CN111793647B - Cd226基因人源化非人动物的构建方法及应用 - Google Patents
Cd226基因人源化非人动物的构建方法及应用 Download PDFInfo
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Abstract
本发明提供了一种CD226基因人源化改造非人动物的构建方法,利用同源重组的方式将编码人CD226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CD226蛋白,可以作为人CD226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CD226嵌合蛋白、一种CD226嵌合基因、一种CD226基因的靶向载体,以及上述构建方法构建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CD226基因改造非人动物模型的构建方法及其在生物医药领域的应用。
背景技术
CD226是一种糖蛋白,属于Ig超家族,主要表达在NK细胞和T细胞表面,同时也在血小板和单核细胞有表达。目前研究表明,CD226的配体是CD112和CD155,二者在很多恶性肿瘤和病毒感染的细胞中都表达,且其过度表达与癌症侵袭和转移有关。因此,CD226在对抗肿瘤免疫反应、控制体内病毒感染起到至关重要作用。研究发现,CD226的Gly307Ser氨基酸的改变还与自身免疫性疾病相关,比如I型糖尿病、类风湿性关节炎、***性红斑狼疮等。抗CD226单克隆抗体可以抑制针对某些肿瘤细胞的T细胞和NK细胞介导的细胞毒性,而对其它细胞的细胞溶解没有影响,这表明CD226诱导的细胞毒性取决于来自肿瘤细胞的特异性配体。CD226与配体CD155和CD112的相互作用触发NK或T细胞介导的细胞毒性,伴随着细胞因子产生的增加。进一步的体外研究表明,具有较高CD155表达的肿瘤细胞更易受到CD226诱导的杀伤。此外,CD226已被发现介导NK细胞抑制CD155阳性肿瘤转移。
实验动物疾病模型对于研究人类疾病发生的病因、发病机制、开发防治技术和开发药物是不可缺少的研究工具。但由于动物与人类的生理结构和代谢***本身的差异,传统的动物模型并不能很好的反映人体的真实状况,在动物体内建立更接近人类的生理特征的疾病模型是生物医药行业的迫切需求。
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***,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。
鉴于CD226在肿瘤及自身免疫性疾病等治疗领域的巨大应用价值,为进一步探索其相关生物学特性,提高临床前期药效试验的有效性,提高研发成功率,使临床前期的试验更有效并使研发失败最小化,本领域急需开发CD226相关信号通路的非人动物模型。
发明内容
本发明的第一方面,提供了一种CD226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人或者人源化CD226核苷酸序列。
优选的,所述的人或者人源化CD226核苷酸序列中包含人CD226核苷酸序列的2号外显子至7号外显子的全部或者部分。优选的包含2号外显子的部分、3号至5号外显子的全部和6号外显子的部分;其中,所述人CD226核苷酸序列的2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2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1-10个胞质区的核苷酸序列。
优选的,所述人或者人源化基因包含人CD226核苷酸序列的CDS序列或人CD226的全长核苷酸序列。
更优选的,所述的人或者人源化CD226基因包含编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列或者包含SEQ IDNO.5的核苷酸序列。
进一步,所述人源化CD226核苷酸序列还包括鼠CD226核苷酸序列的6号外显子的部分和7号外显子的部分。
优选的,所述鼠CD226核苷酸序列包括编码SEQ ID NO.1第279-333位所示氨基酸序列的核苷酸序列或者SEQ IDNO.6的核苷酸序列。
发明的一个具体实施方式中,所述人源化CD226核苷酸序列从5’-3’顺序依次包含SEQ IDNO.5和SEQ IDNO.6的核苷酸序列。
更优选的,在鼠CD226核苷酸序列后***LoxP STOP序列。
在本发明的一个具体实施方式中,所述的人源化CD226核苷酸序列包含下列组中的一种:
a)人源化CD226核苷酸序列中的人CD226核苷酸序列为编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列或者SEQ ID NO.5所示的核苷酸序列的部分或全部;
b)人源化CD226核苷酸序列中的人CD226核苷酸序列与编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列或者SEQ ID NO.5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CD226核苷酸序列中的人CD226核苷酸序列与编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列或者SEQ ID NO.5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CD226核苷酸序列中的人CD226核苷酸序列与编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列或者SEQ ID NO.5所示的核苷酸序列所示的,包括取代、缺失和/或***一个或多个核苷酸的核苷酸序列。
优选的,所述的构建方法包括***、翻转、敲除或替换。优选为***。
优选的,所述的构建方法包括用包含上述人或者人源化CD226核苷酸序列***或替换到非人动物CD226基因座上
在一个具体的实施方式中,所述的构建方法包括用包含编码SEQ ID NO.2第1至278位氨基酸的核苷酸序列、包含SEQ IDNO.5的核苷酸序列或者包含5’-3’顺序依次包含SEQ IDNO.5和SEQ IDNO.6的核苷酸序列***至非人动物CD226基因座。
优选的,所述的***或替换位点为CD226基因的内源调控元件之后。
优选的,所述的***为首先破坏非人动物内源CD226基因的编码框,随后进行***操作,或者所述的***步骤既可在内源CD226基因处造成移码突变也可以实现***人源序列的步骤。
优选的,所述的动物模型中人源化CD226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CD226基因。
优选的,使用基因编辑技术进行CD226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。
优选的,使用靶向载体进行CD226基因人源化改造的动物模型的构建。
优选的,使用sgRNA靶向的靶位点序列如SEQ ID NO.8-15任一项所示;更优选的,使用的sgRNA靶位点序列为SEQ ID NO.8、10、11、13、14和/或15。
优选的,所述的待改变的转换区位于非人动物CD226基因座上。进一步优选的,位于非人动物CD226基因的2号外显子至7号外显子上。更优选的,位于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,鉴定筛选获得动物模型。
优选的,所述的动物模型体内表达人或人源化CD226蛋白,同时内源CD226蛋白的表达降低或缺失。
优选的,所述的人源化CD226蛋白包含人CD226基因的2号外显子至7号外显子编码的氨基酸序列的部分或全部。更优选的,所述的人源化CD226蛋白包含人CD226蛋白的信号肽、胞外区、跨膜区和/或胞质区。
优选的,所述的人源化CD226蛋白还包含非人动物CD226蛋白的部分,更优选为非人动物CD226蛋白的胞质区。进一步,所述非人动物CD226蛋白的部分为SEQ ID NO.1第279-333位所示氨基酸序列。
在本发明的一个具体实施方式中,所述的人源化CD226蛋白包含下列组中的一种:
a)SEQ ID NO.2第1-278位所示氨基酸序列或者SEQ ID NO.32的氨基酸序列的部分或全部;
b)与SEQ ID NO.2第1-278位所示氨基酸或者SEQ ID NO.32的氨基酸序列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第1-278位所示的氨基酸或者SEQ ID NO.32的氨基酸序列的序列差异不超过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第1-278位或者SEQ ID NO.32的氨基酸所示的,包括取代、缺失和/或***一个或多个氨基酸残基的氨基酸序列。
本发明的第二方面,提供了一种CD226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上述人或者人源化CD226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CD226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_000084.6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CD226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_000084.6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***,进一步优选的,所述特异性重组***为Frt重组位点,也可选择常规的LoxP重组***,所述的特异性重组***为具有两个Frt重组位点,分别连接在抗性基因的两侧。
所述靶向载体用于上述CD226基因人源化改造的动物模型的构建方法中。
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CD226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、***或替换。
本发明的第六方面,涉及一种CD226基因改造的人源化细胞,所述的人源化CD226基因改造细胞的基因组中包括如上所述的人或者人源化CD226核苷酸序列。优选的,其通过内源性CD226调控元件调控;该人源化CD226基因改造细胞体内表达人或人源化CD226蛋白,同时内源CD226蛋白的表达降低或缺失。
本发明的第七方面,涉及一种CD226基因缺失的细胞,所述的CD226基因缺失的细胞缺失内源CD226基因的1号外显子至6号外显子。
本发明第八方面,涉及一种sgRNA,所述 sgRNA靶向的靶位点序列如SEQ ID NO.8-15任一项所示;更优选的,所述sgRNA靶位点序列为SEQ ID NO.8、10、11、13、14和/或15。
本发明的第九方面,提供一种载体,所述载体包括上述sgRNA,优选的,所述载体如SEQ ID NO.20所示。
本发明的第十方面,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CD226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、PD-L1、TIGIT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。
本发明的第十一方面,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。
本发明的第十二方面,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CD226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。
本发明的第十三方面,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。
本发明的第十四方面,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CD226基因人源化改造的动物模型、上述的CD226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。
本发明的第十五方面,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CD226基因人源化改造的动物模型、上述的CD226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。
本发明的第十六方面,提供了一种人源化CD226蛋白,所述的人源化CD226蛋白如本发明的第一方面所述。
本发明的第十七方面,提供了一种编码上述人源化CD226蛋白的人源化CD226基因,所述的人源化CD226基因如本发明第一方面所述的人源化CD226核苷酸序列。
本发明的第十八方面,涉及一种表达上述的人源化CD226蛋白的构建体。
本发明的第十九方面,涉及一种包含上述构建体的细胞。
本发明的第二十方面,涉及一种包含上述细胞的组织。
本发明的第二十一方面,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CD226蛋白或上述的人源化CD226基因在制备治疗或预防肿瘤的药物中的应用。
本发明的第二十二方面,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CD226蛋白或上述的人源化CD226基因在CD226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***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CD226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CD226基因功能,研究人CD226抗体,研究针对人CD226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。
所述疾病为与CD226相关的疾病,优选的,所述疾病为肿瘤、自身免疫性疾病等。
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、***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、***性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、I型糖尿病、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。
优选的,所述应用不是疾病的诊断和治疗方法。所述细胞也不能发育成个体。
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。
本发明的有益效果:本发明所述的CD226基因人源化的非人动物体内可以正常表达人或人源化CD226蛋白。可用于针对人CD226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CD226蛋白功能及相关疾病药物筛选提供了有效的保障。
除非本文另有定义,与本发明结合使用的科学和技术术语及其缩略语应具有本发明所属领域的普通技术人员通常理解的含义。以下列举了本文中使用的部分术语和缩略语。
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CD226蛋白”,包含来源于人CD226蛋白的部分和非人CD226蛋白的部分。其中,所述的“人CD226蛋白”同“人CD226蛋白的全部”,即其氨基酸序列与人CD226蛋白的全长氨基酸序列一致。所述的“人CD226蛋白的部分”,为连续或间隔的5-336个氨基酸序列与人CD226蛋白的氨基酸序列一致。优选为连续或间隔10-239,例如为连续5、10、20、30、40、50、60、70、80、90、100、200、240、300个氨基酸序列与人CD226蛋白的氨基酸序列一致。
本发明所述的“人源化CD226基因”,包含来源于人CD226核苷酸序列的部分和非人CD226基因的部分。其中,所述的“人CD226核苷酸序列”同“人CD226核苷酸序列的全部”,即其核苷酸序列与人CD226核苷酸序列的全长核苷酸序列一致。所述的“人CD226核苷酸序列的部分”为连续或间隔的20-1008bp个核苷酸序列与人CD226核苷酸序列一致,优选为20-831个,例如可以为20、50、100、200、300、400、500、600、700、800个核苷酸序列与人CD226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。
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“CD226基因座”表示CD226基因1号至6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。
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:小鼠CD226基因和人CD226基因对比示意图(非按比例);
图2:小鼠CD226基因人源化改造示意图(非按比例);
图3:CD226基因打靶策略及靶向载体设计示意图(非按比例),其中,靶向载体包含5’同源臂、3’同源臂以及包含人CD226和鼠CD226 DNA的A片段;
图4:sgRNA活性检测结果,其中Con为空白对照,PC为阳性对照;
图5:F0代小鼠基因型鉴定结果,其中WT为野生型对照,H2O为水对照;
图6:F1代小鼠基因型鉴定结果,其中WT为野生型对照,PC为阳性对照,H2O为水对照;
图7:Southern blot检测结果,其中WT为野生型对照;
图8:流式细胞检测结果,其中WT为野生型C57BL/6小鼠,H/+为CD226基因人源化小鼠杂合子。
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:
BbsI、EcoRI、BamHI、StuI、BglII酶购自NEB,货号分别为R0539L、R0101M、R0136M、R0187M、R0144M;
C57BL/6小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;
Ambion体外转录试剂盒购自Ambion,货号AM1354;
Cas9mRNA来源SIGMA,货号CAS9MRNA-1EA;
UCA试剂盒来源百奥赛图公司,货号BCG-DX-001;
Brilliant Violet 510™ anti-mouse CD45购自Biolegend,货号103138;
PerCP/Cy5.5 anti-mouse TCR β chain(mTCRb-BV711)购自Biolegend,货号109228;
APC anti-mouse CD226 (DNAM-1) Antibody(mCD226-APC)购自Biolegend,货号128809;
PE anti-human CD226 (DNAM-1) Antibody(hCD226-PE)购自Biolegend,货号338305。
实施例1 CD226基因人源化小鼠
小鼠CD226基因(NCBI Gene ID:225825,Primary source:MGI:3039602,UniProt:Q8K4F0,位于18号染色体NC_000084.6的第89176954至89272232位,基于转录本NM_178687.2及其编码蛋白NP_848802.2(SEQ ID NO.1))和人CD226基因(NCBI Gene ID:10666,Primary source:HGNC:16961,UniProt ID:Q15762,位于18号染色体NC_000018.10的第69853274至69962086位,基于转录本NM_006566.4及其编码蛋白NP_006557.2(SEQ IDNO.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CD226基因座引入编码人CD226蛋白的核苷酸序列,使得该小鼠表达人或人源化CD226蛋白。具体来说,用基因编辑技术,在小鼠CD226基因调节元件的控制下,***包含人CD226基因的2号外显子部分序列至6号外显子部分序列及小鼠6号外显子的部分序列至7号外显子的序列,并在小鼠序列后***LoxP STOP序列,得到人源化CD226基因座示意图如图2所示,实现对小鼠CD38基因的人源化改造。
引入CRISPR/Cas***进行基因编辑,设计如图3所示的打靶策略,图中显示了靶向载体上含有小鼠CD226基因上游和下游的同源臂序列,以及包含人CD226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_000084.6的第89205265至89206595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_000084.6的第89206599至89208210位核苷酸序列具有99%的同源性,区别在于第89206694位上G替换为C;A片段上人CD226、鼠CD226以及LoxP STOP序列按照5’-3’顺序依次排列,其中,人CD226的核苷酸序列(SEQ ID NO.5)与NCBI登录号为NM_006566.4的第73至906位核苷酸序列相同,鼠CD226的序列如SEQ ID NO.6所示。A片段中鼠CD226序列下游与LoxP STOP序列上游的连接设计为5’-gattgcttgaacatttttacgtctcaatttgtgaactgttatta agggttccggatcctcggggacaccaaatatggcgatc-3’(SEQ ID NO.7),其中序列“ctgtt”中的最后一个“t”是小鼠的最后一个核苷酸,序列“attaa”中的第一个 “a”是LoxP STOP序列的第一个核苷酸。改造后的人源化小鼠CD226基因表达的氨基酸序列如SEQ ID NO.32所示。
鉴于人CD226具有多种亚型或转录本,本文所述的方法可应用于其它亚型或转录本。
靶向载体构建可采用常规方法进行,如酶切连接、直接合成等。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用于后续实验。
靶序列决定了sgRNA的靶向特异性和诱导Cas9切割目的基因的效率。因此,高效特异的靶序列选择和设计是构建sgRNA表达载体的前提。设计并合成识别靶位点的sgRNA序列,各sgRNA在CD226基因上的靶位点序列如下:
sgRNA1靶位点序列(SEQ ID NO.8):5’-CAGAGATGGCTTATGTTACTTGG-3’
sgRNA2靶位点序列(SEQ ID NO.9):5’-GTTTTGCTATGGGGAGCACCAGG-3’
sgRNA3靶位点序列(SEQ ID NO.10):5’-CAAGTAACATAAGCCATCTCTGG-3’
sgRNA4靶位点序列(SEQ ID NO.11):5’-TTTTGCTATGGGGAGCACCAGGG-3’
sgRNA5靶位点序列(SEQ ID NO.12):5’-TCCTGAACTGTTTCCAGAGATGG-3’
sgRNA6靶位点序列(SEQ ID NO.13):5’-GCCATCTCTGGAAACAGTTCAGG-3’
sgRNA7靶位点序列(SEQ ID NO.14):5’-GCTGATAAGATCTGAGAGGAAGG-3’
sgRNA8靶位点序列(SEQ ID NO.15):5’-GTTTTGCTATGGGGAGCACCAGG-3’
利用UCA试剂盒检测多个sgRNA的活性,检测结果见图4,虽然有些sgRNA活性相对较低,这可能由于靶位点序列的特殊性导致,但根据我们的实验,这些sgRNA的数值仍显著高于对照组数值,仍可判断它们是具有活性的,活性满足基因打靶实验要求。从中选择sgRNA8进行后续实验。在其5’端及互补链上分别加上酶切位点得到正向寡核苷酸和反向寡核苷酸序列(见表1),退火后将退火产物连接至pT7-sgRNA质粒(质粒先用BbsI线性化),获得表达载体pT7-CD226-8。
表1 sgRNA8序列列表
SEQ ID NO.16 | 上游:5’-GTTTTGCTATGGGGAGCACC-3’ |
SEQ ID NO.17 | 上游:5’-TAGGTTTTGCTATGGGGAGCACC-3’ |
SEQ ID NO.18 | 下游:5’-GGTGCTCCCCATAGCAAAAC--3’ |
SEQ ID NO.19 | 下游:5’-AAACGGTGCTCCCCATAGCAAAAC--3’ |
pT7-sgRNA载体由质粒合成公司合成含有T7启动子及sgRNA scaffold的片段DNA(SEQ ID NO.20)并依次通过酶切(EcoRI及BamHI)连接至骨架载体(来源Takara,货号3299)上,经专业测序公司测序验证,结果表明获得了目的质粒。
取小鼠的原核期受精卵,例如C57BL/6小鼠,利用显微注射仪将pT7-CD226-8质粒的体外转录产物(使用Ambion体外转录试剂盒,按照说明书方法进行转录)、靶向载体与Cas9 mRNA预混好后注射至小鼠受精***质或细胞核中。按照《小鼠胚胎操作实验手册(第三版)》(安德拉斯・纳吉,化学工业出版社,2006)中的方法进行受精卵的显微注射,注射后的受精卵转移至培养液中短暂培养,然后移植至受体母鼠的输卵管中发育,将获得的小鼠(F0代)通过杂交和自交,扩大种群数量,建立稳定的CD226基因突变小鼠品系。
可通过常规检测方法(如PCR分析)鉴定F0代小鼠体细胞的基因型,部分F0代小鼠的鉴定结果见图5。结合5’端引物检测结果和3’端引物检测结果,经测序进一步验证图5中编号为F0-01到F0-06的6只小鼠均为阳性小鼠。PCR分析包括下述引物:
5’端引物:
L-GT-F(SEQ ID NO.21):5’-TCTATTATGGTCATACATACCTCCTGG-3’
L-GT-R(SEQ ID NO.22):5’-AGTGATCCCCATAGTTTTGAGGTAACTA-3’
3’端引物:
R-GT-F(SEQ ID NO.23):5’-TGACTGTAGCCGAGGGTAAAACCGA-3’
R-GT-R(SEQ ID NO.24):5’-AGGTGCTGAAACTAGTGCCAAATGA-3’
其中,引物L-GT-F位置位于5’同源臂左侧,R-GT-R位于3’同源臂右侧,L-GT-R和R-GT-F均位于A片段序列上。
将F0鉴定为阳性的CD226基因人源化小鼠与野生型小鼠交配得到F1代小鼠。可使用同样的PCR方法对F1代小鼠进行基因型鉴定,示例性检测结果见图6,显示编号为F1-01到F1-05的5只小鼠为阳性小鼠。
PCR检测引物序列包括:
L-GT-F(SEQ ID NO.21):5’-TCTATTATGGTCATACATACCTCCTGG-3’,
L-GT-R(SEQ ID NO.22):5’-AGTGATCCCCATAGTTTTGAGGTAACTA-3’;
R-GT-F(SEQ ID NO.23):5’-TGACTGTAGCCGAGGGTAAAACCGA-3’,
R-GT-R(SEQ ID NO.24):5’-AGGTGCTGAAACTAGTGCCAAATGA-3’;
WT-F(SEQ ID NO.25):5’-GCAGCTGATAAGATCTGAGAGGAAGG-3’,
WT-R(SEQ ID NO.26):5’-GATAAGCTCATGGTGAAGACTGACA-3’;
Mut-F(SEQ ID NO.27):5’-TGGGCTGCAGGTCGAGGGACCTA-3’
对这5只F1代PCR鉴定为阳性的小鼠进行Southern blot检测,确认是否存在随机***。剪取鼠尾提取基因组DNA,选用StuI酶或BglII酶消化基因组,转膜,杂交。5’探针和3’探针分别位于5’同源臂外侧及A片段序列上,具体探针及目的片段的长度见表2。Southernblot检测结果见图7,综合3’探针和5’探针的结果表明,5只小鼠均无随机***,证实这5只小鼠为阳性杂合小鼠且不存在随机***。这表明使用本方法能构建出可稳定传代,且无随机***的CD226基因人源化的基因工程小鼠。
表2具体探针及目的片段的长度
限制性内切酶 | 探针 | 野生型片段大小 | 重组序列片段大小 |
StuI | 5’探针(5’ Probe) | 12.8kb | 16.7kb |
BglII | 3’探针(3’ Probe) | —— | 3.8 kb |
探针合成引物如下:
5’ Probe-F(SEQ ID NO.28):5’-GGACCTGACTTCAATGATCACCTGGC-3’
5’ Probe-R(SEQ ID NO.29):5’-TGTGTCCACATGGGGCATGCTTAG-3’
3’ Probe-F(SEQ ID NO.30):5’-AACTGATGAATGGGAGCAGTGGTGG-3’
3’ Probe-R(SEQ ID NO.31):5’-GCAGACACTCTATGCCTGTGTGGAG-3’
将F1代鉴定为阳性的杂合小鼠相互交配,获得F2代CD226基因人源化纯合子小鼠。
可通过常规检测方法确认阳性小鼠体内人源化CD226蛋白的表达情况,例如使用流式细胞术等。具体来说,分别选取野生型C57BL/6雌性小鼠(7周龄)和本实施例制备的CD226基因人源化杂合子小鼠(10周龄)各1只,脱颈安乐死后取脾脏细胞,用抗鼠CD45抗体Brilliant Violet 510™ anti-mouse CD45、鼠特异性T细胞表面抗体PerCP/Cy5.5 anti-mouse TCR β chain(mTCRb-BV711)、抗鼠CD226抗体APC anti-mouse CD226 (DNAM-1)Antibody(mCD226-APC)或抗人CD226抗体PE anti-human CD226 (DNAM-1) Antibody(hCD226-PE)识别染色后进行流式检测,检测结果见图8。从图中可以看出,在野生型C57BL/6小鼠体内仅能检测出鼠CD226蛋白的表达(图8A),检测不到人源化CD226蛋白的表达(图8C);在CD226基因人源化杂合子小鼠体内既能检测到鼠CD226蛋白的表达(图8B),也能检测到人源化CD226蛋白的表达(图8D)。
实施例2 双重人源化或多重双人源化小鼠的制备
利用本方法或制得的CD226小鼠还可以制备双人源化或多人源化小鼠模型。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、TIGIT等其它基因修饰的小鼠,或者,也可在人源化CD226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CD226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CD226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,可有一定机率得到人源化CD226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CD226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。
序列表
<110> 北京百奥赛图基因生物技术有限公司
<120> CD226基因人源化非人动物的构建方法及应用
<130> 1
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 333
<212> PRT
<213> 小鼠(Mus musculus)
<400> 1
Met Ala Tyr Val Thr Trp Leu Leu Ala Ile Leu His Val His Lys Ala
1 5 10 15
Leu Cys Glu Glu Thr Leu Trp Asp Thr Thr Val Arg Leu Ser Glu Thr
20 25 30
Met Thr Leu Glu Cys Val Tyr Pro Leu Thr His Asn Leu Thr Gln Val
35 40 45
Glu Trp Thr Lys Asn Thr Gly Thr Lys Thr Val Ser Ile Ala Val Tyr
50 55 60
Asn Pro Asn His Asn Met His Ile Glu Ser Asn Tyr Leu His Arg Val
65 70 75 80
His Phe Leu Asn Ser Thr Val Gly Phe Arg Asn Met Ser Leu Ser Phe
85 90 95
Tyr Asn Ala Ser Glu Ala Asp Ile Gly Ile Tyr Ser Cys Leu Phe His
100 105 110
Ala Phe Pro Asn Gly Pro Trp Glu Lys Lys Ile Lys Val Val Trp Ser
115 120 125
Asp Ser Phe Glu Ile Ala Ala Pro Ser Asp Ser Tyr Leu Ser Ala Glu
130 135 140
Pro Gly Gln Asp Val Thr Leu Thr Cys Gln Leu Pro Arg Thr Trp Pro
145 150 155 160
Val Gln Gln Val Ile Trp Glu Lys Val Gln Pro His Gln Val Asp Ile
165 170 175
Leu Ala Ser Cys Asn Leu Ser Gln Glu Thr Arg Tyr Thr Ser Lys Tyr
180 185 190
Leu Arg Gln Thr Arg Ser Asn Cys Ser Gln Gly Ser Met Lys Ser Ile
195 200 205
Leu Ile Ile Pro Asn Ala Met Ala Ala Asp Ser Gly Leu Tyr Arg Cys
210 215 220
Arg Ser Glu Ala Ile Thr Gly Lys Asn Lys Ser Phe Val Ile Arg Leu
225 230 235 240
Ile Ile Thr Asp Gly Gly Thr Asn Lys His Phe Ile Leu Pro Ile Val
245 250 255
Gly Gly Leu Val Ser Leu Leu Leu Val Ile Leu Ile Ile Ile Ile Phe
260 265 270
Ile Leu Tyr Asn Arg Lys Arg Arg Arg Gln Val Arg Ile Pro Leu Lys
275 280 285
Glu Pro Arg Asp Lys Gln Ser Lys Val Ala Thr Asn Cys Arg Ser Pro
290 295 300
Thr Ser Pro Ile Gln Ser Thr Asp Asp Glu Lys Glu Asp Ile Tyr Val
305 310 315 320
Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Pro Arg Leu
325 330
<210> 2
<211> 336
<212> PRT
<213> 人(Homo sapiens)
<400> 2
Met Asp Tyr Pro Thr Leu Leu Leu Ala Leu Leu His Val Tyr Arg Ala
1 5 10 15
Leu Cys Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn
20 25 30
Met Ser Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val
35 40 45
Glu Trp Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser
50 55 60
Pro Thr His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr
65 70 75 80
Phe Leu Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg
85 90 95
Asn Ala Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr
100 105 110
Tyr Pro Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp
115 120 125
Ser Phe Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro
130 135 140
Gly Lys Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val
145 150 155 160
Gln Ala Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu
165 170 175
Thr Tyr Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro
180 185 190
Arg Gln Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val
195 200 205
Ile Pro Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu
210 215 220
Gln Ala Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val
225 230 235 240
Ala Glu Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Gly Gly
245 250 255
Thr Val Leu Leu Leu Leu Phe Val Ile Ser Ile Thr Thr Ile Ile Val
260 265 270
Ile Phe Leu Asn Arg Arg Arg Arg Arg Glu Arg Arg Asp Leu Phe Thr
275 280 285
Glu Ser Trp Asp Thr Gln Lys Ala Pro Asn Asn Tyr Arg Ser Pro Ile
290 295 300
Ser Thr Ser Gln Pro Thr Asn Gln Ser Met Asp Asp Thr Arg Glu Asp
305 310 315 320
Ile Tyr Val Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Thr Arg Val
325 330 335
<210> 3
<211> 1331
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cttgaacttc atatgtgatg agtccattta aaagattatc agtgtgaatc aattattttg 60
tgtatcagca tgagtataaa acagggagat gtttttagag aacatgaaat ttcagctgag 120
aataagaaat aacctcattt ataatgtgca cattgtatca tggttctgct aatggggcca 180
aatgtaatat aattcttctt tttataaaga taaaagtttg agttttgatt gtcttgttga 240
aatattcttc acaaattttt aagaattaga attaatattt tattatatat tagttattca 300
tattataatt gtgtgtgttt gtttggtatg tgtggtttca tgtgccaccg tgtacatgtg 360
ctagttcaag cataactttg tgaagttagt tctatcctct tacctttatg taggttctgg 420
agactcaggt catcaggctt ttttaataag cattgttact ggctaaccca tcttgtcaca 480
accctaagtt gtttttataa ttgaagagag aaatcttaga aatgaatctc actttgatag 540
tgtatcacag ttttagatat ataaggtgta acctctaagt tgtaatatct agttttttcg 600
tagtactcta tgcttatttt aaaattaaaa agtggaaaaa aaatcatcag atataatggt 660
atattacatg tgttttggat cgtaatattt tgtagttgga ggtccattca gttgaatatc 720
tttgttcaaa tctaacccag aggtgtggtg cagctggaag gccacacctg agtggttgtc 780
tgatatgcag gctcctccca tcagctccag tcttcacccc atttcacttt ctcagtgagc 840
agttcctgga cttgcctata tgaaaagatc ttgactgtcc tgctttgctt cacttcttct 900
caggtaccaa ccaccttcag cacactatat ctagtacacc tttagcattg tatcaacctc 960
ttcctaacaa aatggcatgg gattttattt ttaagatgca tttttgtata ttcttgttgc 1020
acagagctaa gtctggcaca gaggacacac tcagtaaaca tttgtcaaat gaaagaatct 1080
ttctcgaaaa ttatatcttt taaagtaaac tgattatctg acagagaaca ataactgcag 1140
cctatccatc cctgtacgat agcaaagctt tctttgaagc tgaaacttct ttcctgtgct 1200
catacttcag agaaaaagat gcagctgata agatctgaga ggaaggaaac agtacttcat 1260
gctgagctgt gaatataaca tcatacacat ttttttttta tacagtccaa cctcctgaac 1320
tgtttccaga g 1331
<210> 4
<211> 1612
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcttatgtta cttggctttt ggctattctt catgtgcaca aaggtaaagc cagtcctatg 60
tattcattta aaggttttgc tatggggagc accagcgtca agaagtggga gtagataggt 120
aggggagggt ggaggagggc atagggaact ttcgggatag catttgaaat gtaaatgaag 180
aaaatacata ataaaaaaat tggaaagaaa aaaaagtttt gctttttttt ttgtgacatg 240
tgcaggatct cttaggcagt ttctcacttg tttagaaata ctaaaattta caagaggcaa 300
ttttgtagtt gaaattgtca gtcttcacca tgagcttatc atgctatttg ttaagtctta 360
taagttgtgt ttatttggct tcgtcatcat tggatctaat caatttatgt gattgatata 420
cattttagca ctgtgtgaag agacattgtg ggacacaaca gttcggcttt ctgagactat 480
gactctggaa tgtgtatatc cattgacgca taacttaacc caggtggagt ggaccaagaa 540
cactggcaca aagacagtga gcatagcagt ttacaaccct aaccataata tgcatataga 600
atctaactac ctccatagag tacacttcct aaactcaaca gtggggttcc gcaacatgag 660
cctttccttt tacaatgcct cagaagcaga cattggcatc tactcctgct tgtttcatgc 720
tttcccaaat ggaccttggg aaaagaagat aaaagtagtc tggtcaggta aggaaaaact 780
ttattttctt tttgtctact gacaatgttt aattctaaga aacccaacca aaagcttaaa 840
atgttattgt attcttttcc caattccatt gctagatttt tgatctaatt tttcctcaac 900
gaataatgtc gtaaaccatc tcattttttt cttaattgct gtcatttctg agtgcaagtg 960
tctcccaggt ttcagtgatg tgggaggaaa tgcagattta catagttttt atgatgacat 1020
aaggttagat tgaaataata ttgtcaacat gtagattcag agcaatccct tctttttttt 1080
cattttattt atttattttt taattaggta ttttcttcat ttacatttca aatgctatcc 1140
caaaagtccc tcataccccc ccccactccc ctacccacct actcccaatt cttggtcctg 1200
gcgttcccct gtactgaggc atataaagtt tgcaagacca aggggcctct cttcccaatg 1260
atggccaact aggccatctt ctgatacata tgcagctaga gacacgagct ccgggggtaa 1320
tggttagttc atattgttgt tccacctata aggttgcaga cccctttagc tccttgagta 1380
ctttctctag ctcctccatt ggcagccctg tgttccatcc aatagctgac tgtgagcatc 1440
cacttctgtg tttgccaggc cctggcatag cctcacaaga gacagcaata tcagggtcct 1500
ttcagcaaaa tctttctggt gcatgcaatg gtgtcagcat ttggaggctg attatgggat 1560
ggatccccgg gtatgccagt ctctagatgg tccatccttt actggtaaga ag 1612
<210> 5
<211> 834
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggattatc ctactttact tttggctctt cttcatgtat acagagctct atgtgaagag 60
gtgctttggc atacatcagt tccctttgcc gagaacatgt ctctagaatg tgtgtatcca 120
tcaatgggca tcttaacaca ggtggagtgg ttcaagatcg ggacccagca ggattccata 180
gccattttca gccctactca tggcatggtc ataaggaagc cctatgctga gagggtttac 240
tttttgaatt caacgatggc ttccaataac atgactcttt tctttcggaa tgcctctgaa 300
gatgatgttg gctactattc ctgctctctt tacacttacc cacagggaac ttggcagaag 360
gtgatacagg tggttcagtc agatagtttt gaggcagctg tgccatcaaa tagccacatt 420
gtttcggaac ctggaaagaa tgtcacactc acttgtcagc ctcagatgac gtggcctgtg 480
caggcagtga ggtgggaaaa gatccagccc cgtcagatcg acctcttaac ttactgcaac 540
ttggtccatg gcagaaattt cacctccaag ttcccaagac aaatagtgag caactgcagc 600
cacggaaggt ggagcgtcat cgtcatcccc gatgtcacag tctcagactc ggggctttac 660
cgctgctact tgcaggccag cgcaggagaa aacgaaacct tcgtgatgag attgactgta 720
gccgagggta aaaccgataa ccaatatacc ctctttgtgg ctggagggac agttttattg 780
ttgttgtttg ttatctcaat taccaccatc attgtcattt tccttaacag aagg 834
<210> 6
<211> 1714
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agacggagac aggtgagaat tccacttaaa gagcccaggg ataaacagag taaggtagcc 60
accaactgca gaagtcctac ttctcccatc cagtctacag atgatgaaaa agaggacatt 120
tatgtaaact atccaacttt ctctcgaaga ccaaaaccaa gactctaagc tgctcttttg 180
gcctgaacac attagtgatg acttctatgg catggaattt tacccatgat ttccttacca 240
ctaggatcta cattgataaa aaaaattgat taaatttatt tcatctcata tatagaagta 300
ctttattacc tggaaacatt cttaatagag attcattaga aaacccaaat ctaatgttca 360
tgtgttcaag gaaccttctt ccattatgta acagaacagt ctagagaaga ttaaggacca 420
catggctttc ttgctctact tgaaattaat tgtgagcata agcttgtttc tggagtcttc 480
ttacattgtt ggttctactt acatactact ggtccaactc tcatgctgtt tctctcagat 540
gttcccatga tggttgccaa ggacacttga tagaaagact actggttaaa cacaataaac 600
aaagttcatt attcacttat tagcaagaag gtagcattat cataaaggat tagatgactt 660
aagttagcta taggttcaag acctggacta aagtattact tggaaattct gagtattgct 720
aaaaaggagg atgaaaggga cctagaagtt gagttattac taaaaacttt gagtgcgaag 780
atattactca ttaaccagat aacaagtgaa tatgctgtag catcaacata attcaaaaga 840
gtaaagaaat ggctaggaat gaggtagttg tgtaattatt tcttctctta ctagtttcaa 900
ataaattcat ctctaattct atagagaatt cttgcctccc attcaggact ggccttctat 960
acagtgagat ggtccagtaa gaaataattt ttattagtgt tttttctatt ttgagaatta 1020
ttttaatata tattttaata tataaacttg tgagttaaat tttttttttg caaaattagc 1080
acatgaaaag agattgatgg ttttaagtag tagaacacag tagtgtagga atctgagagc 1140
agagagtttg ggagggggtg aagagaaaac aacatcacca aatagtgata tataagagaa 1200
aatctgtgct tcagagtttg atcagggcca tctctcccaa ctctgctgga actgagagaa 1260
tgcacctgat gttgtctcca ttttagatag agaaaaaaaa aacccgaata tttataaaac 1320
taaataaaac tatagttacc tcaaaactat ggggatcact ataacataga atagaataga 1380
atagaataga atagaataga atagaataga atagatttag tatctatttt ctaatctcta 1440
aaaaatttta aattatcata atctttgaat gctagtgctg ttacatatat tctaaaatgt 1500
aatccatagt tttaaggtag ctgggggata tttcatattt ttaaccttag atccatgcat 1560
aatgaatagg ccaattattt tgccaattat agaaattcag aatgtaataa ataaaaatga 1620
cttttttctt agttgttctt aaataagaat gattaattaa ctcagatttt gactgattgc 1680
ttgaacattt ttacgtctca atttgtgaac tgtt 1714
<210> 7
<211> 82
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gattgcttga acatttttac gtctcaattt gtgaactgtt attaagggtt ccggatcctc 60
ggggacacca aatatggcga tc 82
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cagagatggc ttatgttact tgg 23
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gttttgctat ggggagcacc agg 23
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caagtaacat aagccatctc tgg 23
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ttttgctatg gggagcacca ggg 23
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcctgaactg tttccagaga tgg 23
<210> 13
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gccatctctg gaaacagttc agg 23
<210> 14
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gctgataaga tctgagagga agg 23
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gttttgctat ggggagcacc agg 23
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gttttgctat ggggagcacc 20
<210> 17
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
taggttttgc tatggggagc acc 23
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ggtgctcccc atagcaaaac 20
<210> 19
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aaacggtgct ccccatagca aaac 24
<210> 20
<211> 132
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60
agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120
tttaaaggat cc 132
<210> 21
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tctattatgg tcatacatac ctcctgg 27
<210> 22
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agtgatcccc atagttttga ggtaacta 28
<210> 23
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgactgtagc cgagggtaaa accga 25
<210> 24
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aggtgctgaa actagtgcca aatga 25
<210> 25
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gcagctgata agatctgaga ggaagg 26
<210> 26
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gataagctca tggtgaagac tgaca 25
<210> 27
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tgggctgcag gtcgagggac cta 23
<210> 28
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ggacctgact tcaatgatca cctggc 26
<210> 29
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgtgtccaca tggggcatgc ttag 24
<210> 30
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
aactgatgaa tgggagcagt ggtgg 25
<210> 31
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gcagacactc tatgcctgtg tggag 25
<210> 32
<211> 333
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Met Asp Tyr Pro Thr Leu Leu Leu Ala Leu Leu His Val Tyr Arg Ala
1 5 10 15
Leu Cys Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn
20 25 30
Met Ser Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val
35 40 45
Glu Trp Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser
50 55 60
Pro Thr His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr
65 70 75 80
Phe Leu Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg
85 90 95
Asn Ala Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr
100 105 110
Tyr Pro Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp
115 120 125
Ser Phe Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro
130 135 140
Gly Lys Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val
145 150 155 160
Gln Ala Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu
165 170 175
Thr Tyr Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro
180 185 190
Arg Gln Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val
195 200 205
Ile Pro Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu
210 215 220
Gln Ala Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val
225 230 235 240
Ala Glu Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Gly Gly
245 250 255
Thr Val Leu Leu Leu Leu Phe Val Ile Ser Ile Thr Thr Ile Ile Val
260 265 270
Ile Phe Leu Asn Arg Arg Arg Arg Arg Gln Val Arg Ile Pro Leu Lys
275 280 285
Glu Pro Arg Asp Lys Gln Ser Lys Val Ala Thr Asn Cys Arg Ser Pro
290 295 300
Thr Ser Pro Ile Gln Ser Thr Asp Asp Glu Lys Glu Asp Ile Tyr Val
305 310 315 320
Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Pro Arg Leu
325 330
Claims (9)
1.一种CD226基因人源化改造的非人动物的构建方法,其特征在于,所述的非人动物的基因组中包含人源化CD226核苷酸序列,所述人源化CD226核苷酸序列从5’-3’顺序依次包含人CD226核苷酸序列、鼠CD226核苷酸序列和LoxP STOP序列,其中,所述人CD226核苷酸序列编码的人CD226蛋白序列如SEQ ID NO.2第1至278位所示氨基酸序列,所述鼠CD226 核苷酸序列如SEQ ID NO.6所示;
所述的构建方法包括用所述人源化CD226核苷酸序列***或替换至非人动物CD226基因座上,使用靶向载体或sgRNA进行非人动物的构建;
所述的靶向载体包含所述人源化CD226核苷酸序列, 所述的靶向载体还包含与待改变的转换区5’端和3’端同源的DNA片段,即5’和3’臂,所述5’臂序列如SEQ ID NO.3所示,所述3’臂序列如SEQ ID NO.4所示;
所述sgRNA靶向的靶位点序列如SEQ ID NO.8-15任一项所示。
2.根据权利要求1所述的构建方法,其特征在于,所述人CD226核苷酸序列如SEQ IDNO.5所示的核苷酸序列。
3.根据权利要求1所述的构建方法,其特征在于,所述人源化CD226蛋白如SEQ IDNO.32的氨基酸所示。
4.根据权利要求1-3任一所述的构建方法,其特征在于,所述的非人动物为大鼠或小鼠。
5.一种靶向载体,其特征在于,所述的靶向载体包含人源化CD226核苷酸序列, 所述的靶向载体还包含与待改变的转换区5’端和3’端同源的DNA片段,即5’和3’臂,所述5’臂序列如SEQ ID NO.3所示,所述3’臂序列如SEQ ID NO.4所示,所述人源化CD226核苷酸序列从5’-3’顺序依次包含人CD226核苷酸序列、鼠CD226 核苷酸序列和LoxP STOP序列,其中,所述人CD226核苷酸序列编码的人CD226蛋白序列如SEQ ID NO.2第1至278位所示氨基酸序列,所述鼠CD226 核苷酸序列如SEQ ID NO.6所示。
6.如权利要求5所述的靶向载体,其特征在于,人CD226核苷酸序列如SEQ ID NO.5所示的核苷酸序列。
7.一种人源化CD226蛋白,其特征在于,所述的人源化CD226蛋白包含人CD226蛋白的胞外区、信号肽、跨膜区和胞质区,并且所述人源化CD226蛋白如SEQ ID NO.32的氨基酸序列。
8.一种编码权利要求7所述的人源化CD226蛋白的人源化CD226基因,其特征在于,所述的人源化CD226基因包含SEQ ID NO.5的核苷酸序列。
9.权利要求1-4任一构建方法构建的非人动物,权利要求7所述的人源化CD226蛋白、权利要求8所述的人源化CD226基因在CD226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***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CD226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CD226基因功能,研究人CD226抗体,研究针对人CD226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应用。
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