CN114990145B - 一种高亲和力动态捕获dna双链断裂修复相关蛋白的方法 - Google Patents
一种高亲和力动态捕获dna双链断裂修复相关蛋白的方法 Download PDFInfo
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- CN114990145B CN114990145B CN202210708697.2A CN202210708697A CN114990145B CN 114990145 B CN114990145 B CN 114990145B CN 202210708697 A CN202210708697 A CN 202210708697A CN 114990145 B CN114990145 B CN 114990145B
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Abstract
本发明公开一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法,属于生物技术及细胞生物学领域。所述方法先将归位内切酶I‑sceI与突变型生物素连接酶BirA*连接构建融合表达质粒，然后构建DNA同源重组修复报告模式细胞(以下简称DR‑GFP)，再用融合表达质粒转染DR‑GFP模式细胞，靶向诱导模式细胞发生DNA双链断裂，使得DNA双链断裂修复蛋白募集于损伤位点启动修复，同时融合蛋白中的生物素连接酶将损伤位点及邻近参与修复的内源性蛋白进行生物素化，通过链霉亲和素‑生物素分离法捕获相关蛋白分子。这种方法可以高效捕获DNA双裂断裂修复过程中的所有蛋白分子，弥补了传统蛋白互作技术的不足，具有快捷、高效、廉价等优点。
Description
技术领域
本发明涉及生物技术及细胞生物学领域，特别涉及一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法。
背景技术
DNA双链断裂修复是一个动态的过程，主要包括损伤感应，损伤应答及损伤修复三个过程，其中DNA损伤应答(DNA damage response，DDR)是细胞内抵御外界及内在因素诱导的一种非常保守的DNA损伤机制，由多条信号传导通路构成的网络来监测和传递损伤信号，并形成一个适当的应答机制，对维持细胞的稳态至关重要。然而DNA双链断裂修复缺陷可导致肿瘤发生或基因组不稳定性的增加，且基因组不稳定性是癌变过程的早期阶段，同时肿瘤细胞格外依赖一些DNA修复通路，因此靶向肿瘤细胞依赖的基因修复通路可以达到抗肿瘤的效果。近年来，以肿瘤细胞DNA损伤反应与修复基因缺陷为背景的靶向治疗取得了重大突破，比如PolyADP ribose polymerase抑制剂(PARP1)用于治疗BRCA1/2(Breast CancerSusceptibility Genes1/2)缺陷型肿瘤，免疫检查点抑制剂用于治疗错配修复缺陷(Mismatch repair deficiency,MMR-D)/高微卫星不稳定性(Microsatelliteinstability high,MSI-H)表型肿瘤等，因此，通过表征肿瘤细胞DNA损伤修复分子图谱，以及阐明DNA修复途径的潜在机制，可以为肿瘤的靶向治疗提供新的方向。
在传统蛋白互作研究技术中，一种诱饵蛋白只能捕获与自身相互作用的蛋白分子，捕获通量低，且无法识别弱作用或瞬时相互作用的蛋白分子，再加上DNA双链断裂修复过程中不同修复阶段的DNA修复分子各不相同，使得传统的蛋白质-蛋白质互作研究技术难以表征DNA双链断裂动态修复过程的分子网络。因此，本领域迫切需要开发高捕获通量及高亲和力的蛋白互作研究技术，用于捕获参与DNA双链断裂动态修复过程的蛋白分子。
发明内容
本发明通过将归位内切酶I-sceI和生物素连接酶BirA*连接构建融合表达质粒，以及筛选被pDRGFP质粒转染后的肿瘤细胞来构建DNA同源重组修复报告细胞，建立了一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，以解决现有蛋白互作研究技术捕获通量低的问题。
为了实现上述目的，本发明的技术方案如下：
一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，其特征在于，所述方法包括：
步骤1：将5’端带有核定位序列及HA序列的归位内切酶I-SceI表达序列通过如SEQIDNO.1所示的连接序列与生物素连接酶BirA*(R118G)表达序列连接得到融合表达序列，再将融合表达序列构入哺乳动物过表达质粒中得到I-SceI-BirA*融合表达质粒；
步骤2：采用pDRGFP质粒转染肿瘤细胞后用嘌呤霉素来筛选阳性细胞，构建DNA同源重组修复报告模式细胞；
步骤3：将步骤1构建的I-SceI-BirA*融合表达质粒转染步骤2获得的DNA同源重组修复报告模式细胞，转染后再加入生物素进行培养。
步聚4：提取步骤3模式细胞总蛋白，利用链霉亲和素磁珠纯化捕获生物素化的DNA双链断裂修复蛋白，最后进行质谱鉴定。
具体地，所述步骤1中如SEQ ID NO.1所示的连接序列为5’-ACGCGTGGCGGAGGAGGCTCCGGGGGAGGGGGAAGC-3’，连接序列有效保持了融合蛋白中内切酶I-SceI和生物素连接酶BirA*各自的活性功能，并有效降低捕获过程中产生的背景。
在其中一个实施例中，步骤1中所述5’端带有核定位序列及HA序列的归位内切酶I-SceI表达序列是如SEQ ID NO.2所示的序列，生物素连接酶BirA*表达序列是如SEQ IDNO.3所示的序列。所述归位内切酶I-SceI表达序列的5’端连接有核定位序列及HA序列，能在哺乳动物细胞中稳定表达，具有强的内切酶活性。
在其中一个实施例中，所述生物素连接酶BirA*序列的第352位碱基由胞嘧啶C突变为鸟嘌呤G，突变后的蛋白质序列第118位氨基酸由精氨酸突变为甘氨酸，具有依赖生物素对邻近蛋白进行生物素化的特性。
在其中一个实施例中，所述核定位序列与HA序列相连，核定位序列位于融合表达序列5’最前端。
在其中一个实施例中，所述步骤1中融合表达序列是如SEQ ID NO.4所示的序列。
在其中一个实施例中，所述核定位序列是如SEQ ID NO.5所示的序列，HA序列是如SEQ ID NO.6所示的序列。
在其中一个实施例中，所述步骤1中所述哺乳动物过表达质粒为pCDNA3.1质粒。
在其中一个实施例中，所述步骤2中的肿瘤细胞为人骨肉瘤细胞。
在其中一个实施例中，所述DNA同源重组修复报告模式细胞带有经过修饰的GFP基因SceGFP，其包含一个I-SceI位点和框内终止密码子。归位内切酶I-SceI能识别SceGFP中的I-SceI位点，诱导DNA双链断裂从而激发DNA损伤反应，SceGFP可以用内部iGFP片段作为模板进行同源重组修复，并产生一个功能性GFP基因，表达完整GFP蛋白，报告绿色荧光。
在其中一个实施例中，所述一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法在捕获DNA双链断裂修复蛋白中的应用。
本发明的有益效果如下：
本发明将5’端带有核定位序列及HA序列的归位内切酶I-sceI与生物素连接酶BirA*连接后构入pCDNA3.1质粒中来，构建I-sceI-BirA*融合表达质粒；然后用pDRGFP质粒转染肿瘤细胞来构建DNA同源重组修复报告模式细胞，从而得到了可以动态捕获DNA双链断裂修复相关蛋白的系统。本发明的方法可捕获DNA双链断裂修复过程中动态变化的蛋白分子，具有特异性强，亲和力高等优点，实现了采用一种诱饵蛋白捕获参与DNA双链断裂动态修复过程的所有蛋白分子，同时可识别弱作用或瞬时相互作用的蛋白分子，弥补了传统蛋白互作技术时空性的不足，具有快捷、高效、廉价等优点，还能用于表征肿瘤细胞DNA损伤反应网络，挖掘肿瘤靶向治疗的新靶点。
附图说明
为了更清楚地说明本发明的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施方式，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的附图。
图1是本发明建立动态捕获DNA双链断裂修复相关蛋白方法的实验操作流程图。
图2是本发明建立动态捕获DNA双链断裂修复相关蛋白方法的检测结果图，图中：A是pCDNA3.1质粒或I-sceI-BirA*融合表达质粒转染DNA同源重组修复报告模式细胞后的流式检测结果；B是HRP-链霉亲和素的Western Blot检测结果；C是pCDNA3.1质粒或I-sceI-BirA*融合表达质粒分别转染DNA同源重组修复报告模式细胞或I-sceI缺失型DNA同源重组修复报告模式细胞的Western Blot检测结果；D是本发明所建立方法捕获经典的DNA双链断裂修复蛋白图。
图3是本发明建立的动态捕获DNA双链断裂修复相关蛋白方法所捕获的修复蛋白图。
具体实施方式
下面对本发明实施方式中的技术方案进行清楚、完整地描述。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。下述实施例中的实验方法，如无特殊说明，均为常规说法。
实施例1：一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法
按照图1的实验操作，所述方法具体包括以下步骤：
1、设计及构建I-sceI-BirA*融合表达质粒
设计并合成I-sceI-BirA*融合表达序列，将5’端带有核定位序列(SEQ ID NO.5)及HA序列(SEQ ID NO.6)的归位内切酶I-sceI表达序列(SEQ ID NO.2)通过特定连接序列5’-ACGCGTGGCGGAGGAGGCTCCGGGGGAGGGGGAAGC-3’(SEQ ID NO.1)与生物素连接酶BirA*表达序列(SEQ ID NO.3)相连得到NLS-HA-I-sceI-BirA*融合表达序列(SEQ ID NO.4)。NLS-HA-I-sceI-BirA*融合表达序列合成于北京擎科生物科技有限公司。将NLS-HA-I-sceI-BirA*融合表达序列通过分子克隆的方法构入pCDNA3.1质粒(购于Addgene)中得到NLS-HA-I-sceI-BirA*融合表达质粒。
NLS-HA-I-sceI-BirA*融合表达序列5’端带有特定核定位序列5’-ATGCCAAAAAAGAAGAGAAAGGTGCCGAAGAAGCATGCAGCACCACCAAAAAAAAAACGAAAAGTAGAAGACCCACGATTT-3’(SEQID NO.5)及HA标签序列5’-ATGTACCCATACGATGTTCCTGACTATGCG-3’(SEQ ID NO.6)。
归位内切酶I-sceI表达序列(SEQ ID NO.2)：5’-GGTATGAAAAACATCAAAAAAAACCAGGTAATGAACCTGGGTCCGAACTCTAAACTGCTGAAAGAATACAAATCCCAGCTGATCGAACTGAACATCGAACAGTTCGAAGCAGGTATCGGTCTGATCCTGGGTGATGCTTACATCCGTTCTCGTGATGAAGGTAAAACCTACTGTATGCAGTTCGAGTGGAAAAACAAAGCATACATGGACCACGTATGTCTGCTGTACGATCAGTGGGTACTGTCCCCGCCGCACAAAAAAGAACGTGTTAACCACCTGGGTAACCTGGTAATCACCTGGGGCGCCCAGACTTTCAAACACCAAGCTTTCAACAAACTGGCTAACCTGTTCATCGTTAACAACAAAAAAACCATCCCGAACAACCTGGTTGAAAACTACCTGACCCCGATGTCTCTGGCATACTGGTTCATGGATGATGGTGGTAAATGGGATTACAACAAAAACTCTACCAACAAATCGATCGTACTGAACACCCAGTCTTTCACTTTCGAAGAAGTAGAATACCTGGTTAAGGGTCTGCGTAACAAATTCCAACTGAACTGTTACGTAAAAATCAACAAAAACAAACCGATCATCTACATCGATTCTATGTCTTACCTGATCTTCTACAACCTGATCAAACCGTACCTGATCCCGCAGATGATGTACAAACTGCCGAACACTATCTCCTCCGAAACTTTCCTGAAA-3’。
生物素连接酶BirA*表达序列(SEQ ID NO.3)：5’-ATGAAGGATAACACCGTGCCACTGAAATTGATTGCCCTGTTAGCGAACGGTGAATTTCACTCTGGCGAGCAGTTGGGTGAAACGCTGGGAATGAGCCGGGCGGCTATTAATAAACACATTCAGACACTGCGTGACTGGGGCGTTGATGTCTTTACCGTTCCGGGTAAAGGATACAGCCTGCCTGAGCCTATCCAGTTACTTAATGCTAAACAGATATTGGGTCAGCTGGATGGCGGTAGTGTAGCCGTGCTGCCAGTGATTGACTCCACGAATCAGTACCTTCTTGATCGTATCGGAGAGCTTAAATCGGGCGATGCTTGCATTGCAGAATACCAGCAGGCTGGCCGTGGTGGCCGGGGTCGGAAATGGTTTTCGCCTTTTGGCGCAAACTTATATTTGTCGATGTTCTGGCGTCTGGAACAAGGCCCGGCGGCGGCGATTGGTTTAAGTCTGGTTATCGGTATCGTGATGGCGGAAGTATTACGCAAGCTGGGTGCAGATAAAGTTCGTGTTAAATGGCCTAATGACCTCTATCTGCAGGATCGCAAGCTGGCAGGCATTCTGGTGGAGCTGACTGGCAAAACTGGCGATGCGGCGCAAATAGTCATTGGAGCCGGGATCAACATGGCAATGCGCCGTGTTGAAGAGAGTGTCGTTAATCAGGGGTGGATCACGCTGCAGGAAGCGGGGATCAATCTCGATCGTAATACGTTGGCGGCCATGCTAATACGTGAATTACGTGCTGCGTTGGAACTCTTCGAACAAGAAGGATTGGCACCTTATCTGTCGCGCTGGGAAAAGCTGGATAATTTTATTAATCGCCCAGTGAAACTTATCATTGGTGATAAAGAAATATTTGGCATTTCACGCGGAATAGACAAACAGGGGGCTTTATTACTTGAGCAGGATGGAATAATAAAACCCTGGATGGGCGGTGAAATATCCCTGCGTAGTGCAGAAAAA-3’。
NLS-HA-I-sceI-BirA*融合表达序列(SEQ ID NO.4)：5’-ATGCCAAAAAAGAAGAGAAAGGTGCCGAAGAAGCATGCAGCACCACCAAAAAAAAAACGAAAAGTAGAAGACCCACGATTTATGTACCCATACGATGTTCCTGACTATGCGGGTATGAAAAACATCAAAAAAAACCAGGTAATGAACCTGGGTCCGAACTCTAAACTGCTGAAAGAATACAAATCCCAGCTGATCGAACTGAACATCGAACAGTTCGAAGCAGGTATCGGTCTGATCCTGGGTGATGCTTACATCCGTTCTCGTGATGAAGGTAAAACCTACTGTATGCAGTTCGAGTGGAAAAACAAAGCATACATGGACCACGTATGTCTGCTGTACGATCAGTGGGTACTGTCCCCGCCGCACAAAAAAGAACGTGTTAACCACCTGGGTAACCTGGTAATCACCTGGGGCGCCCAGACTTTCAAACACCAAGCTTTCAACAAACTGGCTAACCTGTTCATCGTTAACAACAAAAAAACCATCCCGAACAACCTGGTTGAAAACTACCTGACCCCGATGTCTCTGGCATACTGGTTCATGGATGATGGTGGTAAATGGGATTACAACAAAAACTCTACCAACAAATCGATCGTACTGAACACCCAGTCTTTCACTTTCGAAGAAGTAGAATACCTGGTTAAGGGTCTGCGTAACAAATTCCAACTGAACTGTTACGTAAAAATCAACAAAAACAAACCGATCATCTACATCGATTCTATGTCTTACCTGATCTTCTACAACCTGATCAAACCGTACCTGATCCCGCAGATGATGTACAAACTGCCGAACACTATCTCCTCCGAAACTTTCCTGAAAACGCGTGGCGGAGGAGGCTCCGGGGGAGGGGGAAGCATGAAGGATAACACCGTGCCACTGAAATTGATTGCCCTGTTAGCGAACGGTGAATTTCACTCTGGCGAGCAGTTGGGTGAAACGCTGGGAATGAGCCGGGCGGCTATTAATAAACACATTCAGACACTGCGTGACTGGGGCGTTGATGTCTTTACCGTTCCGGGTAAAGGATACAG CCTGCCTGAGCCTATCCAGTTACTTAATGCTAAACAGATATTGGGTCAGCTGGATGGCGGTAGTGTAGCCGTGCTGCCAGTGATTGACTCCACGAATCAGTACCTTCTTGATCGTATCGGAGAGCTTAAATCGGGCGATGCTTGCATTGCAGAATACCAGCAGGCTGGCCGTGGTGGCCGGGGTCGGAAATGGTTTTCGCCTTTTGGCGCAAACTTATATTTGTCGATGTTCTGGCGTCTGGAACAAGGCCCGGCGGCGGCGATTGGTTTAAGTCTGGTTATCGGTATCGTGATGGCGGAAGTATTACGCAAGCTGGGTGCAGATAAAGTTCGTGTTAAATGGCCTAATGACCTCTATCTGCAGGATCGCAAGCTGGCAGGCATTCTGGTGGAGCTGACTGGCAAAACTGGCGATGCGGCGCAAATAGTCATTGGAGCCGGGATCAACATGGCAATGCGCCGTGTTGAAGAGAGTGTCGTTAATCAGGGGTGGATCACGCTGCAGGAAGCGGGGATCAATCTCGATCGTAATACGTTGGCGGCCATGCTAATACGTGAATTACGTGCTGCGTTGGAACTCTTCGAACAAGAAGGATTGGCACCTTATCTGTCGCGCTGGGAAAAGCTGGATAATTTTATTAATCGCCCAGTGAAACTTATCATTGGTGATAAAGAAATATTTGGCATTTCACGCGGAATAGACAAACAGGGGGCTTTATTACTTGAGCAGGATGGAATAATAAAACCCTGGATGGGCGGTGAAATATCCCTGCGTAGTGCAGAAAAA-3’。
2、构建DNA同源重组修复报告模式细胞(DR-GFP U2OS)
采用pDRGFP质粒(购自Addgene公司，货号为26475)转染人骨肉瘤细胞(U2OS细胞)。取对数生长期的U2OS细胞铺板至6cm细胞培养皿，使细胞贴壁后的细胞密度约为70％-80％，进行细胞转染，取pDRGFP质粒2.5ug加入250ul Opti-MEM培养基中，取另一份250ulOpti-MEM培养基加入5ul的lipo2000转染试剂，再将二者轻轻混匀，静置10分钟后加入到6cm的U2OS细胞中，补新鲜完全培养基2ml，转染后6小时换新鲜完全培养基。48小时后，用含有10％胎牛血清及1ug/ml嘌呤霉素的DMEM培养基对U2OS细胞进行抗性筛选得到DNA同源重组修复报告模式细胞(DR-GFP U2OS)。pDRGFP质粒中带有经过修饰的GFP基因SceGFP，它包含一个I-SceI位点和框内终止密码子。归位内切酶I-sceI识别SceGFP中的I-SceI位点，诱导DNA双链断裂时，SceGFP可以内部iGFP片段作为模板，进行同源重组修复，并产生一个功能性GFP基因，表达完整GFP蛋白，报告绿色荧光。
3、构建I-SceI位点缺失型DR-GFP U2OS的细胞，并将其作为对照组，构建具体包括以
下步骤：
a.采用步骤(1)中的NLS-HA-I-sceI-BirA*融合表达质粒来转染步骤(2)中建立的DNA同源重组修复报告模式细胞(DR-GFP U2OS)，取对数生长期的DR-GFP U2OS细胞铺板至6cm细胞培养皿，使细胞贴壁后的细胞密度约70％-80％，进行细胞转染，取NLS-HA-I-sceI-BirA*融合表达质粒2.5ug加入250ul Opti-MEM培养基中，取另一份250ul Opti-MEM培养基加入5ul的lipo2000转染试剂，将二者轻轻混匀，静置10分钟后加入到6cm的U2OS细胞中，补新鲜完全培养基2ml，转染后6小时换新鲜完全培养基。48小时后，用含有10％胎牛血清及1ug/ml嘌呤霉素的DMEM培养基对U2OS细胞进行抗性筛选。融合蛋白可靶向切割模式细胞中的I-sceI位点，使细胞发生DNA双链断裂，由于细胞主要是通过非同源末端连接(Non-homologous end joining，NHEJ)方式进行修复，导致I-sceI位点突变或缺失。
b.嘌呤霉素筛选完成后，用胰酶消化并重悬DR-GFP U2OS细胞，细胞计数，再将200个DR-GFP U2OS细胞加入30ml含10％胎牛血清的DMEM培养基中，混匀后铺于96孔培养板中，每孔加入100ul细胞悬液，待孔中单克隆细胞长满后，进行扩大培养并进行流式细胞术鉴定。
c.DR-GFP U2OS细胞部分传代于12孔培养板内，细胞贴壁后，每孔转染2.5ugpCBASceI质粒(采购于Addgene公司)。取2.5ug pCBASceI质粒加入250ul Opti-MEM培养基中，取另一份250ul Opti-MEM培养基加入5ul的lipo2000转染试剂，将二者轻轻混匀，静置10分钟后加入到DR-GFP U2OS细胞中，补新鲜完全培养基500ul，转染后6小时换新鲜完全培养基。48小时后，细胞内表达I-SceI内切酶，用流式细胞仪进行检测。若细胞内I-sceI位点突变或缺失，I-SceI内切酶将无法识别并切割，细胞不报告绿色荧光，为阳性克隆，即I-SceI位点突变或缺失型DR-GFP U2OS细胞。同时对阳性克隆进行测序鉴定，确认I-SceI位点为突变或缺失型；
4、用步骤(1)构建的NLS-HA-I-sceI-BirA*融合表达质粒来转染步骤(2)或步骤(3)制得的DNA同源重组修复报告模式细胞或对照组细胞。取对数生长期的DR-GFP U2OS细胞或对照组细胞铺板至10cm细胞培养皿，使细胞贴壁后的细胞密度约为70％-80％，进行细胞转染，取NLS-HA-I-sceI-BirA*融合表达质粒8ug加入250ul Opti-MEM培养基中，取另一份250ul Opti-MEM培养基加入16ul的lipo2000转染试剂，将二者轻轻混匀，静置10分钟后加入到10cm的U2OS细胞中，补新鲜完全培养基6ml，转染8小时后更换新鲜含10％胎牛血清DMEM培养基并加入终浓度50uM的生物素，继续培养48小时。去除培养基，胰酶消化收集细胞并提取细胞总蛋白。
5、纯化鉴定
提取生物素化后的DNA同源重组修复报告模式细胞或对照组细胞的总蛋白，利用链霉亲和素磁珠(购自Invitrogen，货号为65305)纯化捕获生物素化的DNA双链断裂修复蛋白，最后对所捕获的蛋白通过定量化DIA质谱进行鉴定。
实验结果：
如图2A所示，通过流式细胞技术检测到DNA同源重组修复报告细胞发生同源重组，表达绿色荧光蛋白，细胞携带绿色荧光，证明了I-SceI-BirA*融合表达蛋白可切割模式细胞(DR-GFP U2OS)中的I-SceI位点，具有I-SceI内切酶活性，诱导细胞DNA发生双链断裂。
由图2B所示，I-SceI-BirA*融合表达蛋白在生物素存在的条件下，可促进近端蛋白生物素化并被链霉亲和素所捕获，这是因为生物素与DNA双链断裂修复蛋白结合形成生物素衍生物，而生物素衍生物和链霉亲和素之间具有极高的结合亲和力，从而通过链霉亲和素能有效的捕获生物化素的DNA双链断裂修复蛋白。
如图2C所示，NLS-HA-I-sceI-BirA*融合表达质粒可在DNA同源重组修复报告模式细胞(DR-GFP U2OS)或对照组细胞中成功表达。
链霉亲和素-生物素(Streptavidin-biotin)检测结果如图2D所示，NLS-HA-I-sceI-BirA*融合表达质粒转染对照组I-SceI位点缺失型DR-GFP U2OS的细胞所建立的系统不能有效捕获DNA损伤修复蛋白，而NLS-HA-I-sceI-BirA*融合表达质粒转染DR-GFP U2OS细胞所建立的系统可有效捕获p-Rad51、γH2AX、53BP1、ku80等经典的DNA损伤修复蛋白，说明本方法准确可靠。
如图3所示，本发明建立的系统能对DNA双链断裂动态修复过程中的修复蛋白进行捕获，可有效捕获PARP1、PRKDC、XRCC5、XRCC6、NONO、MSH2、TOP1等经典的DNA修复因子，同时蛋白分子网络互作分析显示，捕获的蛋白分子主要富集于DNA修复通路与DNA复制通路。
综上所述，本发明所建立的系统可有效捕获DNA双链断裂修复蛋白，而且，链霉亲和素可以捕获多种参与DNA双链断裂修复的蛋白，捕获通量高，能用于表征肿瘤细胞DNA损伤反应网络。
以上所述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，这些改进和润饰也视为本发明的保护范围。
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<110> 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所)
<120> 一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法
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35 40 45
Gly Gly Thr Gly Ala Ala Thr Thr Thr Cys Ala Cys Thr Cys Thr Gly
50 55 60
Gly Cys Gly Ala Gly Cys Ala Gly Thr Thr Gly Gly Gly Thr Gly Ala
65 70 75 80
Ala Ala Cys Gly Cys Thr Gly Gly Gly Ala Ala Thr Gly Ala Gly Cys
85 90 95
Cys Gly Gly Gly Cys Gly Gly Cys Thr Ala Thr Thr Ala Ala Thr Ala
100 105 110
Ala Ala Cys Ala Cys Ala Thr Thr Cys Ala Gly Ala Cys Ala Cys Thr
115 120 125
Gly Cys Gly Thr Gly Ala Cys Thr Gly Gly Gly Gly Cys Gly Thr Thr
130 135 140
Gly Ala Thr Gly Thr Cys Thr Thr Thr Ala Cys Cys Gly Thr Thr Cys
145 150 155 160
Cys Gly Gly Gly Thr Ala Ala Ala Gly Gly Ala Thr Ala Cys Ala Gly
165 170 175
Cys Cys Thr Gly Cys Cys Thr Gly Ala Gly Cys Cys Thr Ala Thr Cys
180 185 190
Cys Ala Gly Thr Thr Ala Cys Thr Thr Ala Ala Thr Gly Cys Thr Ala
195 200 205
Ala Ala Cys Ala Gly Ala Thr Ala Thr Thr Gly Gly Gly Thr Cys Ala
210 215 220
Gly Cys Thr Gly Gly Ala Thr Gly Gly Cys Gly Gly Thr Ala Gly Thr
225 230 235 240
Gly Thr Ala Gly Cys Cys Gly Thr Gly Cys Thr Gly Cys Cys Ala Gly
245 250 255
Thr Gly Ala Thr Thr Gly Ala Cys Thr Cys Cys Ala Cys Gly Ala Ala
260 265 270
Thr Cys Ala Gly Thr Ala Cys Cys Thr Thr Cys Thr Thr Gly Ala Thr
275 280 285
Cys Gly Thr Ala Thr Cys Gly Gly Ala Gly Ala Gly Cys Thr Thr Ala
290 295 300
Ala Ala Thr Cys Gly Gly Gly Cys Gly Ala Thr Gly Cys Thr Thr Gly
305 310 315 320
Cys Ala Thr Thr Gly Cys Ala Gly Ala Ala Thr Ala Cys Cys Ala Gly
325 330 335
Cys Ala Gly Gly Cys Thr Gly Gly Cys Cys Gly Thr Gly Gly Thr Gly
340 345 350
Gly Cys Cys Gly Gly Gly Gly Thr Cys Gly Gly Ala Ala Ala Thr Gly
355 360 365
Gly Thr Thr Thr Thr Cys Gly Cys Cys Thr Thr Thr Thr Gly Gly Cys
370 375 380
Gly Cys Ala Ala Ala Cys Thr Thr Ala Thr Ala Thr Thr Thr Gly Thr
385 390 395 400
Cys Gly Ala Thr Gly Thr Thr Cys Thr Gly Gly Cys Gly Thr Cys Thr
405 410 415
Gly Gly Ala Ala Cys Ala Ala Gly Gly Cys Cys Cys Gly Gly Cys Gly
420 425 430
Gly Cys Gly Gly Cys Gly Ala Thr Thr Gly Gly Thr Thr Thr Ala Ala
435 440 445
Gly Thr Cys Thr Gly Gly Thr Thr Ala Thr Cys Gly Gly Thr Ala Thr
450 455 460
Cys Gly Thr Gly Ala Thr Gly Gly Cys Gly Gly Ala Ala Gly Thr Ala
465 470 475 480
Thr Thr Ala Cys Gly Cys Ala Ala Gly Cys Thr Gly Gly Gly Thr Gly
485 490 495
Cys Ala Gly Ala Thr Ala Ala Ala Gly Thr Thr Cys Gly Thr Gly Thr
500 505 510
Thr Ala Ala Ala Thr Gly Gly Cys Cys Thr Ala Ala Thr Gly Ala Cys
515 520 525
Cys Thr Cys Thr Ala Thr Cys Thr Gly Cys Ala Gly Gly Ala Thr Cys
530 535 540
Gly Cys Ala Ala Gly Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr
545 550 555 560
Thr Cys Thr Gly Gly Thr Gly Gly Ala Gly Cys Thr Gly Ala Cys Thr
565 570 575
Gly Gly Cys Ala Ala Ala Ala Cys Thr Gly Gly Cys Gly Ala Thr Gly
580 585 590
Cys Gly Gly Cys Gly Cys Ala Ala Ala Thr Ala Gly Thr Cys Ala Thr
595 600 605
Thr Gly Gly Ala Gly Cys Cys Gly Gly Gly Ala Thr Cys Ala Ala Cys
610 615 620
Ala Thr Gly Gly Cys Ala Ala Thr Gly Cys Gly Cys Cys Gly Thr Gly
625 630 635 640
Thr Thr Gly Ala Ala Gly Ala Gly Ala Gly Thr Gly Thr Cys Gly Thr
645 650 655
Thr Ala Ala Thr Cys Ala Gly Gly Gly Gly Thr Gly Gly Ala Thr Cys
660 665 670
Ala Cys Gly Cys Thr Gly Cys Ala Gly Gly Ala Ala Gly Cys Gly Gly
675 680 685
Gly Gly Ala Thr Cys Ala Ala Thr Cys Thr Cys Gly Ala Thr Cys Gly
690 695 700
Thr Ala Ala Thr Ala Cys Gly Thr Thr Gly Gly Cys Gly Gly Cys Cys
705 710 715 720
Ala Thr Gly Cys Thr Ala Ala Thr Ala Cys Gly Thr Gly Ala Ala Thr
725 730 735
Thr Ala Cys Gly Thr Gly Cys Thr Gly Cys Gly Thr Thr Gly Gly Ala
740 745 750
Ala Cys Thr Cys Thr Thr Cys Gly Ala Ala Cys Ala Ala Gly Ala Ala
755 760 765
Gly Gly Ala Thr Thr Gly Gly Cys Ala Cys Cys Thr Thr Ala Thr Cys
770 775 780
Thr Gly Thr Cys Gly Cys Gly Cys Thr Gly Gly Gly Ala Ala Ala Ala
785 790 795 800
Gly Cys Thr Gly Gly Ala Thr Ala Ala Thr Thr Thr Thr Ala Thr Thr
805 810 815
Ala Ala Thr Cys Gly Cys Cys Cys Ala Gly Thr Gly Ala Ala Ala Cys
820 825 830
Thr Thr Ala Thr Cys Ala Thr Thr Gly Gly Thr Gly Ala Thr Ala Ala
835 840 845
Ala Gly Ala Ala Ala Thr Ala Thr Thr Thr Gly Gly Cys Ala Thr Thr
850 855 860
Thr Cys Ala Cys Gly Cys Gly Gly Ala Ala Thr Ala Gly Ala Cys Ala
865 870 875 880
Ala Ala Cys Ala Gly Gly Gly Gly Gly Cys Thr Thr Thr Ala Thr Thr
885 890 895
Ala Cys Thr Thr Gly Ala Gly Cys Ala Gly Gly Ala Thr Gly Gly Ala
900 905 910
Ala Thr Ala Ala Thr Ala Ala Ala Ala Cys Cys Cys Thr Gly Gly Ala
915 920 925
Thr Gly Gly Gly Cys Gly Gly Thr Gly Ala Ala Ala Thr Ala Thr Cys
930 935 940
Cys Cys Thr Gly Cys Gly Thr Ala Gly Thr Gly Cys Ala Gly Ala Ala
945 950 955 960
Ala Ala Ala
<210> 4
<211> 1818
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Thr Gly Cys Cys Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Ala
1 5 10 15
Gly Ala Ala Ala Gly Gly Thr Gly Cys Cys Gly Ala Ala Gly Ala Ala
20 25 30
Gly Cys Ala Thr Gly Cys Ala Gly Cys Ala Cys Cys Ala Cys Cys Ala
35 40 45
Ala Ala Ala Ala Ala Ala Ala Ala Ala Cys Gly Ala Ala Ala Ala Gly
50 55 60
Thr Ala Gly Ala Ala Gly Ala Cys Cys Cys Ala Cys Gly Ala Thr Thr
65 70 75 80
Thr Ala Thr Gly Thr Ala Cys Cys Cys Ala Thr Ala Cys Gly Ala Thr
85 90 95
Gly Thr Thr Cys Cys Thr Gly Ala Cys Thr Ala Thr Gly Cys Gly Gly
100 105 110
Gly Thr Ala Thr Gly Ala Ala Ala Ala Ala Cys Ala Thr Cys Ala Ala
115 120 125
Ala Ala Ala Ala Ala Ala Cys Cys Ala Gly Gly Thr Ala Ala Thr Gly
130 135 140
Ala Ala Cys Cys Thr Gly Gly Gly Thr Cys Cys Gly Ala Ala Cys Thr
145 150 155 160
Cys Thr Ala Ala Ala Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Ala
165 170 175
Ala Thr Ala Cys Ala Ala Ala Thr Cys Cys Cys Ala Gly Cys Thr Gly
180 185 190
Ala Thr Cys Gly Ala Ala Cys Thr Gly Ala Ala Cys Ala Thr Cys Gly
195 200 205
Ala Ala Cys Ala Gly Thr Thr Cys Gly Ala Ala Gly Cys Ala Gly Gly
210 215 220
Thr Ala Thr Cys Gly Gly Thr Cys Thr Gly Ala Thr Cys Cys Thr Gly
225 230 235 240
Gly Gly Thr Gly Ala Thr Gly Cys Thr Thr Ala Cys Ala Thr Cys Cys
245 250 255
Gly Thr Thr Cys Thr Cys Gly Thr Gly Ala Thr Gly Ala Ala Gly Gly
260 265 270
Thr Ala Ala Ala Ala Cys Cys Thr Ala Cys Thr Gly Thr Ala Thr Gly
275 280 285
Cys Ala Gly Thr Thr Cys Gly Ala Gly Thr Gly Gly Ala Ala Ala Ala
290 295 300
Ala Cys Ala Ala Ala Gly Cys Ala Thr Ala Cys Ala Thr Gly Gly Ala
305 310 315 320
Cys Cys Ala Cys Gly Thr Ala Thr Gly Thr Cys Thr Gly Cys Thr Gly
325 330 335
Thr Ala Cys Gly Ala Thr Cys Ala Gly Thr Gly Gly Gly Thr Ala Cys
340 345 350
Thr Gly Thr Cys Cys Cys Cys Gly Cys Cys Gly Cys Ala Cys Ala Ala
355 360 365
Ala Ala Ala Ala Gly Ala Ala Cys Gly Thr Gly Thr Thr Ala Ala Cys
370 375 380
Cys Ala Cys Cys Thr Gly Gly Gly Thr Ala Ala Cys Cys Thr Gly Gly
385 390 395 400
Thr Ala Ala Thr Cys Ala Cys Cys Thr Gly Gly Gly Gly Cys Gly Cys
405 410 415
Cys Cys Ala Gly Ala Cys Thr Thr Thr Cys Ala Ala Ala Cys Ala Cys
420 425 430
Cys Ala Ala Gly Cys Thr Thr Thr Cys Ala Ala Cys Ala Ala Ala Cys
435 440 445
Thr Gly Gly Cys Thr Ala Ala Cys Cys Thr Gly Thr Thr Cys Ala Thr
450 455 460
Cys Gly Thr Thr Ala Ala Cys Ala Ala Cys Ala Ala Ala Ala Ala Ala
465 470 475 480
Ala Cys Cys Ala Thr Cys Cys Cys Gly Ala Ala Cys Ala Ala Cys Cys
485 490 495
Thr Gly Gly Thr Thr Gly Ala Ala Ala Ala Cys Thr Ala Cys Cys Thr
500 505 510
Gly Ala Cys Cys Cys Cys Gly Ala Thr Gly Thr Cys Thr Cys Thr Gly
515 520 525
Gly Cys Ala Thr Ala Cys Thr Gly Gly Thr Thr Cys Ala Thr Gly Gly
530 535 540
Ala Thr Gly Ala Thr Gly Gly Thr Gly Gly Thr Ala Ala Ala Thr Gly
545 550 555 560
Gly Gly Ala Thr Thr Ala Cys Ala Ala Cys Ala Ala Ala Ala Ala Cys
565 570 575
Thr Cys Thr Ala Cys Cys Ala Ala Cys Ala Ala Ala Thr Cys Gly Ala
580 585 590
Thr Cys Gly Thr Ala Cys Thr Gly Ala Ala Cys Ala Cys Cys Cys Ala
595 600 605
Gly Thr Cys Thr Thr Thr Cys Ala Cys Thr Thr Thr Cys Gly Ala Ala
610 615 620
Gly Ala Ala Gly Thr Ala Gly Ala Ala Thr Ala Cys Cys Thr Gly Gly
625 630 635 640
Thr Thr Ala Ala Gly Gly Gly Thr Cys Thr Gly Cys Gly Thr Ala Ala
645 650 655
Cys Ala Ala Ala Thr Thr Cys Cys Ala Ala Cys Thr Gly Ala Ala Cys
660 665 670
Thr Gly Thr Thr Ala Cys Gly Thr Ala Ala Ala Ala Ala Thr Cys Ala
675 680 685
Ala Cys Ala Ala Ala Ala Ala Cys Ala Ala Ala Cys Cys Gly Ala Thr
690 695 700
Cys Ala Thr Cys Thr Ala Cys Ala Thr Cys Gly Ala Thr Thr Cys Thr
705 710 715 720
Ala Thr Gly Thr Cys Thr Thr Ala Cys Cys Thr Gly Ala Thr Cys Thr
725 730 735
Thr Cys Thr Ala Cys Ala Ala Cys Cys Thr Gly Ala Thr Cys Ala Ala
740 745 750
Ala Cys Cys Gly Thr Ala Cys Cys Thr Gly Ala Thr Cys Cys Cys Gly
755 760 765
Cys Ala Gly Ala Thr Gly Ala Thr Gly Thr Ala Cys Ala Ala Ala Cys
770 775 780
Thr Gly Cys Cys Gly Ala Ala Cys Ala Cys Thr Ala Thr Cys Thr Cys
785 790 795 800
Cys Thr Cys Cys Gly Ala Ala Ala Cys Thr Thr Thr Cys Cys Thr Gly
805 810 815
Ala Ala Ala Ala Cys Gly Cys Gly Thr Gly Gly Cys Gly Gly Ala Gly
820 825 830
Gly Ala Gly Gly Cys Thr Cys Cys Gly Gly Gly Gly Gly Ala Gly Gly
835 840 845
Gly Gly Gly Ala Ala Gly Cys Ala Thr Gly Ala Ala Gly Gly Ala Thr
850 855 860
Ala Ala Cys Ala Cys Cys Gly Thr Gly Cys Cys Ala Cys Thr Gly Ala
865 870 875 880
Ala Ala Thr Thr Gly Ala Thr Thr Gly Cys Cys Cys Thr Gly Thr Thr
885 890 895
Ala Gly Cys Gly Ala Ala Cys Gly Gly Thr Gly Ala Ala Thr Thr Thr
900 905 910
Cys Ala Cys Thr Cys Thr Gly Gly Cys Gly Ala Gly Cys Ala Gly Thr
915 920 925
Thr Gly Gly Gly Thr Gly Ala Ala Ala Cys Gly Cys Thr Gly Gly Gly
930 935 940
Ala Ala Thr Gly Ala Gly Cys Cys Gly Gly Gly Cys Gly Gly Cys Thr
945 950 955 960
Ala Thr Thr Ala Ala Thr Ala Ala Ala Cys Ala Cys Ala Thr Thr Cys
965 970 975
Ala Gly Ala Cys Ala Cys Thr Gly Cys Gly Thr Gly Ala Cys Thr Gly
980 985 990
Gly Gly Gly Cys Gly Thr Thr Gly Ala Thr Gly Thr Cys Thr Thr Thr
995 1000 1005
Ala Cys Cys Gly Thr Thr Cys Cys Gly Gly Gly Thr Ala Ala Ala Gly
1010 1015 1020
Gly Ala Thr Ala Cys Ala Gly Cys Cys Thr Gly Cys Cys Thr Gly Ala
1025 1030 1035 1040
Gly Cys Cys Thr Ala Thr Cys Cys Ala Gly Thr Thr Ala Cys Thr Thr
1045 1050 1055
Ala Ala Thr Gly Cys Thr Ala Ala Ala Cys Ala Gly Ala Thr Ala Thr
1060 1065 1070
Thr Gly Gly Gly Thr Cys Ala Gly Cys Thr Gly Gly Ala Thr Gly Gly
1075 1080 1085
Cys Gly Gly Thr Ala Gly Thr Gly Thr Ala Gly Cys Cys Gly Thr Gly
1090 1095 1100
Cys Thr Gly Cys Cys Ala Gly Thr Gly Ala Thr Thr Gly Ala Cys Thr
1105 1110 1115 1120
Cys Cys Ala Cys Gly Ala Ala Thr Cys Ala Gly Thr Ala Cys Cys Thr
1125 1130 1135
Thr Cys Thr Thr Gly Ala Thr Cys Gly Thr Ala Thr Cys Gly Gly Ala
1140 1145 1150
Gly Ala Gly Cys Thr Thr Ala Ala Ala Thr Cys Gly Gly Gly Cys Gly
1155 1160 1165
Ala Thr Gly Cys Thr Thr Gly Cys Ala Thr Thr Gly Cys Ala Gly Ala
1170 1175 1180
Ala Thr Ala Cys Cys Ala Gly Cys Ala Gly Gly Cys Thr Gly Gly Cys
1185 1190 1195 1200
Cys Gly Thr Gly Gly Thr Gly Gly Cys Cys Gly Gly Gly Gly Thr Cys
1205 1210 1215
Gly Gly Ala Ala Ala Thr Gly Gly Thr Thr Thr Thr Cys Gly Cys Cys
1220 1225 1230
Thr Thr Thr Thr Gly Gly Cys Gly Cys Ala Ala Ala Cys Thr Thr Ala
1235 1240 1245
Thr Ala Thr Thr Thr Gly Thr Cys Gly Ala Thr Gly Thr Thr Cys Thr
1250 1255 1260
Gly Gly Cys Gly Thr Cys Thr Gly Gly Ala Ala Cys Ala Ala Gly Gly
1265 1270 1275 1280
Cys Cys Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Gly Ala Thr Thr
1285 1290 1295
Gly Gly Thr Thr Thr Ala Ala Gly Thr Cys Thr Gly Gly Thr Thr Ala
1300 1305 1310
Thr Cys Gly Gly Thr Ala Thr Cys Gly Thr Gly Ala Thr Gly Gly Cys
1315 1320 1325
Gly Gly Ala Ala Gly Thr Ala Thr Thr Ala Cys Gly Cys Ala Ala Gly
1330 1335 1340
Cys Thr Gly Gly Gly Thr Gly Cys Ala Gly Ala Thr Ala Ala Ala Gly
1345 1350 1355 1360
Thr Thr Cys Gly Thr Gly Thr Thr Ala Ala Ala Thr Gly Gly Cys Cys
1365 1370 1375
Thr Ala Ala Thr Gly Ala Cys Cys Thr Cys Thr Ala Thr Cys Thr Gly
1380 1385 1390
Cys Ala Gly Gly Ala Thr Cys Gly Cys Ala Ala Gly Cys Thr Gly Gly
1395 1400 1405
Cys Ala Gly Gly Cys Ala Thr Thr Cys Thr Gly Gly Thr Gly Gly Ala
1410 1415 1420
Gly Cys Thr Gly Ala Cys Thr Gly Gly Cys Ala Ala Ala Ala Cys Thr
1425 1430 1435 1440
Gly Gly Cys Gly Ala Thr Gly Cys Gly Gly Cys Gly Cys Ala Ala Ala
1445 1450 1455
Thr Ala Gly Thr Cys Ala Thr Thr Gly Gly Ala Gly Cys Cys Gly Gly
1460 1465 1470
Gly Ala Thr Cys Ala Ala Cys Ala Thr Gly Gly Cys Ala Ala Thr Gly
1475 1480 1485
Cys Gly Cys Cys Gly Thr Gly Thr Thr Gly Ala Ala Gly Ala Gly Ala
1490 1495 1500
Gly Thr Gly Thr Cys Gly Thr Thr Ala Ala Thr Cys Ala Gly Gly Gly
1505 1510 1515 1520
Gly Thr Gly Gly Ala Thr Cys Ala Cys Gly Cys Thr Gly Cys Ala Gly
1525 1530 1535
Gly Ala Ala Gly Cys Gly Gly Gly Gly Ala Thr Cys Ala Ala Thr Cys
1540 1545 1550
Thr Cys Gly Ala Thr Cys Gly Thr Ala Ala Thr Ala Cys Gly Thr Thr
1555 1560 1565
Gly Gly Cys Gly Gly Cys Cys Ala Thr Gly Cys Thr Ala Ala Thr Ala
1570 1575 1580
Cys Gly Thr Gly Ala Ala Thr Thr Ala Cys Gly Thr Gly Cys Thr Gly
1585 1590 1595 1600
Cys Gly Thr Thr Gly Gly Ala Ala Cys Thr Cys Thr Thr Cys Gly Ala
1605 1610 1615
Ala Cys Ala Ala Gly Ala Ala Gly Gly Ala Thr Thr Gly Gly Cys Ala
1620 1625 1630
Cys Cys Thr Thr Ala Thr Cys Thr Gly Thr Cys Gly Cys Gly Cys Thr
1635 1640 1645
Gly Gly Gly Ala Ala Ala Ala Gly Cys Thr Gly Gly Ala Thr Ala Ala
1650 1655 1660
Thr Thr Thr Thr Ala Thr Thr Ala Ala Thr Cys Gly Cys Cys Cys Ala
1665 1670 1675 1680
Gly Thr Gly Ala Ala Ala Cys Thr Thr Ala Thr Cys Ala Thr Thr Gly
1685 1690 1695
Gly Thr Gly Ala Thr Ala Ala Ala Gly Ala Ala Ala Thr Ala Thr Thr
1700 1705 1710
Thr Gly Gly Cys Ala Thr Thr Thr Cys Ala Cys Gly Cys Gly Gly Ala
1715 1720 1725
Ala Thr Ala Gly Ala Cys Ala Ala Ala Cys Ala Gly Gly Gly Gly Gly
1730 1735 1740
Cys Thr Thr Thr Ala Thr Thr Ala Cys Thr Thr Gly Ala Gly Cys Ala
1745 1750 1755 1760
Gly Gly Ala Thr Gly Gly Ala Ala Thr Ala Ala Thr Ala Ala Ala Ala
1765 1770 1775
Cys Cys Cys Thr Gly Gly Ala Thr Gly Gly Gly Cys Gly Gly Thr Gly
1780 1785 1790
Ala Ala Ala Thr Ala Thr Cys Cys Cys Thr Gly Cys Gly Thr Ala Gly
1795 1800 1805
Thr Gly Cys Ala Gly Ala Ala Ala Ala Ala
1810 1815
<210> 5
<211> 81
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Thr Gly Cys Cys Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Ala
1 5 10 15
Gly Ala Ala Ala Gly Gly Thr Gly Cys Cys Gly Ala Ala Gly Ala Ala
20 25 30
Gly Cys Ala Thr Gly Cys Ala Gly Cys Ala Cys Cys Ala Cys Cys Ala
35 40 45
Ala Ala Ala Ala Ala Ala Ala Ala Ala Cys Gly Ala Ala Ala Ala Gly
50 55 60
Thr Ala Gly Ala Ala Gly Ala Cys Cys Cys Ala Cys Gly Ala Thr Thr
65 70 75 80
Thr
<210> 6
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ala Thr Gly Thr Ala Cys Cys Cys Ala Thr Ala Cys Gly Ala Thr Gly
1 5 10 15
Thr Thr Cys Cys Thr Gly Ala Cys Thr Ala Thr Gly Cys Gly
20 25 30
Claims (5)
1.一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，其特征在于，所述方法包括：
步骤1：将5’端带有核定位序列及HA序列的归位内切酶I-SceI表达序列通过如SEQ IDNO.1所示的连接序列与生物素连接酶BirA*表达序列连接得到融合表达序列，再将融合表达序列构入哺乳动物过表达质粒中得到I-SceI-BirA*融合表达质粒，所述融合表达序列是如SEQ ID NO.4所示的序列；所述5’端带有核定位序列及HA序列的归位内切酶I-SceI表达序列是如SEQ ID NO.2所示的序列，生物素连接酶BirA*表达序列是如SEQ ID NO.3所示的序列，所述核定位序列是如SEQ ID NO.5所示的序列，HA序列是如SEQ ID NO.6所示的序列；所述生物素连接酶BirA*表达序列的第352位碱基由胞嘧啶C突变为鸟嘌呤G，突变后的蛋白质序列第118位氨基酸由精氨酸突变为甘氨酸；
步骤2：采用pDRGFP质粒转染肿瘤细胞后用嘌呤霉素来筛选阳性细胞，构建DNA同源重组修复报告模式细胞；所述DNA同源重组修复报告模式细胞带有经过修饰的GFP基因SceGFP，其包含一个I-SceI位点和框内终止密码子；
步骤3：将步骤1构建的I-SceI-BirA*融合表达质粒转染步骤2获得的DNA同源重组修复报告模式细胞，转染后再加入生物素进行培养；
步聚4：提取步骤3模式细胞总蛋白，利用链霉亲和素磁珠纯化捕获生物素化的DNA双链断裂修复蛋白，最后进行质谱鉴定。
2.根据权利要求1所述一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，其特征在于，所述核定位序列与HA序列相连，核定位序列位于融合表达序列5’最前端。
3.根据权利要求1所述一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，其特征在于，步骤1中所述哺乳动物过表达质粒为pCDNA3.1质粒。
4.根据权利要求1所述一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法，其特征在于，所述步骤2中的肿瘤细胞为人骨肉瘤细胞。
5.根据权利要求1-4任一项所述一种高亲和力动态捕获DNA双链断裂修复相关蛋白的方法在捕获DNA双链断裂修复蛋白中的应用。
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