CN114134183B - 一种siglec15基因人源化动物模型的构建方法及应用 - Google Patents
一种siglec15基因人源化动物模型的构建方法及应用 Download PDFInfo
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Abstract
本发明通过筛选获得了用于小鼠高效同源重组的CRISPR‑CAS9靶标序列，进一步构建成功包含人Siglec‑15基因片段的小鼠同源重组载体，其可定向重组人Siglec‑15基因到小鼠Siglec‑15基因内，实现对小鼠基因组的人源化改造。通过CRISPR‑CAS9以及同源重组技术成功获得了人源化Siglec‑15基因的小鼠，该小鼠模型体内能正常表达人源的SIGLEC15蛋白。获得的SIGLEC15人源化小鼠可以应用于针对SIGLEC15信号机制研究，针对炎症、自身免疫行疾病、肿瘤和抗病毒的药物筛选和评价，无论对SIGLEC15基因功能机制还是药物研发都具有重要的应用价值。
Description
技术领域
本发明属于生物技术领域，涉及人源化基因修饰动物模型的构建方法及应用，具体而言，涉及基于一种SIGLEC15基因人源化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
肿瘤细胞在发生发展过程中发展出各种免疫逃逸机制来逃避肿瘤微环境(TME)中针对肿瘤特异性和非特异性的攻击。因此，单纯的某一种免疫系统的全身性激活甚至外周肿瘤特异性T细胞的增加并不一定能介导肿瘤消退。同时，目前已知的癌症免疫逃逸策略也被用于维持体内的免疫系统稳态。为了杀伤肿瘤细胞，针对这些免疫逃逸机制，激活的非特异性免疫反应越多，发生不良事件的风险也就越高，进而导致治疗成功的可能性降低。
开发免疫正常化疗法的关键在于靶向肿瘤诱导的免疫逃逸机制，这些机制应限于TME，并选择性地恢复TME中针对肿瘤的免疫反应。因此，肿瘤免疫治疗正常化的靶标需要具备以下特点：1）由于肿瘤生长和/或随后的免疫监视而诱导产生；2）优先在TME中表达，在正常组织中不表达或少量表达；3）TME中的表达能够引发免疫逃逸，阻断或控制其表达可以重置或重编程TME中的抗肿瘤免疫。
肿瘤免疫治疗中，针对PD-L1/PD-1信号通路的免疫检查点抑制剂是最为成功的案例，阻断PD-L1/PD-1信号通路进行抗肿瘤治疗，即为肿瘤免疫治疗正常化的原型。PD-L1蛋白在正常人体组织中的表达水平很低，但在一些肿瘤患者中可以被选择性诱导过度表达，并且这种诱导局限于肿瘤微环境中。因此，阻断PD-L1/PD-1可避免全身过度免疫反应，避免对正常组织的严重损伤，同时又可以选择性地使TME中的免疫功能正常化和复位。然而，PD-L1/PD-1在人类实体肿瘤中只负责部分免疫功能失调，尽管针对该通路的抑制性药物取得了巨大成功，但只有30-40%的患者有响应，大多数患者表现出原发性或获得性耐药性。因此，需要在 TME 中鉴定新的下一代可媲美PD-L1/PD-1的免疫治疗靶标。
Siglec15 (S15) 为Siglec家族(唾液酸结合免疫球蛋白凝集素)的成员之一，该家族是免疫球蛋白(Ig)超家族蛋白的一个独特亚群，参与识别自身和非自身免疫调节。以前的研究表明，Siglec-15在破骨细胞分化和骨重建中的作用，但其免疫功能仍然很大程度上未知。2019年4月，Nature Medicine上刊登了陈列平教授团队的文章（Nature Medicine，volume 25, pages656–666 (2019)）。他们的研究表明，S15是一种关键的免疫抑制因子，其通常仅在某些骨髓细胞上表达，但在人类肿瘤细胞和肿瘤浸润性骨髓细胞上广泛上调，抑制肿瘤免疫，并且这种肿瘤抑制性能够以独立于PD-L1/PD-1通路的方式介导。此外，该研究显示S15敲除小鼠未发生自身免疫或其他疾病，表明Siglec15抑制可能不会引起正常细胞的不良反应。这些证据暗示，S15靶点可成为针对PD-1靶向治疗无应答的癌症患者的潜在治疗靶点，其可能是开发免疫系统正常化下一代免疫药物的一个重要靶点。目前针对S15靶点已有两个药物进入临床阶段，一个是美国NextCure公司开发的NC-318,处于临床II期阶段，一个是日本第一三共株式会社开发的DS-1501，处于临床I期阶段；国内也有多家公司针对S15靶点进行临床前的研发。
在药物临床前研究中，小鼠被广泛的应用于药效评价、药理毒理分析等研究，应用于这些研究的前提是，作用于人源靶点的药物需能够结合或激活小鼠对应的靶点，如受试药物不能和鼠源靶点结合或激活下游信号通路，则不能利用小鼠进行药效、药理毒理等评价。通过对人和小鼠的SIGLEC15蛋白比较发现，人和小鼠的SIGLEC15蛋白氨基酸一致性只有77.9%。这种人鼠蛋白氨基酸序列的差异，很可能造成针对人S15靶点的药物不能识别鼠源S15靶点，尤其是抗体类药物，识别位点一个氨基酸的差异都可能造成抗体无法识别靶位点。因此，很可能无法用野生型小鼠模拟针对人源SIGLEC15的信号转导、疾病模型建立、药物评价和筛选研究，而动物模型实验作为临床前药物研究的必备环节，模型缺失可能成为药物研发的瓶颈。
随着基因工程技术的不断发展和成熟，用人类细胞或基因替代或置换动物的内源性同类细胞或基因，以建立更接近人类的生物体系或疾病模型，建立人源化实验动物模型(humanized animal model)，已经为临床上新的治疗方法或手段提供了重要工具。其中基因人源化动物模型，即利用基因修饰操作技术，用人类正常或突变基因替换动物同源基因，可在动物体内建立更接近人类疾病特征的正常或突变基因的基因人源化动物模型。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提高药物对体内药靶的识别性，更重要的是，由于人类基因片段的存在，动物体内可表达或部分表达人类功能的蛋白质，从而大大减少临床前动物实验与临床实验结果的差异，为在临床前动物体内水平进行药物筛选和验证提供了可能。
发明内容
本发明的目的在于提供一种SIGLEC15基因人源化改造的非人动物模型的构建方法，利用同源重组的方式用人SIGLEC15的相关序列替换动物内源的SIGLEC15序列，使该模型体内能正常表达人源的SIGLEC15蛋白，从而应用于针对SIGLEC15信号机制研究，针对炎症、自身免疫行疾病、肿瘤和抗病毒的药物筛选和评价，无论对SIGLEC15基因功能机制还是药物研发都具有重要的应用价值。
本发明公开了一种SIGLEC15基因人源化非人动物模型的构建方法，所述的构建方法包括使用靶向SIGLEC15基因的gRNA和同源重组载体进行构建。
优选的，所述gRNA靶向5’端的序列如SEQ ID NO:12所示，所述gRNA靶向3’端序列如SEQ ID NO:26所示。
优选的，所述同源重组载体包括5’同源臂、人SIGLEC15基因片段、3’同源臂，所述人SIGLEC15基因片段的编码的蛋白序列如SEQ ID NO:2所示。
优选的，所述的非人动物体内表达人SIGLEC15基因编码的蛋白。
优选的，所述同源重组载体包括的5’同源臂的序列如SEQ ID NO:6所示，所述同源重组载体包括的3’同源臂的序列如SEQ ID NO:7所示。
优选的，所述人SIGLEC15基因部分破坏内源SIGLEC15基因的编码框，使内源SIGLEC15基因不表达。
优选的，所述非人动物为小鼠。
优选的，所述人SIGLEC15基因片段的序列如SEQ ID NO:5所示。
本发明公开了一种用于构建SIGLEC15基因人源化非人动物模型的同源重组载体。
优选的，所述同源重组载体包括5’同源臂、人SIGLEC15基因片段、3’同源臂。
优选的，所述同源重组载体包括的5’同源臂的序列如SEQ ID NO:6所示，所述同源重组载体包括的3’同源臂的序列如SEQ ID NO:7所示。
优选的，所述人SIGLEC15基因片段编码的蛋白序列如SEQ ID NO:2所示。
本发明公开了一种用于构建SIGLEC15基因人源化非人动物模型的gRNA，包括靶向5’端的gRNA和靶向3’端的gRNA。
优选的，所述靶向5’端的gRNA识别的靶位点序列如SEQ ID NO:12所示，所述靶向3’端的gRNA识别的靶位点序列如SEQ ID NO:26所示。
本发明公开了一种用于构建SIGLEC15基因人源化非人动物模型的试剂盒，所述试剂盒包括所述的同源重组载体和所述的gRNA，以及Cas9 mRNA。
本发明公开了所述的构建方法获得的SIGLEC15基因人源化的非人动物在SIGLEC15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，用于病原学研究中的应用；
d）在体内研究人SIGLEC15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SIGLEC15基因功能，研究人SIGLEC15抗体，研究针对人SIGLEC15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，所述应用不是疾病的诊断和治疗方法。
本发明通过筛选获得了用于小鼠高效同源重组的CRISPR-CAS9靶标序列，构建成功包含人Siglec-15基因片段的小鼠同源重组载体，其可定向重组人Siglec-15基因到小鼠Siglec-15基因内，实现对小鼠基因组的人源化改造，通过CRISPR-CAS9以及同源重组技术成功获得了人源化Siglec-15基因的小鼠，并对获得的人源化小鼠中Siglec-15基因的表达量以及药效试验进行了验证。利用本方法可以高效构建获得可稳定传代的SIGLEC15基因人源化小鼠模型，SIGLEC15人源化小鼠可以用于针对人源SIGLEC15靶点药物的药效和安全性进行评价。
附图说明
为了更清楚的说明本发明实施例或现有方法和实验中的技术方案，下面将对实施例中所需使用的附图做简单的介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为小鼠SIGLEC15基因和人SIGLEC15基因结构示意图。
图2为改造后的人源化小鼠SIGLEC15基因结构示意图。
图3为对小鼠SIGLEC15基因进行人源化改造的重组策略示意图。
图4为gRNA活性验证T7核酸内切酶I酶切检测电泳图。A为针对5’端重组位点靶序列的gRNA活性检测电泳结果；B为针对3’端重组位点靶序列的gRNA活性检测电泳结果。数字为对应的gRNA，Con为对照，M为DL2000 DNA marker。
图5为F0代和F1代小鼠同源重组PCR鉴定结果电泳图。A为F0代小鼠5’同源臂同源重组鉴定电泳图；B为F0代3’同源臂同源重组鉴定电泳图；C为F1代小鼠5’和3’同源臂同源重组鉴定电泳图，电泳图中M左侧为5’同源臂同源重组鉴定结果，电泳图中M右侧为3’同源臂同源重组鉴定。上方数字为阳性小鼠号码；M为1kb DNA marker。
图6为SIGLEC15人源化小鼠RNA水平人源SIGLEC15表达验证结果。A为反转录PCR电泳引物位置设计示意图（非按比例）；B为反转录PCR电泳结果图。WT，野生型小鼠；HE，SIGLEC15人源化杂合子小鼠；HO，SIGLEC15人源化纯合子小鼠；M为DL2000 DNA marker。
图7为SIGLEC15人源化纯合子小鼠接种MC38-SIGLEC15结肠癌肿瘤细胞给予抗人SIGLEC15抗体的抗肿瘤药效结果。
图8为SIGLEC15人源化小鼠黑色素瘤细胞侵染，其中A和B为野生型小鼠体内的黑色素瘤细胞，C为SIGLEC15人源化纯合子小鼠体内的黑色素瘤细胞。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 SIGLEC15人源化小鼠模型构建
1）人源化策略设计：
人的SIGLEC15基因含有多个转录本，本实施例的序列设计主要以其中一个转录本为例进行阐述。人源化的总体策略为：从DNA层面，将小鼠Siglec15基因(ENSEMBL Gene ID：ENSMUSG00000091055)第2号外显子的部分编码区到第4号外显子部分编码区之间的序列(基于ENSEMBL登录号为ENSMUST00000170760.3的转录本，该转录本对应的蛋白序列如SEQID NO：1所示)用人SIGLEC15基因(ENSEMBL Gene ID：ENSG00000197046)的第2号外显子的部分编码区到第4号外显子部分编码区之间的序列替换(基于ENSEMBL登录号为ENST00000389474.8的转录本，该转录本对应的蛋白序列如SEQ ID NO：2所示)；对应的蛋白层面，则是将小鼠Siglec15蛋白的胞外区对应的蛋白氨基酸序列替换为人SIGLEC15蛋白的胞外区，从而使小鼠表达编码一个人鼠嵌合的人源化SIGLEC15蛋白，人源化后对应的蛋白序列如SEQ ID NO:3所示。最终被替换的鼠源序列如SEQ ID NO：4所示，置换的人源序列如SEQ ID NO：5所示。鼠SIGLEC15基因与人SIGLEC15基因对比示意图见图1，最终得到的改造后的人源化小鼠SIGLEC15基因示意图见图2。
根据序列设计，本发明进一步的设计了如图3所示的同源重组方案以及包含5’同源臂、包含人SIGLEC15的基因片段、3’同源臂的载体。其中5’同源臂(SEQ ID NO：6)为NCBI登录号为NC_000084.7的第78095478-78092479位核苷酸；3’同源臂(SEQ ID NO：7)为NCBI登录号为NC_000084.7的第78090522-78087523位核苷酸；包含人SIGLEC15的基因片段序列如SEQ ID NO：5。根据设计方案，改造后的人源化小鼠表达的人源SIGLEC15的mRNA序列如SEQ ID NO：8所示，蛋白序列如SEQ ID NO：3所示。根据策略涉及方案，选用CRISPR/Cas9的方式构建SIGLEC15人源化小鼠模型。
2）gRNA靶位点筛选
在用CRISPR/Cas9的方式构建基因修饰小鼠模型过程中，gRNA靶序列决定了其靶向特异性和诱导Cas9切割目的基因的效率。Cas9切割目的基因的效率越高，发生同源重组的效率就越高。因此，高效特异的靶序列选择和设计是高效获得基因修饰小鼠模型的前提。
根据重组方案，在替换序列的5’端和3’端附近设计并合成识别5’端靶位点1-10（SEQ ID NO:9-18）的gRNA(gRNA1-gRNA10)和识别3’端靶位点11-20（SEQ ID NO:19-28）的gRNA(gRNA11-gRNA20)。使用T7核酸内切酶 I检测试剂盒检测，从酶切后凝胶电泳结果可见不同的gRNA活性不同，其中针对5’端的gRNA4和针对3’端的gRNA18切割活性最高，检测结果如图4所示。因此，选择靶向5’端的靶位点4和靶向3’端的靶位点18的gRNA4和gRNA18为后续小鼠受精卵注射实验的gRNA。
3）同源重组载体构建
以C57BL/6小鼠基因组DNA或BAC文库为模板PCR扩增获得5’同源臂、3’同源臂，以合成的人SIGLEC15基因片段为模板，PCR扩增获得人SIGLEC15的基因片段。通过In-fusion试剂盒将片段连接到PBR322-MCS质粒上，获得同源重组载体PBR322-SIGLEC15，载体经酶切、测序验证正确后，用于后续受精卵显微注射。
4）SIGLEC15人源化小鼠获得：取C57BL/6小鼠的受精卵，按照《小鼠胚胎操作实验手册(第三版)》中的方法将PBR322-SIGLEC15重组载体、Cas9 mRNA和gRNA混合后,进行受精卵显微注射，注射后的受精卵经培养箱短暂培养后，移植至受体母鼠的输卵管，获得基因修饰小鼠F0代小鼠。F0代小鼠出生后，剪尾抽提基因组，通过长片段PCR的方式分别对5’同源臂和3’同源臂同源重组阳性克隆进行PCR鉴定，PCR鉴定的引物位置示意如图3所示，F0代小鼠同源重组具体鉴定信息如下所示。
5’臂同源重组鉴定条件如下：
引物序列信息如下：
| 引物名称 | 序列信息（5’→3’） |
| P1 | CAAGCCCTCTCCCCCTTTGG （SEQ ID NO: 29） |
| P2 | GGACTATTCCCCTCTTCGTTTCCTAA （SEQ ID NO: 30） |
PCR反应体系如下：
| PCR反应组成 | 体积 (µl) |
| ddH2O | 13.2 |
| 10xPCR Buffer | 2 |
| 2.5 mM dNTP | 2 |
| 引物P1 (10pmol/µl) | 0.5 |
| 引物P2 (10pmol/µl) | 0.5 |
| DNA Polymerase | 0.8 |
| genomic DNA | 1 |
| 总计 | 20 |
PCR反应程序如下：
| 步骤 | 温度（℃） | 时间 | 备注 |
| 1 | 94 | 3 min | |
| 2 | 98 | 15 sec | |
| 3 | 60 | 15 sec | |
| 4 | 68 | 3 min | 重复步骤2-4共34个循环 |
| 5 | 68 | 5 min | |
| 6 | 12 | 10 min |
3’臂同源重组鉴定条件如下：
引物序列信息如下：
| 引物名称 | 序列信息（5’→3’） |
| P3 | ACCCAAGTGCCCATTCCTC （SEQ ID NO: 31） |
| P4 | TCCCAGACATCCCTACCTCCAA （SEQ ID NO: 32） |
PCR反应体系如下：
| PCR反应组成 | 体积 (µl) |
| ddH2O | 13.2 |
| 10xPCR Buffer | 2 |
| 2.5 mM dNTP | 2 |
| 引物P3 (10pmol/µl) | 0.5 |
| 引物P4 (10pmol/µl) | 0.5 |
| DNA Polymerase | 0.8 |
| genomic DNA | 1 |
| 总计 | 20 |
PCR反应程序如下：
| 步骤 | 温度（℃） | 时间 | 备注 |
| 1 | 94 | 3 min | |
| 2 | 98 | 15 sec | |
| 3 | 60 | 15 sec | |
| 4 | 68 | 3 min | 重复步骤2-4共34个循环 |
| 5 | 68 | 5 min | |
| 6 | 12 | 10 min |
根据设计策略和鉴定方案，5’臂正确重组的阳性F0代小鼠可以扩增出3.7kb大小目的PCR产物条带，阴性克隆无目的大小条带扩增出；3’臂正确重组的阳性F0代小鼠可以扩增出3.7kb大小的目的PCR产物条带，阴性克隆无目的大小条带扩增出。经鉴定，9只F0代小鼠中有5只小鼠5’和3’臂均扩增出目的大小产物条带，鉴定电泳结果如图5中A和B所示，PCR产物经测序确认，5只F0代小鼠为阳性SIGLEC15人源化小鼠，阳性率为55.6%。
F0代小鼠成年后与野生型C57BL/6小鼠交配，获得F1代小鼠。F1代小鼠出生后，剪尾抽提基因组，用和鉴定F0代小鼠同源重组相同的条件，分别针对5’同源臂和3’同源臂进行PCR鉴定，确认获得的 F1代小鼠是否是正确同源重组的SIGLEC15基因人源化小鼠的后代，该阳性小鼠为F1代杂合子小鼠，PCR鉴定结果如图5中C所示，阳性小鼠为2，3，6，8号。F1代杂合子小鼠通过自交获得SIGLEC15人源化纯合子小鼠。
以上结果表明，利用本方法可以高效构建获得可稳定传代的SIGLEC15基因人源化小鼠模型。
实施例2 人源SIGLEC15在人源化小鼠模型中的mRNA水平表达验证
各选取一只SIGLEC15人源化野生型、杂合子和纯合子小鼠（8周龄），分离腹腔巨噬细胞，抽提总RNA，利用反转录试剂盒，反转录为cDNA后，以cDNA为模板进行PCR，验证人源化小鼠中人源SIGLEC15 mRNA水平表达。
该实验分别设计针对鼠源Siglec15和人源化改造后的SIGLEC15 RNA设计检测引物,引物位置如图6中A的P1/P2，P3/P4引物位置所示，针对鼠源Siglec15 RNA表达检测的引物序列为：
P1：5' - GACGCTTCGGGGGATCTGCTCAACACA-3’(SEQ ID NO：33)，和
P2：5’- CAGGGCAGCGGAGCTGTTGCCTG-3’(SEQ ID NO：34),PCR产物为561bp；
针对人源化小鼠中人源SIGLEC15 RNA表达检测的引物序列为：
P3：5’- CACAGAGGTGCACAGCTCGCCA-3’(SEQ ID NO：35)，和
P4：5’- TAGGTGGCCGTGACCCTCACGCG-3’(SEQ ID NO：36)，PCR产物大小为565bp。
P1/P2,P3/P4两对引物的PCR反应体系和反应条件相同，20μL PCR体系，反应条件：94℃，4min；(94℃，15sec；62℃，15sec；72℃，60sec，34个循环)；72℃，5min；12℃保温。
PCR结果如图6中B所示：在野生型和杂合子小鼠中可以检测到鼠源Siglec15 mRNA表达产物，而人源化纯合子小鼠中检测不到鼠源Siglec15 mRNA表达产物；在杂合子和纯合子小鼠中可以检测到人源化改造后的人源SIGLEC15 mRNA表达产物，而野生型小鼠中检测不到人源SIGLEC15 mRNA表达产物。
以上结果表明，SIGLEC15人源化小鼠中可以检测到人源SIGLEC15 mRNA的活跃的表达。
实施例3 SIGLEC15人源化小鼠针对SIGLEC15靶点药物的药效评价
如背景技术描述，SIGLEC15靶点是当前抗肿瘤免疫治疗药物联用的热门靶点，为了验证SIGLEC15人源化小鼠是否可以用于针对SIGLEC15靶点药物的抗肿瘤药效评价，申请人进行了如下应用：SIGLEC15人源化纯合子小鼠，接种人源化改造的MC38-SIGLEC15结肠癌肿瘤细胞，每只小鼠接种5x106的细胞量，待肿瘤生长至60-80mm3的时候开始分组给药，分为三组，分别给予20mg/kg的人IgG1对照、5mg/kg和20mg/kg针对人SIGLEC15的抗体5G12，具体给药设计如下表所示：
肿瘤生长抑制结果如图7中A所示： SIGLEC15人源化纯合子小鼠中给5mg/kg 5G12抗体组的肿瘤生长抑制率为90.53%，给20mg/kg 5G12抗体组的肿瘤生长抑制率为84.03%，低剂量和高剂量给药两组之间没有显著差异，在SIGLEC15人源化小鼠上5mg/kg低剂量给药即可实现非常显著的肿瘤抑制效果。
实验过程中小鼠体重结果如图7中B所示：在给药初期，给予5G12抗体高剂量和低剂量组的小鼠体重略有下降，但较对照组无显著差异，说明5G12抗体无明显的毒性。
以上结果表明，SIGLEC15人源化小鼠中可以用于针对人源SIGLEC15靶点药物的药效和安全性评价。
实施例4 SIGLEC15人源化小鼠黑色素瘤细胞侵染
取野生型小鼠和SIGLEC15人源化纯合子小鼠各两只，进行黑色素瘤细胞侵染试验，具体操作如下。用黑色素瘤细胞系B16 分别移植入野生型小鼠和SIGLEC15人源化纯合子小鼠皮下，在移植后第3、5、7 天，采用抗人SIGLEC15抗体以每只100 μg 的剂量进行3 次腹腔注射，观察肿瘤生长。在第14 天时，野生型小鼠小鼠中肿瘤质量低于SIGLEC15人源化纯合子小鼠(图8)。
在SIGLEC15人源化纯合子小鼠中，由于小鼠自身的SIGLEC15基因已经被破坏，在进一步注射抗人SIGLEC15抗体后，表达的人SIGLEC15蛋白的活性也受到抑制。而野生型小鼠体内仍然表达有小鼠的SIGLEC15蛋白，虽然也被注射了人SIGLEC15抗体，但是黑色素瘤的生长相对于SIGLEC15人源化纯合子小鼠明显收到抑制。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东南模生物科技有限公司
上海南方模式生物科技股份有限公司
上海砥石生物科技有限公司
<120> 一种SIGLEC15基因人源化动物模型的构建方法及应用
<130> 2021
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 328
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro
1 5 10 15
Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu
20 25 30
Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val
35 40 45
Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys
50 55 60
Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile
65 70 75 80
Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala
85 90 95
Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly
100 105 110
Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg
115 120 125
Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val
130 135 140
Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val
145 150 155 160
Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu
165 170 175
Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu
180 185 190
Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu
195 200 205
Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala
210 215 220
Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala
225 230 235 240
Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His
245 250 255
Gly Ala Ser Gly Ala Ser Thr Val Ala Leu Leu Leu Gly Ala Leu Gly
260 265 270
Phe Lys Ala Leu Leu Leu Leu Gly Val Leu Ala Ala Arg Ala Ala Arg
275 280 285
Arg Arg Pro Glu His Leu Asp Thr Pro Asp Thr Pro Pro Arg Ser Gln
290 295 300
Ala Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Asn Pro Arg Ser
305 310 315 320
Pro Pro Ala Thr Met Cys Ser Pro
325
<210> 2
<211> 342
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln
1 5 10 15
Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu
20 25 30
Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val
35 40 45
Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys
50 55 60
Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile
65 70 75 80
Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr
85 90 95
Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly
100 105 110
Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg
115 120 125
Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val
130 135 140
Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val
145 150 155 160
Arg Leu Arg Val Thr Ala Ala Ala Pro Arg Ile Val Asn Ile Ser Val
165 170 175
Leu Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly
180 185 190
Glu Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser
195 200 205
Ser Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu
210 215 220
Leu Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn
225 230 235 240
Ser Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly
245 250 255
Ala Pro Gly Thr Ser Thr Leu Ala Leu Leu Leu Gly Ala Leu Gly Leu
260 265 270
Lys Ala Leu Leu Leu Leu Gly Ile Leu Gly Ala Arg Ala Thr Arg Arg
275 280 285
Arg Leu Asp His Leu Val Pro Gln Asp Thr Pro Pro Arg Ser Gln Ala
290 295 300
Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Ser Pro Pro Gly His
305 310 315 320
Gln Leu Pro Arg Val Cys Cys Glu Glu Leu Leu Ser His His His Leu
325 330 335
Val Ile His His Glu Lys
340
<210> 3
<211> 343
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln
1 5 10 15
Met Gly Ser Leu Val Lys Thr Arg Arg Asp Thr Thr Glu Asn Leu Leu
20 25 30
Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val
35 40 45
Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys
50 55 60
Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile
65 70 75 80
Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala
85 90 95
Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly
100 105 110
Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg
115 120 125
Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val
130 135 140
Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val
145 150 155 160
Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu
165 170 175
Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu
180 185 190
Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu
195 200 205
Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala
210 215 220
Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala
225 230 235 240
Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His
245 250 255
Gly Ala Ser Gly Thr Ser Thr Leu Ala Leu Leu Leu Gly Ala Leu Gly
260 265 270
Leu Lys Ala Leu Leu Leu Leu Gly Ile Leu Gly Ala Arg Ala Thr Arg
275 280 285
Arg Arg Leu Asp His Leu Val Pro Gln Asp Thr Pro Pro Arg Ser Gln
290 295 300
Ala Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Ser Pro Pro Gly
305 310 315 320
His Gln Leu Pro Arg Val Cys Cys Glu Glu Leu Leu Ser His His His
325 330 335
Leu Val Ile His His Glu Lys
340
<210> 4
<211> 1956
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcttcggggg atctgctcaa cacagaggcg cacagtgagt gattttatca ccttagggat 60
cttgtaatac gggataccat agcatctcag caactctttt tgatgggtag tgtgggtcac 120
acgccaggat gaaacaaact gtccaaggaa gtgtctgagg actttctagt ccgaccccca 180
tccttttagt tatgggggag cacagctagg gagatgcttc taaagtcaga aagtggaatt 240
ccagcagaaa ggaactaagt gttgcctgag tcctggtgtt tgcatccctg cctccggaag 300
tcgaggggag tggagtggag atgcgccccc cccccaagag gacaaggacc gggtccccct 360
atgctaagtc tgatgtgtct ctggcccgca ggtgccccgg cgcagcgctg gtccatgcag 420
gtgcccgcgg aggtgaacgc ggaggctggc gacgcggcgg tgctgccctg caccttcacg 480
cacccgcacc gccactacga cgggccgctg acggccatct ggcgctcggg cgagccgtac 540
gcgggcccgc aggtgttccg ctgcaccgcg gcgccgggca gcgagctgtg ccagacggcg 600
ctgagcctgc acggccgctt ccgcctgctg ggcaacccgc gccgcaacga cctgtccctg 660
cgcgtcgagc gcctcgccct ggcggacagc ggccgctact tctgccgcgt ggagttcacc 720
ggcgacgccc acgatcgcta tgagagtcgc catggggtcc gtctgcgcgt gactggtgag 780
acgggctggc gcctgagggg tgcggggggc cccaacttcc cccacattcc cgagagcgta 840
ctgggtgcta gggaggcctc tgagaacttg gtagaaatgg aaaactcagg tcctaggaat 900
ggcccgcctg ggactagtgg gtggtgtcca gatgctccta aacacccata gttggcaagg 960
aaagattgaa aaacaaacag tcaataattc taaagagcta ggggttgcag tagaaagtgc 1020
gcgccagctg ggcggtggtg gcgcacgcct ttaatcccag cacttgggag gcagaggcag 1080
gcggatttct gagttcgagg tcagccagga cagccagggc tatacagaga aaccccgact 1140
cgaaaaaaca aaacaaaaaa agaaaaagaa acaaaaacaa aaaacaaaaa agaaagaaag 1200
aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa agtgcgcgcc 1260
tgcactgaag gatttcccat ccccacccca ccccaccccg tacatccctg ttcaccagag 1320
cactgtgctg caactggccc ctccctacgt tgtgaccctt tagtgggcac cagctgcaag 1380
cccggcagtc taagcaacac acaagtttgc caaatctcaa tgaacccata aaatgagagc 1440
aattgtgatt accgttttac acaaggagaa actgaggcag agaagagaaa taacttgcta 1500
aggccatcgc tgcaaaagga cccaggagcc cagccctggg cattccctta cttcctccct 1560
ccagcttagc agggagcggg ccagggtgct accccttgct taaccaaacc gtgggctctc 1620
taacgctccc gcgagctttg tgctctgcag agtgacccac gccgaccctc ttccccagca 1680
gctgcgccgc ggatcgtcaa catctcggtg ctgccgggcc ccgcgcacgc cttccgcgcg 1740
ctctgcaccg ccgaggggga gcccccgccc gccctcgcct ggtcgggtcc cgccccaggc 1800
aacagctccg ctgccctgca gggccagggt cacggctacc aggtgaccgc cgagttgccc 1860
gcgctgaccc gcgacggccg ctacacgtgc acggcggcca atagcctggg ccgcgccgag 1920
gccagcgtct acctgttccg cttccacggc gccccc 1956
<210> 5
<211> 1944
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
actacggaga acttgctcaa cacagaggtg cacagtaagt gcttttatta ttatcaccat 60
ctcggggatc ttgggagtct gttttaacca cgagatccca gggtttttcc acagggcagg 120
ttttgatggg gaaaaactaa gggtaagaat atggggtcaa ggggcctgca ggtgaattag 180
gaaacgaaga ggggaatagt cccagggaag agccgcggga acgcaggcac tcccaagtcg 240
agggagtgca ggccctgggg gtgcaggcac caggaattgc ccctcaagtc acaaggtggg 300
gttccggctc ccctggaagt gggggacgat ccctgagtcc tggggtttcc aggctagggg 360
ttgggggagc gtttcctggg tcgtggggtt tccaggcccc gggtgcgggc gcctcgaccc 420
cagggccccg agcctgacgc agcccgcccc gccctcaggc tcgccagcgc agcgctggtc 480
catgcaggtg ccacccgagg tgagcgcgga ggcaggcgac gcggcagtgc tgccctgcac 540
cttcacgcac ccgcaccgcc actacgacgg gccgctgacg gccatctggc gcgcgggcga 600
gccctatgcg ggcccgcagg tgttccgctg cgctgcggcg cggggcagcg agctctgcca 660
gacggcgctg agcctgcacg gccgcttccg gctgctgggc aacccgcgcc gcaacgacct 720
ctcgctgcgc gtcgagcgcc tcgccctggc tgacgaccgc cgctacttct gccgcgtcga 780
gttcgccggc gacgtccatg accgctacga gagccgccac ggcgtccggc tgcacgtgac 840
aggcgaggcg gcgtgggagc gggtccccgg cctcccttcc cgccctcccg cctgccccgc 900
cccaagggct acgtgggtgc caggcgctgt gctgagccag gaagggcaac gagacccagc 960
cctctcctct accccaggga tctcacacct gggggtagtt taggaccacc tgggagcttg 1020
acacaaatgc agaatccagg tcccaggaag ggctgaggtg ggcccgggaa taggcattgc 1080
cgtgactctc gtagagtgac tgtccccagt ggctctcaga cgaagaggcg agaaagacaa 1140
gtgaatggca atcctaaata tgccaagagg tgcaatgtgg tgtgtgctac cagcccggaa 1200
agacactcgc agcccctcta cccaggggtg cacagacagc ccaccaagta gtgcctagca 1260
ctttgccaga ccctgatata caaagatgcc tgaaccaggg tcccgtccct agagcagtgg 1320
ctctccactc tagcccccac cctgctctgc gacaataatg gccacttagc atttgctagg 1380
gagccgggac ctagtccaag cacccacaag catgaatttg ccaaatcttt tcagcaacct 1440
cttaaggcaa ctgctatcat gatcctcact ttacacatgg agaagcagaa gcagagatga 1500
tagaatcttt cgcccaaggc cacatctgta ttgggacggg ggcagcctgg cacccaagtg 1560
cccattcctc ccttctgacc agcccccacc cctccggctc tggcgtccaa agggctaagg 1620
ggaggggtgc ccttgtgaca gtcacccgcc ttctcccctg cagccgcgcc gcggatcgtc 1680
aacatctcgg tgctgcccag tccggctcac gccttccgcg cgctctgcac tgccgaaggg 1740
gagccgccgc ccgccctcgc ctggtccggc ccggccctgg gcaacagctt ggcagccgtg 1800
cggagcccgc gtgagggtca cggccaccta gtgaccgccg aactgcccgc actgacccat 1860
gacggccgct acacgtgtac ggccgccaac agcctgggcc gctccgaggc cagcgtctac 1920
ctgttccgct tccatggcgc cagc 1944
<210> 6
<211> 3000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcagtcacca gctgactttg agaagtcgct tgggccttcc aagccctcag tttccccctc 60
tctaaaacgg gccattgaca acatctcctg gggctgttat gtgttttaag tgaggtagag 120
aatgactcac aatgcccccc cccccgcctt cttcagcagt ctggcctcca cagcccctca 180
tgcattctct gccccctctg agagccaggc ttcatgcttg cctttgccag ttttttcgag 240
acagggtccc aagtatcttt aactccctat gtagacccag gctttggtgg cgcatgcgcg 300
cctttaatcc cagcactcgg gaggcagagg caggtggatt tctgagttca aggccagtct 360
ggtctacaga gtgagttcca ggacagccag ggctacacag agaaaccctg tctcaaaaaa 420
acaaaaaaac aaaaaaaaaa caaaaaaacc aaaaaccaaa acccaaaacc caaataaaca 480
aacaaaaaac caacctcccg atgtaggtaa aagtggcctg aacgcctaat aactctcctc 540
ctgaacattg aggctttctg cacaggctgc catgtctttc acagtcgtac cttacagctt 600
ggttccctgc tccccgtgca cagactgtca agcccaggtc tccgtggaat tcattctctg 660
gactctgcat acacatacct cctctctgtt gggctctaca acccctgcct gctatttcca 720
atccctgcaa ctctttccac cagccctcat ccttctgaaa gcctaacccc ctttcatccc 780
agggtacagg ggagagatac cagagctagg ttgggtgctc agagactatt ctggcaaggg 840
tgattcatca gataacagag gttgaatagg agagatctgg caagtgaaag taggctggcg 900
accatgttca gactaaggca atattgtctg tatctttcat cagagcacct ggcagagcac 960
agcctaatta catgttcact gatctttctc cagctcgatg ataggggacc ctgttcatct 1020
ttgtccccac agctcatatc ccagcacctg gcgcagcgtg cggaagtatc caataaatgt 1080
ttgcagaatt gatgaatgca caatagcaga caatgtgtgg gcaatccgtg cctgactcca 1140
tgagcttgca agatgcagaa ttcaagccta aggtcacaga gggtagggag tacaggaaaa 1200
ctgaggtagg gctgagggtg cacactcata gtcccagctg ttaggaagct gagacaggaa 1260
gattgcaagt tgaacttgtg gcttgtttga gctacactgt cagacagaca gacagacaga 1320
cagacagaca gacctaagga aactttcttc agctagacgt cctgacagga tcagcgcaat 1380
catcacacag gtttctcaca tttgaaaggc agccaaacac gttcttacta aggataaacg 1440
tccagccccc gacttgaatt caaacacgta gttcaacttg tccagagcta aaagagatta 1500
agcctaagaa caggccgtgt gaaacaggaa ctggggttta actcgatggt cttccagctc 1560
tgtgcccaca gcaggatcag attggacatt cagcattcca gcccaaggtg cacaagaact 1620
cgctgcactc tgcaggtgca gcacactagg gccacccggg gcgggacttg aatggaaggg 1680
aaagggggat gggggcaggt gagccatcct ctcccatcag gaaaaggtaa tgagtccaag 1740
agccaccata atgagcttct gcaagcggca gctgggacct gtacatgttc ccgaaagcag 1800
tggcagttcc ggtgtggttc tgtcgatcca caggtgtatg ggctctacag gagatggact 1860
agcagcggtg gcttgagggc attccaaccc aagaagtcat tgaggctcac tgaccacaaa 1920
gagcactggg cttagttagc atcaggaaga actggctttt ctctttcctt gcctggatgt 1980
tggccttcat gtacacatct ataacactct ctgccctgtc tttctcctct tgggtgcttt 2040
caggacagat ggcatggtgg tgtggtataa ataaagggac tgtgtataat acaagcccaa 2100
ttactccctg taaaggtccc ttttgtgcac tgtgccccag ggcactgact gggcaggctc 2160
aaccgcctgc ctcctcttgg ggaggtacca cccctggcat aaccttctga gactgaacac 2220
ctccataaac acagcaacac agagcccgat ggtttgccag cactgggtga actggaagca 2280
gggctcagct tccacaaccc ttggctccgc attctgaaga atgtggttca ccaggtggtg 2340
ggcaaagtga ggctggagca gaggtgtcaa cctgtgggcc gcacgccctt ttggggggga 2400
gggtgttgac tgaccctttc ctaggagtca cctaagacta cctgcatatc agatatttat 2460
attatgattc ataacagtaa caaaattaca ccaatgaagt agcaacaaaa ataattttat 2520
ggttggggtc accacaacat gaggaactgt attaaagggt cgcagcatta ggaaggctga 2580
gaaccactgg tcttgaggta caatgcagtg tgtacccacc atgttaaagt acccaccacg 2640
agggtctttc tctgcctttt aagcagccag ccaggacgca gttcacctgg gctggctctg 2700
attctaaagg aacttgtaac tctactggtg tgcttcaaag caccgccccc cccccccccc 2760
ccagggtctg ggtttctcaa ctacaaataa agactgttag cccttcaggt gaatcacagc 2820
ttcatgcctt tagggcgttc taatacaggc tctgggtcca ggcaggtctg agtgggacgc 2880
agactttgcc tcaacatcgc agttactcca attatttaac ttttctgcgc ctcagtgtat 2940
taacatgtaa caggctatct acatttgtct ctaggatccc ttgtgaaaac tagaagagac 3000
<210> 7
<211> 3000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggaacctcga ccctagcgct cctgctgggc gcgctgggcc tcaaggcctt gctgctgctt 60
ggcattctgg gagcgcgtgc cacccgacgc cgactaggtg ggtgcatccc agatctgggt 120
gggtgagagg gggagagaag gctccctgcc ctttctgccg gtccacctga tgtggcacat 180
cagaaatcct ttctgcacct gcatccttct ctctgcctgg agcccatgag atggagctct 240
tcgcagttag gaaatggtgg ctctgcaaag gattttgagt cacacagaga cacaggcctg 300
taggctctcc aacacttctt ttactaacta ccacgccaca aatcagtgtc ctgtaaaggt 360
cgggtctccg gcttctccct tacttttaac tagcgtcttg aggtcctgag gttatggacc 420
aggatgctgg gaacgcaaag atgctcagat gtgattccct gctagccggt aagaggctgg 480
gtctgcagaa ctccttttca tggaggtact gcagaaggca gaaattgcag aagacagggg 540
gaggggggat atcttgcaat tctgaggtct tagtaagtta ggagggctca gccaaaaaaa 600
aaaaaaataa ataaaataaa gccttcagga cacaggagct tggaaatcat aaatggctca 660
cctggcctgg gaacccaggt tacttttaga gtctctccaa gctgctgtcc ctgaccttga 720
tgggtcatct gctgagcacc cagaaaaggg agggaccttc tcagagcctc tcagtcttgg 780
ccaaccaata aggttagcct ccaactctcc agggacacaa cgcagaattt gtctctgggg 840
gccccagctg ccatccttgc tgtgaccctg ccattcttgc tggttcctca aacttatcaa 900
acatgttgtg tttcagggtc cccagaaagg cagttgtgtt ctctcttaat atccccaccc 960
cgcctccccg accggggtca agttaaccct atcatgtatc aggttaagtt gctccagcta 1020
gcaagccatc tgctgcacat ttcctgggga ctgcctctca ctgtggggcg gggcaatgtg 1080
tgaagctgac aggttggctg gagaccactg tgcaaattca cacctcctct ttctccatgt 1140
ccctcataca gatcacctgg tcccccagga cacccctcca cggtacgtac tcactgccct 1200
tcccccatct ctgcctacca cattggccca ggcatgccac tttctcagaa ctgggagttc 1260
acagagaaaa agcaggcata gggcaaggct agggtagtag gctggactag ggcctggatc 1320
atacccaaaa tgccctgccc ctccatccag cacaccccag agtgggccag gctgacctgt 1380
tactgcatcc agtacacaag gagcttgctg tctatgaccc caaaagcgaa taccactaag 1440
ctgtgtcctc ctgttgcctc ccagctttct agaaattagc cccagaagga tgccactgta 1500
ggaagactca tcccccaggc ttaatgctga ctgcttccaa acaaaatcac caagagcctg 1560
gcttagatga aagatctaga aagacattaa gccagagtcc actctttcct cttcagtgtc 1620
taccccagta aggacccttg ttgagggagt aagaacccag ctctggagtg ctgtgcctgg 1680
tgctggctgg gagcacactc tttcctgctg ctcaagtctt gacagcccgc catatgtgca 1740
tctcctttac tgacgtgcaa caagatcaga gtcacaacac tgcctgatgt acactcacct 1800
aacaaatatt taccgaatac ctacgatcta gcaggcaata acatggatga tagagactgg 1860
aaatccaagc ttactctcca atgtgcacag ttttgcacaa acataacaag accatctgag 1920
atctgtaaag ctagggataa agcagtgccc agtgggtagc caggcggcca tgactgcttt 1980
agagctggat actttctttg aagacatggc cctaaagaca ccaagctgga ggctggaacc 2040
tagggtgacc aaggtgatag ggatatccca agagtgaaaa ctgtaaaagg atcttctgtt 2100
ctcctgaagt aactgtcatc tctaaggctc actgcctcca tctgctaacc taagcctagt 2160
tctggaagct tctagccttc gtacaatcta atctaggcct agaatgtttt caggctctga 2220
gactcactgc tgaataagtt caccccttct agctctttct gagctctggc tggctaattc 2280
aactcagctg ttctggctca aactcctctc aaagctgact gatacagtct ggcttctctc 2340
ttggactctg atatttttgc tgtgcttggc gtctaagtct ggcaatctgt tctaaccttc 2400
tggctccttc tccttctctg gcttgtcttg tcttcaccag tgtctagctt gcttgttctc 2460
tcttcaacct gtgtctgtac aactctccca gtaaaactgc ttgcttgctt cctctgtctc 2520
tgtctctctc ttcttcttct tttctcctcc tcctcctcct cctcctcctc ctccttcttc 2580
ttcttcttct tcttcttctc tctctctctc tctctctctc tctctctctc tctctctctc 2640
tctctgcact gctcccttaa gtagcttccc tttcctctct ctcctcatga gagttgggcg 2700
tatcctattc tgtcaaatct ttctccgact catcactttg tctgccactc aattagactt 2760
aactttcaag catgcatgct tccttctaca aactaacttc accttcattg tttgggatta 2820
aaagtgtgta ctaagggttg agctataact agaaacaggc tttttttttt ttcagtaaat 2880
aacacaatct ctcagggttc actgtgtgat caaatatcct gcaacagaaa accttaagta 2940
gaaaggtcag gtgggatttg gaatggacac agacatgggg ggagcatgga aatgtgggtt 3000
<210> 8
<211> 1159
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttagagccca gcggccctgc agacttggca cagagcacac ccacctgcct ttgtcacagc 60
acactaagaa ggttctctgt ggtgaccagg ctgggtagag ggctgctggg tctgcaggcg 120
tcagagcatg gaggggtccc tccaactcct ggcctgcttg gcctgtgtgc tccagatggg 180
atcccttgtg aaaactagaa gagacactac ggagaacttg ctcaacacag aggtgcacag 240
ctcgccagcg cagcgctggt ccatgcaggt gccacccgag gtgagcgcgg aggcaggcga 300
cgcggcagtg ctgccctgca ccttcacgca cccgcaccgc cactacgacg ggccgctgac 360
ggccatctgg cgcgcgggcg agccctatgc gggcccgcag gtgttccgct gcgctgcggc 420
gcggggcagc gagctctgcc agacggcgct gagcctgcac ggccgcttcc ggctgctggg 480
caacccgcgc cgcaacgacc tctcgctgcg cgtcgagcgc ctcgccctgg ctgacgaccg 540
ccgctacttc tgccgcgtcg agttcgccgg cgacgtccat gaccgctacg agagccgcca 600
cggcgtccgg ctgcacgtga cagccgcgcc gcggatcgtc aacatctcgg tgctgcccag 660
tccggctcac gccttccgcg cgctctgcac tgccgaaggg gagccgccgc ccgccctcgc 720
ctggtccggc ccggccctgg gcaacagctt ggcagccgtg cggagcccgc gtgagggtca 780
cggccaccta gtgaccgccg aactgcccgc actgacccat gacggccgct acacgtgtac 840
ggccgccaac agcctgggcc gctccgaggc cagcgtctac ctgttccgct tccatggcgc 900
cagcggaacc tcgaccctag cgctcctgct gggcgcgctg ggcctcaagg ccttgctgct 960
gcttggcatt ctgggagcgc gtgccacccg acgccgacta gatcacctgg tcccccagga 1020
cacccctcca cggtctcagg ctcaggagtc caattatgaa aatttgagcc agatgagtcc 1080
tccaggccac cagctgccac gtgtttgctg tgaggaactc ctcagccatc accatctagt 1140
cattcaccat gagaaataa 1159
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgttacatgt taatacactg agg 23
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcagtgtatt aacatgtaac agg 23
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaaaactaga agagacgctt cgg 23
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aaaactagaa gagacgcttc ggg 23
<210> 13
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gtctcttcta gttttcacaa ggg 23
<210> 14
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgtctcttct agttttcaca agg 23
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aaactagaag agacgcttcg ggg 23
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aactagaaga gacgcttcgg ggg 23
<210> 17
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gggggatctg ctcaacacag agg 23
<210> 18
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cccgtattac aagatcccta agg 23
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
caatagcctg ggccgcgccg agg 23
<210> 20
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gaacaggtag acgctggcct cgg 23
<210> 21
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gaagcggaac aggtagacgc tgg 23
<210> 22
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gggcgccgtg gaagcggaac agg 23
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ttccgggggc gccgtggaag cgg 23
<210> 24
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcgaggttcc gggggcgccg tgg 23
<210> 25
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtctacctgt tccgcttcca cgg 23
<210> 26
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gagcgctagg gtcgaggttc cgg 23
<210> 27
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cgcgcccagc aggagcgcta ggg 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tcgaccctag cgctcctgct ggg 23
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
caagccctct ccccctttgg 20
<210> 30
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ggactattcc cctcttcgtt tcctaa 26
<210> 31
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
acccaagtgc ccattcctc 19
<210> 32
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcccagacat ccctacctcc aa 22
<210> 33
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gacgcttcgg gggatctgct caacaca 27
<210> 34
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
cagggcagcg gagctgttgc ctg 23
<210> 35
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
cacagaggtg cacagctcgc ca 22
<210> 36
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
taggtggccg tgaccctcac gcg 23
Claims (10)
1.一种SIGLEC15基因人源化非人动物模型的构建方法，所述的构建方法包括使用靶向SIGLEC15基因的gRNA和同源重组载体进行构建，其特征在于，所述靶向SIGLEC15基因的gRNA包括靶向5’端的gRNA和靶向3’端的gRNA，所述靶向5’端的gRNA识别的靶位点序列如SEQ IDNO:12所示，所述靶向3’端的gRNA识别的靶位点序列如SEQ ID NO:26所示，所述同源重组载体包括5’同源臂、人SIGLEC15基因片段、3’同源臂，所述人SIGLEC15基因片段编码的蛋白序列如SEQ ID NO:2所示，所述的非人动物体内表达人SIGLEC15基因编码的蛋白。
2.根据权利要求1所述的构建方法，其特征在于，所述同源重组载体包括的5’同源臂的序列如SEQ ID NO:6所示，所述同源重组载体包括的3’同源臂的序列如SEQ ID NO:7所示。
3.根据权利要求2所述的构建方法，其特征在于，所述人SIGLEC15基因部分破坏内源SIGLEC15基因的编码框，使内源的SIGLEC15基因不表达。
4.根据权利要求1-3任一权利要求所述的构建方法，其特征在于，所述非人动物为小鼠。
5.根据权利要求4所述的构建方法，所述人SIGLEC15基因片段的序列如SEQ ID NO:5所示。
6.一种用于构建SIGLEC15基因人源化非人动物模型的同源重组载体，其特征在于，所述同源重组载体包括5’同源臂、人SIGLEC15基因片段、3’同源臂，所述同源重组载体包括的5’同源臂的序列如SEQ ID NO:6所示，所述同源重组载体包括的3’同源臂的序列如SEQ IDNO:7所示。
7.根据权利要求6所述的同源重组载体，其特征在于，所述人SIGLEC15基因片段编码的蛋白序列如SEQ ID NO:2所示。
8.一种用于构建SIGLEC15基因人源化非人动物模型的gRNA，包括靶向5’端的gRNA和靶向3’端的gRNA, 其特征在于，所述靶向5’端的gRNA识别的靶位点序列如SEQ ID NO:12所示，所述靶向3’端的gRNA识别的靶位点序列如SEQ ID NO:26所示。
9.一种用于构建SIGLEC15基因人源化非人动物模型的试剂盒，其特征在于，所述试剂盒包括权利要求6-7任一所述的同源重组载体和权利要求8所述的gRNA，以及Cas9 mRNA。
10.权利要求1-5任一所述的构建方法获得的SIGLEC15基因人源化的非人动物在SIGLEC15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，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SIGLEC15信号通路调节剂的筛选；或者
e）研究SIGLEC15基因功能，研究人SIGLEC15抗体，研究针对人SIGLEC15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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