CN112553213B - Cx3cr1基因人源化的非人动物及其构建方法和应用 - Google Patents
Cx3cr1基因人源化的非人动物及其构建方法和应用 Download PDFInfo
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Abstract
本发明提供了一种CX3CR1基因人源化的非人动物的构建方法,利用同源重组的方式将编码人CX3CR1蛋白的核苷酸序列导入非人动物基因组中,该动物体内能正常表达人源化CX3CR1蛋白,可以作为人CX3CR1信号机理研究、肿瘤及免疫性疾病药物筛选的动物模型,对免疫靶点的新药研发具有重要的应用价值。本发明还提供了一种人源化CX3CR1蛋白、一种人源化CX3CR1基因、一种CX3CR1基因的靶向载体,以及上述构建方法获得的非人动物及其在生物医药领域的应用。
Description
技术领域
本发明属于动物基因工程和基因遗传修饰领域,具体地说,涉及一种CX3CR1基因人源化的非人动物及其构建方法和在生物医药领域的应用。
背景技术
CX3CR1(C-X3-C motif chemokine receptor 1)又名V28,是趋化因子受体,属于7次跨膜蛋白,是G蛋白受体成员之一。CX3CR1主要表达在单核细胞、巨噬细胞、DC细胞、T细胞、NK细胞、小胶质细胞等。根据趋化受体与趋化因子结合部位不同,将其分为4类:CX3CR1、CXCR1-CXCR5、XCR1、CCR1-CCR9。在外周血中,CX3CR1多位于单核细胞和白细胞的细胞膜上,既参与细胞的趋化作用,也与细胞的粘附作用有关。炎症细胞从外周循环中到达炎症部位是一个动态的、多变的过程,在此过程中,CX3CR1起了重要的作用。
随着基因工程技术的不断发展和成熟,用人类基因替代或置换动物的同源性基因已经实现,通过这种方式开发人源化实验动物模型是动物模型未来的发展方向。其中基因人源化动物模型,即,利用基因编辑技术,用人源正常或突变基因替换动物基因组的同源基因,可建立更接近人类生理或疾病特征的正常或突变基因动物模型。基因人源化动物不但本身具有重要应用价值,如通过基因人源化可改进和提升细胞或组织移植人源化动物模型,更重要的是,由于人类基因片段的插入,动物体内可表达或部分表达人源蛋白,可作为仅能识别人蛋白氨基酸序列的药物的靶点,为在动物水平进行抗人抗体及其它药物的筛选提供了可能。然而,由于动物与人类在生理学及病理学方面存在差异,加上基因(即遗传因子)的复杂性,如何能构建出“有效”的人源化动物模型用于新药研发仍是最大的挑战(Scheer N, Snaith M, Wolf CR, Seibler J. Generation and utility ofgenetically humanized mouse models, Drug Discov Today; 18(23-24):1200-11,2013)。
鉴于CX3CR1在免疫性疾病等多种疾病发生过程中的广泛参与性以及靶向该信号通路的巨大应用价值,为了使临床前期的试验更有效并使研发失败最小化,本领域仍急需开发人源化CX3CR1信号通路相关的非人动物模型。
发明内容
本发明的第一方面,提供了一种CX3CR1基因人源化改造的非人动物的构建方法,所述的非人动物的基因组中包括编码SEQ ID NO:2所示氨基酸的核苷酸序列,所述的构建方法包括用包含编码SEQ ID NO:2的核苷酸序列构建至非人动物CX3CR1基因座。
优选的,所述的非人动物的基因组中包括SEQ ID NO:5所示核苷酸序列,所述的构建方法包括用包含SEQ ID NO:5所示核苷酸序列构建至非人动物CX3CR1基因座。
优选的,所述的非人动物的基因组中包含人CX3CR1核苷酸序列的2号外显子的部分;其中,所述人CX3CR1核苷酸序列的2号外显子的部分至少包含2号外显子中编码人CX3CR1蛋白的核苷酸序列,优选的,2号外显子的部分至少包含从2号外显子5’端第10个核苷酸开始至长度为1059bp、1060bp、1061bp、1062bp、1063bp、1064bp、1065bp、1066bp、1067bp或1068bp的核苷酸序列,优选的,为1068bp的核苷酸序列。
优选的,所述的构建方法包括用包含人CX3CR1核苷酸序列的2号外显子的全部或部分核苷酸序列插入或替换到非人动物CX3CR1基因座上,进一步优选的,用包含人CX3CR1核苷酸序列的2号外显子的部分核苷酸序列构建至非人动物CX3CR1基因座;其中,所述人CX3CR1基因的2号外显子的部分至少包含编码人CX3CR1蛋白的核苷酸序列,优选的,2号外显子的部分至少包含从2号外显子5’端第10个核苷酸开始至长度为1059bp、1060bp、1061bp、1062bp、1063bp、1064bp、1065bp、1066bp、1067bp或1068bp的核苷酸序列,优选的,为1068bp的核苷酸序列。
优选的,替换至非人动物CX3CR1基因座为替换非人动物与NCBI登录号为NC_000075.7的第119880336-119881400所示序列相同的核苷酸序列。
优选的,所述的构建方法包括用包含所述人源化CX3CR1基因的核苷酸序列构建至非人动物CX3CR1基因座。
优选的,所述的构建方法包括用包含编码所述人源化CX3CR1蛋白的核苷酸序列构建至非人动物CX3CR1基因座。
优选的,所述的构建方法包括插入、翻转、敲除或替换。
更优选的,所述的构建方法是替换,所述的替换是将非人动物CX3CR1基因中编码SEQ ID NO:1所示氨基酸的核苷酸序列进行替换。
优选的,所述的构建至非人动物CX3CR1基因座为替换至非人动物内源CX3CR1基因中包含编码非人动物2号外显子的部分核苷酸序列,其中,所述非人动物CX3CR1基因的2号外显子的部分至少包含2号外显子中编码非人动物内源CX3CR1蛋白的核苷酸序列,优选的,2号外显子的部分至少包含从2号外显子5’端第10个核苷酸开始至长度为1056bp、1057bp、1058bp、1059bp、1060bp、1061bp、1062bp、1063bp、1064bp或1065bp的核苷酸序列,优选的,为1065bp的核苷酸序列。
本发明所述的非人动物为啮齿类动物;优选的,所述的啮齿类动物为大鼠或小鼠。
优选的,所述的非人动物体内表达人或人源化CX3CR1蛋白,同时内源CX3CR1蛋白的表达降低或缺失。
优选的,所述的人源化CX3CR1蛋白包含人CX3CR1蛋白的全部或部分,进一步优选的,包含与SEQ ID NO:2具有至少70%、80%、85%、90%、95%或至少99%同一性的氨基酸序列或者包含与SEQ ID NO:2一致的氨基酸序列。
在本发明的一个具体实施方式中,所述的人源化CX3CR1蛋白包含下列组中的一种:
a)SEQ ID NO:2所示氨基酸序列的部分或全部;
b)与SEQ ID NO:2所示氨基酸的序列同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)与SEQ ID NO:2所示的氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;
d)具有SEQ ID NO:2所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
优选的,所述的非人动物的基因组中包含人源化CX3CR1基因,所述的人源化CX3CR1基因编码人源化CX3CR1蛋白。
优选的,所述的人源化CX3CR1基因包含SEQ ID NO:5所示的核苷酸序列,进一步优选的,所述的非人动物中包含的CX3CR1基因转录的mRNA序列包含SEQ ID NO:8所示的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化CX3CR1基因包含下列组中的一种:
a)人源化CX3CR1基因的mRNA序列为SEQ ID NO:8所示的序列的部分或全部;
b)人源化CX3CR1基因的mRNA序列与SEQ ID NO:8所示的核苷酸序列的部分或全部的同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)人源化CX3CR1基因的mRNA序列与SEQ ID NO:8所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸;
d)人源化CX3CR1基因的mRNA序列具有SEQ ID NO:8所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
优选的,所述的插入为首先破坏非人动物内源CX3CR1基因的编码框,随后进行插入操作,或者所述的插入步骤既可在内源CX3CR1基因处造成移码突变又可以实现插入人源序列的步骤。
优选的,所述的非人动物中人源化CX3CR1基因是纯合或杂合的。
优选的,所述非人动物的基因组中至少一个染色体上包含人源化CX3CR1基因。
优选的,所述的非人动物中至少一个细胞表达人或人源化CX3CR1蛋白。
优选的,使用基因编辑技术进行CX3CR1基因人源化的非人动物的构建,所述的基因编辑技术包括利用胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。
优选的,使用靶向载体进行CX3CR1基因人源化的非人动物的构建,其中,所述的靶向载体包含人CX3CR1的2号外显子的全部或部分核苷酸序列;进一步优选的,包含2号外显子的部分,其中,所述人CX3CR1的核苷酸序列的2号外显子的部分至少包含编码人CX3CR1蛋白的核苷酸序列,优选的,2号外显子的部分至少包含从2号外显子5’端第10个核苷酸开始至长度为1059bp、1060bp、1061bp、1062bp、1063bp、1064bp、1065bp、1066bp、1067bp或1068bp的核苷酸序列,优选的,为1068bp的核苷酸序列。
优选的,所述靶向载体包含编码SEQ ID NO:2所示氨基酸的核苷酸序列或SEQ IDNO:5所示核苷酸序列。
优选的,所述的靶向载体还包含与待改变的转换区5’端同源的DNA片段,即5’臂,其选自非人动物CX3CR1基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的5’臂与NCBI登录号为NC_000075.7至少具有90%同源性的核苷酸;进一步优选的,所述5’臂序列与SEQ ID NO:3至少具有90%同源性,或者如SEQ ID NO:3所示。
优选的,所述的靶向载体还包含与待改变的转换区3’端同源的DNA片段,即3’臂,其选自非人动物CX3CR1基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的3’臂与NCBI登录号为NC_000075.7至少具有90%同源性的核苷酸;进一步优选的,所述的3’臂序列与SEQ ID NO:4至少具有90%同源性,或者如SEQ ID NO:4所示。
优选的,所述的待改变的转换区位于非人动物CX3CR1基因座上。进一步优选的,位于非人动物CX3CR1基因的2号外显子上。
在本发明的一个具体实施方式中,所述的构建方法包括将上述靶向载体导入非人动物细胞中,培养该细胞(优选为胚胎干细胞),然后将培养后的细胞移植至雌性非人动物输卵管内,允许其发育,鉴定筛选获得非人动物。
本发明的第二方面,提供了一种CX3CR1基因人源化的非人动物,所述的非人动物采用上述构建方法获得。
本发明的第三方面,提供了一种CX3CR1基因的靶向载体,所述的靶向载体包含人CX3CR1核苷酸序列的部分,优选的,所述的人CX3CR1核苷酸序列的部分包含人CX3CR1的2号外显子的全部或部分核苷酸序列;进一步优选的,包含2号外显子的部分,其中,所述人CX3CR1的核苷酸序列的2号外显子的部分至少包含编码人CX3CR1蛋白的核苷酸序列,优选的,2号外显子的部分至少包含从2号外显子5’端第10个核苷酸开始至长度为1059bp、1060bp、1061bp、1062bp、1063bp、1064bp、1065bp、1066bp、1067bp或1068bp的核苷酸序列,优选的,为1068bp的核苷酸序列。
优选的,所述的靶向载体包含编码SEQ ID NO:2所示氨基酸的核苷酸序列或者SEQID NO:5所示核苷酸序列。
优选的,所述的靶向载体还包含与待改变的转换区5’端同源的DNA片段,即5’臂,其选自非人动物CX3CR1基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的5’臂与NCBI登录号为NC_000075.7至少具有90%同源性的核苷酸;进一步优选的,所述5’臂序列与SEQ ID NO:3至少具有90%同源性,或者如SEQ ID NO:3所示。
优选的,所述的靶向载体还包含与待改变的转换区3’端同源的DNA片段,即3’臂,其选自非人动物CX3CR1基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的3’臂与NCBI登录号为NC_000075.7至少具有90%同源性的核苷酸;进一步优选的,所述的3’臂序列与SEQ ID NO:4至少具有90%同源性,或者如SEQ ID NO:4所示。
优选的,所述的待改变的转换区位于非人动物CX3CR1基因座上,进一步优选的,所述的待改变的转换区位于非人动物CX3CR1基因2号外显子上。
本发明所述的非人动物为啮齿类动物;优选的,所述的啮齿类动物为大鼠或小鼠。
优选的,所述的靶向载体还包含标记基因,进一步优选的,所述标记基因为负筛选标记的编码基因,更进一步优选的,所述负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
在本发明的一个具体实施方式中,所述的靶向载体中还包括阳性克隆筛选的抗性基因,进一步优选的,所述阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。
在本发明的一个具体实施方式中,所述的靶向载体中还包括特异性重组系统,进一步优选的,所述特异性重组系统为Frt重组位点(也可选择常规的LoxP重组系统),所述的特异性重组系统为具有两个Frt重组位点,分别连接在抗性基因的两侧。
本发明的第四方面,提供了一种包含上述靶向载体的细胞。
本发明的第五方面,提供了上述靶向载体,或者上述的细胞在CX3CR1基因修饰中的应用,优选的,所述的应用包括但不限于翻转、敲除、插入或替换。
本发明的第六方面,涉及一种CX3CR1基因人源化细胞,所述的CX3CR1基因人源化细胞的基因组中包括人CX3CR1基因的2号外显子。优选的,所述的人CX3CR1基因编码SEQ IDNO:2所示氨基酸的核苷酸序列或包含SEQ ID NO:5所示核苷酸序列,其通过内源性CX3CR1调控元件调控;该CX3CR1基因人源化细胞体内表达人或人源化CX3CR1蛋白,同时内源CX3CR1蛋白的表达降低或缺失。优选的,所述的人CX3CR1基因通过内源性CX3CR1调控元件调控。
本发明的第七方面,涉及一种CX3CR1基因缺失的细胞,所述的CX3CR1基因缺失的细胞缺失内源CX3CR1基因的2号外显子。
本发明的第八方面,涉及一种荷瘤动物模型的制备方法,所述的动物模型的制备方法包括通过上述的CX3CR1基因人源化的非人动物制备荷瘤动物模型的步骤。
优选的,所述的荷瘤动物模型的制备方法还包括在上述基因人源化的非人动物或其后代植入肿瘤细胞的步骤。
本发明的第九方面,提供了一种上述的制备方法获得的荷瘤动物模型。
本发明的第十方面,涉及一种细胞或细胞系或原代细胞培养物,所述细胞或细胞系或原代细胞培养物来源于上述的非人动物或上述的荷瘤动物模型。
本发明的第十一方面,涉及一种组织或器官或其培养物,所述组织或器官或其培养物来源于上述的非人动物或上述的荷瘤动物模型。
优选的,所述的组织或器官或其培养物为脾脏、肿瘤或其培养物。
本发明的第十二方面,提供了一种人源化CX3CR1蛋白,所述的人源化CX3CR1蛋白包含人CX3CR1蛋白的全部或部分,进一步优选的,所述的人源化CX3CR1蛋白包含人CX3CR1蛋白的全部,所述的人源化CX3CR1蛋白包含与SEQ ID NO:2具有至少70%、80%、85%、90%、95%或至少99%同一性的氨基酸序列或者包含与SEQ ID NO:2所示氨基酸序列。
在本发明的一个具体实施方式中,所述的人源化CX3CR1蛋白包含下列组中的一种:
a)SEQ ID NO:2所示氨基酸序列的部分或全部;
b)与SEQ ID NO:2所示氨基酸的序列同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)与SEQ ID NO:2所示的氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;
d)具有SEQ ID NO:2所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明的第十三方面,提供了一种编码上述人源化CX3CR1蛋白的人源化CX3CR1基因,所述的人源化CX3CR1基因包含人CX3CR1基因的2号外显子。
优选的,所述的人源化CX3CR1基因包含SEQ ID NO:5所示的核苷酸序列。
优选的,所述的人源化CX3CR1基因转录的mRNA序列包含SEQ ID NO:8所示的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化CX3CR1基因中包含的人CX3CR1核苷酸序列选自下列组中的一种:
(a)包含SEQ ID NO:5所示核苷酸序列的全部或部分;
(b)包含与SEQ ID NO:5所示核苷酸序列的同一性至少为75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%的核苷酸序列;
(c)包含与SEQ ID NO:5所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列;
(d)具有SEQ ID NO:5所示核苷酸序列的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化CX3CR1基因的核苷酸序列转录的mRNA选自下列组中的一种:
(a)包含SEQ ID NO:8所示核苷酸序列的全部或部分;
(b)包含与SEQ ID NO:8所示核苷酸序列的同一性至少为75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%的核苷酸序列;
(c)包含与SEQ ID NO:8所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列;或
(d)包含SEQ ID NO:8所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
本发明的第十四方面,涉及一种表达上述的人源化CX3CR1蛋白的构建体。
本发明的第十五方面,涉及一种包含上述构建体的细胞。
本发明的第十六方面,涉及一种包含上述细胞的组织。
优选的,上述的细胞或细胞系或原代细胞培养物、组织或器官或其培养物均不能发育为动物个体。
本发明的第十七方面,提供了一种多基因修饰的非人动物的构建方法,所述的构建方法包括:
(a)应用上述的构建方法制备获得非人动物;
(b)将步骤(a)制备获得的非人动物与除CX3CR1外的其他基因修饰的动物交配、体外授精或直接进行基因编辑,并进行筛选,得到多基因人源化修饰的非人动物。
优选的,所述多基因人源化修饰的非人动物为双基因人源化非人动物、三基因人源化非人动物、四基因人源化非人动物、五基因人源化非人动物、六基因人源化非人动物、七基因人源化非人动物、八基因人源化非人动物或九基因人源化非人动物。
优选的,所述的除CX3CR1外的其他基因修饰的动物选自基因PD-1、PD-L1、CTLA4、OX40、LAG3、TIM3或CD73等修饰的动物中的一种或两种以上的组合。
本发明的第十八方面,涉及了一种上述的非人动物、上述的荷瘤动物模型、上述的细胞或细胞系或原代细胞培养物、上述的组织或器官或其培养物、上述的人源化CX3CR1蛋白或上述的人源化CX3CR1基因在制备治疗或预防肿瘤的药物中的应用。
本发明的第十九方面,涉及一种上述的非人动物、上述的荷瘤动物模型、上述的细胞或细胞系或原代细胞培养物、上述的组织或器官或其培养物、上述的人源化CX3CR1蛋白或上述的人源化CX3CR1基因在CX3CR1基因或蛋白中相关研究中的应用,所述的应用包括:
a)涉及人类细胞的免疫过程的产品开发,制造或筛选人类抗体中的应用;
b)作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;
c)涉及人类细胞的免疫过程的生产和利用动物实验疾病模型,用于病原学研究、用于开发诊断策略或用于开发治疗策略中的应用;
d)在体内研究人CX3CR1信号通路调节剂的筛选、药效检测、评估疗效、验证或评价;或者,
e)研究CX3CR1基因功能,研究人CX3CR1抗体,研究针对人CX3CR1靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤或炎症药物方面的用途。
优选的,所述应用包括在制备药物组合物或者检测试剂盒中的用途。
优选的,所述应用不是疾病的诊断和治疗方法。
本发明所述的“肿瘤”包括但不限于淋巴瘤、B细胞肿瘤、T细胞肿瘤、骨髓/单核细胞肿瘤、非小细胞肺癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。其中,所述的白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病;所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤,包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症;所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、及软骨肉瘤。在本发明的一个具体实施方式中,所述的肿瘤选自B细胞肿瘤、T细胞肿瘤、骨髓/单核细胞肿瘤。优选包括B或T细胞急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、非霍奇金淋巴瘤(NHL)和多发性骨髓瘤(MM)、鼻咽癌、肺癌。
本发明所述的“免疫相关疾病”包括但不限于过敏、哮喘、心肌炎、肾炎、肝炎、系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、原发性血小板减少性紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、自身免疫性肝病、糖尿病、疼痛或神经障碍等。在本发明的一个具体实施方式中。所述的免疫相关疾病为类风湿性关节炎。
本发明所述的“炎症”包括急性炎症,也包括慢性炎症。具体的,包括但不限于变质性炎症、渗出性炎症(浆液性炎、纤维素性炎、化脓性炎、出血性炎、坏死性炎、卡他性炎)、增生性炎症、特异性炎症(结核、梅毒、麻疯、淋巴肉芽肿等)。
本发明所述的CX3CR1基因人源化的非人动物体内可以正常表达人或人源化CX3CR1蛋白。可用于针对人CX3CR1靶位点的药物筛选、药效评估、免疫相关疾病和肿瘤治疗,可以加快新药研发过程、节约时间和成本。对于研究CX3CR1蛋白功能及相关疾病药物筛选提供了有效的保障。
本发明所述的“全部或部分”,“全部”为整体,“部分”为整体中的局部,或者组成整体的个体。
本发明所述的“人源化CX3CR1蛋白”,包含来源于人CX3CR1蛋白的部分。其中,所述的“人CX3CR1蛋白”同“人CX3CR1蛋白的全部”,即其氨基酸序列与人CX3CR1蛋白的全长氨基酸序列一致。所述的“人CX3CR1蛋白的部分”,为连续或间隔的5-355个(优选为10-355个)氨基酸序列与人CX3CR1蛋白的氨基酸序列一致或与人CX3CR1蛋白的氨基酸序列具有70%以上同源性。
本发明所述的“人源化CX3CR1基因”,包含来源于人CX3CR1核苷酸序列的部分。其中,所述的“人CX3CR1核苷酸序列”同“人CX3CR1核苷酸序列的全部”,即其核苷酸序列与人CX3CR1核苷酸序列的全长核苷酸序列一致。所述的“人CX3CR1核苷酸序列的部分”为连续或间隔的20-18242bp(优选为20-1068bp)个核苷酸序列与人CX3CR1核苷酸序列一致或与人CX3CR1核苷酸序列具有70%以上同源性。
本发明所述的“外显子的部分”表示连续或间隔几个、几十个或几百个核苷酸序列与全部的外显子核苷酸序列一致。例如人CX3CR1核苷酸序列的2号外显子的部分,优选10-1068bp个核苷酸序列与人CX3CR1核苷酸序列的2号外显子核苷酸序列一致。在本发明的一个具体实施方式中,所述的“人源化CX3CR1基因”中包含的“2号外显子的部分”至少包括2号外显子中编码人CX3CR1蛋白的核苷酸序列。
本发明所述的“基因座”广义上讲代表基因在染色体上所占的位置,狭义上讲代表某一基因上的一段DNA片段,即可以是一个基因也可以是一个基因的一部分。例如所述的“CX3CR1基因座”表示CX3CR1基因1号至2号外显子上的任选一段的DNA片段。优选为1号外显子、2号外显子或其期间的内含子的组合,或一个或两个或多个的全部或部分,更优选为CX3CR1基因的2号外显子上。
本发明所述的“核苷酸序列”包含天然的或经过修饰的核糖核苷酸序列、脱氧核糖核苷酸序列。优选为DNA、cDNA、pre-mRNA、mRNA、rRNA、hnRNA、miRNAs、scRNA、snRNA、siRNA、sgRNA、tRNA。
本发明所述“治疗(treating)”(或“治疗(treat)”或“治疗(treatment)”)表示减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性,但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。术语“治疗(treating)”等是指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预。
本发明所述“同源性”,是指在使用蛋白序列或核苷酸序列的方面,本领域技术人员可以根据实际工作需要对序列进行调整,使使用序列与现有技术获得的序列相比,具有(包括但不限于)1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%的同一性。
本领域的技术人员能够确定并比较序列元件或同一性程度,以区分另外的小鼠和人序列。
在一个方面,所述非人动物是哺乳动物。在一个方面,所述非人动物是小型哺乳动物,例如跳鼠科或鼠总科超家族。在一个实施方式中,所述基因修饰的动物是啮齿动物。在一个实施方式中,所述啮齿动物选自小鼠、大鼠和仓鼠。在一个实施方式中,所述啮齿动物选自鼠家族。在一个实施方式中,所述基因修饰的动物来自选自丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)家族。在一个特定实施方式中,所述基因修饰的啮齿动物选自真小鼠或大鼠(鼠总科)、沙鼠、刺毛鼠和冠毛大鼠。在一个实施方式中,所述基因修饰的小鼠来自鼠科家族成员。在一个实施方式中,所述动物是啮齿动物。在一个特定实施方式中,所述啮齿动物选自小鼠和大鼠。在一个实施方式中,所述非人动物是小鼠。
在一个特定实施方式中,所述非人动物是啮齿动物,其为选自BALB/c、A、A/He、A/J、A/WySN、AKR、AKR/A、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、 C57BL/10ScSn、C57BL/10Cr和C57BL/Ola的C57BL、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st、CBA/H品系的小鼠。
除非特别说明,本发明的实践将采取细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术。这些技术在以下文献中进行了详细的解释。例如:Molecular Cloning A Laboratory Manual,2ndEd.,ed. By Sambrook,FritschandManiatis (Cold Spring Harbor Laboratory Press:1989);DNA Cloning,Volumes I and II (D.N.Glovered.,1985);Oligonucleotide Synthesis (M.J.Gaited.,1984);Mullisetal. U.S. Pat.No.4,683,195;Nucleic Acid Hybridization(B.D.Hames& S.J.Higginseds.1984);Transcription And Translation (B.D.Hames&S.J.Higginseds.1984);Culture Of Animal Cells (R.I.Freshney,AlanR.Liss,Inc.,1987);Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A PracticalGuide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY (J.Abelsonand M.Simon,eds.inchief,Academic Press,Inc.,New York),specifically,Vols. 154and 155 (Wuetal.eds.) and Vol.185,″Gene Expression Technology″ (D.Goeddel,ed.);Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Caloseds.,1987,Cold Spring Harbor Laboratory);Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker,eds.,Academic Press,London,1987);HandbookOf Experimental Immunology,Volumes V (D.M.Weir and C.C.Blackwell,eds.,1986);and Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.,1986)。
以上只是概括了本发明的一些方面,不是也不应该认为是在任何方面限制本发明。
本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。本领域的技术人员应认识到,对本发明可作某些改变并不偏离本发明的构思或范围。
下面的实施例进一步详细说明本发明,不能认为是限制本发明或本发明所说明的具体方法的范围。
附图说明
以下,结合附图来详细说明本发明的实施例,其中:
图1:人和小鼠CX3CR1基因座结构对比示意图(非按比例);
图2:人源化CX3CR1基因座示意图(非按比例);
图3:CX3CR1打靶策略示意图(非按比例);
图4:重组后ES细胞Southern blot结果,其中WT为野生型对照;
图5:人源化CX3CR1小鼠FRT重组过程示意图(非按比例);
图6:F1代小鼠PCR结果,其中WT为野生型对照,H2O为水对照,PC为阳性对照;
图7:人CX3CR1和鼠CX3CR1的流式检测结果,(A)为CX3CR1在骨髓单核细胞中的表达情况;(B)为CX3CR1在骨髓巨噬细胞中的表达情况;(C)为CX3CR1在脾单核细胞中的表达情况;(D)为CX3CR1在脾巨噬细胞中的表达情况,其中WT为野生型C57BL/6小鼠,H/+为CX3CR1人源化杂合子小鼠。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
在下述每一实施例中,设备和材料是从以下所指出的几家公司获得:
Brilliant Violet 510™ anti-mouse CD45 Antibody购自Biolegend,货号:103138;
Brilliant Violet 711™ anti-mouse TCR β chain Antibody购自Biolegend,货号:109243;
PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody购自Biolegend,货号:108426;
FITC anti-mouse F4/80 Antibody购自Biolegend,货号:123108;
V450 Rat Anti- mouse CD11b Antibody购自Biolegend,货号:560455;
APC anti-mouse CX3CR1 Recombinant Antibody购自Biolegend,货号:153707;
PE anti-human CX3CR1 Antibody购自Biolegend,货号:341603;
Zombie NIR™ Fixable Viability Kit购自Biolegend,货号:423106;
APC Mouse IgG1, κ Isotype Ctrl Antibody购自Biolegend,货号:400120;
PE Rat IgG2b, k isotype Ctrl Antibody购自Biolegend,货号:400608。
实施例1 CX3CR1基因人源化小鼠的制备
本实施例对非人动物(如小鼠)进行改造,使该非人动物体内包含编码人源化CX3CR1蛋白的核苷酸序列,得到经遗传修饰的非人动物体内可表达人源化CX3CR1蛋白。小鼠CX3CR1基因(NCBI Gene ID:13051,Primary source:MGI:1333815,UniProt ID:Q9Z0D9,位于9号染色体NC_000075.7的第119877749位至第119897362位,基于转录本NM_009987.4及其编码蛋白NP_034117.3(SEQ ID NO:1))和人CX3CR1基因(NCBI Gene ID:1524,Primarysource:HGNC:2558,UniProt ID:P49238,位于3号染色体NC_000003.12的第39263494-39281735位,基于转录本NM_001337.4及其编码蛋白NP_001328.1(SEQ ID NO:2))。对比示意图如图1所示。
为了达到本发明的目的,可在小鼠内源CX3CR1基因座引入编码人CX3CR1蛋白的基因序列,使得该小鼠表达人或人源化CX3CR1蛋白。具体来说,可以通过基因编辑技术在小鼠内源CX3CR1基因座上用人CX3CR1基因的核苷酸序列(例如DNA序列、cDNA序列等)替换小鼠相应序列,如将至少包含小鼠CX3CR1基因的起始密码子ATG至终止密码子TGA的序列用对应的人DNA序列替换,得到人源化CX3CR1基因座(示意图如图2所示),实现对小鼠CX3CR1基因的人源化改造。
进一步设计如图3所示的打靶策略示意图,图中显示了靶向载体上含有小鼠CX3CR1基因上游和下游的同源臂序列,以及包含人CX3CR1 DNA序列的A片段。其中,上游同源臂序列(5’同源臂,SEQ ID NO:3)与NCBI登录号为NC_000075.7第119881401至119884783位核苷酸序列相同,下游同源臂序列(3’同源臂,SEQ ID NO:4)与NCBI登录号为NC_000075.7第119872544至119877060位核苷酸序列相同;人CX3CR1序列(SEQ ID NO:5)与NCBI登录号为NC_000003.12的第39265442至39266509位核苷酸序列相同。
靶向载体上还包括用于阳性克隆筛选的抗性基因,即新霉素磷酸转移酶编码序列Neo,并在抗性基因的两侧装上两个同向排列的位点特异性重组系统Frt重组位点,组成Neo盒(Neo cassette)。其中,Neo盒5’端与鼠的连接设计为5’-ACATGAACACACATGAAGAAACTCAGAGGACAAATTGCAGAAGTCAGGGCACCCTGTGAAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAAGTATA-3’(SEQ ID NO:6),其中,序列“TCAGGG”的最后一个“G”是鼠的最后一个核苷酸,序列“CACCCT”的第一个“C”是Neo盒的第一个核苷酸;Neo盒3’端与鼠的连接设计为5’-CGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCCTCCCTTTCCACCACATAGGTCCCAGGATCAAACTCCGGTTGAGGGGCTTGGT-3’(SEQ ID NO:7)内,其中序列“TGGATCC”中的最后一个 “C”是Neo盒的最后一个核苷酸,序列“CTCCCTTT”的第一个“C”是鼠的第一个核苷酸。此外,还在靶向载体3’同源臂下游构建了具有负筛选标记的编码基因(白喉毒素A亚基的编码基因(DTA))。改造后的人源化小鼠CX3CR1的mRNA序列如SEQ ID NO:8所示,表达的蛋白序列如SEQ ID NO:2所示。
靶向载体构建可采用常规方法进行,如酶切连接等。构建好的靶向载体通过酶切进行初步验证后,再送测序公司进行测序验证。将测序验证正确的靶向载体电穿孔转染入C57BL/6小鼠的胚胎干细胞中,利用阳性克隆筛选标记基因对得到的细胞进行筛选,并利用PCR(PCR引物详见表1)和Southern Blot技术进行检测确认外源基因的整合情况,筛选出正确的阳性克隆细胞,经PCR鉴定为阳性的克隆再进行Southern Blot(分别用SpeI或ScaI或BclI消化细胞DNA并使用3个探针进行杂交,酶、探针及目的片段长度如表2所示)检测,Southern Blot检测结果如图4所示,表明7个经PCR验证为阳性的胚胎干细胞(ES-01至ES-07)均为阳性的克隆,且无随机插入。
表1 PCR检测引物序列及目的片段长度
表2 Southern Blot酶和探针表
Southern Blot检测包括如下探针引物:
5’探针(5’Probe):
5’Probe-F:5’-ACGGGCATCATACTCTCCTCCACTT-3’(SEQ ID NO:13),
5’Probe-R:5’-AGAGAGCCTAGTCCAAGGTTACAGCA-3’(SEQ ID NO:14);
3’探针(3’Probe):
3’Probe-F:5’-AGCCCATTTTAACTCCCACCACCTG-3’(SEQ ID NO:15),
3’Probe-R:5’-CCAGGTGTACATTCCTAGCCTGCAC-3’(SEQ ID NO:16);
Neo探针(Neo Probe):
Neo Probe-F:5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO:17),
Neo Probe-R:5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO:18)。
将筛选出的正确阳性克隆细胞(黑色鼠)按照本领域已知的技术导入已分离好的囊胚中(白色鼠),得到的嵌合囊胚转移至培养液中短暂培养后移植至受体母鼠(白色鼠)的输卵管,可生产F0代嵌合体鼠(黑白相间)。将F0代嵌合鼠与野生型鼠回交获得F1代鼠,再将F1代杂合小鼠互相交配即可获得F2代纯合子鼠。还可将阳性鼠与Flp工具鼠交配去除阳性克隆筛选标记基因(该过程示意图见图5)后,再通过互相交配即可得到人源化CX3CR1基因纯合子小鼠。示例性的F1代小鼠的鉴定结果见图6,其中,编号为F1-01至F1-07的小鼠均为阳性杂合小鼠。PCR测定引物如表3所示,其中,图6A中的引物WT-F如SEQ ID NO:19所示,图6B中的引物WT-F如SEQ ID NO:21所示。
表3 PCR检测引物序列及目的片段长度
这表明使用本方法能构建出可稳定传代且无随机插入的CX3CR1人源化基因工程小鼠。可通过常规检测方法确认阳性小鼠体内人源化CX3CR1蛋白的表达情况,如流式细胞术检测(FACS)等。可通过常规检测方法确认阳性小鼠体内人源化CX3CR1蛋白的表达情况,如流式细胞术检测(FACS)等。选取9周龄野生型C57BL/6小鼠和CX3CR1基因人源化杂合子小鼠各1只,取脾脏细胞和骨髓细胞,用抗鼠CD45抗体Brilliant Violet 510™ anti-mouseCD45Antibody、鼠源T细胞表面抗体Brilliant Violet 711™ anti-mouse TCR β chainAntibody、抗鼠Gr-1抗体PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody、抗鼠F4/80抗体FITC anti-mouse F4/80 Antibody、抗鼠CD11b 抗体V450 Rat Anti- mouse CD11bantibody、抗鼠CX3CR1抗体APC anti-mouse CX3CR1 Recombinant Antibody(mCX3CR1)(以AP小大鼠抗体IgG1同型对照抗体 APC Mouse IgG1, κ Isotype Ctrl(IgG1)抗体作参照),或抗人CX3CR1抗体PE anti-human CX3CR1 Antibody(hCX3CR1)(以鼠抗体PE RatIgG2b, k isotype CtrlAntibody为参照)进行染色,将染色后的细胞进行流式检测,hCX3CR1在骨髓细胞中的检测结果如图7A和7B所示,野生C57BL/6小鼠(WT)骨髓细胞的单核细胞和巨噬细胞中只检测到mCX3CR1表达,杂合鼠(H/+)骨髓细胞的单核细胞和巨噬细胞中检测到mCX3CR1和hCX3CR1表达;hCX3CR1在脾细胞中的检测结果如图7C和7D所示,野生C57BL/6小鼠(WT)脾细胞的单核细胞和巨噬细胞中只检测到mCX3CR1表达,杂合鼠(H/+)脾细胞的单核细胞和巨噬细胞中检测到mCX3CR1和hCX3CR1表达,以上表明本实施例成功构建出可表达人CX3CR1蛋白的CX3CR1基因人源化小鼠。
实施例2 体内药效验证
利用本方法制得的CX3CR1人源化小鼠可以用于评估靶向人CX3CR1的调节剂的药效。例如,取CX3CR1人源化小鼠纯合子皮下接种小鼠结肠癌细胞MC38,待肿瘤体积生长到约100mm3后根据肿瘤体积分为对照组或治疗组,治疗组随机选择靶向人CX3CR1的药物X、Y、Z等,对照组注射等体积的生理盐水。定期测量肿瘤体积并称量小鼠的体重,可通过比较小鼠体重变化和肿瘤大小即可有效评估化合物的体内安全性和体内药效。
实施例3双基因或多基因人源化小鼠
利用本方法或制得的CX3CR1基因人源化小鼠还可以制备双基因修饰或多基因修饰的小鼠模型。如前述实施例1中,囊胚显微注射使用的胚胎干细胞可选择来源于含有PD-1、PD-L1、CTLA4、OX40、LAG3、TIM3、CD73等其它基因修饰的小鼠,或者,也可在人源化CX3CR1小鼠的基础上,利用分离小鼠ES胚胎干细胞和基因重组打靶技术,获得CX3CR1与其它基因修饰的双基因或多基因修饰的小鼠模型。也可将本方法得到的CX3CR1小鼠纯合子或杂合子与其它基因修饰的纯合或杂合小鼠交配,对其后代进行筛选,根据孟德尔遗传规律,可有一定机率得到CX3CR1基因与其它基因修饰的双基因或多基因修饰的杂合小鼠,再将杂合子相互交配可以得到双基因或多基因修饰的纯合子,利用这些双基因或多基因修饰的小鼠可以进行靶向人CX3CR1和其它基因调节剂的体内药效验证等。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
序列表
<110> 百奥赛图(北京)医药科技股份有限公司
<120> CX3CR1基因人源化的非人动物及其构建方法和应用
<130> 1
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 354
<212> PRT
<213> 小鼠(Mouse)
<400> 1
Met Ser Thr Ser Phe Pro Glu Leu Asp Leu Glu Asn Phe Glu Tyr Asp
1 5 10 15
Asp Ser Ala Glu Ala Cys Tyr Leu Gly Asp Ile Val Ala Phe Gly Thr
20 25 30
Ile Phe Leu Ser Val Phe Tyr Ala Leu Val Phe Thr Phe Gly Leu Val
35 40 45
Gly Asn Leu Leu Val Val Leu Ala Leu Thr Asn Ser Arg Lys Pro Lys
50 55 60
Ser Ile Thr Asp Ile Tyr Leu Leu Asn Leu Ala Leu Ser Asp Leu Leu
65 70 75 80
Phe Val Ala Thr Leu Pro Phe Trp Thr His Tyr Leu Ile Ser His Glu
85 90 95
Gly Leu His Asn Ala Met Cys Lys Leu Thr Thr Ala Phe Phe Phe Ile
100 105 110
Gly Phe Phe Gly Gly Ile Phe Phe Ile Thr Val Ile Ser Ile Asp Arg
115 120 125
Tyr Leu Ala Ile Val Leu Ala Ala Asn Ser Met Asn Asn Arg Thr Val
130 135 140
Gln His Gly Val Thr Ile Ser Leu Gly Val Trp Ala Ala Ala Ile Leu
145 150 155 160
Val Ala Ser Pro Gln Phe Met Phe Thr Lys Arg Lys Asp Asn Glu Cys
165 170 175
Leu Gly Asp Tyr Pro Glu Val Leu Gln Glu Met Trp Pro Val Leu Arg
180 185 190
Asn Ser Glu Val Asn Ile Leu Gly Phe Ala Leu Pro Leu Leu Ile Met
195 200 205
Ser Phe Cys Tyr Phe Arg Ile Ile Gln Thr Leu Phe Ser Cys Lys Asn
210 215 220
Arg Lys Lys Ala Arg Ala Val Arg Leu Ile Leu Leu Val Val Phe Ala
225 230 235 240
Phe Phe Leu Phe Trp Thr Pro Tyr Asn Ile Met Ile Phe Leu Glu Thr
245 250 255
Leu Lys Phe Tyr Asn Phe Phe Pro Ser Cys Asp Met Lys Arg Asp Leu
260 265 270
Arg Leu Ala Leu Ser Val Thr Glu Thr Val Ala Phe Ser His Cys Cys
275 280 285
Leu Asn Pro Phe Ile Tyr Ala Phe Ala Gly Glu Lys Phe Arg Arg Tyr
290 295 300
Leu Gly His Leu Tyr Arg Lys Cys Leu Ala Val Leu Cys Gly His Pro
305 310 315 320
Val His Thr Gly Phe Ser Pro Glu Ser Gln Arg Ser Arg Gln Asp Ser
325 330 335
Ile Leu Ser Ser Phe Thr His Tyr Thr Ser Glu Gly Asp Gly Ser Leu
340 345 350
Leu Leu
<210> 2
<211> 355
<212> PRT
<213> 人(human)
<400> 2
Met Asp Gln Phe Pro Glu Ser Val Thr Glu Asn Phe Glu Tyr Asp Asp
1 5 10 15
Leu Ala Glu Ala Cys Tyr Ile Gly Asp Ile Val Val Phe Gly Thr Val
20 25 30
Phe Leu Ser Ile Phe Tyr Ser Val Ile Phe Ala Ile Gly Leu Val Gly
35 40 45
Asn Leu Leu Val Val Phe Ala Leu Thr Asn Ser Lys Lys Pro Lys Ser
50 55 60
Val Thr Asp Ile Tyr Leu Leu Asn Leu Ala Leu Ser Asp Leu Leu Phe
65 70 75 80
Val Ala Thr Leu Pro Phe Trp Thr His Tyr Leu Ile Asn Glu Lys Gly
85 90 95
Leu His Asn Ala Met Cys Lys Phe Thr Thr Ala Phe Phe Phe Ile Gly
100 105 110
Phe Phe Gly Ser Ile Phe Phe Ile Thr Val Ile Ser Ile Asp Arg Tyr
115 120 125
Leu Ala Ile Val Leu Ala Ala Asn Ser Met Asn Asn Arg Thr Val Gln
130 135 140
His Gly Val Thr Ile Ser Leu Gly Val Trp Ala Ala Ala Ile Leu Val
145 150 155 160
Ala Ala Pro Gln Phe Met Phe Thr Lys Gln Lys Glu Asn Glu Cys Leu
165 170 175
Gly Asp Tyr Pro Glu Val Leu Gln Glu Ile Trp Pro Val Leu Arg Asn
180 185 190
Val Glu Thr Asn Phe Leu Gly Phe Leu Leu Pro Leu Leu Ile Met Ser
195 200 205
Tyr Cys Tyr Phe Arg Ile Ile Gln Thr Leu Phe Ser Cys Lys Asn His
210 215 220
Lys Lys Ala Lys Ala Ile Lys Leu Ile Leu Leu Val Val Ile Val Phe
225 230 235 240
Phe Leu Phe Trp Thr Pro Tyr Asn Val Met Ile Phe Leu Glu Thr Leu
245 250 255
Lys Leu Tyr Asp Phe Phe Pro Ser Cys Asp Met Arg Lys Asp Leu Arg
260 265 270
Leu Ala Leu Ser Val Thr Glu Thr Val Ala Phe Ser His Cys Cys Leu
275 280 285
Asn Pro Leu Ile Tyr Ala Phe Ala Gly Glu Lys Phe Arg Arg Tyr Leu
290 295 300
Tyr His Leu Tyr Gly Lys Cys Leu Ala Val Leu Cys Gly Arg Ser Val
305 310 315 320
His Val Asp Phe Ser Ser Ser Glu Ser Gln Arg Ser Arg His Gly Ser
325 330 335
Val Leu Ser Ser Asn Phe Thr Tyr His Thr Ser Asp Gly Asp Ala Leu
340 345 350
Leu Leu Leu
355
<210> 3
<211> 3383
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcatacaagt ctctcaggat aagcaacaat tcctgtttgc tcgtctgtca actatagata 60
tatgaaatat cacattttag gggatactta tttatttact tatttatttg taagacaaag 120
tctgaaatat cccaaattgg actgaagctt gctgaagctt gctgtgtagc caagaatgag 180
gtaaccttga actctgccag ttctcctgtc tccacctcct gattgctggg attacacatg 240
catactaata tgcctggttt gtgtgacgct gaggtccaag cccaaggctt catgctccct 300
aggcaagtac tcacaccgca gttacatcct cagccctgaa tgacacccgt tgggtccagc 360
gagtgctgtc agccagcagt ctggtactga ccagcttgaa cgctcctgag taagatcacc 420
acaaaggccc tctgcagaga aaggggcccc gtggcctgaa gcagagactg cctatgcaat 480
ccccgcagcc tgaactccat cacagcccga ggtggtccag agcagacagc ctcagcccag 540
tccactaggg gagagattgg ctggctggct ggctgacatg caaattcaca gacaggcaat 600
gcaggaagaa gccatggctg ggtgggtggg ctgcggtgag accattgtga gctgggtgcg 660
ctgggtgtgg aaactccctg ttaaaacccg tgagctttgc tttccattgc aatggggaag 720
gaggcaaagg cagccaggaa gtgcactctg agatgacttt agacttctga agggtctgtc 780
tgtggtttca agaggcagag gaggaagccc agagcagctt gacagagtgg ccaaccaaca 840
catcctgcct ggagcgtgag gggaagggga caggtgttat tacccaactt gtggggaaag 900
aatagggata ggctaggggt gagaatatcc caatagatga gccactgaaa gcaagagcag 960
caggctggct gagcccatcc tctgcctggt gtcagctgat gcttctctgg ctacagagcc 1020
catgctacaa caatgcttcc tttccaccta gagctgccag atggccaacc cagtgcgatt 1080
gggatggagg aactttccac acagaattaa ccctgacaaa atcctgggta ctatgagaca 1140
agagtgtagg gactacactg gttattttcc tcacaccagt gaccaaattc caggctagaa 1200
gcaactaagg atgaaagaag ttacagcttc ttctatgata gcctgggctg aggctgaggc 1260
tgaggctctg gaccacctcg ggccatgatg gaggtcaggc tgcaggacaa gcatagcagt 1320
ctttgcttca ggctgcagga ccttcttttt tttttaatta ggtattttct tcatttacat 1380
ttcaaatgct atcccaaaag tccccccgta ctgccccccc ccattcccct acccacccac 1440
tcccacttct tggccctggc gttcccctgt actgaggcat ataaagttta caagaccaag 1500
gggcctctct tcccaatgat ggcctactaa gccatcttct gatacatatg cagctagaga 1560
catgaactcg gggggtactg gttagttcat actgttgttc cacctatagg gttgcagttc 1620
ccttcagctc cttgggtact ttctctagct cctccattgg ggaccctgtg ttccatccaa 1680
tagctgactg tgagcatcca cttctgtgtt tgctaggccc cagcatagtc tcacaagaga 1740
cagctatatc agggtccttt cagcaaaatc ttgcttgtgt atgcaatggt gtcagtgttt 1800
ggaggctgat tatgggatgg atccccgggt gtggcagtct ctagatggtc catccttttg 1860
tctcagctcc aaactttgtc tctgtaactc cttccatggg tgttttgttc ccaattctaa 1920
gaaggggcaa ggtgtccaca ctttggtctt ccttcttctt gagtttcatg tgttttgcaa 1980
attgtatctt gtatcttggg tattctaaag gaccttcttc tctgagcacc tctgctgtga 2040
tcttagtaag ctggtcagta agctgcctca cagctggtgt gtccttcctc cccgaaagga 2100
agtggcatac tgactggtgg gatcactctg gtgtgaagtg gtgtgaagtc ctggcaggcg 2160
tgggaggcag caggttccta tgtgcctgta aatgggaaag ctcactcact ccttttctag 2220
gaggtttggg accctaacct atgcaatggt tccatccacg tttagagtgt tttggttttg 2280
tttttgtttt accaagacag aatttcactg tgtagccctg gatggcctgt aactcactct 2340
tgtagaccag gctggcctca gactcagaaa tctgccagcc tctgcctacc aagtgctaag 2400
attaaaggcg tgtactacca ggcccctggc tacagtgggt ctccctacct tattaactca 2460
atatttaaaa tccctcccat ggggctggag agatggctca ggagtttaga gcattagctg 2520
ctcttccaga ggacttaggt tcaattccca gaacctacat ggtggctccc aaccatctgt 2580
tttcaaaggt tccaacaccc tcttctggtc tgggcaccag gcacaaccat ggtgctggcc 2640
agtcatgcac acatgtaaat aaataaatct ttgtaaaaat tataaataaa ttaggcccct 2700
cacagatgtg ctgagatgtg catttccatg gagattctaa attccatcaa agagctgaga 2760
gatgcaaatg ctattctcaa agaggaatca gtccgggtgg ctcccatgtc agggtggccc 2820
ccatgtcagg gtggcctaca aacacctgta agtccaactc taggggatcc attgccccct 2880
tcaggactca acaaacacct gcactccatg tgccttccca cacacaaacc cacatgcaca 2940
taattttaaa taaaatcata tttaaaaaaa aaagaaccag cgtagtatgg gctcatatct 3000
ttgatcccag cattcaggag acagagacag acagatctct gtgagttgca gaccaacagg 3060
gttccaagca agtcagggtt tcacaatgag accctgttgc aaaaataaaa ataaaaacag 3120
aaatgtttta aaagatagga tgagtgaaga caaaatctag ttccaattgt tcaccctttc 3180
agtgttttct cccgcttgct gcatgcagcc agtgagaacc gcgatcctct aagactcacg 3240
tgatctggtt tgctgcatac agccagtgag aactgcgatc ctctaagact cacgtggacc 3300
tgcttactgc atgcagccag tgagaactac aatcctttaa ggctcacgtg atctggtttc 3360
tccttcccct ccaggacctc acc 3383
<210> 4
<211> 4517
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctccctttcc accacatagg tcccaggatc aaactccggt tgaggggctt ggtggcaatt 60
gtctttacct gatgaaccat ctctccaggc ccgctttagt ttttgcatac acatgacctt 120
catttgataa tgacaatttg ttacaagtct ttacgaagca catatcctag cgggctccag 180
agatgtttcc acagcagtga gaccacacct ctgttctcag aggttttgat ttattttttg 240
agatagagtc tcactttgta gaccaggctg ccctggaact cggaaatctg cctgcctctt 300
cccctggagt gttgggatta aaggtgtgcg ccaccaccct gggcttgata tctgtggaca 360
gaaaaggcct ggctgtgttt aacttgcttg ctgtaagaca gagcagaggt aataagatct 420
gacccaagac ttcagggata tgagcttctc caggagaagc aggggctgtt tgctaggagg 480
cagggatgtg gaattcgtgt gagagagcac gaggttcctt cagtgtcagt ccctcattcc 540
aactgatgag cctgagagtt gtcatcaaag gttaatctgt tcctggactc ccatgtagag 600
aagaaagact tctggcctga gaagcaaaca tagggaagca aacattttct tttctacaaa 660
caaactcatt gcactgagca agtgagagag gccagtcaat aaaagggctt gccaccgagt 720
ctgtcaacct gagtcccatc ctcaggactc ccgtggggca aggagagaaa cacctgcact 780
tagcgccaca caggtaccat ggtacacgca cacgcacatg caataaaaat aaaataataa 840
atccgtatcc ttcattttct catttgcttt gctgaacaag acaggatggg acaaacacaa 900
agagtttcgt tggaaatccc cacatggcaa ggacacattt gaagcaactg gcctttgaca 960
tataactggg tcaggcctac tgctgcctct gagcttggag ctccaaatag caaggagtga 1020
atggcggtgt gctctggagg caggagacat ggaggcagcg cacaccagag gccttcagca 1080
agttctgcac cttctcagga gctcaaagtt cttatcagag agaagggaaa acagagcttg 1140
ttctagtgtc tcaaggcatt aataagaccc tagactcaca tctaactaca aagctcgagc 1200
aagcagccct gcttttcttg gccctccctt tctgcgggag gacacaccca acagctagtt 1260
tttgcttctg tttttcttag ttccagggtc acagattact gagtggtcag aagttaactc 1320
agtctatcgc aggctttagc taggcttcct tttgtgaagg tgtggtttgg gctctgtgtg 1380
tgtgtttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgcaaa 1440
tgtcagtgtg tatgtatgtg agtgtgtgtg tgcatatgtg tgtgggtatg tatgtatatg 1500
agagagtgtg tgtgaatgtc agtgtgtatg tatgagagta tgtgtgagtg tgtgtgtgtt 1560
tgtgtgtgag tgtatgtatg tgttcatgta tgtgagagtg tgtgtgtttg tgtctatgtg 1620
tgagtgcgag tgttcctgtg cgtgcttgag aaagagagac agaggcagag gtcagtcttg 1680
ggtttcattc tgggagtcct ccatcttgct ttttgagaca gagtctgagt ccctctccct 1740
ctctccctcc cactgtcccc ctctctctgg ggtctgagtt tccacaatta ggctaggatg 1800
gctggcaagg agcccaaggg atctgcctgt ctctgactcc tctgtatggg acaaagcttg 1860
cataccagcc ttttacactt ggggtcaagc tcgggtcctt gagattgcac agtggacact 1920
ctaccgacca ggttacatcc gcagccccca aatgcatact ttgtagctaa ggaaaaggta 1980
tccagccagt caggactcag agcacagtag gaaaaggtgc atcctagctc cccatctgcc 2040
ctgcctgccc tgcctgccct gcctgccctg cctgccctgc ctgccctgcc tgccctgcct 2100
gccctgcctg cctccatctt ggtcacagta tatatcatcc agcctttagg agtggagacc 2160
acaggcggat aacaggaaaa aaaaaaaaac tggagaagat ggtctgcagg tgtgtagaca 2220
cagagaggct gaggcatatg acccggacca caggtaagtc ttgcccacag gtgtgggctg 2280
tgctgcaacc ttccctgatg tcagtatgca cagagatcat cagcaaaagg acaggatgcc 2340
cagaggcaaa tgcagtcagc agccttggga agggcccaga cctcacacag gcacggtaga 2400
tgctggggag agctatgccc aggtgctgca taatgtcaag tccacccact tctgccttag 2460
tccatcctca ttcagggatg gtccacagac tgacttccca ccaccacccc cgggggcctt 2520
gccactgaga aagatggacc atgtgttgtt tctgttcctg ggaggaactt ttggttcata 2580
ctgtgcattg cagctggggt cctaatttcc catccatcgg tctctgaagc tttgtccttg 2640
cgtgtccaga gagtctactt ctggccccat aaatgacaac tacccatggg cgtacacata 2700
gcagcttgat tgagaggccc atcataggaa caccaacccc actcctgccc tcagaaacgc 2760
caggggagtc tttcctgact cagaacctat ttttctttcc cactgcaccc cacggggacc 2820
actgcagcta cagtttgact ggaagctgtt gtttatataa ctggcagctt catttgggat 2880
aatcccaata ctggtgtata gaaggtggtc tgtagccagt ttcctgtcac tggagcctct 2940
gaggtccagg gaggcgccag ttgttttcta gagtccgtga ggcgccatgc ctccacatct 3000
cagctagtaa agcagaaggg cctgagttca agtctcaaat cctgtgtaaa agagatgggt 3060
gcagggcagg tgagagggcg caacaagtca aggtgcttgc tgccaagcct gaggacctga 3120
gttcgatccc tgataaccgc atgggggagg gaaagaactg actcccacaa gctgtcttct 3180
gaccttacat gtacacacat gtacatacaa taaataagca ggtacacaca tgtacgtaca 3240
ataaataaat aggtaatgtg gaagaggagg aaaggaaaag ctggatgtga tgcgcaagtt 3300
tgtaatctgc gtgtgtgcat gcgtgtgtgt gtgtctgtgt ctgtgtcatg tgtgtgtgtc 3360
tgtgtgtgtg tctatatgtg tgtgtcatgt gtgtgtatgt gtgtctctgt gtgtatgtgt 3420
gtcatgtgtg tgtctgtgtg tctgtgtgtg tgtctttgtg tcggtgtgtg tctgtgtgcg 3480
tgtgatgtac atgacacaca cacatactcc tctgcctctg actgagcaaa acccagaaca 3540
aagagtgtcc atgcatcata tgtggaacaa taggcccttt gaaaaaagac aaagagaaaa 3600
gggaggccat cgttgcctct cttagactta gctgagaagg cctgaggaga ggacagaaga 3660
tggggtagga aaacctgtct tatagttttg gttgtaagcc taacctttaa tggctgggcc 3720
atcttcagcc caggaaagtt gagctttgag gaggttgtca ggtaattttg ggagcctgag 3780
gctgctggaa acagaacttt ggggcacaag ggttgggaag ggccatttgt gggtgaagaa 3840
cagggagagg gtaacctgga ggattggctg cacaaagtag gtgggaaaag cagggctgaa 3900
gggacatgtc acgacaagct attagagtcc tgcagaataa gtccagattg acaaagtcaa 3960
gcacattcct gacagtgtct gaagctgttt tctcctgcct tctagccagt gtgcatcttc 4020
aagtatgcac ctgctgtttt ccacacttct ctgatagccc tcttccattc cggcatccat 4080
ctggcacacc acacaaaaag ctgcacttgt cagagtgtta gggaaaacac ttaggaagtt 4140
gggcagctgg gtgaagcggg agccttcaga ccacgaacat gcccaagtgt tctctccctg 4200
ggcccctttc tctatgtgtg ttagtccttt cctacacctg ccctgaagcc atgcttccct 4260
ccctcaacag gccaaggagg ggaaggggag aggctatgta ggatgatggg ggatgggaga 4320
gggatagtgt ctgacttgga caccctatgt tctggctccc aggcctcctg cttccccgcc 4380
tggaccttca gcaatgtggt ccaagcagga gtttccccca gaacccaggc cccgtgtgct 4440
tggaagagca gaaactgagc agccctgttt gcacgggacg gtaggcaact tcacccaccc 4500
agcaccagcc ttcactc 4517
<210> 5
<211> 1068
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggatcagt tccctgaatc agtgacagaa aactttgagt acgatgattt ggctgaggcc 60
tgttatattg gggacatcgt ggtctttggg actgtgttcc tgtccatatt ctactccgtc 120
atctttgcca ttggcctggt gggaaatttg ttggtagtgt ttgccctcac caacagcaag 180
aagcccaaga gtgtcaccga catttacctc ctgaacctgg ccttgtctga tctgctgttt 240
gtagccactt tgcccttctg gactcactat ttgataaatg aaaagggcct ccacaatgcc 300
atgtgcaaat tcactaccgc cttcttcttc atcggctttt ttggaagcat attcttcatc 360
accgtcatca gcattgatag gtacctggcc atcgtcctgg ccgccaactc catgaacaac 420
cggaccgtgc agcatggcgt caccatcagc ctaggcgtct gggcagcagc cattttggtg 480
gcagcacccc agttcatgtt cacaaagcag aaagaaaatg aatgccttgg tgactacccc 540
gaggtcctcc aggaaatctg gcccgtgctc cgcaatgtgg aaacaaattt tcttggcttc 600
ctactccccc tgctcattat gagttattgc tacttcagaa tcatccagac gctgttttcc 660
tgcaagaacc acaagaaagc caaagccatt aaactgatcc ttctggtggt catcgtgttt 720
ttcctcttct ggacacccta caacgttatg attttcctgg agacgcttaa gctctatgac 780
ttctttccca gttgtgacat gaggaaggat ctgaggctgg ccctcagtgt gactgagacg 840
gttgcattta gccattgttg cctgaatcct ctcatctatg catttgctgg ggagaagttc 900
agaagatacc tttaccacct gtatgggaaa tgcctggctg tcctgtgtgg gcgctcagtc 960
cacgttgatt tctcctcatc tgaatcacaa aggagcaggc atggaagtgt tctgagcagc 1020
aattttactt accacacgag tgatggagat gcattgctcc ttctctga 1068
<210> 6
<211> 103
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acatgaacac acatgaagaa actcagagga caaattgcag aagtcagggc accctgtgaa 60
gcttgatatc gaattccgaa gttcctattc tctagaaagt ata 103
<210> 7
<211> 115
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgaagttcct attctctaga aagtatagga acttcatcag tcaggtacat aatggtggat 60
ccctcccttt ccaccacata ggtcccagga tcaaactccg gttgaggggc ttggt 115
<210> 8
<211> 3756
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
tagagaggga agcacttgcc tctggtggag tctgcgtgag actgggtgag tgactggcac 60
ttcctgcaga agttcccttc ccatctgctc aggacctcac catggatcag ttccctgaat 120
cagtgacaga aaactttgag tacgatgatt tggctgaggc ctgttatatt ggggacatcg 180
tggtctttgg gactgtgttc ctgtccatat tctactccgt catctttgcc attggcctgg 240
tgggaaattt gttggtagtg tttgccctca ccaacagcaa gaagcccaag agtgtcaccg 300
acatttacct cctgaacctg gccttgtctg atctgctgtt tgtagccact ttgcccttct 360
ggactcacta tttgataaat gaaaagggcc tccacaatgc catgtgcaaa ttcactaccg 420
ccttcttctt catcggcttt tttggaagca tattcttcat caccgtcatc agcattgata 480
ggtacctggc catcgtcctg gccgccaact ccatgaacaa ccggaccgtg cagcatggcg 540
tcaccatcag cctaggcgtc tgggcagcag ccattttggt ggcagcaccc cagttcatgt 600
tcacaaagca gaaagaaaat gaatgccttg gtgactaccc cgaggtcctc caggaaatct 660
ggcccgtgct ccgcaatgtg gaaacaaatt ttcttggctt cctactcccc ctgctcatta 720
tgagttattg ctacttcaga atcatccaga cgctgttttc ctgcaagaac cacaagaaag 780
ccaaagccat taaactgatc cttctggtgg tcatcgtgtt tttcctcttc tggacaccct 840
acaacgttat gattttcctg gagacgctta agctctatga cttctttccc agttgtgaca 900
tgaggaagga tctgaggctg gccctcagtg tgactgagac ggttgcattt agccattgtt 960
gcctgaatcc tctcatctat gcatttgctg gggagaagtt cagaagatac ctttaccacc 1020
tgtatgggaa atgcctggct gtcctgtgtg ggcgctcagt ccacgttgat ttctcctcat 1080
ctgaatcaca aaggagcagg catggaagtg ttctgagcag caattttact taccacacga 1140
gtgatggaga tgcattgctc cttctctgaa ggggtctccc cgaccctagc tccactagga 1200
acccagagtt cttgcatcag atttccctgc cgctccccct gcatcttatg tgcaagaaat 1260
atggaccaga tgcctgcaaa ccaaccccgt ggtgtttttt tgaaaaattt atgttcaatg 1320
tgtgaaaaac acacgtatct cttactgcaa atgttgaaca ttggggctta ctggtgacaa 1380
aaattctaac cagattagtg caattacaaa ggggtttggt gagtcctggt tgcatgatca 1440
tgtgataaag gacaactaag tcctcagact gagtggaaac caaggcttgg ctccaatgtc 1500
ccctctctga ccttcagatc cttcatagtg acagatcatc caggttctat catcagagaa 1560
ggaccacatc tctctgattt caaaattggt attcctaggg aacacctccg ttggccgagt 1620
gtgtcgggtg tccattcctt gactaggtgg tgttattgaa atagaaggga tacctaagat 1680
gctgttggaa tctcaaggtt agcggttgag agagacacat ctctcagaag ctggggggtg 1740
ggagctactc tgacagcaag aactgctgac tcgccttacc atggagctca ttcaggctcc 1800
ccttcagtaa ctagcagtat ctgttgctag cttctttaat cttctgttga gaatgtcctg 1860
aactctccaa gggttagaat ttgggttact gctcacagca tcaaattcaa tcccaaggcc 1920
ctgtcctcca agaccaggaa gataggatgc agttctaaca agagactcca cgctgactcc 1980
tcattccaca ggactccgtc cacccagttg gccatgtccc tttttcttgt cttgacccac 2040
atccctccac tcttctcagc ccaggggaag aaatagaaag agccatgccc cagagtgaag 2100
ggatcgattc atatcccaac tctgttgctt gcacattggg gagactggag ccaacagagc 2160
ttcagtccct tcaatataga aaggggactg tggtgccttc atccattcta gcatagtacc 2220
tcagcaagga ctttagcatt ccctggatca ggaactgtta ccctgcctct gagaaatgga 2280
gctggtggag tgggaggaac ctgcaggctt tgctgcagga cacagccaga caagaggaga 2340
cctattccta cagcatcccc caacatgtgc ctagatctac aaagccattc ccatgtcccc 2400
gctgtcaggg caagacccag gccacgagag cagcttgctt tctcccctga ccctgcaagc 2460
atcacgtagg atgtagccac aagaatgtct tcatgcattc actgactccg gtctcatttg 2520
caggcttaca ttgaaagttc cagttctgca acttgctttt taaaaaacac tggatttcag 2580
gggctggaga gatggctcag tggttaagag cacagtctgc tcctacagag gtcccaagtt 2640
caattcccat gtcatgtctg aagacaagga cagtgtagtc aaaaaaaaca ctggaattca 2700
aatagctccc tgttcctgtg ttggttgtga taaccatttc agaagtctct cccagcctgt 2760
tgctcacggc ggcatgtctg atatctcctt ggcagtctgt atgtttgtgt cgaggatgac 2820
agacagactg agtcatatcc cccacaggca tatccaaccc tttgttattg caacaccatg 2880
ctgtcatatt caaaggtcac aaatgagaac ctcaaaattg agttaccaaa gtgggagcct 2940
ggagagatgg ctcagtggtt aatagcactg gctgctcttc cagaggtctg gggttggatt 3000
cccagcaagc atagtagctt acaatggtct ataatataac tctagttcca ggtgacccaa 3060
caccctcttc taatttgtga gggtagcaga catgtgcatt gtgaacattc aggcaaacac 3120
ccatacatac aaaataagta aaaaagaaaa agaaaggaag gaatggagag agagaaagag 3180
ggggagagag agagggagag ggagagggag agggagaggg agggagggag agggagggag 3240
ggagggaggg agggagggag ggagggaggg aaaggaaaga aagaaagaaa gaaaggaagg 3300
aaggaaggaa ggaaggaagg aaggaagaaa gaaagaaaga gggagagaga gaaagagaga 3360
aagagagaaa gagaaagaga gaaagagaga aagagaaaga gagaaagaga gaaagaagaa 3420
agaaagacag aaaattggtt tttcaagata ggttctctct atgtagcctc ggctgtcctg 3480
aaactctctg tgtagaccag gctggcctag agctcaaaga aatccacctg cctctgcctc 3540
ctgagtactg ggatcgaagg tctgtggcac catggcaggc ttagtcaata aatgttttat 3600
tttcaaaagt atcctataat gttttaagta agtttatgct ttggtgttgg tctgtatttc 3660
ccgctgtctc gggtcacatg gttaagcgtg cctagagtgt gtctatccca cttgtaattc 3720
tgtcaataaa cattggtttc cttccagctc ttggca 3756
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctctccttag actagctcgt ggt 23
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggccaggt acctatcaat gctg 24
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cgcattgtct gagtaggtgt c 21
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgggggatta gctgtcccct aagag 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
acgggcatca tactctcctc cactt 25
<210> 14
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agagagccta gtccaaggtt acagca 26
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
agcccatttt aactcccacc acctg 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccaggtgtac attcctagcc tgcac 25
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggatcggcca ttgaacaaga t 21
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cagaagaact cgtcaagaag gc 22
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcctacaaac acctgtaagt ccaac 25
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aggccctcat ggctgatgag g 21
<210> 21
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cctttcagtg ttttctcccg cttgc 25
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gccaatggca aagatgacgg ag 22
<210> 23
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
acacacatga agaaactcag aggaca 26
<210> 24
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
actgctgtgg aaacatctct ggagc 25
<210> 25
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gacaagcgtt agtaggcaca tatac 25
<210> 26
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gctccaattt cccacaacat tagt 24
Claims (6)
1.一种CX3CR1基因人源化的非人动物的构建方法,其特征在于,所述的构建方法包括使用靶向载体进行非人动物的构建,其中,所述的靶向载体包含编码SEQ ID NO:2所示氨基酸的核苷酸序列,所述的靶向载体还包含5’臂和3’臂,所述的5’臂的核苷酸序列如SEQ IDNO:3所示,所述的3’臂的核苷酸序列如SEQ ID NO:4所示,所述的构建方法包括使用靶向载体替换非人动物CX3CR1基因中编码SEQ ID NO:1所示氨基酸的核苷酸序列,所述非人动物的编码SEQ ID NO:2所示氨基酸的核苷酸序列通过内源性调控元件调控,所述的非人动物内源CX3CR1蛋白的表达降低或缺失,所述的非人动物为小鼠。
2.根据权利要求1所述的构建方法,其特征在于,其中所述的编码SEQ ID NO:2所示氨基酸的核苷酸序列如SEQ ID NO:5所示。
3.根据权利要求1或2任一所述的构建方法,其特征在于,所述的非人动物体内表达人源化CX3CR1蛋白,所述的人源化CX3CR1蛋白的氨基酸序列如SEQ ID NO:2所示。
4.根据权利要求1或2任一所述的构建方法,其特征在于,所述的非人动物的基因组中包含人源化CX3CR1基因,所述的人源化CX3CR1基因转录的mRNA的核苷酸序列如SEQ ID NO:8所示。
5.一种CX3CR1基因的靶向载体,其特征在于,所述的靶向载体包含编码SEQ ID NO:2所示氨基酸的核苷酸序列或包含SEQ ID NO:5所示核苷酸序列,所述的靶向载体还包含5’臂和3’臂,所述的5’臂的核苷酸序列如SEQ ID NO:3所示,所述的3’臂的核苷酸序列如SEQ IDNO:4所示。
6.权利要求1-4任一所述的构建方法获得的CX3CR1基因人源化的非人动物在CX3CR1基因或蛋白相关研究中的应用,所述应用不是疾病的诊断和治疗方法,所述的应用包括:
a)涉及人类细胞的免疫过程的产品开发,制造或筛选人类抗体中的应用;
b)作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;
c)涉及人类细胞的免疫过程的生产和利用动物实验疾病模型,用于病原学研究中的应用;
d)在体内研究人CX3CR1信号通路调节剂的筛选、药效检测、评估疗效、验证或评价;或者,
e)研究CX3CR1基因功能,研究人CX3CR1抗体,研究针对人CX3CR1靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤或炎症药物方面的用途。
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