CN111808882A - Il7r基因人源化改造的动物模型的构建方法及其应用 - Google Patents
Il7r基因人源化改造的动物模型的构建方法及其应用 Download PDFInfo
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- CN111808882A CN111808882A CN202010901786.XA CN202010901786A CN111808882A CN 111808882 A CN111808882 A CN 111808882A CN 202010901786 A CN202010901786 A CN 202010901786A CN 111808882 A CN111808882 A CN 111808882A
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Abstract
本发明提供了一种IL7R基因人源化改造的动物模型的构建方法、一种人源化IL7R蛋白、一种人源化IL7R基因、一种IL7R基因的靶向载体和其在生物医药领域的应用,利用同源重组的方式将部分编码人IL7R蛋白的核苷酸序列导入非人动物基因组中,该动物模型体内能正常表达人源化IL7R蛋白,可以用于人IL7R信号机理研究、肿瘤及免疫相关疾病药物筛选,对免疫靶点的新药研发具有重要的应用价值。
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IL7R基因人源化改造的动物模型的构建方法和在生物医药领域的应用。
背景技术
白细胞介素7受体(interleukin 7receptor,IL7R)属于Ⅰ型细胞因子受体家族成员的跨膜受体,由α链(IL7Rα,CD127)和γ链(γc,CD132)组成,α链是IL7R的特异性组分;γ链是IL2、IL4、IL7、IL9、IL15、IL21等细胞因子受体的共同组分,也被称为共有γ链,是启动IL7R下游信号表达的必需成分。生理情况下,IL7R是淋巴细胞增殖和分化的必需关键因子,IL7R在细胞表面缺失会导致B系和T系淋巴细胞生成障碍;病理情况下,IL7R功能缺陷可导致重症联合免疫缺陷病,而IL7R获得性突变可引起急性T淋巴细胞白血病等。
IL7R主要在淋巴前体细胞、B祖细胞、T细胞、胸腺细胞、髓样细胞及单核细胞表达。此外,IL7R还在多种肿瘤组织、自身免疫性疾病及其进展过程中异常表达。已有研究表明干预IL7R的信号会影响和预防急性T淋巴细胞白血病复发。小鼠实验表明施用IL-7R抗体可缓解II型胶原诱导性关节炎模型(CIA)的临床症状,减轻关节炎性细胞浸润以及骨和软骨的破坏,这为IL-7R作为治疗新靶点提供了支持。
实验动物疾病模型对于研究人类疾病发病机制、开发防治技术及药物是不可缺少的研究工具。由于人IL7R序列与啮齿类动物中的对应蛋白存在显著差异,如人IL7R和小鼠IL7R蛋白序列的一致性仅为64%,识别人IL7R蛋白的抗体通常无法识别小鼠IL7R,即无法用普通小鼠来筛选和评价靶向IL7R药物的药效。鉴于IL7R相关药物全球研发进展及靶向该信号通路的巨大应用价值,为了使临床前期试验更有效并使研发失败率最小化,本领域急需开发人源化IL7R相关的非人动物模型,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。
发明内容
本发明的第一方面,提供了一种IL7R基因人源化改造的动物模型的构建方法,所述的动物模型的基因组中包括编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列。
进一步优选的,所述的动物模型的基因组中包括NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
优选的,所述的动物模型的基因组中包含人IL7R核苷酸序列的1号外显子的部分、2号至5号外显子的全部和6号外显子的部分,进一步优选的,包含1-2号内含子和/或5-6号内含子,更优选的,包含1-6号外显子之间的任一内含子;其中,所述人IL7R核苷酸序列的1号外显子的部分至少包含从编码信号肽的核苷酸序列开始至1号外显子最后1个核苷酸序列为止,6号外显子的部分至少包含从6号外显子第一个核苷酸开始至编码胞外区的最后一个核苷酸序列为止。
优选的,所述的构建方法包括插入、翻转、敲除或替换。优选为原位替换。
优选的,所述的构建方法包括用包含人IL7R核苷酸序列的1号至6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IL7R基因座上,进一步优选的,用包含人IL7R核苷酸序列的1号外显子的部分、2号至5号外显子的全部和6号外显子的部分核苷酸序列插入或替换到非人动物IL7R基因座上,更优选的,包含1-2号内含子和/或5-6号内含子,更进一步优选的,包含1-6号外显子之间的任一内含子;其中,所述人IL7R基因的1号外显子的部分至少包含从编码信号肽的核苷酸序列开始至1号外显子最后1个核苷酸序列为止,6号外显子的部分至少包含从6号外显子第一个核苷酸开始至编码胞外区的最后一个核苷酸序列为止。
优选的,所述的构建方法包括用包含编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或者包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列替换至非人动物IL7R基因座。
在本发明的一个具体实施方式中,所述的构建方法包括用包含编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或者包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列替换至非人动物IL7R基因的相应区域。
优选的,所述的构建方法包括用包含人IL7R核苷酸序列的1号至6号外显子全部或部分替换非人动物IL7R核苷酸序列的1号至6号外显子的全部或部分;其中,所述非人动物IL7R核苷酸序列包含编码非人动物1号外显子的部分、2号至5号外显子的全部、6号外显子的部分核苷酸序列,优选的,包含1-2号内含子和/或5-6号内含子,进一步优选的,包含1-6号外显子之间的任一内含子,其中,所述非人动物IL7R核苷酸序列的1号外显子的部分至少包含从编码信号肽的核苷酸序列开始至1号外显子最后1个核苷酸为止,6号外显子的部分至少包含从1号外显子第1个核苷酸开始至编码胞外区最后1个核苷酸为止。
优选的,所述的构建方法包括用包含所述人源化IL7R基因的核苷酸序列插入或替换到非人动物IL7R基因座上。
优选的,所述的构建方法包括用包含编码所述人源化IL7R蛋白的核苷酸序列插入或替换到非人动物IL7R基因座上。
优选的,所述的插入或替换位点为IL7R基因的内源调控元件之后。
优选的,所述的插入为首先破坏非人动物内源IL7R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IL7R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,所述的动物模型中人源化IL7R基因是纯合或杂合的。优选的,所述动物模型的基因组中至少一个染色体上包含人源化IL7R基因。
优选的,所述的动物模型中至少一个细胞表达人或人源化IL7R蛋白。
优选的,使用基因编辑技术进行IL7R基因人源化改造的动物模型的构建,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。
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,使用靶向载体进行IL7R基因人源化改造的动物模型的构建,其中,所述的靶向载体包含人IL7R的1号至6号外显子的全部或部分核苷酸序列;进一步优选的,包含1号外显子的部分、2号至5号外显子的全部和6号外显子的部分,更优选的,包含1-2号内含子和/或5-6号内含子,更进一步优选的,包含1-6号外显子之间的任一内含子,其中,所述人IL7R的核苷酸序列的1号外显子的部分至少包含从编码信号肽开始至1号外显子最后一个核苷酸为止,6号外显子的部分至少包含从6号外显子第一个核苷酸开始至编码胞外区最后1个核苷酸为止,更进一步优选的,所述靶向载体包含编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
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IL7R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_000081.6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IL7R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_000081.6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IL7R基因座上。进一步优选的,位于非人动物IL7R基因的1号外显子至6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,鉴定筛选获得动物模型。
优选的,所述的动物模型体内表达人或人源化IL7R蛋白,同时内源IL7R蛋白的表达降低或缺失。
优选的,所述的人源化IL7R蛋白包含人IL7R蛋白的信号肽和/或胞外区,更优选的,包含与SEQ ID NO.2第1至239位或SEQ ID NO.8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第1至239位或SEQ ID NO.8所示氨基酸序列一致的氨基酸序列。
优选的,所述的人源化IL7R蛋白还包含非人动物IL7R蛋白的部分,优选为非人动物IL7R蛋白的跨膜区和/或胞质区。
在本发明的一个具体实施方式中,所述的人源化IL7R蛋白包含下列组中的一种:
a)SEQ ID NO.8或SEQ ID NO.2第1至239位所示氨基酸序列的部分或全部;
b)与SEQ ID NO.8或SEQ ID NO.2第1至239位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.8或SEQ ID NO.2第1至239位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.8或SEQ ID NO.2第1至239位所示的,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,所述的动物模型的基因组中包含人源化IL7R基因,所述的人源化IL7R基因编码人源化IL7R蛋白。
优选的,所述的人源化IL7R基因包含NCBI登录号为NC_000005.10的第35856978-35874459 位的核苷酸序列,进一步优选的,所述的动物模型中包含的IL7R基因转录的mRNA序列包含SEQ ID NO.7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IL7R基因包含下列组中的一种:
a)人源化IL7R基因的mRNA序列为SEQ ID NO.7所示的序列的部分或全部;
b)人源化IL7R基因的mRNA序列与SEQ ID NO.7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IL7R基因的mRNA序列与SEQ ID NO.7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IL7R基因的mRNA序列具有SEQ ID NO.7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。
本发明的第二方面,提供了一种IL7R基因人源化改造的动物模型,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,提供了一种靶向载体,所述的靶向载体包含人IL7R核苷酸序列的部分,优选的,所述的靶向载体包含编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或者NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
优选的,所述的人IL7R核苷酸序列的部分包含人IL7R的1号至6号外显子的全部或部分核苷酸序列;进一步优选的,包含1号外显子的部分、2号至5号外显子的全部和6号外显子的部分,更优选的,包含1-2号内含子和/或5-6号内含子,更进一步优选的,包含1-6号外显子之间的任一内含子,其中,所述人IL7R的核苷酸序列的1号外显子的部分至少包含从编码信号肽开始至1号外显子最后一个核苷酸为止,6号外显子的部分至少包含从6号外显子第一个核苷酸开始至编码胞外区最后1个核苷酸为止。
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IL7R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_000081.6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IL7R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_000081.6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IL7R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IL7R基因1号至6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IL7R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IL7R基因改造的人源化细胞,所述的人源化IL7R基因改造细胞的基因组中包括人IL7R基因的1号外显子至6号外显子。优选的,所述的人IL7R基因编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列,其通过内源性IL7R调控元件调控;该人源化IL7R基因改造细胞体内表达人或人源化IL7R蛋白,同时内源IL7R蛋白的表达降低或缺失。优选的,所述的人IL7R基因通过内源性IL7R调控元件调控。
本发明的第七方面,涉及一种IL7R基因缺失的细胞,所述的IL7R基因缺失的细胞缺失内源IL7R基因的1号外显子至6号外显子。
本发明的第八方面,提供了一种多基因修饰的非人动物的构建方法,包括如下步骤:
(a)应用上述的构建方法制备获得动物模型;
(b)将步骤(a)制备获得的动物模型与除IL7R外的人源化动物交配、体外授精或直接进行基因编辑,并进行筛选,得到多基因人源化改造动物。
优选的,所述的其他基因修饰的非人动物包括但不限于基因PD-1人源化的非人动物。
优选的,所述的多基因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,所述的多基因修饰的非人动物的基因组中人源化的多个基因中的每一个基因均可以是纯合或杂合的。
优选的,所述的其他基因优选为PD-1。
本发明的第九方面,涉及上述的制备多基因人源化改造动物的方法制备获得的多基因修饰非人动物或其后代。
本发明的第十方面,涉及一种荷瘤动物模型,所述的动物模型的制备方法包括通过上述的人源化IL7R基因改造动物模型或上述的制备多基因人源化改造动物的方法制备动物的步骤。
优选的,所述的荷瘤动物模型的制备方法还包括在上述方法制备的人源化基因改造动物或其后代植入肿瘤细胞的步骤。
本发明的第十一方面,提供了一种上述的构建方法获得的动物模型或者上述的构建方法获得的多基因修饰非人动物在制备荷瘤动物模型中的应用。
本发明的第十二方面,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构建方法获得的IL7R基因人源化改造的动物模型、上述的IL7R基因人源化改造的动物模型、上述的构建方法制备获得的多基因修饰非人动物、上述的多基因修饰非人动物或其后代或上述的荷瘤动物模型。
本发明的第十三方面,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构建方法获得的IL7R基因人源化改造的动物模型、上述的IL7R基因人源化改造的动物模型、上述的构建方法制备获得的多基因修饰非人动物、上述的多基因修饰非人动物或其后代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。
本发明的第十四方面,提供了一种人源化IL7R蛋白,所述的人源化IL7R蛋白包含人IL7R蛋白的全部或部分。进一步优选的,所述的人源化IL7R蛋白包含人IL7R蛋白的信号肽和/或胞外区。
优选的,所述的人源化IL7R蛋白包含与SEQ ID NO.2第1至239位或SEQ ID NO.8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第1至239位或SEQ ID NO.8所示氨基酸序列一致的氨基酸序列。
优选的,所述的人源化IL7R蛋白还包含非人动物IL7R蛋白的部分,优选为非人动物IL7R蛋白的跨膜区、胞质区。
优选的,所述的人源化IL7R蛋白包含人IL7R基因的1号外显子至6号外显子编码的氨基酸序列,和非人动物IL7R蛋白的氨基酸序列。
在本发明的一个具体实施方式中,所述的人源化IL7R蛋白包含下列组中的一种:
a)SEQ ID NO.8或SEQ ID NO.2第1至239位所示氨基酸序列的部分或全部;
b)与SEQ ID NO.8或SEQ ID NO.2第1至239位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.8或SEQ ID NO.2第1至239位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.8或SEQ ID NO.2第1至239位所示的,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。
本发明的第十五方面,提供了一种编码上述人源化IL7R蛋白的人源化IL7R基因,所述的人源化IL7R基因包含人IL7R基因的1号外显子至6号外显子,和非人动物IL7R基因的核苷酸序列。
优选的,所述的人源化IL7R基因包含NCBI登录号为NC_000005.10的第35856978-35874459 位的核苷酸序列。
优选的,所述的人源化IL7R基因转录的mRNA序列包含SEQ ID NO.7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IL7R基因中包含的人IL7R核苷酸序列的部分选自下列组中的一种:
(A)包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列的全部或部分;
(B)包含与NCBI登录号为NC_000005.10的第35856978-35874459 位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NCBI登录号为NC_000005.10的第35856978-35874459 位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NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列的,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IL7R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.7所示核苷酸序列的全部或部分;
(b)包含与SEQ ID NO.7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.7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.7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。
本发明的第十六方面,涉及一种表达上述的人源化IL7R蛋白的构建体。
本发明的第十七方面,涉及一种包含上述构建体的细胞。
本发明的第十八方面,涉及一种包含上述细胞的组织。
本发明的第十九方面,涉及了一种上述的构建方法获得的动物模型、上述的构建方法获得的多基因修饰非人动物、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IL7R蛋白或上述的人源化IL7R基因在制备治疗或预防肿瘤的药物中的应用。
本发明的第二十方面,涉及一种上述的构建方法获得的动物模型、上述的构建方法获得的多基因修饰非人动物、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IL7R蛋白或上述的人源化IL7R基因在IL7R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IL7R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IL7R基因功能,研究人IL7R抗体,研究针对人IL7R靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤药物方面的用途。
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IL7R基因人源化的非人动物体内可以正常表达人或人源化IL7R蛋白。可用于针对人IL7R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IL7R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IL7R蛋白”,包含来源于人IL7R蛋白的部分和非人IL7R蛋白的部分。其中,所述的“人IL7R蛋白”同“人IL7R蛋白的全部”,即其氨基酸序列与人IL7R蛋白的全长氨基酸序列一致。所述的“人IL7R蛋白的部分”,为连续或间隔的5-459个氨基酸序列与人IL7R蛋白的氨基酸序列一致。优选为连续或间隔10-239,可以为连续5、10、20、30、40、50、60、70、80、90、100、200、239、300、400、410、420、430、440、450、459个氨基酸序列与人IL7R蛋白的氨基酸序列一致。
本发明所述的“人源化IL7R基因”,包含来源于人IL7R核苷酸序列的部分和非人IL7R基因的部分。其中,所述的“人IL7R核苷酸序列”同“人IL7R核苷酸序列的全部”,即其核苷酸序列与人IL7R核苷酸序列的全长核苷酸序列一致。所述的“人IL7R核苷酸序列的部分”为连续或间隔的20-22713bp个核苷酸序列与人IL7R核苷酸序列一致,优选为20-17482个,可以为20、50、100、200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000、6000、7000、8000、9000、10000、11000、12000、13000、14000、15000、16000、17000、17482、18000、19000、20000、21000、22000、22500、22713bp个核苷酸序列与人IL7R核苷酸序列一致。
本发明所述的“xx号至xxx号外显子”或“xx号至xxx号外显子的全部”包含外显子及其期间的内含子的核苷酸序列,例如所述的“1号至6号外显子”包含1号外显子、1-2号内含子、2号外显子、2-3号内含子、3号外显子、3-4号内含子、4号外显子、4-5号内含子、5号外显子、5-6号内含子、6号外显子的全部核苷酸序列。
本发明所述的“x-xx号内含子”表示x号外显子与xx号外显子之间的内含子。例如“1-2号内含子”表示1号外显子与2号外显子之间的内含子。
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IL7R核苷酸序列的1号外显子的部分,包含连续或间隔的5-169bp个,优选10-82bp个核苷酸序列与人IL7R核苷酸序列的1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IL7R基因”中包含的“1号外显子的部分”至少包括从1号外显子编码信号肽的核苷酸开始至1号外显子的最后一个核苷酸序列为止。
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“IL7R基因座”表示IL7R基因1号至8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、3号外显子、4号外显子、5号外显子、6号外显子、7号外显子、8号外显子的任一个或两个或多个的组合,或一个或两个或多个的全部或部分。
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:鼠IL7R基因和人IL7R基因对比示意图(非按比例);
图2:人源化IL7R小鼠基因示意图,其中,用人IL7R基因1号外显子的部分序列至6号外显子的部分序列替换小鼠的1号至6号外显子区域获得人源化IL7R小鼠基因(非按比例);
图3:IL7R基因打靶策略及靶向载体设计示意图,其中,靶向载体包含5’同源臂、3’同源臂以及含有人IL7R DNA片段、FRT和NeoR的敲进片段(KI片段),3’同源臂下游还有DTA片段;
图4:采用5’探针、3’探针和Neo探针进行Southern Blot鉴定的结果图,其中,WT为野生型C57BL/6小鼠,编号为1-D07的克隆为阳性克隆;
图5:去Neo过程示意图;
图6:IL7R人源化小鼠F1代鼠尾PCR鉴定结果,其中,图(A)使用引物对WT-F和WT-R用于扩增小鼠内源的野生型IL7R基因片段;图(B)使用引物对WT-F和Mut-R用于扩增修饰后的IL7R基因片段,用以验证靶向载体是否正确插入小鼠基因组位点,其中编号为F1-17、F1-20和F1-21的小鼠为阳性杂合小鼠,PC为阳性对照组,WT为野生型小鼠,M为Marker组,H2O为水;
图7:小鼠体内脾脏细胞中T细胞上IL7R蛋白表达流式分析结果,其中H/+为IL7R基因人源化杂合子小鼠,WT为野生型C57BL/6小鼠。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
在下述实施例中,设备和材料是从以下所指出的几家公司获得:
BglII、KpnI和MfeI酶购自NEB,货号分别为R0144M、R3142M和R3589L;
C57BL/6小鼠和Flp工具鼠购自中国食品药品检定研究院国家啮齿类实验动物种子中心;
Zombie NIR™ Fixable Viability Kit来源Biolegend,货号423106
Brilliant Violet 510™ anti-mouse CD45 Antibody来源Biolegend,货号为:103138;
PerCP/Cy5.5 anti-mouse TCR β chain(mTCRβ-PerCP/Cy5.5)来源Biolegend,货号为:109228;
APC anti-mouse CD127 (IL-7Rα) Antibody(mIL7R APC)来源Biolegend,货号为:135011
PE anti-human CD127 (IL-7Rα) Antibody(hIL7R PE)来源Biolegend,货号为:351303。
实施例1 IL7R基因人源化小鼠
小鼠IL7R基因(NCBI Gene ID:16197,Primary source:MGI: 96562,UniProtKB:P16872,位于15号染色体NC_000081.6 的第9506159至9529941位,基于转录本NM_008372.4及其编码蛋白NP_032398.3(SEQ ID NO.1))和人IL7R基因(NCBI Gene ID:3575,Primarysource:HGNC: 6024,UniProtKB:P16871,位于5号染色体NC_000005.10的第35856891至35879603位,基于转录本NM_002185.5及其编码蛋白NP_002176.2(SEQ ID NO.2所示))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IL7R基因座引入编码人IL7R蛋白的基因序列,使得该小鼠表达人或人源化IL7R蛋白。可以采取在小鼠内源IL7R基因座上直接插入含有人IL7R的基因序列的方法,例如含有人IL7R的DNA序列或cDNA序列,并可在插入序列中加入辅助序列(例如终止密码子或含有终止功能的序列等)或其他方法(例如,翻转,或敲除)使得插入位点后的小鼠内源IL7R基因组序列不能正常表达;也可以采取原位替换的策略,即,在小鼠内源IL7R基因座上直接用人IL7R的基因序列(例如,人IL7R的DNA序列或cDNA序列)进行替换。本实施例将以DNA序列的原位替换策略来阐述如何对小鼠IL7R基因进行人源化改造。
具体而言,用基因编辑技术对小鼠细胞进行修饰,在小鼠内源IL7R基因座上用人IL7R基因的序列替换特定小鼠IL7R基因序列。在小鼠IL7R基因调节元件的控制下,如将至少包含小鼠IL7R基因的1号外显子部分序列至6号外显子的部分序列的19494bp的序列用对应的人基因序列17482bp替换,得到小鼠人源化IL7R基因座示意图如图2所示。
进一步的,设计如图3所示的打靶策略。其中图3所示的靶向载体上含有5’同源臂(SEQ ID NO.3)、3’同源臂(SEQ ID NO.4)以及含有人IL7R DNA片段的敲进片段(KI片段),其中5’同源臂与NCBI登录号为NC_000081.6的第9529743-9534180位核苷酸序列相同;3’同源臂与NCBI登录号为NC_000081.6的第9505623-9509583位核苷酸序列相同;敲进片段上的人IL7R DNA片段与NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列相同。其中,含有人IL7R DNA序列的片段上游与5’同源臂直接连接,含有人IL7R DNA序列的片段下游与鼠基因座的连接设计为5’-CACAATCTATTCTTGCTTTCCAGGGGAGATGGATCCTGTCTTGCCAAGTGTCACCATTCT-3’(SEQ ID NO.6),其中序列“TGGAT”的最后一个“T”是人序列的最后一个核苷酸,序列“CCTG”的第一个“C”是小鼠序列的第一个核苷酸。
改造后的人源化小鼠IL7R的mRNA序列如SEQ ID NO.7所示,其表达的氨基酸序列如SEQ ID NO.8所示。
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具有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盒上游与小鼠IL7R基因座的连接设计为5’-CCTGTCAGATTAATTGGTCAGTAAACTCTGAGTATCTGTCTAAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAA -3’(SEQ ID NO.9),其中序列“GTCT”的最后一个“T”是小鼠序列的最后一个核苷酸,序列“AAGC”的第一个“A”是NEO盒的第一个核苷酸。NEO盒下游与小鼠IL7R基因座的连接设计为5’-AAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCGCCTCCACCACTCCACATATCATAGAGTTACAGATACATG-3’(SEQ IDNO.10),其中序列“ATCC”的最后一个“C”是NEO盒的最后一个核苷酸,“GCCT”的“G”是小鼠序列的第一个核苷酸。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,即白喉毒素A亚基的编码基因(DTA)。
使用常规方法构建靶向载体,如通过酶切连接、直接合成等构建用人IL7R基因替换小鼠基因的靶向载体。小鼠和人IL7R DNA分别获自细菌人工染色体(BAC)克隆RP23-288H22和CH17-142K4。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KpnI或BglII或MfeI消化细胞DNA并使用3个探针进行杂交)检测,示例性检测结果如图4所示,检测结果表明PCR鉴定为阳性的克隆1-D07为阳性杂合克隆且无随机插入。
表1 PCR引物及目的条带大小如下:
Southern Blot检测包括如下探针引物:
5’探针:
F:5’-TGCTAAGACAATGCTGTTCAGTTGG-3’(SEQ ID NO.15)
R:5’- GGAAGTCACTTACCTTGCTCTTGGG-3’(SEQ ID NO.16)
3’探针:
F:5’- TTGAAGCAGAAAGAGAGTTTCC-3’(SEQ ID NO.17)
R:5’-TCATTGTATATTGGGAACCCATCCC-3’(SEQ ID NO.18)
Neo探针:
F:5’-GGATCGGCCATTGAACAAGAT -3’(SEQ ID NO.19)
R:5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO.20)
表2
按照本领域已知的技术将筛选出的阳性克隆细胞(黑色鼠)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后(见图5),再通过互相交配即可得到表达人源化IL7R蛋白的IL7R基因人源化纯合子小鼠。可通过PCR鉴定子代小鼠体细胞的基因型,示例性的F1代小鼠(已去除Neo标记基因)的鉴定结果见图6,其中,编号为F1-17、F1-20和F1-21的小鼠为阳性杂合小鼠。
表3 PCR引物及目的条带大小
其中,WT为野生型,Mut为IL7R人源化条带
通过流式方法确认小鼠体内人源化IL7R蛋白的表达情况。选取野生型C57BL/6小鼠和IL7R基因人源化杂合小鼠各1只,后取脾脏细胞,在流式细胞仪上,通过排除用活力染料(Zombie NIR,BioLegend)标记的死细胞并用荧光染料标记的抗体Brilliant Violet 510™ anti-mouse CD45 Antibody、PerCP/Cy5.5 anti-mouse TCRβchain(mTCRβ-PerCP/Cy5.5)、抗鼠IL7R抗体APC anti-mouse CD127 (IL-7Rα) Antibody(mIL7R APC)、或抗人IL7R抗体PE anti-human CD127 (IL-7Rα) Antibody(hIL7R PE)染色来标记细胞。
流式分析结果(见图7)表明,与野生型C57BL/6小鼠相比,抗人IL7R抗体可以检测到人源化小鼠脾脏内的T细胞中存在表达人源化IL7R蛋白的细胞,而在C57BL/6对照鼠的脾脏内未检测到表达人或人源化IL7R蛋白的细胞。
实施例2 双基因人源化小鼠和多基因人源化小鼠
利用制得的IL7R小鼠还可以制备含有IL7R双人源化或多人源化小鼠模型。如,前述实施例1中,电转时使用的ES细胞选择含有其他基因修饰小鼠的ES细胞,例如来源于专利CN201710505554.0得到的PD-1基因人源化阳性克隆细胞,可以进一步得到IL7R人源化与PD-1基因修饰的双基因人源化小鼠。也可将本方法得到的IL7R小鼠纯合或杂合子与其它基因修饰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,可有一定几率得到IL7R人源化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。
以双重人源化IL7R/PD-1小鼠的为例。由于鼠IL7R位于15号染色体,鼠PD-1基因位于1号染色体上,可选择IL7R基因人源化小鼠与PD-1基因人源化小鼠交配,通过阳性子代小鼠的筛选,最终得到人源化IL7R/PD-1小鼠。
实施例3 动物模型体内药效验证
利用本方法制备的含有IL7R人源化的小鼠建立疾病模型后可用于验证抗人抗体的药效。以IL7R单基因人源化小鼠及肿瘤模型为例,先在 IL7R人源化小鼠皮下接种小鼠肿瘤细胞(例如结肠癌细胞MC38),待肿瘤生长达到规定体积后随机分为对照组或治疗组。治疗组使用抗人IL7R抗体,对照组注射空白溶剂。通过测量肿瘤体积并称量小鼠的体重可以评估待测抗体的毒性、药效及联用方案的效果。
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。
序列表
<110> 北京百奥赛图基因生物技术有限公司
<120> IL7R基因人源化改造的动物模型的构建方法及其应用
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<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 459
<212> PRT
<213> 小鼠(Mouse)
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Gln Arg Gln Pro Ile Ser Thr Ser Ser Val Leu Asn Gln Glu Glu Ala
435 440 445
Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Lys
450 455
<210> 2
<211> 459
<212> PRT
<213> 人(human)
<400> 2
Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln
1 5 10 15
Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp
20 25 30
Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val
35 40 45
Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val
50 55 60
Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val
65 70 75 80
Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr
85 90 95
Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly
100 105 110
Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys
115 120 125
Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn
130 135 140
Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val
145 150 155 160
Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn
165 170 175
Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln
180 185 190
Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile
195 200 205
Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr
210 215 220
Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro
225 230 235 240
Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu
245 250 255
Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val
260 265 270
Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys
275 280 285
Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu
290 295 300
Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val
305 310 315 320
Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu
325 330 335
Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu
340 345 350
Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr
355 360 365
Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser
370 375 380
Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val
385 390 395 400
Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro
405 410 415
Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala
420 425 430
Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala
435 440 445
Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln
450 455
<210> 3
<211> 4438
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taaaagctca gttctcaagg accacagtga aaggtttatg tatgaggaca gtttacaaca 60
caaaaataag aaccttttct atatgtcttg tctgtttcat agacacccta aatgattaca 120
aaagcagaga caattccaga cacttgcaca aggggtactg gaaaatctat gaaatcctcc 180
aattctgctc ccatacaaga tatgtgatcc tttctaggga ttcacatttg aagaacacat 240
gagaaaagga ctatagaaga agaataagaa atggaagaga tgactgaaga gaggagtttg 300
acaatctgag caaattgcta aggatcaaaa ccaaggaata aacccaagga ccttcccttg 360
ggtcctggct tctcaggtgc ccaaagactc ttttttcatc ttcctttcaa acaactctga 420
caaagcagtg ccatccatgt ctgtcatgtg gaggtcaggg aggacagata agaaaaggga 480
agttctttgc ctgctttcgg cttcaaagga tcctacgtta agaaacagag ttaccattgc 540
tcacccacaa tctctagaag tattagtgat gtttctcatt tttctttaag cttcccgcac 600
tctatttaga tttctctctg atgtcctttc agcatctcct gccccaccca aaaggggtaa 660
gcacaccagt ggaaatcccc tgagcaaact agcacatgct gtaccaaaat attatgtctt 720
aactttgttc ttttacatct tcacaactaa aggaaagaga tacaatcaaa atgatggtcc 780
acttaaatga taaacaagag aatttaaaga gacatgatac aattgttcac catggagtga 840
tagcccatga aaggctgatg aggttggggt tagtagatat aatacatatg aatttctcct 900
taaacatttt cacatagctt tgagaattta tcattgtaga tttctagaaa gcacattcaa 960
cttagtgtgc aaaacaggct gattttgcgg tatgtttttc tgatgaagct ttcaaagcaa 1020
gattacaagc cagagtgtaa ataattacct gattccccaa atcatttctt ttgttgcttg 1080
ggagaaaatt tgaaattctc caaaagaggc ccaaaatgca tggcagagat agaatatgct 1140
acctccaact gaattctttc tctaacattt ctttatggat tgtagtcaag aacttgatgt 1200
tataacagat gaaaatcaca taacacctct aagatcccct caacctgagg aggcattttc 1260
agagaacagc acaaataata catctccttt cttttcttat tagatcatct cattaagaca 1320
tgttgaggga agttaagtca agttgtagtc aaactctaaa actctcaggt actaatgcta 1380
ttcaacctct gggtggcatt gatcatctac tctaacttag tcattttata gactcaagta 1440
actgattctt ccagaaatct agctataaca gaagttcata aaatcctatc cactcagatg 1500
ttttgaactc cttctattat aatcattctt actgcttctt tacaaacagt gtgattatga 1560
atcaaaattt aaaaaaaaac tgtactctat ggtctggatc cagatttctt gattccttca 1620
aagaactgaa aaggggccac tcatctatct agttatgaca ggaccctgat aatgaactta 1680
aagaaaataa tccaacttta tcaaaatctc agccattctt aacaaaaagc accaattcat 1740
cttgaggcca tgttaaaaac aaagtggatt ggcatacaag tttagccaca gtctgaataa 1800
tgactgcctc caacacctat cctacatgcc ttggagccaa taaagtttat gtgaagcctt 1860
tgttaaagaa aaattgataa ccagtatgat gaaactctct tttaaatatt ctgagattgt 1920
acttatgtag ctgtttagag atcagaagtt ttctaaagtc agatggtgta agttttacta 1980
tttatatatc atctctttgc ccactcatct ctactatatg tatgagaaca gctgcagata 2040
gtacacaaca aatgggtaac attggactcc aacaacgaat tacttatgaa agaaggtaaa 2100
ggatcaaatt ccattcctga gtcataattt gccaacactt gcatttaata atttctccgt 2160
gcctgtactt aatagttaag tacaggtgta tactagagat atatagaact caaaggagaa 2220
aaatggctac atttgtacac tgaaagattg agtgtaaaaa atatttttaa atgatctttt 2280
aatagcttat gaaagatgaa atctttattt tgtacatttc tttgttttta gaaactgtat 2340
atatgtattg aggatattga aaccataacc accctcctgt cttccctacc taccgcattc 2400
caacacctac tctccttctc agtttcatgt cctctagttc tttaattatc cagactccat 2460
atccccattg ctaggagtcg tagctagggt caccctcata gactcctggg agttttcatt 2520
gcccttgttt ctagctagtc ccagagatgc ccatctcctg taaccagaaa agacttccaa 2580
tggaggaact gggacaccaa cccactcaca aaatctttag ctcatagttt gtcctaccta 2640
caagatgtgc gggcataaaa gatgaaacag aattagagag aagggcaaac caatgactgg 2700
cccagcttga cacctatgcc atgagaggga gcccactccg acactcttaa taatattctc 2760
ctatacatgc agacaggagc ctagcatagc tatcatctga gaggtttcac ctagcattga 2820
tggaaacaaa tgcagtgacc cacagccaaa cgttaagtgg aacttgggga atcctatgga 2880
agagagtgag gatggattga agatgcccag aggggtcatg gtcacattga gaaatactac 2940
ggaatcaact aacctgggca cataggtttt catatagact aaactttcaa ccagaaatca 3000
tgcacaagac aagcctagac cctctgcaca tatgtaacag ttgtgcagct gggtcttcat 3060
gtgggactcc taaaaccgaa gccagggctg tctctgacta cattgcctgc ctttggaagc 3120
ctttctgcta actgggctgc cttttctagt ctcgagagaa gaagatgtgc ctagtcttac 3180
tgcagcttga tatgtcaagg ctggttgata tccaagggag gcctcccctt ttctgtggag 3240
aaatggaaga atgttagaga aggttggagg gaagggagaa agagggattg ggagcaaagg 3300
atgggggggg gggcgctgca gtcagaattt aggataaata aataacttaa tcaattctta 3360
aaatgattta agaaaaggat ccacagaacc tacttagttc agctcttgtg ctcatgagtg 3420
tataactgcc cactagagct tgagcaacct accatgggac acacttttaa agaaaactaa 3480
ctcttaatcg cctggtattt gtcaacctgc caggagggcc tcagctaggg atgtggcctc 3540
ttgaacacct gctctgtcca tattggaatg tggattggtg tgatctcatg ccagtcttgt 3600
gtcacaacca tagcttctgt gaaattatga acacaaagat cctattattt ttataataga 3660
taatttcact gtatttctct ttggctcctg cctcttctga tctttccttc tctcctccat 3720
ggtgttccct gagttcttga ggggagtgtt tcatggctgg tactataaat ctgaccacga 3780
acccgtgcca gggaggtctt agagcatagg ggaaaacaca ggactattgt ttgtttaaaa 3840
gacagagtat caaactgtct tctagacatt tgtccttata cccatagaat agtgcagcct 3900
tgcctctcat tagagaagct cttttctcac ttctctctca gagagcagag aactgggcgt 3960
gaccattgag ggttcagccc taagtgggac aactctatcc tagcatcatt attcctaaaa 4020
caagagcatc cccaaccaac tgaggagcat cgtaagtgtg gattttggcc ttgtgatcct 4080
gttacattgg accctgagat catctgaagc atcacttgaa gtctaccacc ttgctcaaaa 4140
gtaagcagtt aagttcagga gcttcaggga ataccaagat gcaggaaaaa aaaaagtcac 4200
actctacctc tccgttttct ctacctctgc ttacagcagc aatccttttg ctcagacttc 4260
ctgtttctgg cacttgcctt cttctgtgat gttcatagtt ccctgtctgt ggtttagcag 4320
gcacagagct ggtttgggtc tccctctctc tcattcactt gcacatacaa gcgtgcttct 4380
tctctattct ttctctctct ctctctctct ctctctctct ctctctctct ctctcaga 4438
<210> 4
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcctccacca ctccacatat catagagtta cagatacatg ttatcataca ttgctatcca 60
aactcagttc ttcaaaattg tatgacaaga ggtttattta ttggattatt tccctaggcc 120
atgacgcccc cccccaaaaa aagaattctt taagaagtgt tttccaaata ttttttccac 180
agcctcctaa aggacttaga ggtgattacc catgctaaca tggagtctat ttgatctcat 240
gactttctgc acacaaattc acatgatttt tgtattttgc tctgtggtag aaccatgccc 300
tgtgaattac tcagtgttct aggaagttgc cctcggcaat tttgtattcc taggaccaaa 360
agtgctctaa tttgaaaaac gctaccataa aataattttc ttgaatagct tagaacgtat 420
tcccaatttc cactggaatt aaagtaaaac ctttacttcc agtaaagaca gtggataaga 480
tgacaatacc aacagtgagc ataaaagacc aggtctcatt gtagctccat ataaacacca 540
atactgcctc cctgcaaacc tcactcttcg ctttagggta ctttgcaaca taattacatt 600
gtctcattca caatagatcg ttagcaagga gctctgttct atccactcct taaaccaaga 660
gcatgatact gtcagagaaa ataaggtgtt tgtccagtaa cagaaatgtt ttcacaatct 720
acctcaaata aggtgaaaga gttgtttgtg tgccttctcc ttctgccgtt tgtttcaagt 780
atgaatgttc tctggtttta ggattaaacc tgtcgtatgg cctagtctcc ccgatcataa 840
gaaaactctg gaacaactat gtaagaagcc aaaaacggta attgcttgag gtggggaaag 900
aaacaccata atgttgaaat cttagtctaa gaatgattaa gactgacact caacttacgg 960
tcttttatat atcacataaa tgaaagtcct tttaagactc tgaagaataa agccaagata 1020
tgccacaggg cagggggttg gggaaaaatc aatatttact tcaaagttgg agtatcacag 1080
ctcagtcaga agtgaagcca actgtcattt tttcacatcg tgtgtcaatt ttacaagaaa 1140
gtttcgtaaa cgttttagtt tcctgaatca aatgtatagc agcgcctctt tgccacgcct 1200
ctaacgcttc tgcctttctc tgcagagtct gaatgtgagt ttcaatcccg aaagtttcct 1260
ggactgccag attcatgagg tgaaaggcgt tgaagccagg gacgaggtgg aaagttttct 1320
gcccaatgat cttcctgcac agccagagga gttggagaca cagggacaca gagccgctgt 1380
acacagtgca aaccgctcgc ctgagacttc agtcagccca ccagaaacag ttagaagaga 1440
gtcaccctta agatgcctgg ctagaaatct gagtacctgc aatgcccctc cactcctttc 1500
ctctaggtcc cctgactaca gagatggtga cagaaatagg cctcctgtgt atcaagactt 1560
gctgccaaac tctggaaaca caaatgtccc tgtccctgtc cctcaaccat tgcctttcca 1620
gtcgggaatc ctgataccag tttctcagag acagcccatc tccacttcct cagtactgaa 1680
tcaagaagaa gcgtatgtca ccatgtctag tttttaccaa aacaaatgaa ttataagaaa 1740
acccttccat cgacaaccaa atgatcactg agatggaaag tctggaatgc ttgctctccc 1800
ccgtagctca cagaagagaa agtcaacgtg accttgctac acatcttcag cattctaaga 1860
aatcattttg ctcttctagc tcagaagcat ttgcacaaag caggaagaat ctgttttccc 1920
tgttgttgga ttagtcataa gagtccatat gacccaatta aaattgcaaa actcagttaa 1980
gtgaagaaag aaagatagac aaaagaagat agaaggatgt ggtgaatgca ggaagaagaa 2040
aatgaaagat gtgagtggtg ggtctatcat tcaaattgac tatttatcca gcactatacc 2100
actcttctca tttcttcctc acaataatat tacaatgtgg gcttatccat tataactttt 2160
attttctttg tcatagatgc tgaagttgaa agtagagatt ttaagtgata tccaaatttt 2220
tctttcagct acagatgagg cacacattcc aacttcaacc ctctcttgcc atgaacctgt 2280
cctattgttg agtgtcaaac atcaccacta agtggatggt tatgtagtcc attatccaaa 2340
ctgagtcgtt ttggaaagaa aaagttagac ataattaaca gtaagcataa actgtatatg 2400
tctaagagag atgtggatgg atggtcattt tacttaaagt ggctataggg atgaacatga 2460
aggacaaagt acatttatgg gtgtggcata ccatgaccat gtgtcaaagg aagtgggaaa 2520
aagaaaaaaa aagcaccaag atcatttgat tttgttttgt tgttttgttt gaaaacaaac 2580
tcaagaagca atgagttaga agccgagaag ttccagagtc agttatcaag accatgattt 2640
tcctgctgct attatccatt ggcttctctg tgacattgta ggaggaacta tggccaatct 2700
acaggagttc aacatttaac agtgaatgga gtcctcctat gtgagtcctc ctatgtgtgg 2760
agacaccatt aagaactacc ccaagttcta catctctgga tattgcctga actacagaaa 2820
aagggggctg cgcacaccac aatgagtgcc ctacctgaaa ctatgctcac agaaacacaa 2880
agaagatggg taagttattc aaattcaaat gttgatttat gactgcaagt cacaattttg 2940
aatccctgct gtgtataacc aatctcctga agaaaacaac aaataactga aagatactgt 3000
ggttgggtgc cttagcatta aaattctgtt taagtgttga cattgtttat ttggattgga 3060
gtgtctgtcc ggtcatgtat tgtatccatg cattatattc agataaccac aacagctgct 3120
aatgcttgat tatattctca gggactgcat gcaatgtaac attactggtt ggttctgcca 3180
attttcctct tggtatttat aaaggaaaac caaaactctt ggtcagagac aatatgcaaa 3240
acagagatgt caagtactat gtccaaatac tgtgaaatat aatgagaaat aggtaacaaa 3300
tttatcaatc aactatgttt ggatccaggg aatctcaagt tattcaattc attctctgta 3360
agcctttgtc tctctcttca tccagacttt tgccttcaaa tacaagcatg cgctattttc 3420
tggaattgat acagcactgt tcattttgtg tgtgtgtgtg tgtatgtgtg tgtgtgtgtg 3480
tgtgtgtgtg tgtgtgtgtg tgtatgtgag agagagagag agagagagag agagagagag 3540
agagagagag agagctcagc agtttttctc tggtgttctt taatacatcc accattcact 3600
acagtgaaga tctctgggtt gactgtacac tttttaaaat gaattagtag tattctctaa 3660
tgatgttaca cagagaaatg tatggtgtgt gttaccattt catgttcaac taaatgctaa 3720
ggtactctgt ggctaaaata ctctctgtaa ctgtggcaac tacatgtaaa taaatgggct 3780
ctacagttca gcacagactt cttgagctac tgaaaatatt cttagaccat ttaaaaggtt 3840
aagattgtca gaaagatagt tttaaattaa ttcaataatg aactaatagc catttattga 3900
gaagccataa tgccttactt aaacactatg ctggctacta aacaatggaa cgagatactc 3960
c 3961
<210> 5
<211> 27
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaaatgactc accattttga gcatagc 27
<210> 6
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cacaatctat tcttgctttc caggggagat ggatcctgtc ttgccaagtg tcaccattct 60
<210> 7
<211> 3227
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtctgtggtt tagcaggcac agagctggtt tgggtctccc tctctctcat tcacttgcac 60
atacaagcgt gcttcttctc tattctttct ctctctctct ctctctctct ctctctctct 120
ctctctctct cagaatgaca attctaggta caacttttgg catggttttt tctttacttc 180
aagtcgtttc tggagaaagt ggctatgctc aaaatggaga cttggaagat gcagaactgg 240
atgactactc attctcatgc tatagccagt tggaagtgaa tggatcgcag cactcactga 300
cctgtgcttt tgaggaccca gatgtcaaca tcaccaatct ggaatttgaa atatgtgggg 360
ccctcgtgga ggtaaagtgc ctgaatttca ggaaactaca agagatatat ttcatcgaga 420
caaagaaatt cttactgatt ggaaagagca atatatgtgt gaaggttgga gaaaagagtc 480
taacctgcaa aaaaatagac ctaaccacta tagttaaacc tgaggctcct tttgacctga 540
gtgtcgtcta tcgggaagga gccaatgact ttgtggtgac atttaataca tcacacttgc 600
aaaagaagta tgtaaaagtt ttaatgcacg atgtagctta ccgccaggaa aaggatgaaa 660
acaaatggac gcatgtgaat ttatccagca caaagctgac actcctgcag agaaagctcc 720
aaccggcagc aatgtatgag attaaagttc gatccatccc tgatcactat tttaaaggct 780
tctggagtga atggagtcca agttattact tcagaactcc agagatcaat aatagctcag 840
gggagatgga tcctgtcttg ccaagtgtca ccattctgag tttgttctct gtgtttttgt 900
tggtcatctt agcccatgtg ctatggaaaa aaaggattaa acctgtcgta tggcctagtc 960
tccccgatca taagaaaact ctggaacaac tatgtaagaa gccaaaaacg agtctgaatg 1020
tgagtttcaa tcccgaaagt ttcctggact gccagattca tgaggtgaaa ggcgttgaag 1080
ccagggacga ggtggaaagt tttctgccca atgatcttcc tgcacagcca gaggagttgg 1140
agacacaggg acacagagcc gctgtacaca gtgcaaaccg ctcgcctgag acttcagtca 1200
gcccaccaga aacagttaga agagagtcac ccttaagatg cctggctaga aatctgagta 1260
cctgcaatgc ccctccactc ctttcctcta ggtcccctga ctacagagat ggtgacagaa 1320
ataggcctcc tgtgtatcaa gacttgctgc caaactctgg aaacacaaat gtccctgtcc 1380
ctgtccctca accattgcct ttccagtcgg gaatcctgat accagtttct cagagacagc 1440
ccatctccac ttcctcagta ctgaatcaag aagaagcgta tgtcaccatg tctagttttt 1500
accaaaacaa atgaattata agaaaaccct tccatcgaca accaaatgat cactgagatg 1560
gaaagtctgg aatgcttgct ctcccccgta gctcacagaa gagaaagtca acgtgacctt 1620
gctacacatc ttcagcattc taagaaatca ttttgctctt ctagctcaga agcatttgca 1680
caaagcagga agaatctgtt ttccctgttg ttggattagt cataagagtc catatgaccc 1740
aattaaaatt gcaaaactca gttaagtgaa gaaagaaaga tagacaaaag aagatagaag 1800
gatgtggtga atgcaggaag aagaaaatga aagatgtgag tggtgggtct atcattcaaa 1860
ttgactattt atccagcact ataccactct tctcatttct tcctcacaat aatattacaa 1920
tgtgggctta tccattataa cttttatttt ctttgtcata gatgctgaag ttgaaagtag 1980
agattttaag tgatatccaa atttttcttt cagctacaga tgaggcacac attccaactt 2040
caaccctctc ttgccatgaa cctgtcctat tgttgagtgt caaacatcac cactaagtgg 2100
atggttatgt agtccattat ccaaactgag tcgttttgga aagaaaaagt tagacataat 2160
taacagtaag cataaactgt atatgtctaa gagagatgtg gatggatggt cattttactt 2220
aaagtggcta tagggatgaa catgaaggac aaagtacatt tatgggtgtg gcataccatg 2280
accatgtgtc aaaggaagtg ggaaaaagaa aaaaaaagca ccaagatcat ttgattttgt 2340
tttgttgttt tgtttgaaaa caaactcaag aagcaatgag ttagaagccg agaagttcca 2400
gagtcagtta tcaagaccat gattttcctg ctgctattat ccattggctt ctctgtgaca 2460
ttgtaggagg aactatggcc aatctacagg agttcaacat ttaacagtga atggagtcct 2520
cctatgtgag tcctcctatg tgtggagaca ccattaagaa ctaccccaag ttctacatct 2580
ctggatattg cctgaactac agaaaaaggg ggctgcgcac accacaatga gtgccctacc 2640
tgaaactatg ctcacagaaa cacaaagaag atgggtaagt tattcaaatt caaatgttga 2700
tttatgactg caagtcacaa ttttgaatcc ctgctgtgta taaccaatct cctgaagaaa 2760
acaacaaata actgaaagat actgtggttg ggtgccttag cattaaaatt ctgtttaagt 2820
gttgacattg tttatttgga ttggagtgtc tgtccggtca tgtattgtat ccatgcatta 2880
tattcagata accacaacag ctgctaatgc ttgattatat tctcagggac tgcatgcaat 2940
gtaacattac tggttggttc tgccaatttt cctcttggta tttataaagg aaaaccaaaa 3000
ctcttggtca gagacaatat gcaaaacaga gatgtcaagt actatgtcca aatactgtga 3060
aatataatga gaaataggta acaaatttat caatcaacta tgtttggatc cagggaatct 3120
caagttattc aattcattct ctgtaagcct ttgtctctct cttcatccag acttttgcct 3180
tcaaatacaa gcatgcgcta ttttctggaa aaaaaaaaaa aaaaaaa 3227
<210> 8
<211> 459
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln
1 5 10 15
Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp
20 25 30
Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val
35 40 45
Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val
50 55 60
Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val
65 70 75 80
Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr
85 90 95
Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly
100 105 110
Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys
115 120 125
Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn
130 135 140
Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val
145 150 155 160
Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn
165 170 175
Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln
180 185 190
Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile
195 200 205
Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr
210 215 220
Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro
225 230 235 240
Val Leu Pro Ser Val Thr Ile Leu Ser Leu Phe Ser Val Phe Leu Leu
245 250 255
Val Ile Leu Ala His Val Leu Trp Lys Lys Arg Ile Lys Pro Val Val
260 265 270
Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu Gln Leu Cys Lys
275 280 285
Lys Pro Lys Thr Ser Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu
290 295 300
Asp Cys Gln Ile His Glu Val Lys Gly Val Glu Ala Arg Asp Glu Val
305 310 315 320
Glu Ser Phe Leu Pro Asn Asp Leu Pro Ala Gln Pro Glu Glu Leu Glu
325 330 335
Thr Gln Gly His Arg Ala Ala Val His Ser Ala Asn Arg Ser Pro Glu
340 345 350
Thr Ser Val Ser Pro Pro Glu Thr Val Arg Arg Glu Ser Pro Leu Arg
355 360 365
Cys Leu Ala Arg Asn Leu Ser Thr Cys Asn Ala Pro Pro Leu Leu Ser
370 375 380
Ser Arg Ser Pro Asp Tyr Arg Asp Gly Asp Arg Asn Arg Pro Pro Val
385 390 395 400
Tyr Gln Asp Leu Leu Pro Asn Ser Gly Asn Thr Asn Val Pro Val Pro
405 410 415
Val Pro Gln Pro Leu Pro Phe Gln Ser Gly Ile Leu Ile Pro Val Ser
420 425 430
Gln Arg Gln Pro Ile Ser Thr Ser Ser Val Leu Asn Gln Glu Glu Ala
435 440 445
Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Lys
450 455
<210> 9
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cctgtcagat taattggtca gtaaactctg agtatctgtc taagcttgat atcgaattcc 60
gaagttccta ttctctagaa a 81
<210> 10
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaagtatagg aacttcatca gtcaggtaca taatggtgga tccgcctcca ccactccaca 60
tatcatagag ttacagatac atg 83
<210> 11
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
gctcgactag agcttgcgga 20
<210> 12
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgtcatccac agtctgtagc tctgt 25
<210> 13
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
tgaggaaggg ggagtactgt gga 23
<210> 14
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
ctcaaactgc attcaaaccc agtgc 25
<210> 15
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
tgctaagaca atgctgttca gttgg 25
<210> 16
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 16
ggaagtcact taccttgctc ttggg 25
<210> 17
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttgaagcaga aagagagttt cc 22
<210> 18
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 18
tcattgtata ttgggaaccc atccc 25
<210> 19
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 19
ggatcggcca ttgaacaaga t 21
<210> 20
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 20
cagaagaact cgtcaagaag gc 22
<210> 21
<211> 24
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 21
tctggcactt gccttcttct gtga 24
<210> 22
<211> 24
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgtcatcatt ccctctgtcc tggt 24
Claims (10)
1.一种IL7R基因人源化改造的动物模型的构建方法,其特征在于,所述的动物模型的基因组中包含编码SEQ ID NO.2第1至239位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第1至239位氨基酸的核苷酸序列替换至非人动物IL7R基因座。
2.根据权利要求1所述的构建方法,其特征在于,所述的动物模型的基因组中包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列;所述的构建方法包括用包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列插入或替换至非人动物IL7R基因座上。
3.根据权利要求1或2所述的构建方法,其特征在于,所述的动物模型体内表达人或人源化IL7R蛋白,所述的人源化IL7R蛋白包括人IL7R蛋白的胞外区和/或信号肽,所述的人源化IL7R蛋白包含SEQ ID NO.2第1至239位氨基酸或包含SEQ ID NO.8所示的氨基酸序列。
4.根据权利要求1或2所述的构建方法,其特征在于,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第1至239位氨基酸的核苷酸序列或包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
5.根据权利要求1或2所述的构建方法,其特征在于,所述的非人动物为啮齿类动物,所述的啮齿类动物为大鼠或小鼠。
6.一种靶向载体,其特征在于,所述的靶向载体包含编码SEQ ID NO.2第1至239位氨基酸的核苷酸序列或包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
7.一种人源化IL7R蛋白,其特征在于,所述的人源化IL7R蛋白包含人IL7R蛋白的胞外区和/或信号肽,所述的人源化IL7R蛋白包含SEQ ID NO.2第1至239位氨基酸。
8.根据权利要求7所述的人源化IL7R蛋白,其特征在于,所述的人源化IL7R蛋白包含SEQ ID NO.8所示的氨基酸序列。
9.一种编码权利要求7或8所述的人源化IL7R蛋白的人源化IL7R基因,其特征在于,所述的人源化IL7R基因包含NCBI登录号为NC_000005.10的第35856978-35874459 位核苷酸序列。
10.权利要求1-5任一构建方法构建的非人动物,权利要求7-8任一所述的人源化IL7R蛋白、权利要求9所述的人源化IL7R基因在IL7R基因或蛋白相关研究中的应用,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IL7R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IL7R基因功能,研究人IL7R抗体,研究针对人IL7R靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤药物方面的用途。
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CN115043929A (zh) * | 2021-06-02 | 2022-09-13 | 百奥赛图(北京)医药科技股份有限公司 | Vsig4基因人源化非人动物的构建方法及应用 |
WO2023046201A1 (en) * | 2021-09-27 | 2023-03-30 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric genes |
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