CN112608942B - Tnfrsf1b基因人源化动物模型的构建方法及应用 - Google Patents
Tnfrsf1b基因人源化动物模型的构建方法及应用 Download PDFInfo
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Abstract
本申请提供一种TNFRSF1B基因人源化动物模型的构建方法及应用。本申请构建的人源化TNFRSF1B基因改造动物模型能够加速与人TNFRSF1B基因或蛋白相关的领域的研究进展，例如利用人源化TNFRSF1B基因改造动物模型代替人试药，为进行TNFRSF1B靶点药物的临床前实验提供有效的模型和有力的工具。
Description
技术领域
本申请涉及基因工程技术领域，尤其涉及一种TNFRSF1B基因人源化动物模型的构建方法及应用。
背景技术
TNFRSF1B（TNF receptor superfamily member 1b）为TNF受体超家族成员，也称为TNFR2，表达仅限于抑制性免疫细胞，尤其是调节性T细胞（Tregs）、髓系抑制性细胞（MDSCs）、内皮细胞和生长过程中的选择神经元。其配体TNF主要识别TNFR1进行凋亡，依赖TNFRSF1B进行与T细胞存活相关的功能——通过激活NFκB诱导激酶（NIK）诱导NFκB的非规范性激活，从而促进Tregs细胞增殖和生存。许多实验表明TNFRSF1B拮抗型抗体可以抑制肿瘤细胞和Tregs的增殖，理论上可用于多种肿瘤的治疗；而激动型抗体可以促进Tregs增殖和活化，进而抑制效应T细胞（Teffs），从而治疗自身免疫疾病，为免疫治疗提供了新的靶点。
目前，使用TNFRSF1B拮抗型抗体抑制Tregs，恢复肿瘤免疫应答并消除肿瘤，已经成为了肿瘤免疫治疗的有效策略。而人源TNFRSF1B靶点药物的筛选及临床前试验需要在动物模型上进行评价，但由于种属性的差异，人源TNFRSF1B与鼠源Tnfrsf1b蛋白的同源性只有62%，在小鼠上筛选的靶点药物并不完全适用于人类，同样针对人源TNFRSF1B蛋白的药物也不一定能够识别小鼠的Tnfrsf1b蛋白。因此，构建具有人源TNFRSF1B靶点的人源化小鼠模型，在筛选评价人源TNFRSF1B靶点药物中具有非常高的应用价值。
发明内容
本申请的目的在于提供一种TNFRSF1B基因人源化动物细胞的构建方法、TNFRSF1B基因人源化动物细胞、TNFRSF1B基因人源化动物模型的构建方法以及由该方法制得的TNFRSF1B基因人源化动物模型的应用、来源于人源化TNFRSF1B基因改造动物模型或其子代的细胞或细胞系或细胞培养物的组织或器官及其应用，以加速与人TNFRSF1B基因或蛋白相关的领域的研究进展。
为实现以上目的，本申请提供一种TNFRSF1B基因人源化动物细胞的构建方法，包括：
在非人动物细胞内导入人TNFRSF1B基因，使所述人TNFRSF1B基因在非人动物细胞中表达产生人源化的TNFRSF1B蛋白，同时降低或消除所述非人动物细胞内的内源TNFRSF1B基因的表达。
在一些实施例中，利用基因编辑技术对非人动物细胞内的内源TNFRSF1B基因进行改造，得到人源化TNFRSF1B基因；
优选地，所述非人动物为啮齿类动物；更优选地，所述啮齿类动物为小鼠；
优选地，所述细胞为受精卵细胞。
在一些实施例中，所述人源化TNFRSF1B基因选自下列组中的至少一种：（a）所述人源化TNFRSF1B基因的CDS编码序列如SEQ ID NO：8所示；（b）所述人源化TNFRSF1B基因转录的mRNA序列如SEQ ID NO：9所示；（c）所述人源化TNFRSF1B基因编码的蛋白序列如SEQ IDNO：10所示。
在一些实施例中，所述构建方法采用CRISPR/Cas9基因编辑技术实现；所述构建方法具体包括：提供人TNFRSF1B基因同源重组载体、sgRNA以及Cas9的混合物，所述Cas9包括Cas9 mRNA和/或Cas9蛋白，将所述混合物注射到所述非人动物细胞中，将所述非人动物细胞转移至培养液中进行培养。
在一些实施例中，所述非人动物为小鼠，所述人TNFRSF1B基因同源重组载体包括从5’端到3’端依次排列的5’同源臂、人TNFRSF1B基因、小鼠Tnfrsf1b基因、polyA以及3’同源臂。
在一些实施例中，所述人TNFRSF1B基因的序列包括人TNFRSF1B基因内含子1的部分序列与人TNFRSF1B基因的部分编码区序列，所述人TNFRSF1B基因内含子1的部分序列如SEQ ID NO：1所示，所述人TNFRSF1B基因的部分编码区序列如SEQ ID NO：2所示；
优选地，所述小鼠Tnfrsf1b基因的序列如SEQ ID NO：3所示；
优选地，所述小鼠Tnfrsf1b基因与所述polyA之间还设置有 WPRE调控元件，所述WPRE调控元件的序列如SEQ ID NO：4所示；优选地，所述polyA的序列如SEQ ID NO：5所示；
优选地，所述5’同源臂的DNA序列如SEQ ID NO：11所示；
优选地，所述3’同源臂的DNA序列如SEQ ID NO：12所示。
具体的，插入人TNFRSF1B基因内含子1的部分序列的目的在于： mRNA前体到成熟mRNA需要依赖剪切体（spliceosome）将mRNA前体中的内含子切除，直接用人源化TNFRSF1B基因编码区及WPRE-polyA序列替换小鼠Tnfrsf1b基因的外显子2时，由于人TNFRSF1B基因和鼠Tnfrsf1b基因的内含子1和外显子2之间的转录剪切位点存在差异，因此可能会影响人源化TNFRSF1B基因的mRNA的正常剪切，本申请在用人源化TNFRSF1B基因的编码区及WPRE-polyA序列替换小鼠Tnfrsf1b基因的外显子2的同时，用人TNFRSF1B基因的内含子1与外显子2交界处的一段内含子1的序列替换与之对应长度的鼠Tnfrsf1b序列，可以更好的保证人源化TNFRSF1B基因的mRNA的正确剪切。
具体的，如SEQ ID NO：2所示的所述人TNFRSF1B基因的部分编码区序列为TNFRSF1B蛋白的胞外区序列，可作为抗体结合位点或者药物作用靶点。
具体的，如SEQ ID NO：3所示的所述小鼠Tnfrsf1b基因的序列为小鼠Tnfrsf1b蛋白的跨膜和胞内区序列，插入该序列的目的在于：小鼠Tnfrsf1b是一个跨膜蛋白，其胞外段感受到的细胞外信号刺激，需要通过跨膜和胞内部分与其它蛋白（鼠源蛋白）相互作用进行信号转导，因此在人源化时，保留跨膜和胞内区序列为鼠源序列，可以更好地保证人源化后Tnfrsf1b正常的信号转导不受影响。
具体的，通过插入WPRE调控元件，能够提高插入的融合基因片段（包括人TNFRSF1B基因与小鼠Tnfrsf1b基因）的表达能力。
在一些实施例中，所述sgRNA的靶位点序列如SEQ ID NO：20-29任一项所示；
优选地，所述sgRNA的靶位点序列如SEQ ID NO：29所示。
本申请还提供一种TNFRSF1B基因人源化动物细胞，所述TNFRSF1B基因人源化动物细胞由上述TNFRSF1B基因人源化动物细胞的构建方法获得。
本申请还提供一种TNFRSF1B基因人源化动物模型的构建方法，将上述TNFRSF1B基因人源化动物细胞或者由所述TNFRSF1B基因人源化动物细胞发育得到的胚胎移植至代孕母体内进行发育，得到F0代动物；
对F0代动物进行检验，获得基因型鉴定正确的F0代阳性动物。
在一些实施例中，所述TNFRSF1B基因人源化动物模型的构建方法还包括：还包括：将所述F0代阳性动物与野生型动物交配，扩大种群数量，同时杂合子动物与杂合子动物交配获得纯合子动物，建立稳定的TNFRSF1B基因人源化动物品系。
本申请还提供一种细胞或细胞系或细胞培养物，所述细胞或细胞系或细胞培养物来源于TNFRSF1B基因人源化动物模型或其子代，所述TNFRSF1B基因人源化动物模型由上述TNFRSF1B基因人源化动物模型的构建方法获得。
本申请还提供一种组织或器官，所述组织或器官来源于TNFRSF1B基因人源化动物模型或其子代，所述TNFRSF1B基因人源化动物模型由上述TNFRSF1B基因人源化动物模型的构建方法获得。
本申请还提供一种上述TNFRSF1B基因人源化动物模型的构建方法获得的TNFRSF1B基因人源化动物模型、来源于所述TNFRSF1B基因人源化动物模型的细胞、细胞系或细胞培养物、组织、器官在与人TNFRSF1B基因或蛋白相关的领域中的应用；
优选地，所述应用包括人TNFRSF1B基因功能研究、人TNFRSF1B抗体研究、针对人TNFRSF1B靶位点的药物制备和药效研究中的至少一种。
本申请的有益效果：
本申请构建的人源化TNFRSF1B基因改造动物模型能够加速与人TNFRSF1B基因或蛋白相关的领域的研究进展，例如利用人源化TNFRSF1B基因改造动物模型代替人试药，为进行TNFRSF1B靶点药物的临床前实验提供有效的模型和有力的工具。
进一步的，本申请利用CRISPR/Cas9基因编辑技术，在免疫系统健全的小鼠上，将鼠源的TNFRSF1B基因替换为人源的TNFRSF1B基因，构建能与抗人源TNFRSF1B抗体相互作用的小鼠模型，该模型与普通小鼠相比，实现了关键靶分子的人源化改造，并且保留了完整的免疫系统，可用于筛选和评价针对人TNFRSF1B基因的药物，是非常理想的临床前药物测试模型。
进一步的，本申请构建的TNFRSF1B人源化小鼠拥有人源胞外区，可筛选人源TNFRSF1B靶点药物，而鼠源胞内区则保证其胞内信号传导不受影响，将外部刺激忠实地转化为胞内行为。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本申请的某些实施例，因此不应被看作是对本申请范围的限定。
图1为鼠Tnfrsf1b基因与人TNFRSF1B基因的对比示意图。
图2为改造后的人源化小鼠Tnfrsf1b基因示意图。
图3为对小鼠Tnfrsf1b基因进行人源化改造的方案示意图。
图4为本申请实施例3中的酶切结果；S：SacII酶切鉴定结果，理论条带大小为9058bp、6023bp；M：1 kb DNA ladder。
图5为本申请实施例5中多个sgRNA的活性比较图。
图6为本申请实施例6中sgRNA体外转录产物的电泳结果。
图7为本申请实施例8中F0代小鼠的PCR鉴定结果。A图为5'同源臂PCR鉴定结果，B图为3'同源臂PCR鉴定结果。数字：F0代小鼠编号；M为1 kb DNA marker。
图8为本申请实施例8中F1代小鼠的PCR鉴定结果。A图为F1代小鼠5'同源臂PCR鉴定电泳图，B图为F1代小鼠3'同源臂PCR鉴定电泳图。数字：F1代小鼠编号。M：1kb DNAladder。
图9为本申请实施例9中流式细胞术检测不同基因型小鼠脾脏细胞鼠源、人源TNFRSF1B蛋白表达结果。横坐标mTNFR2表示抗鼠源Tnfr2抗体信号；纵坐标hTNFR2表示抗人源TNFR2抗体信号。
图10为本申请实施例10中流式细胞术检测不同基因型小鼠外周血细胞中鼠源、人源TNFRSF1B表达结果。
图11为本申请实施例10中针对人源TNFRSF1B抗体抗肿瘤药效结果。
具体实施方式
下面将结合具体实施例对本申请的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本申请，而不应视为限制本申请的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请实施例所用的小鼠品系、生化试剂、实验仪器为：
引物合成和测序服务购自生工生物工程（上海）股份有限公司；
PCR产物凝胶回收试剂盒为QIAquick Gel Extraction Kit，购自QIAGEN，货号：28706；
In-FusionHDCloning Kits购自Takara，货号为：639650；
大肠杆菌DH5α感受态细胞购自TaKaRa公司，货号为9057；
SacII限制性内切酶购自Takara公司，货号为：R0157V；
sgRNA体外转录试剂盒购自Ambion，货号AM1354；
T7核酸内切酶 I检测试剂盒，购自NEB公司，货号为M0302S；
Cas9 mRNA来源SIGMA，货号CAS9MRNA-1EA；
C57BL/6，ICR小鼠购自上海灵畅生物科技有限公司；
FITC anti-mouse CD3抗体购自Biolegend，货号为：100204；
Human TNF RII/TNFRSF1B PE-conjugated抗体购自R&D，货号为：FAB226P；
Mouse TNF RII/TNFRSF1B APC-conjugated抗体购自R&D，货号为：FAB426A；
流式细胞仪品牌为BECKMAN COULTER，型号为CytoFLEX Flow Cytometer。
实施例1 序列设计
小鼠的Tnfrsf1b基因有多个转录本，人的TNFRSF1B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，人对应的转录本为人TNFRSF1B基因NCBI登录号为NM_001066.3的转录本（对应的TNFRSF1B基因，其Gene ID：7133）；小鼠对应的转录本为小鼠Tnfrsf1b基因NCBI登录号为NM_011610.3的转录本（对应的Tnfrsf1b基因，其NCBI Gene ID：21938）。
在人源化策略中，用人TNFRSF1B基因内含子1的部分序列（NCBI登录序列NC_000001.11中的12188496- 12188795，序列信息如SEQ ID NO：1）、人TNFRSF1B的部分编码区（对应NM_001066.3转录本的180-888，序列信息如SEQ ID NO：2）、小鼠Tnfrsf1b的部分编码区（对应NM_011610.3转录本的860-1494，序列信息如SEQ ID NO：3）及土拨鼠肝炎病毒转录后调控元件--WPRE（序列如SEQ ID NO：4）和牛生长激素基因多聚腺苷酸--polyA（序列如SEQ ID NO：5），取代小鼠Tnfrsf1b基因部分内含子1和外显子2的序列（对应的DNA序列为NCBI登录序列NC_000070.7中的144955976-144955574，序列信息如SEQ ID NO：6）。
其中，鼠Tnfrsf1b基因与人TNFRSF1B基因对比示意图见图1，最终得到的改造后的人源化小鼠TNFRSF1B基因示意图见图2，人源化改造后的小鼠Tnfrsf1b的基因组序列信息如SEQ ID NO：7所示：
AAGGACAGAGGGACAGATGGACAGACATCCTAAAAAGAGAAGCTTTGTGTttctggatatctgtcccatgcagtgttaggggtcacccagcactggagaaggccagcagagtcagtgcttctgcctgcagacatgactagggtacactgaggtggggaggcaggggaggtaaaagaaggcacgagctctccttcctgtaccctgctccagggggagaaacctccccagccatcatcagtgcagactggcagggggagggccaaacatttgcagggcggggacctgggcatcaggcatggcagaacccaggggcggccctgttgatggcagtcttcccttcttccttccagGTGGCATTTACACCCTACGCCC CGGAGCCCGGGAGCACATGCCGGCTCAGAGAATACTATGACCAGACAGCTCAGATGTGCTGCAGCAAATGCTCGCCG GGCCAACATGCAAAAGTCTTCTGTACCAAGACCTCGGACACCGTGTGTGACTCCTGTGAGGACAGCACATACACCCA GCTCTGGAACTGGGTTCCCGAGTGCTTGAGCTGTGGCTCCCGCTGTAGCTCTGACCAGGTGGAAACTCAAGCCTGCA CTCGGGAACAGAACCGCATCTGCACCTGCAGGCCCGGCTGGTACTGCGCGCTGAGCAAGCAGGAGGGGTGCCGGCTG TGCGCGCCGCTGCGCAAGTGCCGCCCGGGCTTCGGCGTGGCCAGACCAGGAACTGAAACATCAGACGTGGTGTGCAA GCCCTGTGCCCCGGGGACGTTCTCCAACACGACTTCATCCACGGATATTTGCAGGCCCCACCAGATCTGTAACGTGG TGGCCATCCCTGGGAATGCAAGCATGGATGCAGTCTGCACGTCCACGTCCCCCACCCGGAGTATGGCCCCAGGGGCA GTACACTTACCCCAGCCAGTGTCCACACGATCCCAACACACGCAGCCAACTCCAGAACCCAGCACTGCTCCAAGCAC CTCCTTCCTGCTCCCAATGGGCCCCAGCCCCCCAGCTGAAGGGAGCACTGGCGACTTCGCTCTTCCAGTTGGTCTGATTGTTGGAGTGACATCACTGGGTCTGCTGATGTTAGGACTGGTGAACTGCATCATCCTGGTGCAGAGGAAAAAGAAGCCCTCCTGCCTACAAAGAGATGCCAAGGTGCCTCATGTGCCTGATGAGAAATCCCAGGATGCAGTAGGCCTTGAGCAGCAGCACCTGTTGACCACAGCACCCAGTTCCAGCAGCAGCTCCCTAGAGAGCTCAGCCAGCGCTGGGGACCGAAGGGCGCCCCCTGGGGGCCATCCCCAAGCAAGAGTCATGGCGGAGGCCCAAGGGTTTCAGGAGGCCCGTGCCAGCTCCAGGATTTCAGATTCTTCCCACGGAAGCCACGGGACCCACGTCAACGTCACCTGCATCGTGAACGTCTGTAGCAGCTCTGACCACAGTTCTCAGTGCTCTTCCCAAGCCAGCGCCACAGTGGGAGACCCAGATGCCAAGCCCTCAGCGTCCCCAAAGGATGAGCAGGTCCCCTTCTCTCAGGAGGAGTGTCCGTCTCAGTCCCCGTGTGAGACTACAGAGACACTGCAGAGCCATGAGAAGCCCTTGCCCCTTGGTGTGCCGGATATGGGCATGAAGCCCAGCCAAGCTGGCTGGTTTGATCAGATTGCAGTCAAAGTGGCCTGATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTT ACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTT GTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGT TTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTC CCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGA CGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCT CTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGATCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGATGGA GGTTGTTGAGGGAGGCATATGTCTCCTGGCACCCCTGGTGTGAGAGGCAGCTGCTGGGGCTTTGGAAGGTGGTGCCATGGAGGGAGTGCTTGTCTGGGA
SEQ ID NO：7仅列出涉及改造部分的DNA序列，其中两端的正常字体大写序列为未改变的小鼠Tnfrsf1b基因组序列，小写字母对应的序列为人TNFRSF1B基因内含子1的部分序列，下划线区域为人源化TNFRSF1B蛋白部分编码序列（其中下划线正常字体标记对应的是人源TNFRSF1B蛋白编码序列---主要是胞外区，下划线粗体对应的是鼠源Tnfrsf1b蛋白的跨膜和胞内区编码序列），斜体为WPRE序列，粗体为polyA序列，粗体斜体为元件插入时引入的其他序列。
改造后的人源化小鼠表达的人源化TNFRSF1B蛋白对应的CDS区、mRNA序列及其编码的蛋白序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示。人源化TNFRSF1B蛋白为一嵌合蛋白，含有鼠源Tnfrsf1b蛋白的信号肽，经过人源化改造的胞外区和鼠源Tnfrsf1b蛋白的跨膜区和胞内区。
实施例2 重组载体PBR322-Tnfrsf1b的设计及构建
根据序列设计，发明人进一步设计了如图3所示的打靶方案和包含5’同源臂、人TNFRSF1B CDS-WPRE-polyA的表达框、3’同源臂的载体。其中5’同源臂（SEQ ID NO：11）为NCBI登录号为NC_000070.6的第145229407-145233271位核苷酸，3’同源臂（SEQ ID NO：12）为NCBI登录号为NC_000070.6的第145226171-145229003位核苷酸，人TNFRSF1B CDS-WPRE-polyA的表达框序列信息（SEQ ID NO：13）。
载体的构建过程如下：设计扩增3段同源重组片段（LA，KI，RA）的上游引物和与其匹配的下游引物以及相关序列。其中，5’同源臂对应LA片段，人TNFRSF1B CDS-WPRE-polyA的表达框对应KI片段，3’同源臂对应RA片段，引物序列如下：
LA（3865bp）：
F：5’-CGCGGTCGACAAGCTCCCTAATTCTGCTGTGTACCTTC-3’（SEQID NO：14）
R：5’- gacagatatccagaaACACAAAGCTTCTCTTTTTAGGAT-3’（SEQ ID NO：15）
KI（2474bp）：
F：5’-ttctggatatctgtcccatgcagt-3’（SEQ ID NO：16）
R：5’- GCCTCCCTCAACAACCTCCATCCCATAGAGCCCACCGCATCCCC-3’（SEQ ID NO：17）
RA（2822bp）：
F：
5’-GTTGTTGAGGGAGGCATATGTCTCCTGGCACCCCTGGTGTGAGAGGCAGCTGCTGGGGC-3’（SEQID NO：18）
R：5’- CGACTCTAGAGGATCTCTGGGAGTTCAGGTCTGTATTC-3’（SEQ ID NO：19）
以C57BL/6小鼠基因组DNA或BAC文库为模板PCR扩增获得LA、RA片段，以全基因合成的人TNFRSF1B CDS-WPRE-polyA的表达框为模板PCR扩增获得KI片段。通过In-fusion试剂盒将片段连接到PBR322-MCS质粒上，最终获得载体PBR322-Tnfrsf1b。
实施例3 载体PBR322-Tnfrsf1b的验证
随机挑选5个PBR322-Tnfrsf1b克隆，使用限制性内切酶SacII进行酶切验证，酶切产物电泳应出现9058bp、6023bp大小片段，酶切正确的克隆进行质粒扩增。扩增的质粒经测序公司测序验证正确和酶切正确双重验证，进行后续实验。用于后续实验质粒的酶切结果参见图4。
实施例4 Tnfrsf1b基因sgRNA设计
sgRNA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。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。
根据打靶方案，设计并合成识别插入位点附近的靶位点（sgRNA1-sgRNA10）。
以小鼠为例，根据插入位点附近序列特征，各sgRNA在Tnfrsf1b基因上的靶位点序列如下：
sgRNA-1靶位点序列（SEQ ID NO：20）：5’-GTACCCAGGTTCCGGTTTGT AGG-3’
sgRNA-2靶位点序列（SEQ ID NO：21）：5’-TACCCAGGTTCCGGTTTGTA GGG-3’
sgRNA-3靶位点序列（SEQ ID NO：22）：5’-ACACCCTACAAACCGGAACCTGG-3’
sgRNA-4靶位点序列（SEQ ID NO：23）：5’-CACCCTACAAACCGGAACCT GGG-3’
sgRNA-5靶位点序列（SEQ ID NO：24）：5’-GGCAGCCATCAGTATGACTCTGG-3’
sgRNA-6靶位点序列（SEQ ID NO：25）：5’-TGTCTTGACACCCTACAAAC CGG-3’
sgRNA-7靶位点序列（SEQ ID NO：26）：5’-GTACGAGTGCCAGATCTCAC AGG-3’
sgRNA-8靶位点序列（SEQ ID NO：27）：5’-TGGCACTCGTACCCAGGTTC CGG-3’
sgRNA-9靶位点序列（SEQ ID NO：28）：5’-GAGATCTGGCACTCGTACCC AGG-3’
sgRNA-10靶位点序列（SEQ ID NO：29）：5’-TGTAGGGTGTCAAGACAACCTGG-3’
实施例5 针对不同靶位点sgRNA筛选
利用试剂盒检测多个sgRNA的活性。使用T7核酸内切酶 I检测试剂盒检测，检测结果参见图5，从图5可见多个sgRNA的活性不同，其中sgRNA-10具有最高的切割效率，因此优选sgRNA-10作为后续实验的sgRNA。
实施例6 sgRNA体外转录
sgRNA的合成通过体外PCR扩增sgRNA模板，以该模板为底物，利用HiScribe™ T7High Yield RNA Synthesis Kit试剂盒进行体外转录。
sgRNA-10的体外转录模板DNA的PCR引物分别为：
SgRNA-F4（SEQ ID NO：30）：5’-gcTAATACGACTCACTATAgTGTAGGGTGTCAAGACAACCGTTTTAGAGCTAGAAATAGCAAG-3’
下游PCR通用引物：
SgRNA-R （SEQ ID NO：31）：5’-aaaAGCACCGACTCGGTGCC-3’
由DNA合成公司合成含有sgRNA scaffold的片段DNA，序列信息为：
sgRNA scaffold（SEQ ID NO：32）
5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3'
使用含有sgRNA scaffold的片段DNA作为模板，引物对（SgRNA-F4和SgRNA-R）扩增出pT7-sgRNA-S4 PCR产物，利用凝胶回收试剂盒，回收PCR产物作为转录模板。根据体外转录试剂盒操作说明，体外转录sgRNA-10。体外转录产物电泳结果请见图6。
实施例7 受精卵显微注射及胚胎移植
取小鼠的受精卵，如C57BL/6小鼠的受精卵，利用显微注射仪将预混好的pT7-sgRNA-S10的体外转录产物（使用Ambion体外转录试剂盒，按照说明书提供方法进行转录）、Cas9 mRNA和PBR322-TNFRSF1B质粒注射至小鼠受精卵细胞质或细胞核中。按照《小鼠胚胎操作实验手册（第三版）》中的方法进行胚胎的显微注射，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，获得基因改造人源化小鼠，得到首建鼠（即founder鼠，为F0代）。将获得的F0代小鼠通过与野生型小鼠杂交，扩大种群数量，然后杂合子小鼠自交获得纯合子小鼠，建立稳定的hTNFRSF1B人源化小鼠品系。
实施例8 TNFRSF1B人源化小鼠的鉴定
1、F0代同源重组阳性小鼠基因型鉴定
分别使用两对引物对得到的F0代hTNFRSF1B小鼠的鼠尾基因组DNA进行PCR鉴定，引物I位于5’同源臂左侧，引物IV位于3’同源臂右侧，引物II和引物III均位于插入的片段末尾上，具体序列如下：
5’同源臂重组鉴定引物：
引物I：5’-GCTGTTTGAGGAAGTAGAGGAGGGT-3’（SEQ ID NO：33）
引物II：5’-ACCAGGGGTGCCAGGAGACAT-3’（SEQ ID NO：34）
3’同源臂重组鉴定引物：
引物III：5’-TGGAGGTTGTTGAGGGAGGCAT-3’（SEQ ID NO：35）
引物IV：5’-CTCGGTTCTGCTGTTTAGTGC-3’（SEQ ID NO：36）
PCR反应体系（20μL）如表3所示：
表3 PCR反应体系（20µL）
PCR扩增反应条件如表4所示：
表4 PCR扩增反应条件
如果重组载体插入位置正确，5'同源臂鉴定引物对I和II，阳性小鼠应有1条6.6kb长度的PCR条带，阴性小鼠应有1条8.3kb的PCR条带。3'同源臂鉴定引物对III和IV，阳性小鼠应有1条3.6kb长度的PCR条带，阴性小鼠应有1条8.4kb长度的PCR条带。
F0代小鼠PCR鉴定结果见图7，其中，A图为5'同源臂PCR鉴定结果，B图为3'同源臂PCR鉴定结果，从中可以看出，编号为1、6、7的3只小鼠为阳性小鼠。
F1代小鼠基因型鉴定：
将F0代阳性的小鼠与野生型小鼠交配得到F1代小鼠。对F1代鼠尾基因组DNA进行PCR鉴定。PCR条件及引物同F0代基因型鉴定。F1代小鼠PCR实验结果见图8，其中，A图为5'同源臂PCR鉴定结果，B图为3'同源臂PCR鉴定结果，从中可以看出，有8只F1代小鼠为阳性，具体编号为3、4、5、10、11、14、15、17。
将获得的F1代小鼠分别与野生型小鼠交配，扩繁种群。同时杂合子小鼠自交获得纯合子小鼠。
实施例9 流式细胞术检测不同基因型人源化小鼠TNFRSF1B（hTNFR2）蛋白水平表达
选取一只TNFRSF1B杂合子小鼠，一只同窝野生型小鼠（6-7周龄），分别提取脾脏细胞和外周血细胞分别检测鼠源和人源TNFRSF1B蛋白表达。
脾脏细胞流式细胞术检测：将脾脏在70µm滤网上研磨后，用PBS将脾脏细胞洗涤下来，400g，5分钟离心获得细胞沉淀，在细胞沉淀中加入1ml红细胞裂解液，重悬后冰上裂解5分钟，然后400g，5分钟离心获得细胞沉淀，再用PBS洗涤一次，离心后PBS重悬细胞，调整细胞浓度至107/ml，取100μl，用CD16/CD32 Fc receptor封闭试剂封闭5分钟后，分别用不同的抗体进行标记。根据文献报道，TNFR2蛋白主要在调节性T细胞（Tregs细胞）和髓系抑制性细胞中表达。通过CD4+/Foxp3+两个标记将Tregs细胞群选择出来，分别用针对鼠源Tnfr2和针对人源TNFR2的抗体进行分析；同时，通过CD11b标记将髓系细胞选择出来，进一步利用Gr1和F4/80标记对髓系抑制性细胞进行进一步分类，分别用针对鼠源Tnfr2和针对人源TNFR2的抗体进行分析。结果如图9所显示：在野生型小鼠的Treg细胞和髓系抑制性细胞中只能检测到鼠源Tnfr2阳性的细胞，不能检测到人源TNFR2阳性的细胞，即检测不到人源TNFR2蛋白的表达；在杂合子小鼠中，因为鼠源和人源化的基因均存在，理论上应既可以检测到鼠源Tnfr2的表达，也可以检测到人源化TNFR2的表达，和理论预期一致，流式细胞术检测结果显示：在杂合子小鼠的Tregs细胞和髓系抑制性细胞中均即可以检测到鼠源Tnfr2的表达，也可以检测到人源TNFR2蛋白的表达。
外周血细胞流式细胞术检测：外周血细胞加入红细胞裂解液，裂解5min后离心弃上清，用PBS清洗细胞1次，调整细胞浓度至107/ml，取100μl，用CD16/CD32 Fc receptor封闭试剂封闭5分钟后，分别用不同的抗体进行标记。与脾脏细胞检测结果相类似，如图10所示，在野生型小鼠的Treg细胞和髓系抑制性细胞中只能检测到鼠源Tnfr2阳性的细胞，检测不到人源TNFR2蛋白的表达；在杂合子小鼠中，无论是Tregs细胞，还是髓系抑制性细胞中均即能检测到鼠源Tnfrsf1b的表达，也能检测到人源化TNFR2的表达；并且在杂合子小鼠中检测到的人源信号和鼠源信号强度相近，说明人源化改造并没有影响鼠源Tnfr2基因的表达。
实施例10 TNFRSF1B人源化动物模型体内药效验证
通过杂合子小鼠交配获得hTNFRSF1B人源化纯合子小鼠，6-8周龄小鼠皮下接种小鼠结肠癌细胞MC38-OVA细胞（1x106/100μlPBS），待肿瘤生长至体积约50-80立方毫米后，随机分为对照组、给药组1和给药组2，每组7-8只小鼠。给药组1和给药组2分别给予特异性针对人源TNFR2的单克隆抗体1和2，给药频率为分组后的1、5、9天，给药方式为腹腔给药，给药剂量为15mg/kg；对照组腹腔注射等体积的PBS。每周分别在第三天和第七天共测量肿瘤体积和小鼠体重2次，3周结束试验。
结果如图11所显示：3次给药后，到21天实验结束时，相对于对照组（PBS），以肿瘤体积为指标，抗体1（hTNFR2Ab1）的肿瘤抑制效率为78%，抗体2（hTNFR2Ab2）的肿瘤抑制效率为49%，均显示出明显的肿瘤抑制效率。通过对给药过程中的体重检测显示，抗体2在实验第10天的时候，小鼠体重有明显的下降，给药结束后体重逐步回升，暗示可能抗体2有一定的毒副作用，还有待进一步检测。
该结果显示，TNFRSF1B人源化小鼠模型可以作为针对人源TNFRSF1B靶点的抗体药物抗肿瘤药效评价模型进行应用。
最后应说明的是：以上各实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述各实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的范围。
此外，本领域的技术人员能够理解，尽管在此的一些实施例包括其它实施例中所包括的某些特征而不是其它特征，但是不同实施例的特征的组合意味着处于本申请的范围之内并且形成不同的实施例。例如，在上面的权利要求书中，所要求保护的实施例的任意之一都可以以任意的组合方式来使用。公开于该背景技术部分的信息仅仅旨在加深对本申请的总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域技术人员所公知的现有技术。
序列表
<110> 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
广东南模生物科技有限公司
<120> TNFRSF1B基因人源化动物模型的构建方法及应用
<130> PA20029104
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> DNA
<213> 人(human)
<400> 1
ttctggatat ctgtcccatg cagtgttagg ggtcacccag cactggagaa ggccagcaga 60
gtcagtgctt ctgcctgcag acatgactag ggtacactga ggtggggagg caggggaggt 120
aaaagaaggc acgagctctc cttcctgtac cctgctccag ggggagaaac ctccccagcc 180
atcatcagtg cagactggca gggggagggc caaacatttg cagggcgggg acctgggcat 240
caggcatggc agaacccagg ggcggccctg ttgatggcag tcttcccttc ttccttccag 300
<210> 2
<211> 709
<212> DNA
<213> 人(human)
<400> 2
gtggcattta caccctacgc cccggagccc gggagcacat gccggctcag agaatactat 60
gaccagacag ctcagatgtg ctgcagcaaa tgctcgccgg gccaacatgc aaaagtcttc 120
tgtaccaaga cctcggacac cgtgtgtgac tcctgtgagg acagcacata cacccagctc 180
tggaactggg ttcccgagtg cttgagctgt ggctcccgct gtagctctga ccaggtggaa 240
actcaagcct gcactcggga acagaaccgc atctgcacct gcaggcccgg ctggtactgc 300
gcgctgagca agcaggaggg gtgccggctg tgcgcgccgc tgcgcaagtg ccgcccgggc 360
ttcggcgtgg ccagaccagg aactgaaaca tcagacgtgg tgtgcaagcc ctgtgccccg 420
gggacgttct ccaacacgac ttcatccacg gatatttgca ggccccacca gatctgtaac 480
gtggtggcca tccctgggaa tgcaagcatg gatgcagtct gcacgtccac gtcccccacc 540
cggagtatgg ccccaggggc agtacactta ccccagccag tgtccacacg atcccaacac 600
acgcagccaa ctccagaacc cagcactgct ccaagcacct ccttcctgct cccaatgggc 660
cccagccccc cagctgaagg gagcactggc gacttcgctc ttccagttg 709
<210> 3
<211> 635
<212> DNA
<213> 小鼠(mouse)
<400> 3
gtctgattgt tggagtgaca tcactgggtc tgctgatgtt aggactggtg aactgcatca 60
tcctggtgca gaggaaaaag aagccctcct gcctacaaag agatgccaag gtgcctcatg 120
tgcctgatga gaaatcccag gatgcagtag gccttgagca gcagcacctg ttgaccacag 180
cacccagttc cagcagcagc tccctagaga gctcagccag cgctggggac cgaagggcgc 240
cccctggggg ccatccccaa gcaagagtca tggcggaggc ccaagggttt caggaggccc 300
gtgccagctc caggatttca gattcttccc acggaagcca cgggacccac gtcaacgtca 360
cctgcatcgt gaacgtctgt agcagctctg accacagttc tcagtgctct tcccaagcca 420
gcgccacagt gggagaccca gatgccaagc cctcagcgtc cccaaaggat gagcaggtcc 480
ccttctctca ggaggagtgt ccgtctcagt ccccgtgtga gactacagag acactgcaga 540
gccatgagaa gcccttgccc cttggtgtgc cggatatggg catgaagccc agccaagctg 600
gctggtttga tcagattgca gtcaaagtgg cctga 635
<210> 4
<211> 588
<212> DNA
<213> 土拨鼠肝炎病毒(Woodchuck hepatitis virus)
<400> 4
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 60
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 120
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 180
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 240
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 300
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 360
gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 420
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 480
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 540
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgca 606
<210> 5
<211> 227
<212> DNA
<213> 牛(
cattle)
<400> 5
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggga 233
<210> 6
<211> 403
<212> DNA
<213> 小鼠(mouse)
<400> 6
ggaggtcctt gtcacagtga gtcaagccac tgtcttaaaa aaactacatc ttctcagagc 60
cttgctgggt ctcacccagc aggcaggagg gaagccctaa agtaacccac ttcctggccc 120
agcaaactgc agacacaagc gtgccacgct gaagaggaag gccaagaagg gaaaaagcct 180
gggctctact ctctcaagcc ttcctcgtcc tccaacgcaa aaacccatcc atgaggcctg 240
gtgacaggta tgctggagcc cagagtcata ctgatggctg cctcccttgt tcccttccag 300
gttgtcttga caccctacaa accggaacct gggtacgagt gccagatctc acaggaatac 360
tatgacagga aggctcagat gtgctgtgct aagtgtcctc ctg 403
<210> 7
<211> 2627
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaggacagag ggacagatgg acagacatcc taaaaagaga agctttgtgt ttctggatat 60
ctgtcccatg cagtgttagg ggtcacccag cactggagaa ggccagcaga gtcagtgctt 120
ctgcctgcag acatgactag ggtacactga ggtggggagg caggggaggt aaaagaaggc 180
acgagctctc cttcctgtac cctgctccag ggggagaaac ctccccagcc atcatcagtg 240
cagactggca gggggagggc caaacatttg cagggcgggg acctgggcat caggcatggc 300
agaacccagg ggcggccctg ttgatggcag tcttcccttc ttccttccag gtggcattta 360
caccctacgc cccggagccc gggagcacat gccggctcag agaatactat gaccagacag 420
ctcagatgtg ctgcagcaaa tgctcgccgg gccaacatgc aaaagtcttc tgtaccaaga 480
cctcggacac cgtgtgtgac tcctgtgagg acagcacata cacccagctc tggaactggg 540
ttcccgagtg cttgagctgt ggctcccgct gtagctctga ccaggtggaa actcaagcct 600
gcactcggga acagaaccgc atctgcacct gcaggcccgg ctggtactgc gcgctgagca 660
agcaggaggg gtgccggctg tgcgcgccgc tgcgcaagtg ccgcccgggc ttcggcgtgg 720
ccagaccagg aactgaaaca tcagacgtgg tgtgcaagcc ctgtgccccg gggacgttct 780
ccaacacgac ttcatccacg gatatttgca ggccccacca gatctgtaac gtggtggcca 840
tccctgggaa tgcaagcatg gatgcagtct gcacgtccac gtcccccacc cggagtatgg 900
ccccaggggc agtacactta ccccagccag tgtccacacg atcccaacac acgcagccaa 960
ctccagaacc cagcactgct ccaagcacct ccttcctgct cccaatgggc cccagccccc 1020
cagctgaagg gagcactggc gacttcgctc ttccagttgg tctgattgtt ggagtgacat 1080
cactgggtct gctgatgtta ggactggtga actgcatcat cctggtgcag aggaaaaaga 1140
agccctcctg cctacaaaga gatgccaagg tgcctcatgt gcctgatgag aaatcccagg 1200
atgcagtagg ccttgagcag cagcacctgt tgaccacagc acccagttcc agcagcagct 1260
ccctagagag ctcagccagc gctggggacc gaagggcgcc ccctgggggc catccccaag 1320
caagagtcat ggcggaggcc caagggtttc aggaggcccg tgccagctcc aggatttcag 1380
attcttccca cggaagccac gggacccacg tcaacgtcac ctgcatcgtg aacgtctgta 1440
gcagctctga ccacagttct cagtgctctt cccaagccag cgccacagtg ggagacccag 1500
atgccaagcc ctcagcgtcc ccaaaggatg agcaggtccc cttctctcag gaggagtgtc 1560
cgtctcagtc cccgtgtgag actacagaga cactgcagag ccatgagaag cccttgcccc 1620
ttggtgtgcc ggatatgggc atgaagccca gccaagctgg ctggtttgat cagattgcag 1680
tcaaagtggc ctgatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 1740
aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 1800
attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 1860
tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 1920
gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 1980
ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 2040
ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 2100
ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 2160
ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 2220
cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 2280
catcgatacc gtcgatcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc 2340
cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 2400
aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 2460
cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 2520
gggatggagg ttgttgaggg aggcatatgt ctcctggcac ccctggtgtg agaggcagct 2580
gctggggctt tggaaggtgg tgccatggag ggagtgcttg tctggga 2713
<210> 8
<211> 1422
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggcgcccg ccgccctctg ggtcgcgctg gtcttcgaac tgcagctgtg ggccaccggg 60
cacacagtgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc 120
cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc 180
caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac 240
agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt 300
agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc 360
aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg 420
cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg 480
tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg 540
ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc 600
acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg 660
tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc 720
ttcctgctcc caatgggccc cagcccccca gctgaaggga gcactggcga cttcgctctt 780
ccagttggtc tgattgttgg agtgacatca ctgggtctgc tgatgttagg actggtgaac 840
tgcatcatcc tggtgcagag gaaaaagaag ccctcctgcc tacaaagaga tgccaaggtg 900
cctcatgtgc ctgatgagaa atcccaggat gcagtaggcc ttgagcagca gcacctgttg 960
accacagcac ccagttccag cagcagctcc ctagagagct cagccagcgc tggggaccga 1020
agggcgcccc ctgggggcca tccccaagca agagtcatgg cggaggccca agggtttcag 1080
gaggcccgtg ccagctccag gatttcagat tcttcccacg gaagccacgg gacccacgtc 1140
aacgtcacct gcatcgtgaa cgtctgtagc agctctgacc acagttctca gtgctcttcc 1200
caagccagcg ccacagtggg agacccagat gccaagccct cagcgtcccc aaaggatgag 1260
caggtcccct tctctcagga ggagtgtccg tctcagtccc cgtgtgagac tacagagaca 1320
ctgcagagcc atgagaagcc cttgcccctt ggtgtgccgg atatgggcat gaagcccagc 1380
caagctggct ggtttgatca gattgcagtc aaagtggcct ga 1468
<210> 9
<211> 4910
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 9
agucaccagc uagagcgcag cugaggcacu agagcuccag gcacaagggc gggagccacc 60
gcugccccua uggcgcccgc cgcccucugg gucgcgcugg ucuucgaacu gcagcugugg 120
gccaccgggc acacagugcc cgcccaggug gcauuuacac ccuacgcccc ggagcccggg 180
agcacaugcc ggcucagaga auacuaugac cagacagcuc agaugugcug cagcaaaugc 240
ucgccgggcc aacaugcaaa agucuucugu accaagaccu cggacaccgu gugugacucc 300
ugugaggaca gcacauacac ccagcucugg aacuggguuc ccgagugcuu gagcuguggc 360
ucccgcugua gcucugacca gguggaaacu caagccugca cucgggaaca gaaccgcauc 420
ugcaccugca ggcccggcug guacugcgcg cugagcaagc aggaggggug ccggcugugc 480
gcgccgcugc gcaagugccg cccgggcuuc ggcguggcca gaccaggaac ugaaacauca 540
gacguggugu gcaagcccug ugccccgggg acguucucca acacgacuuc auccacggau 600
auuugcaggc cccaccagau cuguaacgug guggccaucc cugggaaugc aagcauggau 660
gcagucugca cguccacguc ccccacccgg aguauggccc caggggcagu acacuuaccc 720
cagccagugu ccacacgauc ccaacacacg cagccaacuc cagaacccag cacugcucca 780
agcaccuccu uccugcuccc aaugggcccc agccccccag cugaagggag cacuggcgac 840
uucgcucuuc caguuggucu gauuguugga gugacaucac ugggucugcu gauguuagga 900
cuggugaacu gcaucauccu ggugcagagg aaaaagaagc ccuccugccu acaaagagau 960
gccaaggugc cucaugugcc ugaugagaaa ucccaggaug caguaggccu ugagcagcag 1020
caccuguuga ccacagcacc caguuccagc agcagcuccc uagagagcuc agccagcgcu 1080
ggggaccgaa gggcgccccc ugggggccau ccccaagcaa gagucauggc ggaggcccaa 1140
ggguuucagg aggcccgugc cagcuccagg auuucagauu cuucccacgg aagccacggg 1200
acccacguca acgucaccug caucgugaac gucuguagca gcucugacca caguucucag 1260
ugcucuuccc aagccagcgc cacaguggga gacccagaug ccaagcccuc agcgucccca 1320
aaggaugagc agguccccuu cucucaggag gaguguccgu cucagucccc gugugagacu 1380
acagagacac ugcagagcca ugagaagccc uugccccuug gugugccgga uaugggcaug 1440
aagcccagcc aagcuggcug guuugaucag auugcaguca aaguggccug accccugaca 1500
gggguaacac ccugcaaagg gacccccgag acccugaacc cauggaacuu caugacuuuu 1560
gcuggaucca uuucccuuag uggcuuccag agccccaguu gcaggucaag ugagggcuga 1620
gacagcuaga guggucaaaa acugccaugg uguuuuaugg gggcaguccc aggaaguugu 1680
ugcucuucca ugaccccucu ggaucuccug ggcucuugcc ugauucuugc uucugagagg 1740
ccccaguauu uuuuccuucu aaggagcuaa cauccucuuc caugaauagc acagcucuuc 1800
agccugaaug cugacacugc agggcgguuc cagcaaguag gagcaagugg uggccuggua 1860
gggcacagag gcccuucagg uuagugcuaa acucuuagga aguacccucu ccaagcccac 1920
cgaaauucuu uugaugcaag aaucagaggc cccaucaggc agaguugcuc uguuauagga 1980
ugguagggcu guaacucagu gguccagugu gcuuuuagca ugcccugggu uugauccuca 2040
gcaacacaug caaaacguaa guagacagca gacagcagac agcacagcca gcccccugug 2100
ugguuugcag ccucugccuu ugacuuuuac ucuggugggc acacagaggg cuggagcucc 2160
uccuccugac cuucuaauga gcccuuccaa ggccacgccu uccuucaggg aaucucaggg 2220
acuguagagu ucccaggccc cugcagccac cugucucuuc cuaccucagc cuggagcacu 2280
cccucuaacu ccccaacggc uugguacugu acuugcugug accccaagug cauguccggg 2340
uuaggcacug ugaguuggaa cagcugauga caucgguuga aaggcccacc cggaaacagc 2400
ugaagccagc ucuuuugcca aaggauucau gccgguuuuc uaaucaaccu gcuccccuag 2460
caugccugga aggaaagggu ucaggagacu ccucaagaag caaguucagu cucaggugcu 2520
uggaugccau gcucaccgau uccacuggau augaacuugg cagaggagcc uaguuguugc 2580
cauggagacu uaaagagcuc agcacucugg aaucaagaua cuggacacuu ggggccgacu 2640
uguuaaggcu cugcagcauc agacuguaga ggggaaggaa cacgucugcc cccugguggc 2700
ccguccuggg augaccucgg gccuccuagg caacaaaaga augaauugga aaggacuguu 2760
ccugggugug gccucagcuc cugugcuugu guggaucccu aaagggugug cuaaggagca 2820
auugcacugu gugcuggaca gaauuccugc uuauaaaugc uuuuuguugu uguuuuguac 2880
acugagcccu ggcugagcca ccccacccca ccucccaucc caccuuuaca gccacucuug 2940
cagagaaccu ggcugucucc cacuuguagc cuguggaugc ugaggaaaca cccagccaag 3000
uagacuccag gcuugccccu aucuccugcu cugagucugg ccuccucauu guguuguggg 3060
aaggagacgg guucugucau cucggaagcc cacaccgugg augugaacaa uggcuguacu 3120
agcuuagacc agcuuagggc ucugcaauca gaggaggggg agcagggaac aauuugagug 3180
cugaccuaua acacauuccu aaaggauggg caguccagaa ucucccuccu ucagugugug 3240
ugugugugug ugugugugug ugugugugug uguguccaug uuugcaugua ugugugugcc 3300
agugugugga ggccagaggu uggcuuuggg uguguuugau cacucucagu uacugaggca 3360
gggcucucau cuguacccag agcuugcaca uuuucuaguc uaacuugcuu cagggaucuc 3420
ugucugccua uggagugcuc agguuacagg caggcugcca uaccugcccg acauuuacau 3480
gaauacuaga gaucugaauu cugguccuca cacuuguaua ccugcauuuu auccacuaag 3540
acaucucucc aagggcuccc ccuuccuauu uaauaaguua guuuugaacu ggcaagaugg 3600
cucagugggu aaggcaguuu gcggacaaac cugaugaccu gaguuggauc ccugaccaua 3660
agguagaaga gaccugauuc cugcaaguug uccucugacc accaccccau acaugcuucu 3720
gcauaugugc acacaucaca uucuugcaca cacacucaca uaccauaaau guaauaaauu 3780
uuuuuaaaua aauugauuuu aucuuuuaau cauuauuuuu gcuuaucugu uuguucauac 3840
auggaguuuu gcuguauacc cagucuggcu gcauccucca ggcaccuuga acuuggauau 3900
ucuccugccu caguuucgag agugcugagu uauaggcaug ugcaaccgug ucaggacccu 3960
uggcguuaca uuauguuuuc gaaucuuauu uuuccaucua agacugacaa ccaccuugcc 4020
aaaggaugac cuugacuuuu guauccucuu gccucuacuu cccaaguauu gggaugacaa 4080
gucuguccau ccuaugauuu guguggugcu ggggcuugaa cccaggggcc ucgugaaugc 4140
uaggcaaacg caaucagcug cccaaccccg aacuucacuu uucaccugug auuucuuaac 4200
augguuccug agcagacagc augauccugu ugccuugagg agcucugcuc guugcuuuga 4260
caaugcucua uuuacuaaag aauacuuaua gaguucaaga cuccaaagca gcaguuccca 4320
accugugggu augaccucuu ggguauccca uaucagauau uuacauuaaa cuuauaacag 4380
aagcaaaauu acaguuaugg gguagcaaca aaauaauuuu uuuuugaggc aggguuuauc 4440
uguguagcuc acuuuguaca gacuaggcug gccuugaacu uaaagaucug ccugccucug 4500
ccuucugaau gcugggauua aaugugugug ccaacaugcc cagugcagug aaauaauuuu 4560
auuguugggu caccaaaaca ugagaaagag uauuaaaagg ucauagcguu aggaagguug 4620
agaaccacug ccccaaaaga uaguagaaau agguaauagc auuuuaaaaa aagauuaauu 4680
ucuuuuuccg ugugugugug ugugugugug ugugugugug ugugugugug ugugugugug 4740
ugugaagggu gcugaauucc cuaaaucugg gauuacagac aauugugcca aauggguauu 4800
gggaaccaaa guuggguuuu cugaaagagc agcaagugcc cuuaacugcu gagcccucuc 4860
ucuagcuaua auaaacuuuu uuuuaaaaaa auuaaaaaaa uuuagcacuc 5072
<210> 10
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu
1 5 10 15
Trp Ala Thr Gly His Thr Val Pro Ala Gln Val Ala Phe Thr Pro Tyr
20 25 30
Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln
35 40 45
Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys
50 55 60
Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp
65 70 75 80
Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
85 90 95
Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
100 105 110
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu
115 120 125
Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg
130 135 140
Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val
145 150 155 160
Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr
165 170 175
Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly
180 185 190
Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser
195 200 205
Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser
210 215 220
Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser
225 230 235 240
Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly
245 250 255
Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ser Leu Gly
260 265 270
Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg Lys
275 280 285
Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val Pro
290 295 300
Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu Leu
305 310 315 320
Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser
325 330 335
Ala Gly Asp Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg Val
340 345 350
Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg Ile
355 360 365
Ser Asp Ser Ser His Gly Ser His Gly Thr His Val Asn Val Thr Cys
370 375 380
Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser
385 390 395 400
Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala Ser
405 410 415
Pro Lys Asp Glu Gln Val Pro Phe Ser Gln Glu Glu Cys Pro Ser Gln
420 425 430
Ser Pro Cys Glu Thr Thr Glu Thr Leu Gln Ser His Glu Lys Pro Leu
435 440 445
Pro Leu Gly Val Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly Trp
450 455 460
Phe Asp Gln Ile Ala Val Lys Val Ala
465 470
<210> 11
<211> 3865
<212> DNA
<213> 小鼠(mouse)
<400> 11
ccctaattct gctgtgtacc ttcctcactc ctttctccca acttacctta ctttctccat 60
gggataggat actcagacca cattgagtag agcagaggag tctctaatcc ccaggaagag 120
ttctttctta ttttatcttt cctctttatg agcagtgtgg ctccccagcc cagagactct 180
ccccccttag atgtgaaagg gtcagccagc cattgatgta ccctttgatg tgtttgttac 240
attagcagtt ctcatctgaa cacaacacac tgggtccgag cggttccacg tgtgagatgt 300
ttattgggag gggggcgcag gggagaaggt gacagcagaa agagaagggg aaaaagagag 360
agagggcctc cttagaagtg ggaacaaaac acccttggaa ggagttacag agacaaagtt 420
tggagctgag attaaagggt ggaccatgta gagactgcct tatccaggga tccaccccat 480
aatcagcatc caaacgctga caccattgca tacactagca agattttatc gaaaggaccc 540
agatgtagct gtctcttgtg agactatgcc ggggcctagc aaacacagaa gtggatgccc 600
acagtcagct aatggatgga tcacagggct cccaatggag gagctagaga aagtacccaa 660
ggagctaaag ggatctgcaa ccctataggt ggatcaacat tatgaactaa ccagtacccc 720
ggagctcttg actctagctg cacatgtatc aaaagatggc ctagtcggcc atcactggaa 780
agagaggccc attggacaca caaactttat atgccccaga acaggggaat gccagggcca 840
aaaaggggga gtgggcaggt aggggagtgg gggtgggtgg gtatggggga cttttggtat 900
agcattggaa atgtaaatga actaaatacc taataaaaaa tggaaaaaaa aaaaggaaaa 960
aaaaagagat attaaggaaa agtaaaaaaa aaaaaagaga gagagagagg gagggctgtt 1020
ccccttattt atatgaaaaa aatgacgtaa cacaggtaaa ggtgggaggt gagccaagtg 1080
gattctggga atatgctgca ggtcgcgctg tcacctatgt gatgtcatag gtttgggagg 1140
tcctgatgca aagacccttg accttgagaa cagtcagtga ggtcccagac agtgtggaac 1200
acccttattt gtgtgctcag actcaactgg ttgttttgtc aagaaatgga acggggaagt 1260
gccaggatat tgtgtgtagg catgtgcgca tgcgcttgga tgtgtgtgca gctgtccaga 1320
gggagctgca gtggcttttt cagactgggt actcagcatg gttctgcgac ttccctcacc 1380
cagctgtctg catgaccttc ctgaaaagga ttgccttgat ggaggaactc agagataggc 1440
cagggcagca gatgcatcta tgttcaagct tcttggccct ggtgcccagg gggaggctct 1500
cgcctcaagg aaatgacatt agaattatta ccttactcag ggtagcaact attgattact 1560
cttttctgtc tggctctgga tagggtagct cgcactgcag cctccaatgg gccctcatct 1620
ggggttactg ggtgagcatg aagacaaggc agggggctgg ctgtattggg ttgcttatcc 1680
agggcacggt gtgcacagtc gcctgaagag ggaagcactc tgtgtggact tttggccaaa 1740
ggcgcaagag gcagctaggg tactgtggca ggtccagaag agccaaagac tgtccacagc 1800
atagaacccc agtcccttga gtcatggctt ctggtggctt tcagctgtta tcactgagtg 1860
gctgagcaaa gagaaacatg ataggctcag aggtcactca gtgcacagca ctaggcaagg 1920
tagggtgatg ctctccagac acagctgggg atgggaaggg gaaagacaga aagcagagcc 1980
actgcccaca gatcatggga gtttgtgcag tctatacaga ccaaatgagc ctaatagtaa 2040
ggtcggcatc ttctctgatt ggcttgtttg cagtggcttt tcagagactg atccatgcct 2100
ttgctgtgct aattgttgga tattttatgt atctctcttg ctcccataga attgagtcag 2160
gaaccgatgt ccagagagat agggacactt gattgactat gaccatacag cttctggatg 2220
gaagaataag aaaagaagac cacatctctc taactctcca cctttatctg aggagtcagg 2280
ggattcattt gggttactgg tttaataaac atttattaag ctcctgctga ccatatgacg 2340
gcagctgctg gtacaatcaa gagtgcggag cagccttcct ctttaaccta gaaccacccc 2400
ttggtcattt ccaggtgaca tcagcagagg ctgcaagggt gttgagtgtg tgttcacacc 2460
ctctttgctc ttgtagatcc actcccatgt ttgctcatga gttcacattt gtgcagaaat 2520
ggctaattca tgcttgtccc taaagtttcc aattgctgtt ccaccttctg gaagaaaggc 2580
tggggcttag gcaggtctct gaggtactgg gatagaccca gtggtgacag ttgctgctgc 2640
tgtacacttc tgagaactga ggacctcttg gagatctgag ggagggctct gtgtttctca 2700
cagttggttc tgtgccatgt tgggtgtttt tttgtttttt gttttttctc ctccccattt 2760
gtctcccttt cataattatt tatttaatta aaaaatgtga gtactcctga gtatatgtat 2820
gtgaaccaca cacattcagg agtatgcaga acttagaagg gagcgtcagg ttacctggaa 2880
ctggagtgac agacagttgt gagctgccat gttggtgctg ggaactgatc ccaagttgtc 2940
tccaagaata gtaagtgccc ctaaccactg cgctatctct ggagctcctc ctccccatcc 3000
tccccatcgc tccctgcttt cctggtctct ccctggaagt gcttagccca gtcattgaaa 3060
tcatggccta gagagacaga caggacccag gctcaaatct ctgtcactgc cttaggtggg 3120
taacctgtgc tcgttatgcc tgtttccttt ctatacaatg gtcggagtcc aacatggctg 3180
gttgttagag aactgaatgc aataatacat ccatctcagg tccaggctct ggaagcacag 3240
cctgaggcat ggcttcttgt aacaggagaa ggctctcagg caagagggtg gggtggcagg 3300
atgtggcagc agggtcacat gttttccagc caaaacctat gagtgttggt cttgggttga 3360
ggccaaggta ctgcctttga gttggagact tgtcggccag tgctgaggct attctcctgg 3420
cagttggagt ctgagcatgg ctaggagccc ccactggtcc aaaccttcta agcccatagg 3480
tgtaaggcag cactgatgtg aggaggtgca caggtttgtt ttcattctgt attaacttcc 3540
tgaggcagct ctgccatggc ctgtgtcaga agcttgggtc tagaggtggc gcagccatta 3600
tctgtgtcat aggtggcagc atatggacac atgtatgtac acctgtgtgc atatgcatgg 3660
atgtgagtgc aaggacacac ggtgccaatt cagaacaatg acatgaaggt cagaaggggc 3720
catgtctata ttcagagaga acccagagga ggaagagcct acattaggcc caggagagta 3780
gagggtactt ctgggagaag gaagacctca gccacaagga cagagggaca gatggacaga 3840
catcctaaaa agagaagctt tgtgt 3993
<210> 12
<211> 2833
<212> DNA
<213> 小鼠(mouse)
<400> 12
gtgagaggca gctgctgggg ctttggaagg tggtgccatg gagggagtgc ttgtctggga 60
atgagggcct tcagctctca ctggctgctt tatacatgct agggttcatg attcatcttg 120
ccctgggctc ttgcatgtgc tgtgccctcc actgagcaca cttctcagtg tcttctcctg 180
gttactgcct acctacattt tgaccctatc ctcttgtcca ggaagcctcc tcaatactgc 240
aatgatgtcc tagtactttt catagcccca ctatgtgcta tgtctacccc tattgagtag 300
tgagtgtagc ttggtccctg tggagttcca agcacacggc tggcctatat tgatgtcctg 360
tacttaattg tcaagtaaat gaatggatag ccatatcata gatggcggat ctgagccctg 420
gcctcattgg cgaggactga gtagctggcc cccagtgctg agtagacaat caagtgtatg 480
cattcaatta agtcgtattt ataatgcatc tactctgtgc tcaatctgct gagaggaaag 540
ccacacaaac acgggataca gcaggctggg aagtaagtgc aaagccctaa agcaggggcc 600
tgttttaatg ggtccaaaga agcttgaggc tggtgtttgt tgcccagagt gaaagaagag 660
tgtgatggaa gatgggtatg aaggggtgag ggttggtgag atggctctgt atgtaaaggc 720
acttgctgtg caagcctgag aacctgaacc aatggtggaa ggagaaaaca ggttacacac 780
acacaccacc accaccacca ccaccaccac catcgtaata atatacattt gattaagcga 840
gatgagtggt gtcaccatga agaattcaga tttcatttac tggaggcttt gagtcctgtg 900
cttgtgaccc agtgggcagg aacacagggc aatgtcatcc tccacagtca cagccagggg 960
acagtggctt ttctagtgac gagcagagtg tctttacgaa gttacaagct tagagtagca 1020
ggaacagagg agacgagctg gttccagaag agaaaacctt aaagatttgt cctgttactc 1080
ttctatcctc caggctctta ggagccctat gcccttggca ctgacaggct accagacatg 1140
acgcccttat agccacttat atttagtaca tcaggataga gaattttaac caactatcaa 1200
gaagacagga gctagcaaac ccattctccc ttaagtattc ttgactgtta ataggttttt 1260
cgagtggtgg tgtttcaact acctggagag agcagaggga agatatgttc agaggtagag 1320
agatagctag ggccaccaga catgcttcat cccaggggct tcctggcaag ggtcttctgc 1380
tgagacctct ggtccttgct tcctcaggcc aatatgtgaa acatttctgc aacaagacct 1440
cggacaccgt gtgtgcggac tgtgaggcaa gcatgtatac ccaggtctgg aaccagtttc 1500
gtacatgttt gagctgcagt tcttcctgta ccactggtga gtggcatgag cactgtgatc 1560
ccaacatctt ccccagtcac tcgccgtcca gcaagtaagg tggatggggt taccctgggc 1620
cagccgtttt cgtcttccag gacttgagtt gcgggagact gcttttttta aaatattttt 1680
ttattacata ttttcctcaa ttacatttcc aatgctatcc caaaagtccc ccacaccctc 1740
ccccccactc ccctacccac ccattcccat tttttggccc tggtgttccc ctgtactggg 1800
gcatataaag tttgcctgtc caatgggcct ctctttccag tgatggccaa ctaggccatc 1860
ctttgataca tatgcagcta gagttaagag ctccggggta ctggttagtt cataatgttg 1920
cacctacagg gttgcagatc tctttagctc cttggatact ttctctagct cctcccttgg 1980
gggccctgtg atctatccaa tagttgactg tgagcatcca cttctgtgtt tgctaggccc 2040
cagcctagtc tcacaagaga tggctatatc acggtccttg caccaaacgc ttgctagtgt 2100
atgcaatggt gtcatcgttt ggaggctaat tatgggatgg atccctggat atggcagtct 2160
ctagatggtc catccttttg gggagactgc tttttttcaa cttgtatgta gttatgtgat 2220
tggaacctgg gacttcatgc aggtgatgca aacacttttt tttttttttt tggttttttg 2280
agacagggtt tctctgtgta gccctggctg tcctggaact cactttgtag accaggctgg 2340
cctcgaactc agaaatctgc ctgcctctgc ctcccaagtg ctgggattaa aggcgtgcgc 2400
caccacgcct ggctgatgca aacacttttt aacagaacta catcaatccc cagatatttt 2460
ttttattaaa ggtgtattaa ttaattaatt aattaattaa ttaattaact tacttatttt 2520
ggtttttcaa gacagcattt ctctgtgtat cctcggctgt cctggaagtc actctgtaga 2580
ccaggctggc ctcgaactca gagatctgcc tgactctgta tcccaagtgc taggactaaa 2640
ggcgtgcgcc gccccaaccc aataagattt atttattttt attgtatgta tatgggtatt 2700
tttactggca tgtagtcagg atctattttg ccttgagtca ggatctctct aggtagctca 2760
gaatggcttt gaagttatga actgcctaac tcagcttcct gagtagctga gaatacagac 2820
ctgaactccc aga 2927
<210> 13
<211> 2295
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atggcgcccg ccgccctctg ggtcgcgctg gtcttcgaac tgcagctgtg ggccaccggg 60
cacacagtgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc 120
cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc 180
caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac 240
agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt 300
agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc 360
aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg 420
cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg 480
tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg 540
ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc 600
acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg 660
tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc 720
ttcctgctcc caatgggccc cagcccccca gctgaaggga gcactggcga cttcgctctt 780
ccagttggtc tgattgttgg agtgacatca ctgggtctgc tgatgttagg actggtgaac 840
tgcatcatcc tggtgcagag gaaaaagaag ccctcctgcc tacaaagaga tgccaaggtg 900
cctcatgtgc ctgatgagaa atcccaggat gcagtaggcc ttgagcagca gcacctgttg 960
accacagcac ccagttccag cagcagctcc ctagagagct cagccagcgc tggggaccga 1020
agggcgcccc ctgggggcca tccccaagca agagtcatgg cggaggccca agggtttcag 1080
gaggcccgtg ccagctccag gatttcagat tcttcccacg gaagccacgg gacccacgtc 1140
aacgtcacct gcatcgtgaa cgtctgtagc agctctgacc acagttctca gtgctcttcc 1200
caagccagcg ccacagtggg agacccagat gccaagccct cagcgtcccc aaaggatgag 1260
caggtcccct tctctcagga ggagtgtccg tctcagtccc cgtgtgagac tacagagaca 1320
ctgcagagcc atgagaagcc cttgcccctt ggtgtgccgg atatgggcat gaagcccagc 1380
caagctggct ggtttgatca gattgcagtc aaagtggcct gatcaacctc tggattacaa 1440
aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 1500
cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 1560
cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 1620
tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 1680
ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 1740
cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 1800
ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 1860
tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 1920
ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 1980
tcggatctcc ctttgggccg cctccccgca tcgataccgt cgatcctgtg ccttctagtt 2040
gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 2100
ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 2160
ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 2220
ggcatgctgg ggatgcggtg ggctctatgg gatggaggtt gttgagggag gcatatgtct 2280
cctggcaccc ctggt 2371
<210> 14
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgcggtcgac aagctcccta attctgctgt gtaccttc 38
<210> 15
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gacagatatc cagaaacaca aagcttctct ttttaggat 39
<210> 16
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttctggatat ctgtcccatg cagt 24
<210> 17
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcctccctca acaacctcca tcccatagag cccaccgcat cccc 44
<210> 18
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gttgttgagg gaggcatatg tctcctggca cccctggtgt gagaggcagc tgctggggc 59
<210> 19
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cgactctaga ggatctctgg gagttcaggt ctgtattc 38
<210> 20
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gtacccaggt tccggtttgt agg 23
<210> 21
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tacccaggtt ccggtttgta ggg 23
<210> 22
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
acaccctaca aaccggaacc tgg 23
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
caccctacaa accggaacct ggg 23
<210> 24
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggcagccatc agtatgactc tgg 23
<210> 25
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgtcttgaca ccctacaaac cgg 23
<210> 26
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gtacgagtgc cagatctcac agg 23
<210> 27
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tggcactcgt acccaggttc cgg 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gagatctggc actcgtaccc agg 23
<210> 29
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgtagggtgt caagacaacc tgg 23
<210> 30
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gctaatacga ctcactatag tgtagggtgt caagacaacc gttttagagc tagaaatagc 60
aag 65
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
aaaagcaccg actcggtgcc 20
<210> 32
<211> 80
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt 82
<210> 33
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gctgtttgag gaagtagagg agggt 25
<210> 34
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
accaggggtg ccaggagaca t 21
<210> 35
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
tggaggttgt tgagggaggc at 22
<210> 36
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ctcggttctg ctgtttagtg c 21
Claims (4)
1.一种TNFRSF1B基因人源化动物细胞的构建方法，其特征在于，包括：
在非人动物细胞内导入人TNFRSF1B基因，使所述人TNFRSF1B基因在非人动物细胞中表达产生人源化的TNFRSF1B蛋白，同时降低或消除所述非人动物细胞内的内源TNFRSF1B基因的表达；
利用基因编辑技术对非人动物细胞内的内源TNFRSF1B基因进行改造，得到人源化TNFRSF1B基因；
所述非人动物为小鼠；
所述细胞为受精卵细胞；
所述构建方法采用CRISPR/Cas9基因编辑技术实现；所述构建方法具体包括：提供包括人TNFRSF1B基因同源重组载体、sgRNA以及Cas9的混合物，所述Cas9包括Cas9 mRNA和/或Cas9蛋白，将所述混合物注射到所述非人动物细胞中，将所述非人动物细胞转移至培养液中进行培养；
所述人TNFRSF1B基因同源重组载体包括从5’端到3’端依次排列的5’同源臂、人TNFRSF1B基因、小鼠Tnfrsf1b基因、polyA以及3’同源臂；
所述人TNFRSF1B基因的序列由人TNFRSF1B基因内含子1的部分序列与人TNFRSF1B基因的部分编码区序列组成；所述人TNFRSF1B基因内含子1的部分序列如SEQ ID NO：1所示，所述人TNFRSF1B基因的部分编码区序列如SEQ ID NO：2所示；
所述小鼠Tnfrsf1b基因的序列如SEQ ID NO：3所示；
所述小鼠Tnfrsf1b基因与所述polyA之间还设置有 WPRE调控元件，所述WPRE调控元件的序列如SEQ ID NO：4所示；
所述polyA的序列如SEQ ID NO：5所示；
所述5’同源臂的DNA序列如SEQ ID NO：11所示；
所述3’同源臂的DNA序列如SEQ ID NO：12所示；
所述sgRNA的靶位点序列如SEQ ID NO：29所示。
2.根据权利要求1所述的TNFRSF1B基因人源化动物细胞的构建方法，其特征在于，所述人源化TNFRSF1B基因选自下列组中的至少一种：（a）所述人源化TNFRSF1B基因的CDS编码序列如SEQ ID NO：8所示；（b）所述人源化TNFRSF1B基因转录的mRNA序列如SEQ ID NO：9所示；（c）所述人源化TNFRSF1B基因编码的蛋白序列如SEQ ID NO：10所示。
3.一种TNFRSF1B基因人源化动物模型的构建方法，其特征在于，将如权利要求1-2任一项所述TNFRSF1B基因人源化动物细胞的构建方法得到的所述TNFRSF1B基因人源化动物细胞或者由所述TNFRSF1B基因人源化动物细胞发育得到的胚胎移植至代孕母体内进行发育，得到F0代动物；
对F0代动物进行检验，获得基因型鉴定正确的F0代阳性动物。
4.根据权利要求3所述的TNFRSF1B基因人源化动物模型的构建方法，其特征在于，还包括：将所述F0代阳性动物与野生型动物交配，扩大种群数量，同时杂合子动物与杂合子动物交配获得纯合子动物，建立稳定的TNFRSF1B基因人源化动物品系。
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