgt 5760
tggacagcaa cttggcgatg gagcgcaggg tttggttttt gtcgcgatcg gcgcgctcct 5820
tggccgcgat gtttagctgc acgtattcgc gcgcaacgca ccgccattcg ggaaagacgg 5880
tggtgcgctc gtcgggcacc aggtgcacgc gccaaccgcg gttgtgcagg gtgacaaggt 5940
caacgctggt ggctacctct ccgcgtaggc gctcgttggt ccagcagagg cggccgccct 6000
tgcgcgagca gaatggcggt agggggtcta gctgcgtctc gtccgggggg tctgcgtcca 6060
cggtaaagac cccgggcagc aggcgcgcgt cgaagtagtc tatcttgcat ccttgcaagt 6120
ctagcgcctg ctgccatgcg cgggcggcaa gcgcgcgctc gtatgggttg agtgggggac 6180
cccatggcat ggggtgggtg agcgcggagg cgtacatgcc gcaaatgtcg taaacgtaga 6240
ggggctctct gagtattcca agatatgtag ggtagcatct tccaccgcgg atgctggcgc 6300
gcacgtaatc gtatagttcg tgcgagggag cgaggaggtc gggaccgagg ttgctacggg 6360
cgggctgctc tgctcggaag actatctgcc tgaagatggc atgtgagttg gatgatatgg 6420
ttggacgctg gaagacgttg aagctggcgt ctgtgagacc taccgcgtca cgcacgaagg 6480
aggcgtagga gtcgcgcagc ttgttgacca gctcggcggt gacctgcacg tctagggcgc 6540
agtagtccag ggtttccttg atgatgtcat acttatcctg tccctttttt ttccacagct 6600
cgcggttgag gacaaactct tcgcggtctt tccagtactc ttggatcgga aacccgtcgg 6660
cctccgaacg gtaagagcct agcatgtaga actggttgac ggcctggtag gcgcagcatc 6720
ccttttctac gggtagcgcg tatgcctgcg cggccttccg gagcgaggtg tgggtgagcg 6780
caaaggtgtc cctgaccatg actttgaggt actggtattt gaagtcagtg tcgtcgcatc 6840
cgccctgctc ccagagcaaa aagtccgtgc gctttttgga acgcggattt ggcagggcga 6900
aggtgacatc gttgaagagt atctttcccg cgcgaggcat aaagttgcgt gtgatgcgga 6960
agggtcccgg cacctcggaa cggttgttaa ttacctgggc ggcgagcacg atctcgtcaa 7020
agccgttgat gttgtggccc acaatgtaaa gttccaagaa gcgcgggatg cccttgatgg 7080
aaggcaattt tttaagttcc tcgtaggtga gctcttcagg ggagctgagc ccgtgctctg 7140
aaagggccca gtctgcaaga tgagggttgg aagcgacgaa tgagctccac aggtcacggg 7200
ccattagcat ttgcaggtgg tcgcgaaagg tcctaaactg gcgacctatg gccatttttt 7260
ctggggtgat gcagtagaag gtaagcgggt cttgttccca gcggtcccat ccaaggttcg 7320
cggctaggtc tcgcgcggca gtcactagag gctcatctcc gccgaacttc atgaccagca 7380
tgaagggcac gagctgcttc ccaaaggccc ccatccaagt ataggtctct acatcgtagg 7440
tgacaaagag acgctcggtg cgaggatgcg agccgatcgg gaagaactgg atctcccgcc 7500
accaattgga ggagtggcta ttgatgtggt gaaagtagaa gtccctgcga cgggccgaac 7560
actcgtgctg gcttttgtaa aaacgtgcgc agtactggca gcggtgcacg ggctgtacat 7620
cctgcacgag gttgacctga cgaccgcgca caaggaagca gagtgggaat ttgagcccct 7680
cgcctggcgg gtttggctgg tggtcttcta cttcggctgc ttgtccttga ccgtctggct 7740
gctcgagggg agttacggtg gatcggacca ccacgccgcg cgagcccaaa gtccagatgt 7800
ccgcgcgcgg cggtcggagc ttgatgacaa catcgcgcag atgggagctg tccatggtct 7860
ggagctcccg cggcgtcagg tcaggcggga gctcctgcag gtttacctcg catagacggg 7920
tcagggcgcg ggctagatcc aggtgatacc taatttccag gggctggttg gtggcggcgt 7980
cgatggcttg caagaggccg catccccgcg gcgcgactac ggtaccgcgc ggcgggcggt 8040
gggccgcggg ggtgtccttg gatgatgcat ctaaaagcgg tgacgcgggc gagcccccgg 8100
aggtaggggg ggctccggac ccgccgggag agggggcagg ggcacgtcgg cgccgcgcgc 8160
gggcaggagc tggtgctgcg cgcgtaggtt gctggcgaac gcgacgacgc ggcggttgat 8220
ctcctgaatc tggcgcctct gcgtgaagac gacgggcccg gtgagcttga gcctgaaaga 8280
gagttcgaca gaatcaattt cggtgtcgtt gacggcggcc tggcgcaaaa tctcctgcac 8340
gtctcctgag ttgtcttgat aggcgatctc ggccatgaac tgctcgatct cttcctcctg 8400
gagatctccg cgtccggctc gctccacggt ggcggcgagg tcgttggaaa tgcgggccat 8460
gagctgcgag aaggcgttga ggcctccctc gttccagacg cggctgtaga ccacgccccc 8520
ttcggcatcg cgggcgcgca tgaccacctg cgcgagattg agctccacgt gccgggcgaa 8580
gacggcgtag tttcgcaggc gctgaaagag gtagttgagg gtggtggcgg tgtgttctgc 8640
cacgaagaag tacataaccc agcgtcgcaa cgtggattcg ttgatatccc ccaaggcctc 8700
aaggcgctcc atggcctcgt agaagtccac ggcgaagttg aaaaactggg agttgcgcgc 8760
cgacacggtt aactcctcct ccagaagacg gatgagctcg gcgacagtgt cgcgcacctc 8820
gcgctcaaag gctacagggg cctcttcttc ttcttcaatc tcctcttcca taagggcctc 8880
cccttcttct tcttctggcg gcggtggggg aggggggaca cggcggcgac gacggcgcac 8940
cgggaggcgg tcgacaaagc gctcgatcat ctccccgcgg cgacggcgca tggtctcggt 9000
gacggcgcgg ccgttctcgc gggggcgcag ttggaagacg ccgcccgtca tgtcccggtt 9060
atgggttggc ggggggctgc catgcggcag ggatacggcg ctaacgatgc atctcaacaa 9120
ttgttgtgta ggtactccgc cgccgaggga cctgagcgag tccgcatcga ccggatcgga 9180
aaacctctcg agaaaggcgt ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc 9240
gggcggcagc gggcggcggt cggggttgtt tctggcggag gtgctgctga tgatgtaatt 9300
aaagtaggcg gtcttgagac ggcggatggt cgacagaagc accatgtcct tgggtccggc 9360
ctgctgaatg cgcaggcggt cggccatgcc ccaggcttcg ttttgacatc ggcgcaggtc 9420
tttgtagtag tcttgcatga gcctttctac cggcacttct tcttctcctt cctcttgtcc 9480
tgcatctctt gcatctatcg ctgcggcggc ggcggagttt ggccgtaggt ggcgccctct 9540
tcctcccatg cgtgtgaccc cgaagcccct catcggctga agcagggcta ggtcggcgac 9600
aacgcgctcg gctaatatgg cctgctgcac ctgcgtgagg gtagactgga agtcatccat 9660
gtccacaaag cggtggtatg cgcccgtgtt gatggtgtaa gtgcagttgg ccataacgga 9720
ccagttaacg gtctggtgac ccggctgcga gagctcggtg tacctgagac gcgagtaagc 9780
cctcgagtca aatacgtagt cgttgcaagt ccgcaccagg tactggtatc ccaccaaaaa 9840
gtgcggcggc ggctggcggt agaggggcca gcgtagggtg gccggggctc cgggggcgag 9900
atcttccaac ataaggcgat gatatccgta gatgtacctg gacatccagg tgatgccggc 9960
ggcggtggtg gaggcgcgcg gaaagtcgcg gacgcggttc cagatgttgc gcagcggcaa 10020
aaagtgctcc atggtcggga cgctctggcc ggtcaggcgc gcgcaatcgt tgacgctcta 10080
ccgtgcaaaa ggagagcctg taagcgggca ctcttccgtg gtctggtgga taaattcgca 10140
agggtatcat ggcggacgac cggggttcga gccccgtatc cggccgtccg ccgtgatcca 10200
tgcggttacc gcccgcgtgt cgaacccagg tgtgcgacgt cagacaacgg gggagtgctc 10260
cttttggctt ccttccaggc gcggcggctg ctgcgctagc ttttttggcc actggccgcg 10320
cgcagcgtaa gcggttaggc tggaaagcga aagcattaag tggctcgctc cctgtagccg 10380
gagggttatt ttccaagggt tgagtcgcgg gacccccggt tcgagtctcg gaccggccgg 10440
actgcggcga acgggggttt gcctccccgt catgcaagac cccgcttgca aattcctccg 10500
gaaacaggga cgagcccctt ttttgctttt cccagatgca tccggtgctg cggcagatgc 10560
gcccccctcc tcagcagcgg caagagcaag agcagcggca gacatgcagg gcaccctccc 10620
ctcctcctac cgcgtcagga ggggcgacat ccgcggttga cgcggcagca gatggtgatt 10680
acgaaccccc gcggcgccgg gcccggcact acctggactt ggaggagggc gagggcctgg 10740
cgcggctagg agcgccctct cctgagcggt acccaagggt gcagctgaag cgtgatacgc 10800
gtgaggcgta cgtgccgcgg cagaacctgt ttcgcgaccg cgagggagag gagcccgagg 10860
agatgcggga tcgaaagttc cacgcagggc gcgagctgcg gcatggcctg aatcgcgagc 10920
ggttgctgcg cgaggaggac tttgagcccg acgcgcgaac cgggattagt cccgcgcgcg 10980
cacacgtggc ggccgccgac ctggtaaccg catacgagca gacggtgaac caggagatta 11040
actttcaaaa aagctttaac aaccacgtgc gtacgcttgt ggcgcgcgag gaggtggcta 11100
taggactgat gcatctgtgg gactttgtaa gcgcgctgga gcaaaaccca aatagcaagc 11160
cgctcatggc gcagctgttc cttatagtgc agcacagcag ggacaacgag gcattcaggg 11220
atgcgctgct aaacatagta gagcccgagg gccgctggct gctcgatttg ataaacatcc 11280
tgcagagcat agtggtgcag gagcgcagct tgagcctggc tgacaaggtg gccgccatca 11340
actattccat gcttagcctg ggcaagtttt acgcccgcaa gatataccat accccttacg 11400
ttcccataga caaggaggta aagatcgagg ggttctacat gcgcatggcg ctgaaggtgc 11460
ttaccttgag cgacgacctg ggcgtttatc gcaacgagcg catccacaag gccgtgagcg 11520
tgagccggcg gcgcgagctc agcgaccgcg agctgatgca cagcctgcaa agggccctgg 11580
ctggcacggg cagcggcgat agagaggccg agtcctactt tgacgcgggc gctgacctgc 11640
gctgggcccc aagccgacgc gccctggagg cagctggggc cggacctggg ctggcggtgg 11700
cacccgcgcg cgctggcaac gtcggcggcg tggaggaata tgacgaggac gatgagtacg 11760
agccagagga cggcgagtac taagcggtga tgtttctgat cagatgatgc aagacgcaac 11820
ggacccggcg gtgcgggcgg cgctgcagag ccagccgtcc ggccttaact ccacggacga 11880
ctggcgccag gtcatggacc gcatcatgtc gctgactgcg cgcaatcctg acgcgttccg 11940
gcagcagccg caggccaacc ggctctccgc aattctggaa gcggtggtcc cggcgcgcgc 12000
aaaccccacg cacgagaagg tgctggcgat cgtaaacgcg ctggccgaaa acagggccat 12060
ccggcccgac gaggccggcc tggtctacga cgcgctgctt cagcgcgtgg ctcgttacaa 12120
cagcggcaac gtgcagacca acctggaccg gctggtgggg gatgtgcgcg aggccgtggc 12180
gcagcgtgag cgcgcgcagc agcagggcaa cctgggctcc atggttgcac taaacgcctt 12240
cctgagtaca cagcccgcca acgtgccgcg gggacaggag gactacacca actttgtgag 12300
cgcactgcgg ctaatggtga ctgagacacc gcaaagtgag gtgtaccagt ctgggccaga 12360
ctattttttc cagaccagta gacaaggcct gcagaccgta aacctgagcc aggctttcaa 12420
aaacttgcag gggctgtggg gggtgcgggc tcccacaggc gaccgcgcga ccgtgtctag 12480
cttgctgacg cccaactcgc gcctgttgct gctgctaata gcgcccttca cggacagtgg 12540
cagcgtgtcc cgggacacat acctaggtca cttgctgaca ctgtaccgcg aggccatagg 12600
tcaggcgcat gtggacgagc atactttcca ggagattaca agtgtcagcc gcgcgctggg 12660
gcaggaggac acgggcagcc tggaggcaac cctaaactac ctgctgacca accggcggca 12720
gaagatcccc tcgttgcaca gtttaaacag cgaggaggag cgcattttgc gctacgtgca 12780
gcagagcgtg agccttaacc tgatgcgcga cggggtaacg cccagcgtgg cgctggacat 12840
gaccgcgcgc aacatggaac cgggcatgta tgcctcaaac cggccgttta tcaaccgcct 12900
aatggactac ttgcatcgcg cggccgccgt gaaccccgag tatttcacca atgccatctt 12960
gaacccgcac tggctaccgc cccctggttt ctacaccggg ggattcgagg tgcccgaggg 13020
taacgatgga ttcctctggg acgacataga cgacagcgtg ttttccccgc aaccgcagac 13080
cctgctagag ttgcaacagc gcgagcaggc agaggcggcg ctgcgaaagg aaagcttccg 13140
caggccaagc agcttgtccg atctaggcgc tgcggccccg cggtcagatg ctagtagccc 13200
atttccaagc ttgatagggt ctcttaccag cactcgcacc acccgcccgc gcctgctggg 13260
cgaggaggag tacctaaaca actcgctgct gcagccgcag cgcgaaaaaa acctgcctcc 13320
ggcatttccc aacaacggga tagagagcct agtggacaag atgagtagat ggaagacgta 13380
cgcgcaggag cacagggacg tgccaggccc gcgcccgccc acccgtcgtc aaaggcacga 13440
ccgtcagcgg ggtctggtgt gggaggacga tgactcggca gacgacagca gcgtcctgga 13500
tttgggaggg agtggcaacc cgtttgcgca ccttcgcccc aggctgggga gaatgtttta 13560
aaaaaaaaaa agcatgatgc aaaataaaaa actcaccaag gccatggcac cgagcgttgg 13620
ttttcttgta ttccccttag tatgcggcgc gcggcgatgt atgaggaagg tcctcctccc 13680
tcctacgaga gtgtggtgag cgcggcgcca gtggcggcgg cgctgggttc tcccttcgat 13740
gctcccctgg acccgccgtt tgtgcctccg cggtacctgc ggcctaccgg ggggagaaac 13800
agcatccgtt actctgagtt ggcaccccta ttcgacacca cccgtgtgta cctggtggac 13860
aacaagtcaa cggatgtggc atccctgaac taccagaacg accacagcaa ctttctgacc 13920
acggtcattc aaaacaatga ctacagcccg ggggaggcaa gcacacagac catcaatctt 13980
gacgaccggt cgcactgggg cggcgacctg aaaaccatcc tgcataccaa catgccaaat 14040
gtgaacgagt tcatgtttac caataagttt aaggcgcggg tgatggtgtc gcgcttgcct 14100
actaaggaca atcaggtgga gctgaaatac gagtgggtgg agttcacgct gcccgagggc 14160
aactactccg agaccatgac catagacctt atgaacaacg cgatcgtgga gcactacttg 14220
aaagtgggca gacagaacgg ggttctggaa agcgacatcg gggtaaagtt tgacacccgc 14280
aacttcagac tggggtttga ccccgtcact ggtcttgtca tgcctggggt atatacaaac 14340
gaagccttcc atccagacat cattttgctg ccaggatgcg gggtggactt cacccacagc 14400
cgcctgagca acttgttggg catccgcaag cggcaaccct tccaggaggg ctttaggatc 14460
acctacgatg atctggaggg tggtaacatt cccgcactgt tggatgtgga cgcctaccag 14520
gcgagcttga aagatgacac cgaacagggc gggggtggcg caggcggcag caacagcagt 14580
ggcagcggcg cggaagagaa ctccaacgcg gcagccgcgg caatgcagcc ggtggaggac 14640
atgaacgatc atgccattcg cggcgacacc tttgccacac gggctgagga gaagcgcgct 14700
gaggccgaag cagcggccga agctgccgcc cccgctgcgc aacccgaggt cgagaagcct 14760
cagaagaaac cggtgatcaa acccctgaca gaggacagca agaaacgcag ttacaaccta 14820
ataagcaatg acagcacctt cacccagtac cgcagctggt accttgcata caactacggc 14880
gaccctcaga ccggaatccg ctcatggacc ctgctttgca ctcctgacgt aacctgcggc 14940
tcggagcagg tctactggtc gttgccagac atgatgcaag accccgtgac cttccgctcc 15000
acgcgccaga tcagcaactt tccggtggtg ggcgccgagc tgttgcccgt gcactccaag 15060
agcttctaca acgaccaggc cgtctactcc caactcatcc gccagtttac ctctctgacc 15120
cacgtgttca atcgctttcc cgagaaccag attttggcgc gcccgccagc ccccaccatc 15180
accaccgtca gtgaaaacgt tcctgctctc acagatcacg ggacgctacc gctgcgcaac 15240
agcatcggag gagtccagcg agtgaccatt actgacgcca gacgccgcac ctgcccctac 15300
gtttacaagg ccctgggcat agtctcgccg cgcgtcctat cgagccgcac tttttgagca 15360
agcatgtcca tccttatatc gcccagcaat aacacaggct ggggcctgcg cttcccaagc 15420
aagatgtttg gcggggccaa gaagcgctcc gaccaacacc cagtgcgcgt gcgcgggcac 15480
taccgcgcgc cctggggcgc gcacaaacgc ggccgcactg ggcgcaccac cgtcgatgac 15540
gccatcgacg cggtggtgga ggaggcgcgc aactacacgc ccacgccgcc accagtgtcc 15600
acagtggacg cggccattca gaccgtggtg cgcggagccc ggcgctatgc taaaatgaag 15660
agacggcgga ggcgcgtagc acgtcgccac cgccgccgac ccggcactgc cgcccaacgc 15720
gcggcggcgg ccctgcttaa ccgcgcacgt cgcaccggcc gacgggcggc catgcgggcc 15780
gctcgaaggc tggccgcggg tattgtcact gtgcccccca ggtccaggcg acgagcggcc 15840
gccgcagcag ccgcggccat tagtgctatg actcagggtc gcaggggcaa cgtgtattgg 15900
gtgcgcgact cggttagcgg cctgcgcgtg cccgtgcgca cccgcccccc gcgcaactag 15960
attgcaagaa aaaactactt agactcgtac tgttgtatgt atccagcggc ggcggcgcgc 16020
aacgaagcta tgtccaagcg caaaatcaaa gaagagatgc tccaggtcat cgcgccggag 16080
atctatggcc ccccgaagaa ggaagagcag gattacaagc cccgaaagct aaagcgggtc 16140
aaaaagaaaa agaaagatga tgatgatgaa cttgacgacg aggtggaact gctgcacgct 16200
accgcgccca ggcgacgggt acagtggaaa ggtcgacgcg taaaacgtgt tttgcgaccc 16260
ggcaccaccg tagtctttac gcccggtgag cgctccaccc gcacctacaa gcgcgtgtat 16320
gatgaggtgt acggcgacga ggacctgctt gagcaggcca acgagcgcct cggggagttt 16380
gcctacggaa agcggcataa ggacatgctg gcgttgccgc tggacgaggg caacccaaca 16440
cctagcctaa agcccgtaac actgcagcag gtgctgcccg cgcttgcacc gtccgaagaa 16500
aagcgcggcc taaagcgcga gtctggtgac ttggcaccca ccgtgcagct gatggtaccc 16560
aagcgccagc gactggaaga tgtcttggaa aaaatgaccg tggaacctgg gctggagccc 16620
gaggtccgcg tgcggccaat caagcaggtg gcgccgggac tgggcgtgca gaccgtggac 16680
gttcagatac ccactaccag tagcaccagt attgccaccg ccacagaggg catggagaca 16740
caaacgtccc cggttgcctc agcggtggcg gatgccgcgg tgcaggcggt cgctgcggcc 16800
gcgtccaaga cctctacgga ggtgcaaacg gacccgtgga tgtttcgcgt ttcagccccc 16860
cggcgcccgc gccgttcgag gaagtacggc gccgccagcg cgctactgcc cgaatatgcc 16920
ctacatcctt ccattgcgcc tacccccggc tatcgtggct acacctaccg ccccagaaga 16980
cgagcaacta cccgacgccg aaccaccact ggaacccgcc gccgccgtcg ccgtcgccag 17040
cccgtgctgg ccccgatttc cgtgcgcagg gtggctcgcg aaggaggcag gaccctggtg 17100
ctgccaacag cgcgctacca ccccagcatc gtttaaaagc cggtctttgt ggttcttgca 17160
gatatggccc tcacctgccg cctccgtttc ccggtgccgg gattccgagg aagaatgcac 17220
cgtaggaggg gcatggccgg ccacggcctg acgggcggca tgcgtcgtgc gcaccaccgg 17280
cggcggcgcg cgtcgcaccg tcgcatgcgc ggcggtatcc tgcccctcct tattccactg 17340
atcgccgcgg cgattggcgc cgtgcccgga attgcatccg tggccttgca ggcgcagaga 17400
cactgattaa aaacaagttg catgtggaaa aatcaaaata aaaagtctgg actctcacgc 17460
tcgcttggtc ctgtaactat tttgtagaat ggaagacatc aactttgcgt ctctggcccc 17520
gcgacacggc tcgcgcccgt tcatgggaaa ctggcaagat atcggcacca gcaatatgag 17580
cggtggcgcc ttcagctggg gctcgctgtg gagcggcatt aaaaatttcg gttccaccgt 17640
taagaactat ggcagcaagg cctggaacag cagcacaggc cagatgctga gggataagtt 17700
gaaagagcaa aatttccaac aaaaggtggt agatggcctg gcctctggca ttagcggggt 17760
ggtggacctg gccaaccagg cagtgcaaaa taagattaac agtaagcttg atccccgccc 17820
tcccgtagag gagcctccac cggccgtgga gacagtgtct ccagaggggc gtggcgaaaa 17880
gcgtccgcgc cccgacaggg aagaaactct ggtgacgcaa atagacgagc ctccctcgta 17940
cgaggaggca ctaaagcaag gcctgcccac cacccgtccc atcgcgccca tggctaccgg 18000
agtgctgggc cagcacacac ccgtaacgct ggacctgcct ccccccgccg acacccagca 18060
gaaacctgtg ctgccaggcc cgaccgccgt tgttgtaacc cgtcctagcc gcgcgtccct 18120
gcgccgcgcc gccagcggtc cgcgatcgtt gcggcccgta gccagtggca actggcaaag 18180
cacactgaac agcatcgtgg gtctgggggt gcaatccctg aagcgccgac gatgcttctg 18240
atagctaacg tgtcgtatgt gtgtcatgta tgcgtccatg tcgccgccag aggagctgct 18300
gagccgccgc gcgcccgctt tccaagatgg ctaccccttc gatgatgccg cagtggtctt 18360
acatgcacat ctcgggccag gacgcctcgg agtacctgag ccccgggctg gtgcagtttg 18420
cccgcgccac cgagacgtac ttcagcctga ataacaagtt tagaaacccc acggtggcgc 18480
ctacgcacga cgtgaccaca gaccggtccc agcgtttgac gctgcggttc atccctgtgg 18540
accgtgagga tactgcgtac tcgtacaagg cgcggttcac cctagctgtg ggtgataacc 18600
gtgtgctgga catggcttcc acgtactttg acatccgcgg cgtgctggac aggggcccta 18660
cttttaagcc ctactctggc actgcctaca acgccctggc tcccaagggt gccccaaatc 18720
cttgcgaatg ggaagagaaa aagaatggag gaggaagcga tgctaatcaa atgcaaactc 18780
acgtatttgg gcaggcgcct tattctggta taaatattac aaaggagggt attcaaatag 18840
gtattgatgc aaccaaagag gaagataatg gaaaggaaat atatgccgat aaaacatttc 18900
aacctgaacc tcaaatagga gaatctcagt ggcaggatag tgataattac tatggaggga 18960
gagtccttaa aaagactacc ccaatgaaac catgttacgg ttcatatgca aaacccacaa 19020
atgaaaatgg agggcaagct aaattcaaaa cacctgaaaa agaaggtgaa gaacccaaag 19080
aaagtcaagt ggaaatgcaa tttttcgata ttcccagtac tggcacaggt ggtaatggaa 19140
caaatgttaa tttcaaacct aaagtggtat tgtacagtga agatgtagat atagaaaccc 19200
cagacactca tatttcttac atgcccggca aggaagatgc aagttcacga gaactaatgg 19260
gccaacaatc tatgcccaac aggcctaatt acattgcttt tagggacaat tttattggtc 19320
taatgtatta caacagcacg ggtaatatgg gtgttctggc gggccaagca tcgcagttga 19380
atgctgttgt agatttgcaa gacagaaaca cagagctttc ataccagctt ttgcttgatt 19440
ccattggtga tagaaccagg tacttttcta tgtggaatca ggctgttgac agctatgatc 19500
cagatgttag aattattgaa aatcatggaa ctgaagatga acttccaaat tactgctttc 19560
cactggatgg cgctggaact aacgcagtgt accaaggtgt aaaagttaaa actactaaca 19620
atacagaatg ggaaaaagac actgcagtat ctgaacacaa tcagataaga gttggaaata 19680
attttgccat ggaaatcaat ctaaatgcca acctgtggag aaatttcctg tactccaaca 19740
tagcgctgta tttgcccgac aagctaaagt acagtccttc caacgtaaaa atttctgata 19800
acccaaacac ctacgactac atgaacaagc gagtggtggc tcccgggtta gtggactgct 19860
acattaacct tggagcacgc tggtcccttg actatatgga caacgtcaac ccatttaacc 19920
accaccgcaa tgctggcctg cgctaccgct caatgttgct gggcaatggt cgctatgtgc 19980
ccttccacat ccaggtgcct cagaagttct ttgccattaa aaacctcctt ctcctgccgg 20040
gctcatacac ctacgagtgg aacttcagga aggatgttaa catggttctg cagagctccc 20100
taggaaatga cctaagggtt gacggagcca gcattaagtt tgatagcatt tgcctttacg 20160
ccaccttctt ccccatggcc cacaacaccg cctccacgct tgaggccatg cttagaaacg 20220
acaccaacga ccagtccttt aacgactatc tctccgccgc caacatgctc taccctatac 20280
ccgccaacgc taccaacgtg cccatatcca tcccctcccg caactgggcg gctttccgcg 20340
gctgggcctt cacgcgcctt aagactaagg aaaccccatc actgggctcg ggctacgacc 20400
cttattacac ctactctggc tctataccct acctagatgg aaccttttac ctcaaccaca 20460
cctttaagaa ggtggccatt acctttgact cttctgtcag ctggcctggc aatgaccgcc 20520
tgcttacccc caacgagttt gaaattaagc gctcagttga cggggagggt tacaacgttg 20580
cccagtgtaa catgaccaaa gactggttcc tggtacaaat gctagctaac tacaacattg 20640
gctaccaggg cttctatatc ccagagagct acaaggaccg catgtactcc ttctttagaa 20700
acttccagcc catgagccgt caggtggtgg atgatactaa atacaaggac taccaacagg 20760
tgggcatcct acaccaacac aacaactctg gatttgttgg ctaccttgcc cccaccatgc 20820
gcgaaggaca ggcctaccct gctaacttcc cctatccgct tataggcaag accgcagttg 20880
acagcattac ccagaaaaag tttctttgcg atcgcaccct ttggcgcatc ccattctcca 20940
gtaactttat gtccatgggc gcactcacag acctgggcca aaaccttctc tacgccaact 21000
ccgcccacgc gctagacatg acttttgagg tggatcccat ggacgagccc acccttcttt 21060
atgttttgtt tgaagtcttt gacgtggtcc gtgtgcaccg gccgcaccgc ggcgtcatcg 21120
aaaccgtgta cctgcgcacg cccttctcgg ccggcaacgc cacaacataa agaagcaagc 21180
aacatcaaca acagctgccg ccatgggctc cagtgagcag gaactgaaag ccattgtcaa 21240
agatcttggt tgtgggccat attttttggg cacctatgac aagcgctttc caggctttgt 21300
ttctccacac aagctcgcct gcgccatagt caatacggcc ggtcgcgaga ctgggggcgt 21360
acactggatg gcctttgcct ggaacccgca ctcaaaaaca tgctacctct ttgagccctt 21420
tggcttttct gaccagcgac tcaagcaggt ttaccagttt gagtacgagt cactcctgcg 21480
ccgtagcgcc attgcttctt cccccgaccg ctgtataacg ctggaaaagt ccacccaaag 21540
cgtacagggg cccaactcgg ccgcctgtgg actattctgc tgcatgtttc tccacgcctt 21600
tgccaactgg ccccaaactc ccatggatca caaccccacc atgaacctta ttaccggggt 21660
acccaactcc atgctcaaca gtccccaggt acagcccacc ctgcgtcgca accaggaaca 21720
gctctacagc ttcctggagc gccactcgcc ctacttccgc agccacagtg cgcagattag 21780
gagcgccact tctttttgtc acttgaaaaa catgtaaaaa taatgtacta gagacacttt 21840
caataaaggc aaatgctttt atttgtacac tctcgggtga ttatttaccc ccacccttgc 21900
cgtctgcgcc gtttaaaaat caaaggggtt ctgccgcgca tcgctatgcg ccactggcag 21960
ggacacgttg cgatactggt gtttagtgct ccacttaaac tcaggcacaa ccatccgcgg 22020
cagctcggtg aagttttcac tccacaggct gcgcaccatc accaacgcgt ttagcaggtc 22080
gggcgccgat atcttgaagt cgcagttggg gcctccgccc tgcgcgcgcg agttgcgata 22140
cacagggttg cagcactgga acactatcag cgccgggtgg tgcacgctgg ccagcacgct 22200
cttgtcggag atcagatccg cgtccaggtc ctccgcgttg ctcagggcga acggagtcaa 22260
ctttggtagc tgccttccca aaaagggcgc gtgcccaggc tttgagttgc actcgcaccg 22320
tagtggcatc aaaaggtgac cgtgcccggt ctgggcgtta ggatacagcg cctgcataaa 22380
agccttgatc tgcttaaaag ccacctgagc ctttgcgcct tcagagaaga acatgccgca 22440
agacttgccg gaaaactgat tggccggaca ggccgcgtcg tgcacgcagc accttgcgtc 22500
ggtgttggag atctgcacca catttcggcc ccaccggttc ttcacgatct tggccttgct 22560
agactgctcc ttcagcgcgc gctgcccgtt ttcgctcgtc acatccattt caatcacgtg 22620
ctccttattt atcataatgc ttccgtgtag acacttaagc tcgccttcga tctcagcgca 22680
gcggtgcagc cacaacgcgc agcccgtggg ctcgtgatgc ttgtaggtca cctctgcaaa 22740
cgactgcagg tacgcctgca ggaatcgccc catcatcgtc acaaaggtct tgttgctggt 22800
gaaggtcagc tgcaacccgc ggtgctcctc gttcagccag gtcttgcata cggccgccag 22860
agcttccact tggtcaggca gtagtttgaa gttcgccttt agatcgttat ccacgtggta 22920
cttgtccatc agcgcgcgcg cagcctccat gcccttctcc cacgcagaca cgatcggcac 22980
actcagcggg ttcatcaccg taatttcact ttccgcttcg ctgggctctt cctcttcctc 23040
ttgcgtccgc ataccacgcg ccactgggtc gtcttcattc agccgccgca ctgtgcgctt 23100
acctcctttg ccatgcttga ttagcaccgg tgggttgctg aaacccacca tttgtagcgc 23160
cacatcttct ctttcttcct cgctgtccac gattacctct ggtgatggcg ggcgctcggg 23220
cttgggagaa gggcgcttct ttttcttctt gggcgcaatg gccaaatccg ccgccgaggt 23280
cgatggccgc gggctgggtg tgcgcggcac cagcgcgtct tgtgatgagt cttcctcgtc 23340
ctcggactcg atacgccgcc tcatccgctt ttttgggggc gcccggggag gcggcggcga 23400
cggggacggg gacgacacgt cctccatggt tgggggacgt cgcgccgcac cgcgtccgcg 23460
ctcgggggtg gtttcgcgct gctcctcttc ccgactggcc atttccttct cctataggca 23520
gaaaaagatc atggagtcag tcgagaagaa ggacagccta accgccccct ctgagttcgc 23580
caccaccgcc tccaccgatg ccgccaacgc gcctaccacc ttccccgtcg aggcaccccc 23640
gcttgaggag gaggaagtga ttatcgagca ggacccaggt tttgtaagcg aagacgacga 23700
ggaccgctca gtaccaacag aggataaaaa gcaagaccag gacaacgcag aggcaaacga 23760
ggaacaagtc gggcgggggg acgaaaggca tggcgactac ctagatgtgg gagacgacgt 23820
gctgttgaag catctgcagc gccagtgcgc cattatctgc gacgcgttgc aagagcgcag 23880
cgatgtgccc ctcgccatag cggatgtcag ccttgcctac gaacgccacc tattctcacc 23940
gcgcgtaccc cccaaacgcc aagaaaacgg cacatgcgag cccaacccgc gcctcaactt 24000
ctaccccgta tttgccgtgc cagaggtgct tgccacctat cacatctttt tccaaaactg 24060
caagataccc ctatcctgcc gtgccaaccg cagccgagcg gacaagcagc tggccttgcg 24120
gcagggcgct gtcatacctg atatcgcctc gctcaacgaa gtgccaaaaa tctttgaggg 24180
tcttggacgc gacgagaagc gcgcggcaaa cgctctgcaa caggaaaaca gcgaaaatga 24240
aagtcactct ggagtgttgg tggaactcga gggtgacaac gcgcgcctag ccgtactaaa 24300
acgcagcatc gaggtcaccc actttgccta cccggcactt aacctacccc ccaaggtcat 24360
gagcacagtc atgagtgagc tgatcgtgcg ccgtgcgcag cccctggaga gggatgcaaa 24420
tttgcaagaa caaacagagg agggcctacc cgcagttggc gacgagcagc tagcgcgctg 24480
gcttcaaacg cgcgagcctg ccgacttgga ggagcgacgc aaactaatga tggccgcagt 24540
gctcgttacc gtggagcttg agtgcatgca gcggttcttt gctgacccgg agatgcagcg 24600
caagctagag gaaacattgc actacacctt tcgacagggc tacgtacgcc aggcctgcaa 24660
gatctccaac gtggagctct gcaacctggt ctcctacctt ggaattttgc acgaaaaccg 24720
ccttgggcaa aacgtgcttc attccacgct caagggcgag gcgcgccgcg actacgtccg 24780
cgactgcgtt tacttatttc tatgctacac ctggcagacg gccatgggcg tttggcagca 24840
gtgcttggag gagtgcaacc tcaaggagct gcagaaactg ctaaagcaaa acttgaagga 24900
cctatggacg gccttcaacg agcgctccgt ggccgcgcac ctggcggaca tcattttccc 24960
cgaacgcctg cttaaaaccc tgcaacaggg tctgccagac ttcaccagtc aaagcatgtt 25020
gcagaacttt aggaacttta tcctagagcg ctcaggaatc ttgcccgcca cctgctgtgc 25080
acttcctagc gactttgtgc ccattaagta ccgcgaatgc cctccgccgc tttggggcca 25140
ctgctacctt ctgcagctag ccaactacct tgcctaccac tctgacataa tggaagacgt 25200
gagcggtgac ggtctactgg agtgtcactg tcgctgcaac ctatgcaccc cgcaccgctc 25260
cctggtttgc aattcgcagc tgcttaacga aagtcaaatt atcggtacct ttgagctgca 25320
gggtccctcg cctgacgaaa agtccgcggc tccggggttg aaactcactc cggggctgtg 25380
gacgtcggct taccttcgca aatttgtacc tgaggactac cacgcccacg agattaggtt 25440
ctacgaagac caatcccgcc cgccaaatgc ggagcttacc gcctgcgtca ttacccaggg 25500
ccacattctt ggccaattgc aagccatcaa caaagcccgc caagagtttc tgctacgaaa 25560
gggacggggg gtttacttgg acccccagtc cggcgaggag ctcaacccaa tccccccgcc 25620
gccgcagccc tatcagcagc agccgcgggc ccttgcttcc caggatggca cccaaaaaga 25680
agctgcagct gccgccgcca cccacggacg aggaggaata ctgggacagt caggcagagg 25740
aggttttgga cgaggaggag gaggacatga tggaagactg ggagagccta gacgaggaag 25800
cttccgaggt cgaagaggtg tcagacgaaa caccgtcacc ctcggtcgca ttcccctcgc 25860
cggcgcccca gaaatcggca accggttcca gcatggctac aacctccgct cctcaggcgc 25920
cgccggcact gcccgttcgc cgacccaacc gtagatggga caccactgga accagggccg 25980
gtaagtccaa gcagccgccg ccgttagccc aagagcaaca acagcgccaa ggctaccgct 26040
catggcgcgg gcacaagaac gccatagttg cttgcttgca agactgtggg ggcaacatct 26100
ccttcgcccg ccgctttctt ctctaccatc acggcgtggc cttcccccgt aacatcctgc 26160
attactaccg tcatctctac agcccatact gcaccggcgg cagcggcagc ggcagcaaca 26220
gcagcggcca cacagaagca aaggcgaccg gatagcaaga ctctgacaaa gcccaagaaa 26280
tccacagcgg cggcagcagc aggaggagga gcgctgcgtc tggcgcccaa cgaacccgta 26340
tcgacccgcg agcttagaaa caggattttt cccactctgt atgctatatt tcaacagagc 26400
aggggccaag aacaagagct gaaaataaaa aacaggtctc tgcgatccct cacccgcagc 26460
tgcctgtatc acaaaagcga agatcagctt cggcgcacgc tggaagacgc ggaggctctc 26520
ttcagtaaat actgcgcgct gactcttaag gactagtttc gcgccctttc tcaaatttaa 26580
gcgcgaaaac tacgtcatct ccagcggcca cacccggcgc cagcacctgt cgtcagcgcc 26640
attatgagca aggaaattcc cacgccctac atgtggagtt accagccaca aatgggactt 26700
gcggctggag ctgcccaaga ctactcaacc cgaataaact acatgagcgc gggaccccac 26760
atgatatccc gggtcaacgg aatccgcgcc caccgaaacc gaattctctt ggaacaggcg 26820
gctattacca ccacacctcg taataacctt aatccccgta gttggcccgc tgccctggtg 26880
taccaggaaa gtcccgctcc caccactgtg gtacttccca gagacgccca ggccgaagtt 26940
cagatgacta actcaggggc gcagcttgcg ggcggctttc gtcacagggt gcggtcgccc 27000
gggcagggta taactcacct gacaatcaga gggcgaggta ttcagctcaa cgacgagtcg 27060
gtgagctcct cgcttggtct ccgtccggac gggacatttc agatcggcgg cgccggccgt 27120
ccttcattca cgcctcgtca ggcaatccta actctgcaga cctcgtcctc tgagccgcgc 27180
tctggaggca ttggaactct gcaatttatt gaggagtttg tgccatcggt ctactttaac 27240
cccttctcgg gacctcccgg ccactatccg gatcaattta ttcctaactt tgacgcggta 27300
aaggactcgg cggacggcta cgactgaatg ttaagtggag aggcagagca actgcgcctg 27360
aaacacctgg tccactgtcg ccgccacaag tgctttgccc gcgactccgg tgagttttgc 27420
tactttgaat tgcccgagga tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc 27480
cagggagagc ttgcccgtag cctgattcgg gagtttaccc agcgccccct gctagttgag 27540
cgggacaggg gaccctgtgt tctcactgtg atttgcaact gtcctaacct tggattacat 27600
caagatcttt gttgccatct ctgtgctgag tataataaat acagaaatta aaatatactg 27660
gggctcctat cgccatcctg taaacgccac cgtcttcacc cgcccaagca aaccaaggcg 27720
aaccttacct ggtactttta acatctctcc ctctgtgatt tacaacagtt tcaacccaga 27780
cggagtgagt ctacgagaga acctctccga gctcagctac tccatcagaa aaaacaccac 27840
cctccttacc tgccgggaac gtacgagtgc gtcaccggcc gctgcaccac acctaccgcc 27900
tgaccgtaaa ccagactttt tccggacaga cctcaataac tctgtttacc agaacaggag 27960
gtgagcttag aaaaccctta gggtattagg ccaaaggcgc agctactgtg gggtttatga 28020
acaattcaag caactctacg ggctattcta attcaggttt ctctagaatc ggggttgggg 28080
ttattctctg tcttgtgatt ctctttattc ttatactaac gcttctctgc ctaaggctcg 28140
ccgcctgctg tgtgcacatt tgcatttatt gtcagctttt taaacgctgg ggtcgccacc 28200
caagatgatt aggtacataa tcctaggttt actcaccctt gcgtcagccc acggtaccac 28260
ccaaaaggtg gattttaagg agccagcctg taatgttaca ttcgcagctg aagctaatga 28320
gtgcaccact cttataaaat gcaccacaga acatgaaaag ctgcttattc gccacaaaaa 28380
caaaattggc aagtatgctg tttatgctat ttggcagcca ggtgacacta cagagtataa 28440
tgttacagtt ttccagggta aaagtcataa aacttttatg tatacttttc cattttatga 28500
aatgtgcgac attaccatgt acatgagcaa acagtataag ttgtggcccc cacaaaattg 28560
tgtggaaaac actggcactt tctgctgcac tgctatgcta attacagtgc tcgctttggt 28620
ctgtacccta ctctatatta aatacaaaag cagacgcagc tttattgagg aaaagaaaat 28680
gccttaattt actaagttac aaagctaatg tcaccactaa ctgctttact cgctgcttgc 28740
aaaacaaatt caaaaagtta gcattataat tagaatagga tttaaacccc ccggtcattt 28800
cctgctcaat accattcccc tgaacaattg actctatgtg ggatatgctc cagcgctaca 28860
accttgaagt caggcttcct ggatgtcagc atctgacttt ggccagcacc tgtcccgcgg 28920
atttgttcca gtccaactac agcgacccac cctaacagag atgaccaaca caaccaacgc 28980
ggccgccgct accggactta catctaccac aaatacaccc caagtttctg cctttgtcaa 29040
taactgggat aacttgggca tgtggtggtt ctccatagcg cttatgtttg tatgccttat 29100
tattatgtgg ctcatctgct gcctaaagcg caaacgcgcc cgaccaccca tctatagtcc 29160
catcattgtg ctacacccaa acaatgatgg aatccataga ttggacggac tgaaacacat 29220
gttcttttct cttacagtat gattaaatga gacatgattc ctcgagtttt tatattactg 29280
acccttgttg cgcttttttg tgcgtgctcc acattggcta agtaatagtt aattaaacaa 29340
gtttgtacaa aaaagcaggc ttcgaaggag atagaaccaa ttctctaagg aaatacttaa 29400
ccatggctct agagatatac tgagtcatta gggactttcc aatgggtttt gcccagtaca 29460
taaggtcaat aggggtgaat caacaggaaa gtcccattgg agccaagtac actgagtcaa 29520
tagggacttt ccattgggtt ttgcccagta caaaaggtca atagggggtg agtcaatggg 29580
tttttcccat tattggcacg tacataaggt caataggggt gagtcattgg gtttttccag 29640
ccatttaatt aaaacgccat gtactttccc accattgacg tcaatgggct attgaaacta 29700
atgcaacgtg acctttaaac ggtactttcc catagctgat taatgggaaa gtaccgttct 29760
cgagccaata cacgtcaatg ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt 29820
ttcccctgga aattccatat tggcactcat tctattggct gagctgcgtt ctacgtgggt 29880
ataagaggcg cgaccagcgt cggtaccgtc gcagtcttcg gtctgaccac cgtagaacgc 29940
agatcgaatt actagtcagg gaattcgcca ccatgtgtca ccagcagttg gtcatctctt 30000
ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg aagaaagatg 30060
tttatgtcgt agaattggat tggtatccgg atgcccctgg agaaatggtg gtcctcacct 30120
gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt gaggtcttag 30180
gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc cagtacacct 30240
gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa aaggaagatg 30300
gaatttggtc cactgatatt ttaaaggacc agaaagaacc caaaaataag acctttctaa 30360
gatgcgaggc caagaattat tctggacgtt tcacctgctg gtggctgacg acaatcagta 30420
ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa ggggtgacgt 30480
gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag tatgagtact 30540
cagtggagtg ccaggaggac agtgcctgcc cagctgctga ggagagtctg cccattgagg 30600
tcatggtgga tgccgttcac aagctcaagt atgaaaacta caccagcagc ttcttcatca 30660
gggacatcat caaacctgac ccacccaaga acttgcagct gaagccatta aagaactctc 30720
ggcaggtgga ggtcagctgg gagtaccctg acacctggag tactccacat tcctacttct 30780
ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa gatagagtct 30840
tcacggacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt agcgtgcggg 30900
cccaggaccg ctactatagc tcatcttgga gcgaatgggc atctgtgccc tgcagtgttc 30960
ctggagtagg ggtacctggg gtgggcgcca gaaacctccc cgtggccact ccagacccag 31020
gaatgttccc atgccttcac cactcccaaa acctgctgag ggccgtcagc aacatgctcc 31080
agaaggccag acaaactcta gaattttacc cttgcacttc tgaagagatt gatcatgaag 31140
acatcacaaa agataaaacc agcacagtgg aggcctgttt accattggaa ttaaccaaga 31200
atgagagttg cctaaattcc agagagacct ctttcataac taatgggagt tgcctggcct 31260
ccagaaagac ctcttttatg atggccctgt gccttagtag tatttatgaa gacttgaaga 31320
tgtaccaggt ggagttcaag accatgaatg caaagcttct gatggatcct aagaggcaga 31380
tctttctaga tcaaaacatg ctggcagtta ttgatgagct gatgcaggcc ctgaatttca 31440
acagtgagac tgtgccacaa aaatcctccc ttgaagaacc ggatttttat aaaactaaaa 31500
tcaagctctg catacttctt catgctttca gaatacgggc agtgactatt gatagagtga 31560
tgagctatct gaatgcttcc taattcgagc aacttgttta ttgcagctta taatggttac 31620
aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 31680
tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcgtcta gcatcgaaga 31740
tccaaataaa ggatccttta tttggatctt cgatgctaga cgatccagac atgataagat 31800
acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 31860
aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgctc 31920
gaatcagctc atctccaaag agttgatgta ctcccgaacc catttcttct ctgggttggc 31980
acacacttgg cggttctttc gggtgacaaa gacgactgct gggttggagc acttgccact 32040
ggtgtagaaa tactccttga tgtgggcacg gggcagtggg cgggcaatgt aggcaaagca 32100
gcagggtgtg gtgtccgagg aatatgggga ggcagatgca ggagcgcaga gggcagtagc 32160
aatgaggatg acagcgaggg ctgccgcgga gaccttcatg gggccagggt tctcttccac 32220
gtcgccacat gtcagcaggc tgcctctgcc ctctccagat ccctgggatg ctcttcgacc 32280
tcgaaacagc atctgactcc tttttcgctt ccctgtttta gctgctggcg acagttcagc 32340
catcacttgg atgagttcat gtattgcttt gcgttggaca ttcaagtcag ttaccgaata 32400
attagtcagc ttttcgaagt catctcgttt ctttttgttg ctattgaaaa acttgacatt 32460
catgtcttcc ttgatggtct ccacactctt ttggatgctc tggtcatctt taaagttttt 32520
aaaaagtttg aagtaaaagg agacaatttg gctctgcatt atttttctgt cactctcctc 32580
tttccaattc ttcaaaatgc ctaagaaaag agttccatta tccgctacat ctgaatgacc 32640
tgcattaaaa tatttcttaa ggttttctgc ttcttttaca tatgggtcct ggcagtaaca 32700
gccaagagaa cccaaaacga tgcagagctg aaaagccaag atataacttg tatatttcat 32760
ggtggccaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact aaacgagctc 32820
tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt 32880
tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt 32940
caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt 33000
actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag 33060
taggaaagtc ccataaggtc atgtactggg cataatgcca gggaattaga tatctagacc 33120
cagctttctt gtacaaagtg gttaattaag atcttattcc ctttaactaa taaaaaaaaa 33180
taataaagca tcacttactt aaaatcagtt agcaaatttc tgtccagttt attcagcagc 33240
acctccttgc cctcctccca gctctggtat tgcagcttcc tcctggctgc aaactttctc 33300
cacaatctaa atggaatgtc agtttcctcc tgttcctgtc catccgcacc cactatcttc 33360
atgttgttgc agatgaagcg cgcaagaccg tctgaagata ccttcaaccc cgtgtatcca 33420
tatgacacgg aaaccggtcc tccaactgtg ccttttctta ctcctccctt tgtatccccc 33480
aatgggtttc aagagagtcc ccctggagtt cttactttaa aatgtttaac cccactaaca 33540
accacaggcg gatctctaca gctaaaagtg ggagggggac ttacagtgga tgacaccaac 33600
ggttttttga aagaaaacat aagtgccacc acaccactcg ttaagactgg tcactctata 33660
ggtttaccac taggagccgg attgggaacg aatgaaaata aactttgtat caaattagga 33720
caaggactta cattcaattc aaacaacatt tgcattgatg acaatattaa caccttatgg 33780
acaggagtca accccaccga agccaactgt caaatcatga actccagtga atctaatgat 33840
tgcaaattaa ttctaacact agttaaaact ggagcactag tcactgcatt tgtttatgtt 33900
ataggagtat ctaacaattt taatatgcta actacacaca gaaatataaa ttttactgca 33960
gagctgtttt tcgattctaa ctggtaattt aactaactag actctcatcc cctcaaaact 34020
ccacttaatc ataaatcagg acaaaacatg gctactggtg ccattactaa tgctaaaggt 34080
ttcatgccca gcacgactgc ctatcctttc aatgataatt ctagagaaaa agaaaactac 34140
atttacggaa cttgttacta cacagctagt gatcgcactg cttttcccat tgacatatct 34200
gtcatgctta accgaagagc aataaatgac gagacatcat attgtattcg tataacttgg 34260
tcctggaaca caggagatgc cccagaggtg caaacctctg ctacaaccct agtcacctcc 34320
ccatttacct tttactacat cagagaagac gactgagccc aagaataaag aatcgtttgt 34380
gttatgtttc aacgtgttta tttttcaatt gcagaaaatt tcaagtcatt tttcattcag 34440
tagtatagcc ccaccaccac atagcttata cagatcaccg taccttaatc aaactcacag 34500
aaccctagta ttcaacctgc cacctccctc ccaacacaca gagtacacag tcctttctcc 34560
ccggctggcc ttaaaaagca tcatatcatg ggtaacagac atattcttag gtgttatatt 34620
ccacacggtt tcctgtcgag ccaaacgctc atcagtgata ttaataaact ccccgggcag 34680
ctcacttaag ttcatgtcgc tgtccagctg ctgagccaca ggctgctgtc caacttgcgg 34740
ttgcttaacg ggcggcgaag gagaagtcca cgcctacatg ggggtagagt cataatcgtg 34800
catcaggata gggcggtggt gctgcagcag cgcgcgaata aactgctgcc gccgccgctc 34860
cgtcctgcag gaatacaaca tggcagtggt ctcctcagcg atgattcgca ccgcccgcag 34920
cataaggcgc cttgtcctcc gggcacagca gcgcaccctg atctcactta aatcagcaca 34980
gtaactgcag cacagcacca caatattgtt caaaatccca cagtgcaagg cgctgtatcc 35040
aaagctcatg gcggggacca cagaacccac gtggccatca taccacaagc gcaggtagat 35100
taagtggcga cccctcataa acacgctgga cataaacatt acctcttttg gcatgttgta 35160
attcaccacc tcccggtacc atataaacct ctgattaaac atggcgccat ccaccaccat 35220
cctaaaccag ctggccaaaa cctgcccgcc ggctatacac tgcagggaac cgggactgga 35280
acaatgacag tggagagccc aggactcgta accatggatc atcatgctcg tcatgatatc 35340
aatgttggca caacacaggc acacgtgcat acacttcctc aggattacaa gctcctcccg 35400
cgttagaacc atatcccagg gaacaaccca ttcctgaatc agcgtaaatc ccacactgca 35460
gggaagacct cgcacgtaac tcacgttgtg cattgtcaaa gtgttacatt cgggcagcag 35520
cggatgatcc tccagtatgg tagcgcgggt ttctgtctca aaaggaggta gacgatccct 35580
actgtacgga gtgcgccgag acaaccgaga tcgtgttggt cgtagtgtca tgccaaatgg 35640
aacgccggac gtagtcatat ttcctgaagc aaaaccaggt gcgggcgtga caaacagatc 35700
tgcgtctccg gtctcgccgc ttagatcgct ctgtgtagta gttgtagtat atccactctc 35760
tcaaagcatc caggcgcccc ctggcttcgg gttctatgta aactccttca tgcgccgctg 35820
ccctgataac atccaccacc gcagaataag ccacacccag ccaacctaca cattcgttct 35880
gcgagtcaca cacgggagga gcgggaagag ctggaagaac catgtttttt tttttattcc 35940
aaaagattat ccaaaacctc aaaatgaaga tctattaagt gaacgcgctc ccctccggtg 36000
gcgtggtcaa actctacagc caaagaacag ataatggcat ttgtaagatg ttgcacaatg 36060
gcttccaaaa ggcaaacggc cctcacgtcc aagtggacgt aaaggctaaa cccttcaggg 36120
tgaatctcct ctataaacat tccagcacct tcaaccatgc ccaaataatt ctcatctcgc 36180
caccttctca atatatctct aagcaaatcc cgaatattaa gtccggccat tgtaaaaatc 36240
tgctccagag cgccctccac cttcagcctc aagcagcgaa tcatgattgc aaaaattcag 36300
gttcctcaca gacctgtata agattcaaaa gcggaacatt aacaaaaata ccgcgatccc 36360
gtaggtccct tcgcagggcc agctgaacat aatcgtgcag gtctgcacgg accagcgcgg 36420
ccacttcccc gccaggaacc ttgacaaaag aacccacact gattatgaca cgcatactcg 36480
gagctatgct aaccagcgta gccccgatgt aagctttgtt gcatgggcgg cgatataaaa 36540
tgcaaggtgc tgctcaaaaa atcaggcaaa gcctcgcgca aaaaagaaag cacatcgtag 36600
tcatgctcat gcagataaag gcaggtaagc tccggaacca ccacagaaaa agacaccatt 36660
tttctctcaa acatgtctgc gggtttctgc ataaacacaa aataaaataa caaaaaaaca 36720
tttaaacatt agaagcctgt cttacaacag gaaaaacaac ccttataagc ataagacgga 36780
ctacggccat gccggcgtga ccgtaaaaaa actggtcacc gtgattaaaa agcaccaccg 36840
acagctcctc ggtcatgtcc ggagtcataa tgtaagactc ggtaaacaca tcaggttgat 36900
tcatcggtca gtgctaaaaa gcgaccgaaa tagcccgggg gaatacatac ccgcaggcgt 36960
agagacaaca ttacagcccc cataggaggt ataacaaaat taataggaga gaaaaacaca 37020
taaacacctg aaaaaccctc ctgcctaggc aaaatagcac cctcccgctc cagaacaaca 37080
tacagcgctt cacagcggca gcctaacagt cagccttacc agtaaaaaag aaaacctatt 37140
aaaaaaacac cactcgacac ggcaccagct caatcagtca cagtgtaaaa aagggccaag 37200
tgcagagcga gtatatatag gactaaaaaa tgacgtaacg gttaaagtcc acaaaaaaca 37260
cccagaaaac cgcacgcgaa cctacgccca gaaacgaaag ccaaaaaacc cacaacttcc 37320
tcaaatcgtc acttccgttt tcccacgtta cgtaacttcc cattttaaga aaactacaat 37380
tcccaacaca tacaagttac tccgccctaa aacctacgtc acccgccccg ttcccacgcc 37440
ccgcgccacg tcacaaactc caccccctca ttatcatatt ggcttcaatc caaaataagg 37500
tatattattg atgat 37515
Claims (2)
1.一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒,其特征在于,所述溶瘤腺病毒同时表达IL-12、IFN-γ和CCL5三个因子,在所述溶瘤腺病毒基因组中,IFN-γ和CCL5基因cDNA序列共有一个表达框,以人巨细胞病毒(hCMV)启动子驱动,IL-12基因cDNA序列表达框,以鼠巨细胞病毒(mCMV)启动子驱动,两个表达框呈“脚对脚”排列,同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示;所述溶瘤腺病毒其外壳蛋白Hexon由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成,序列如SEQ ID NO:3的第18327-21170bp所示,其纤毛蛋白Fiber由Ad5和Ad11两个血清型病毒的Fiber序列嵌合而成,序列如SEQ ID NO:3的第33373-34356bp所示;所述溶瘤腺病毒同时采用肿瘤特异性强势启动子和氧依赖元件开关,并删除Elb-55kD基因;所述溶瘤腺病毒同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列,所述溶瘤腺病毒的全基因组序列如 SEQ ID NO:3所示。
2.权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒在制备治疗和/或预防和/或辅助治疗癌症或者抗肿瘤的药物中的用途;所述癌症或者肿瘤为乳腺癌、肝癌、胆囊癌、胃癌、结肠癌、肺癌、***癌、卵巢癌、***、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、膀胱癌或头颈癌。
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