CN111617261B - 靶向cd45的抗体药物偶联物及其制备和应用 - Google Patents

靶向cd45的抗体药物偶联物及其制备和应用 Download PDF

Info

Publication number
CN111617261B
CN111617261B CN202010438614.3A CN202010438614A CN111617261B CN 111617261 B CN111617261 B CN 111617261B CN 202010438614 A CN202010438614 A CN 202010438614A CN 111617261 B CN111617261 B CN 111617261B
Authority
CN
China
Prior art keywords
seq
dta
sequence shown
vector
furin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010438614.3A
Other languages
English (en)
Other versions
CN111617261A (zh
Inventor
盛剑鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qianyuan Kangan Suzhou Biotechnology Co ltd
Original Assignee
Qianyuan Kangan Suzhou Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qianyuan Kangan Suzhou Biotechnology Co ltd filed Critical Qianyuan Kangan Suzhou Biotechnology Co ltd
Priority to CN202010438614.3A priority Critical patent/CN111617261B/zh
Publication of CN111617261A publication Critical patent/CN111617261A/zh
Application granted granted Critical
Publication of CN111617261B publication Critical patent/CN111617261B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

本发明公开了一种靶向CD45的抗体药物偶联物及其制备和应用方法，通过将CD45抗体和药物偶联，通过靶向CD45用于高度选择性的清除正常、病变、癌变的免疫细胞，本发明所述的靶向CD45抗体药物偶联物可用于清除病变或癌变免疫细胞，代替化疗药物，治疗血液系统疾病。本发明所述的靶向CD45抗体药物偶联物只针对免疫细胞，不会杀伤其他活跃分裂细胞，靶向清除免疫系统能够避免移植物抗宿主病(GVHD)，因此可同时移植大量的赠体成熟免疫细胞，快速建立免疫系统。

Description

靶向CD45的抗体药物偶联物及其制备和应用
技术领域
本发明属于生物技术领域，具体涉及靶向CD45抗体药物偶联物的构建与应用。
技术背景
血液病包括原发于血液系统的疾病和继发于其他疾病所致血液系统异常的疾病，在医学上血液病也是一种很“庞大”的疾病，它的病种很多，分类很广，主要分为四个大类：造血干细胞疾病、红细胞疾病、粒细胞疾病与出血性疾病,其中前三者可以通过骨髓移植来治疗。
骨髓移植分为两类：一类为异基因骨髓移植，一类为自体骨髓移植。异基因与自体骨髓移植各有优缺点。自体骨髓移植的最大缺点为复发率高，原因是自体骨髓中的基因缺陷并未得到修复。异基因骨髓移植需有与患者人类白细胞抗原(HLA)相匹配的父母、子女、同胞以及极少数的无亲缘关系的供髓者所输入的异体骨髓。异基因骨髓移植受到诸多因素及时间影响，其成败的关键之一是HLA(人类白细胞抗原)配型问题，如果骨髓供者与患者(受者)的HLA不同，易发生轻重不等的移植物抗宿主病(GVHD)，甚至危及患者的生命。
上述自体和异体骨髓移植方面的缺陷大大影响了血液系统疾病的治愈率。
发明内容
抗体偶联药物(antibody-drug conjugate，ADC)是将单克隆抗体药物的高特异性和细胞毒药的高活性相结合，用以提高药物的靶向性、减少毒副作用。和传统的完全或部分人源化抗体或抗体片段相比，ADC因为能在肿瘤组织内释放高活性的细胞毒素，而不依赖抗体对靶点蛋白功能的阻断功能。而CD45是一种主要表达于免疫细胞表面的信号分子，其广泛表达于T细胞、B细胞、粒细胞等免疫细胞，因此CD45靶向免疫细胞的靶点。
本发明旨在提供一种针对免疫细胞的抗体药物偶联物，通过靶向CD45实现，用于清除正常、病变、癌变的免疫细胞，与自体、异体骨髓移植联用，治疗血液系统疾病、免疫系统疾病，包括但不限于艾滋病。
为了达到上述目的，本发明提供的技术方案为：
靶向CD45的抗体药物偶联物，由靶向部分与药物部分组成；所述靶向部分由单抗或多抗构成，靶向CD45；所述药物部分包括但不限于小分子药物、大分子毒素、生物活性肽、细胞裂解免疫活性蛋白、酶或者放射性核素中的一种或者多种。
进一步的，上述靶向CD45的抗体药物偶联物，所述小分子药物包括Maytansine、Dolastatin、Cemadotin Tasidotin、Cryptophycin、Eribulin meslyat、(+)-CC-1065、Adozelesin、Carzelesin、Bizelesin、KW-2189、SJG-136、vedotin、emtansine、ozogamicin、auristatins,taxol derivatives中的一种或者多种；所述大分子毒素包括白喉毒素、假单胞菌外毒素、绿脓杆菌毒素、绿脓杆菌外毒素、菌外毒素中的一种或者多种；所述生物活性肽包括GLP-1；所述细胞裂解免疫活性蛋白包括Fas ligands。
进一步的，上述靶向CD45的抗体药物偶联物，所述靶向部分为CD45单抗，所述药物部分为白喉毒素，所述CD45单抗与所述白喉毒素通过包含Furin蛋白剪切酶识别位点序列的氨基酸片段连接。
进一步的，上述靶向CD45的抗体药物偶联物，所述抗体药物偶联物为CD45 Ab-DTA重组蛋白，其氨基酸序列如SEQ ID.3所示。
进一步的，包括上述靶向CD45的抗体药物偶联物的药物组合物。
进一步的，上述靶向CD45的抗体药物偶联物，所述生产载体包括编码以下部分氨基酸的核酸序列：靶向CD45抗体轻链、靶向CD45抗体重链、白喉毒素、白喉毒素与重链之间由Furin蛋白剪切酶识别位点的氨基酸以及6 X His。
进一步的，上述靶向CD45的抗体药物偶联物，所述生产载体为pTrioz-CD45 Ab-Furin-DTA-6 X His，其序列为SEQ ID NO.2。
进一步的，上述生产载体为pTrioz-CD45 Ab-Furin-DTA-6 X His的构建方法，包括以下步骤:
(1)以pTrioz空白质粒为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR，扩增出序列如SEQ ID NO.6所示的基因片段IgHC；
(2)以pIRES-proHB EGF WT质粒为模板，利用序列如SEQ ID NO.7所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行PCR，扩增出序列如SEQ ID NO.9所示的基因片段DTA；
(3)以步骤2和3得到的基因片段IgHC与DTA为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行Overlap PCR得到序列如SEQ IDNO.10所示的融合基因片段IgHC-Furin-DTA-6Xhis；
(4)将目的基因片段IgHC-Furin-DTA-6XHis利用酶切位点NheI、AvrII克隆至载体pTrioz，得到载体pTrioz-Furin-DTA-6 X His，如SEQ ID NO.1所示；
(5)合成如SEQ ID NO.11所示序列CD45 Ab轻链；以合成产物为模板，利用序列如SEQ ID NO.12所示的正向引物和序列如SEQ ID NO.13所示的反向引物进行PCR，扩增出序列如SEQ ID NO.14所示的基因片段CD45 Ab L；将PCR产物利用酶切位点NcoI、BsiWI克隆至载体ppTrioz-Furin-DTA-6 X His，得到载体pTrioz-L-Furin-DTA-6 X His,如SEQ IDNO.15所示。
(6)合成如SEQ ID NO.16所示序列CD45 Ab重链；以合成产物为模板，利用序列如SEQ ID NO.17所示的正向引物和序列如SEQ ID NO.18所示的反向引物进行PCR，扩增出序列如SEQ ID NO.19所示的基因片段CD45 Ab H；将PCR产物利用酶切位点NheI、EcoRV克隆至载体ppTrioz-L-Furin-DTA-6 X His，得到生产载体pTrioz-CD45 Ab-Furin-DTA-6 X His,如SEQ ID NO.2所示。
进一步的，使用上述生产载体生产CD45 Ab-DTA重组蛋白的方法，包括以下步骤：
(1)培养TR细胞(所述TR细胞的构建见实施例8)；
(2)取pTrioz-CD45 Ab-Furin-DTA-6 X His生产载体转染至TR细胞；
(3)培养转染后的TR细胞并收取CD45 Ab DTA重组蛋白
进一步的，上述CD45 Ab DTA重组蛋白的纯化方法，包括以下步骤:
(1)使用Ni-NTA Superflow Resin纯化CD45 Ab DTA；
(2)使用NanoDrop测定CD45 Ab DTA重组蛋白浓度，并调整至2mg/Ml。
进一步的，上述CD45 Ab DTA重组蛋白清楚免疫细胞的测试方法包括如下步骤:
(1)在6周大C57小鼠注射CD45 Ab DTA 200ug，一周三次，持续一周
(2)用流式细胞仪检测免疫细胞清除程度
进一步的，上述靶向CD45的抗体药物偶联物在制备用于清除正常、病变、癌变的免疫细胞方面的药物、制备治疗血液系统疾病方面的药物、制备辅助自体、异体骨髓移植方面的药物、制备治疗免疫系统疾病方面的药物中的应用；所述免疫系统疾病包括但不限于艾滋病
综合上述方案，本发明提供了靶向CD45的抗体药物偶联物及其制备和应用，至少有以下方面的有益效果：本发明所述的靶向CD45抗体药物偶联物可用于清除病变或癌变免疫细胞，代替化疗药物，治疗血液系统疾病；与传统骨髓移植相比，具有四大优势：
(1)无需化疗、放疗即可清除受体原有免疫细胞，副作用低，减轻病人负担；
(2)靶向清除免疫系统能够避免移植物抗宿主病(GVHD),无需HLA配对，使得骨髓赠体的范围大大增加
(3)治疗白血病或淋巴瘤时，非HLA配对的异基因骨髓移植可起到杀伤癌细胞作用
(4)肠道上皮细胞形成的物理屏障被破坏，肠道菌群转移，造成感染；以及赠体骨髓重新建立免疫细胞的空窗期时微生物入侵造成的感染。靶向CD45抗体药物偶联物只针对免疫细胞，不会杀伤其他活跃分裂细胞。靶向清除免疫系统能够避免移植物抗宿主病(GVHD)，因此可同时移植大量的赠体成熟免疫细胞，快速建立免疫系统。从以上两个方面，靶向清除免疫系统避免感染的发生。
附图说明
图1：如图1所示靶向CD45抗体药物偶联物的结构；
图2：空质粒pTrioz的结构示意图；
图3：空质粒pTrioz-Furin-DTA-6 X His构建流程；
图4：质粒pTrioz-Furin-DTA-6 X His结构示意图
图5：质粒pTrioz-L-Furin-DTA-6 X His构建流程；
图6：质粒pTrioz-CD45 Ab-Furin-DTA-6 X His构建流程:
图7：靶向CD45抗体药物偶联物生产质粒pTrioz-CD45 Ab-Furin-DTA-6 X His的机构示意图；
图8：流式细胞仪检测免疫细胞清除程度；
图9：9a小鼠实验流程示意图，9b:野生型Balb/C小鼠，未做骨髓移植处理；9c:野生型Balb/C小鼠注射CD45 Ab-DTA后；
图10：基因改造载体pX260a-GFP-Dph7的构建流程示意图；
图11：pX260a-GFP空载体的质粒结构示意图；
图12：基因改造载体pX260a-GFP-Dph7的质粒结构示意图；
图13：pMyc-IRES-GFP空质粒示意图；
图14：pMyc-DTA-IRES-GFP示意图；
图15：TR细胞株鉴定结果图；
图16：TR细胞株检测结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例1
构建pTrioz-Furin-DTA-6 X His的生产载体，整体流程如附图3所示。
(1)以pTrioz空白质粒为模板，如附图2所示，其包含人IgG1轻链C区、人IgG1重链C区，利用序列如SEQ ID NO.4所示的正向引物
IgHC-F-NheI-GCTAGCACCAAGGGCCCATCGGTCTTCCCCC和序列如SEQ ID NO.5所示的反向引物
IgHC-R-Furin-TGACAATGAGCTACCTACTGATCGCCTGACACGATTTCCTGCTTTACCCGGAGACAGGGAGAGGC进行PCR，扩增出序列如SEQ ID NO.6所示的基因片段IgHC；下划线序列为酶切位点。
(2)以pIRES-proHB EGF WT质粒(addgene)为模板，利用序列如SEQ ID NO.7所示的正向引物
DTA-F-Furin-GCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATTGTCAGGCGCTGATGATGTTGTTGATTC和序列如SEQ ID NO.8所示的反向引物DTA-R-6XHis-AvrII-CCTAGGGTGGTGATGATGGTGATGAAGAAATGGTTGCGTTTTATGCCC进行PCR，扩增出序列如SEQ ID NO.9所示的基因片段DTA；下划线序列为酶切位点
PCR反应体系如下：
PCR扩增条件如下：
将目的基因片段IgHC和DTA产物经琼脂糖凝胶电泳分离后，分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。
利用序列如SEQ ID NO.4所示的正向引物IgHC-F-NheI-GCTAGCACCAAGGGCCCATCGGTCTTCCCCC和SEQ ID NO.8所示的反向引物DTA-R-6XHis-AvrII-CCTAGGGTGGTGATGATGGTGATGAAGAAATGGTTGCGTTTTATGCCC进行PCR；以IgHC、DTA基因片段为模板，进行Overlap PCR，合成融合基因片段IgHC-DTA(SEQ ID NO.10)，具体步骤如下：
Overlap PCR反应体系如下：
Overlap PCR扩增条件为：
将目的基因片段IgHC-DTA克隆至空载体pTrizo：PCR产物经琼脂糖凝胶电泳分离后，分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的IgHC-DTA和空载体pTrizo分别经MheI、AvrII(NEB)双酶切,空载体pTrizo酶切时加入Alkaline phosphatase(NEB)。PCR酶切产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收，酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA GelExtraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接，接着转化大肠杆菌DH5α，挑取单个菌落培养，提取质粒DNA，经酶切初步鉴定后送商业公司测序。测序结果显示IgHC-DTA插入成功并命名为载体pTrioz-Furin-DTA-6 X His(SEQ ID NO.1)，如附图4所示，其包含以下部分，人IgG1轻链C区、人IgG1重链C区、Furin识别位点、白喉毒素以及6 X His tag。
实施例2
商业公司合成如SEQ ID NO.11所示序列CD45 Ab轻链；
以合成产物为模板，利用序列如SEQ ID NO.12CD45 Ab-L-F-NcoI-CCATGGCAATGATGTCCTCTGCTCAG所示的正向引物和序列如SEQ ID NO.13 CD45 Ab-L-R-BsiWI-CGTACGTTTGATTTCCAGCTTGGTGC所示的反向引物进行PCR，扩增出序列如SEQ IDNO.14所示的基因片段CD45 Ab L；
将PCR产物利用酶切位点NcoI、BsiWI克隆至载体pTrioz-Furin-DTA-6 X His，得到中间载体pTrioz-L-Furin-DTA-6 X His,如SEQ ID NO.15所示，构建流程如附图5所示。
PCR反应体系如下：
PCR扩增条件为：
将目的基因片段CD45 L克隆至载体pTrioz-Furin-DTA-6 X His：PCR产物经琼脂糖凝胶电泳分离后，分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的CD45 Ab和载体pTrioz-Furin-DTA-6 X His分别经NcoI、BsiWI(NEB)双酶切,载体pTrioz-Furin-DTA-6 X His酶切时加入Alkaline phosphatase(NEB)。PCR酶切产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收，酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNAligase(NEB)将载体与目的片段连接，接着转化大肠杆菌DH5α，挑取单个菌落培养，提取质粒DNA，经酶切初步鉴定后送商业公司测序。测序结果显示CD45 Ab L插入成功并命名为载体pTrioz-CD45 Ab-L-Furin-DTA-6 X His。
实施例3
插入CD45 Ab重链包括如下步骤：
商业公司合成如SEQ ID NO.16所示序列CD45 Ab重链；
以合成产物为模板，利用序列如SEQ ID NO.17 CD45 Ab-H-F-EcoRV所示的正向引物GATATCATGAGCGTGCTGATTCTTTTGTGGC和序列如SEQ ID NO.18 CD45 Ab-H-R-NheI-GCTAGCTACTGGTACTTCGATGTCTG所示的反向引物进行PCR，扩增出序列如SEQ ID NO.19所示的基因片段CD45 Ab H；
PCR反应体系如下：
PCR扩增条件为：
将目的基因片段CD45 H克隆至载体pTrioz-L-Furin-DTA-6 X His：PCR产物经琼脂糖凝胶电泳分离后，分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的CD45 Ab H和载体pTrioz-L-Furin-DTA-6 X His分别经EcoRV、NheI(NEB)双酶切,载体pTrioz-L-Furin-DTA-6 X His酶切时加入Alkaline phosphatase(NEB)。PCR酶切产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收，酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接，接着转化大肠杆菌DH5α，挑取单个菌落培养，提取质粒DNA，经酶切初步鉴定后送商业公司测序。测序结果显示CD45 Ab H插入成功,并命名为生产载体pTrioz-CD45 Ab-Furin-DTA-6 X His(SEQ ID NO.2)，构建流程如附图6所示，载体结构见附图7。
实施例4
CD45 Ab-DTA重组蛋白的生产方法如下：
所使用的毒素耐受细胞株(Toxin resistant,TR)用于生产靶向CD45抗体药物偶联物CD45 Ab-DTA。TR细胞株由本公司开发，TR细胞株具体构建方法见实施例8。
(1)将TR细胞用FreeStyleTM293培养基调整至1×106cells/mL，共30mL
(2)取载体pTrioz-CD45 Ab-Furin-DTA-6 X His生产载体37.5微克与0.6mLOptiPROTMSFM培养基混合均匀；
(3)取37.5微升FreeStyleTMMAX Reagent反应物与in OptiPROTMSFM0.6mLOptiPROTMSFM培养基混合均匀，静置5分钟；
(4)将步骤2与3获得培养基混合均匀，静置30分钟；
(5)将步骤4获得1.2mL培养基加入TR细胞中并培养7天，7天后收取CD45Ab-DTA重组蛋白。
实施例5
CD45 Ab-DTA的纯化方法如下：
(1)将30mLNi-NTA Superflow Resin 700g离心2分钟，弃上清；
(2)加入60mL平衡液与Ni-NTA Superflow Resin混合均匀，700g离心2分钟，弃上清；
(3)将实施例步骤5获得的细胞培养基300g离心5分钟，取上清加入步骤2获得Ni-NTA Superflow Resin中，轻柔混合30分钟后700g离心2分钟，弃上清；
(4)使用60mL清洗液与Ni-NTA Superflow Resin混合均匀，700g离心2分钟，弃上清，并清洗3次；
(5)使用30mL洗脱液与Ni-NTA Superflow Resin混合均匀，轻柔混合10分钟后700g离心2分钟，取上清，上清中包含CD45 Ab-DTA重组蛋白；
(6)使用NanoDrop测定CD45 Ab-DTA，并调整至2mg/mL。
实施例6
测试CD45 Ab-DTA对小鼠免疫细胞清除程度方法如下：
(1)使用NanoDrop测定CD45 Ab-DTA浓度，并调整至2mg/mL；
(2)在6周大C57小鼠注射CD45 Ab DTA 200ug，一周三次，持续一周。
(3)用流式细胞仪检测免疫细胞清除程度，结果如附图8所示。
实施例7
CD45 Ab DTA应用于小鼠异体骨髓移植的实施例，流程见附图9a。
(1)在6周大Balb/C(CD45.2基因型)中，注射CD45 Ab DTA 200ug每次，一周三次；
(2)注射一周后移植同种异体小鼠C57(CD45.1基因型)小鼠的骨髓细胞；
(3)四个月后用流式细胞仪检测骨髓移植情况，如图9b所示：野生型Balb/C小鼠，未做骨髓移植处理，其免疫细胞(T细胞、B细胞、粒细胞)均来源于本身，均带有自身免疫标志CD45.2；如图9c所示，野生型Balb/C小鼠注射CD45Ab-DTA后，自身免疫细胞(免疫标志CD45.2)消亡，骨髓移植后，赠体(免疫标志CD45.1)免疫细胞T细胞、B细胞、粒细胞)替代原有骨髓细胞。并实现长期存活。充分说明本发明所述的抗体药物偶联物可以应用于骨髓移植，并效果良好。
实施例8
毒素耐受细胞株(Toxin resistant,TR)的构建。
1:构建基因工程改造载体pX260a-GFP-Dph7:整体流程如附图10所示。
设计正向引物Dph7-gRNA-F-TGTTGATGCTTCTCTTTCTCCA(SEQ ID NO.20)，反向引物Dph7-gRNA-R-TCTGAACCAGTGTCCAGCAC(SEQ ID NO.21)，退火结合为Dph7 gRNA(SEQ IDNO.30)。
Annealing反应体系如下：
Annealing反应条件如下：
将Dph7 gRNA克隆至空载体pX260a-GFP(载体结构如附图11所示)：空载体pX260a-GFP BbsI(NEB)酶切。酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与Dph7连接，接着转化大肠杆菌DH5α，挑取单个菌落培养，提取质粒DNA，经酶切初步鉴定后送商业公司测序。测序结果显示Dph7 gRNA插入成功并命名为基因改造载体pX260a-GPF-Dph 7(SEQ ID NO.22)，如附图12所示。
2:药物毒性耐受生产细胞株(Toxin resistant,TR)的构建方法如下：
(1)将FreeStyle 293-F细胞用FreeStyleTM293培养基调整至106cells/mL，共1mL，于6孔板中培养；
(2)取实施例1中获得pX260a-GFP-Dph7基因改造载体5微克与125微升P3000TMReagent(thermofisher)混合；
(3)取Medium(thermofisher)125微升与/>3000Reagent7.5微升混合；
(4)将步骤2与步骤3获得培养基与DNA混合均匀，静置5分钟；
(5)将步骤4获得的混合物加入细胞；
(6)转染后细胞培养3天；
(7)使用FACS Aria III分选GFP阳性细胞至96孔板，每个孔含有一个单细胞；
(8)培养3天后，使用胰酶消化，扩增至24孔板；
(9)培养3天后，取一半细胞利用dneasy-blood-and-tissue-kit提取DNA，另一半细胞扩增至6孔板。
随后通过对步骤9提取的DNA检测，验证转化是否成功，包括以下步骤：
利用步骤9得到DNA作为模板，正向引物Dph7-Screen-F-
CTTCCTCACAGACAGAGTCACCCAG(SEQ ID NO.23)，反向引物Dph7-Screen-R-
CGAGAAGGGCCCGAGAGCCTCC(SEQ ID NO.24)作PCR扩增得到Dph7 Screen基因片段(SEQ ID NO.25)。
PCR反应体系如下：
PCR扩增条件如下：
将目的基因片段Dph7 Screen PCR产物经琼脂糖凝胶电泳分离后，切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收，经Surveyor Enzyme(NEB)酶切。酶切的PCR产物经琼脂糖凝胶电泳分离后拍照，如附图15所示。
3:检测药物毒性耐受生产细胞株(Toxin resistant,TR)
以pIRES-proHB EGF WT质粒(addgene)为模板，利用正向引物DTA-F-BamHI-ACCACC
GGATCCATGGGCGCTGATGATGTTGTTGATTCTTC(SEQ ID NO.26)，反向引物DTA-R(EcoRI)-ACCACCGAATTCTTAGTGGGAATTAGTCATGCCC(SEQ ID NO.27)作PCR扩增DTA基因片段(SEQ ID NO.28)。下划线序列为酶切位点。PCR反应体系同上，PCR扩增条件同上。
将目的基因片段DTA克隆至空载体pMyc-IRES-GFP(载体结构如附图13所示)：PCR产物经琼脂糖凝胶电泳分离后，分别切出目的条带并利用AxyPrep DNA Gel ExtractionKit(Axygen)回收。纯化后的DTA和空载体pMyc-IRES-GFP分别经EcoRI、BamHI(NEB)双酶切,空载体pMyc-IRES-GFP酶切时加入Alkaline phosphatase(NEB)。PCR酶切产物经AxyPrepPCR Clean-Up Kit(Axygen)纯化回收，酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接，接着转化大肠杆菌DH5α，挑取单个菌落培养，提取质粒DNA，经酶切初步鉴定后送商业公司测序。测序结果显示DTA插入成功并命名为检测载体pMyc-DTA-IRES-GFP(SEQ ID NO.29)，载体结构如附图14所示。
(1)将实施例2中获得的药物毒性耐受生产细胞株用FreeStyleTM293培养基调整至106cells/mL，共1mL，于6孔板中培养；
(2)取pMyc-DTA-IRES-GFP检测载体5微克与125微升P3000TMReagent(thermofisher)混合；
(3)取Medium(thermofisher)125微升与/>3000Reagent7.5微升混合；
(4)将步骤2和3获得的培养基与DNA混合均匀，静置5分钟；
(5)将步骤4获得的混合物加入细胞中；
(6)转染后细胞培养3天；
(7)使用Cyto Flex检测,发现GFP阳性，如附图16所示，即DTA表达，TR细胞株成功表达DTA，表明TR细胞株对毒性耐受。
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，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 乾元康安(苏州)生物科技有限公司
<120> 靶向CD45的抗体药物偶联物及其制备和应用
<130> 2020
<160> 30
<170> PatentIn version 3.3
<210> 1
<211> 9195
<212> DNA
<213> 人工
<223> 质粒pTrioz-Furin-DTA-6 X His
<400> 1
cctgcaggcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 60
cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 120
tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 180
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 240
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 300
gctattacca tgatgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 360
tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttgactagtc 420
agggccccaa cccccccaag cccccatttc acaacacgct ggcgctacag gcgcgtgact 480
tccccttgct ttggggcggg gggctgagac tcctatgtgc tccggattgg tcaggcacgg 540
ccttcggccc cgcctcctgc caccgcagat tggccgctag gcctccccga gcgccctgcc 600
tccgagggcc ggcgcaccat aaaagaagcc gccctagcca cgtcccctcg cagttcggcg 660
gtcccgcggg tctgtctcaa gcttgccgcc agaacacagg taagtgccgt gtgtggttcc 720
cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccatgccc 780
ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 840
gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcttgggcg 900
ctggggccgc cgcgtgctaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgctaa 960
gtctctagcc atttaaaatt tttgataacc agctgcgacg ctttttttct ggcgagatag 1020
tcttgtaaat gcgggccagg atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 1080
cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 1140
accgagaatc ggacgggggt agtctcaaac tggccggcct gctctggtgc ctggcctcgc 1200
gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 1260
agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 1320
cccgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cttcctcatc 1380
cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttgtc 1440
gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 1500
ccacactgag tgggtggaga ctgaagagtt aggccagctt ggcacttgat gtaattctcc 1560
ttggaatttg ccctttttga gtttggatct tgcctcattc tcaagcctca gacagtggtt 1620
caaagttttt ttcttccatt tcaggtgtcg tgaaaactac ccctaaaagc caccggcgag 1680
gcgcgccaag tttaaacacc atggaacgta cggtggctgc accatctgtc ttcatcttcc 1740
cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact 1800
tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa tcgggtaact 1860
cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc agcagcaccc 1920
tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa gtcacccatc 1980
agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag agggacctag 2040
ctggccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 2100
aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 2160
ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 2220
ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg aaatgttaat 2280
taactagcca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 2340
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 2400
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 2460
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 2520
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 2580
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 2640
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 2700
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 2760
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 2820
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 2880
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 2940
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3000
tttgctcaca tgttcttaat taacctgcag gcgaaactca gcttccttcg tttccgactt 3060
ttccatccgc gtcctccact tccccgttcc gccctccccc attgccaaca ttctggctga 3120
gtcacggcgc cccagagcgc gccaggctgg gggaaaggag cagaagggag ggccctagcg 3180
acccgcggga tgtggtccga gtcacgtccg aggggggtgg ggagggatcg tgttctcggc 3240
gcccgcccct tcctagcgcg gcctctgggc tgcgcctctc gggggcggcc cgtagcccag 3300
tccgtcgact agttccgcca gagcgcgcga gggcctccag cggccgcccc tcccccacag 3360
caggggcggg gtcccgcgct caccggaagg agcgggctcg gggcgggcgg cgctgattgg 3420
ccggggcggg cctgacgccg acgcggctat aagagaccac aagcgacccg cagggccaga 3480
cgttcttcgc cgaagcttgc cgtcagaacg caggtgaggg gcgggtgtgg cttccgcggg 3540
ccgccgagct ggaggtcctg ctccgagcgg gccgggcccc gctgtcgtcg gcggggatta 3600
gctgcgagca ttcccgcttc gagttgcggg cggcgcggga ggcagagtgc gaggcctagc 3660
ggcaaccccg tagcctcgcc tcgtgtccgg cttgaggcct agcgtggtgt ccgcgccgcc 3720
gccgcgtgct actccggccg cactctggtc tttttttttt ttgttgttgt tgccctgctg 3780
ccttcgattg ccgttcagca ataggggcta acaaagggag ggtgcggggc ttgctcgccc 3840
ggagcccgga gaggtcatgg ttggggagga atggagggac aggagtggcg gctggggccc 3900
gcccgccttc ggagcacatg tccgacgcca cctggatggg gcgaggcctg gggtttttcc 3960
cgaagcaacc aggctggggt tagcgtgccg aggccatgtg gccccagcac ccggcacgat 4020
ctggcttggc ggcgccgcgt tgccctgcct ccctaactag ggtgaggcca tcccgtccgg 4080
caccagttgc gtgcgtggaa agatggccgc tcccgggccc tgttgcaagg agctcaaaat 4140
ggaggacgcg gcagcccggt ggagcgggcg ggtgagtcac ccacacaaag gaagagggcc 4200
tggtccctca ccggctgctg cttcctgtga ccccgtggtc ctatcggccg caatagtcac 4260
ctcgggcttt tgagcacggc tagtcgcggc ggggggaggg gatgtaatgg cgttggagtt 4320
tgttcacatt tggtgggtgg agactagtca ggccagcctg gcgctggaag tcatttttgg 4380
aatttgtccc cttgagtttt gagcggagct aattctcggg cttcttagcg gttcaaaggt 4440
atcttttaaa ccctttttta ggtgttgtga aaaccaccgc taattcaaag caaccggtcg 4500
cacgcgtaga tatcacgtca tgaaagctag caccaagggc ccatcggtct tccccctggc 4560
accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta 4620
cttccccgag ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac 4680
cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 4740
ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac 4800
caaggtggac aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg 4860
cccagcacct gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga 4920
caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga 4980
agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac 5040
aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct 5100
gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc 5160
agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta 5220
caccctgccc ccatcccggg acgagctgac caagaaccag gtcagcctga cctgcctggt 5280
caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa 5340
caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa 5400
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca 5460
tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaagcagg 5520
aaatcgtgtc aggcgatcag taggtagctc attgtcaggc gctgatgatg ttgttgattc 5580
ttctaaatct tttgtgatgg aaaacttttc ttcgtaccac gggactaaac ctggttatgt 5640
agattccatt caaaaaggta tacaaaagcc aaaatctggt acacaaggaa attatgacga 5700
tgattggaaa gggttttata gtaccgacaa taaatacgac gctgcgggat actctgtaga 5760
taatgaaaac ccgctctctg gaaaagctgg aggcgtggtc aaagtgacgt atccaggact 5820
gacgaaggtt ctcgcactaa aagtggataa tgccgaaact attaagaaag agttaggttt 5880
aagtctcact gaaccgttga tggagcaagt cggaacggaa gagtttatca aaaggttcgg 5940
tgatggtgct tcgcgtgtag tgctcagcct tcccttcgct gaggggagtt ctagcgtttc 6000
ctatattaat aactgggaac aggcgaaagc gttaagcgta gaacttgaga ttaattttga 6060
aacccgtgga aaacgtggcc aagatgcgat gtatgagtat atggctcaag cctgtgcagg 6120
aaatcgtgtc aggcgatcag taggtagctc attgtcatgc ataaatcttg attgggatgt 6180
cataagggat aaaactaaga caaagataga gtctttgaaa gagcatggcc ctatcaaaaa 6240
taaaatgagc gaaagtccca ataaaacagt atctgaggaa aaagctaaac aatacctaga 6300
agaatttcat caaacggcat tagagcatcc tgaattgtca gaacttaaaa ccgttactgg 6360
gaccaatcct gtattcgctg gggctaacta tgcggcgtgg gcagtaaacg ttgcgcaagt 6420
tatagatctc gaaacagctg ataatttgga aaagacaact gctgctcttt cgatacttcc 6480
tggtatcggt agcgtaatgg gcattgcaga cggtgccgtt caccacaata cagaagagat 6540
agtggcacaa tcaatagctt tatcgtcttt aatggttgct caagctattc cattggtagg 6600
agagctagtt gatattggtt tcgctgcata taattttgta gagagtatta tcaatttatt 6660
tcaagtagtt cataattcgt ataatcgtcc cgcgtattct ccggggcata aaacgcaacc 6720
atttcttcat caccatcatc accaccctag gagcaggttt ccccaatgac acaaaacgtg 6780
caacttgaaa ctccgcctgg tctttccagg tctagaagct cgctttcttg ctgtccaatt 6840
tctattaaag gttcctttgt tccctaagtc caactactaa actgggggat attatgaagg 6900
gccttgagca tctggattct gcctaataaa aaacatttat tttcattgca atgatgtatt 6960
taaattattt ctgaatattt tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca 7020
taaagaaatg aagagctagt tcaaaccttg ggaaaataca ctatatctta aactccatga 7080
aagaaggtga ggctgcaaac agctaatgca cattggcaac agcccctgat gcctatgcct 7140
tattcatccc tcagaaaagg attcaagtag aggcttgatt tggaggttaa agttttgcca 7200
tgctgtattt tagaattcct gcaggagtca atgggaaaaa cccattggag ccaagtacac 7260
tgactcaata gggactttcc attgggtttt gcccagtaca taaggtcaat agggggtgag 7320
tcaacaggaa agtcccattg gagccaagta cattgagtca atagggactt tccaatgggt 7380
tttgcccagt acataaggtc aatgggaggt aagccaatgg gtttttccca ttactgacat 7440
gtatactgag tcattaggga ctttccaatg ggttttgccc agtacataag gtcaataggg 7500
gtgaatcaac aggaaagtcc cattggagcc aagtacactg agtcaatagg gactttccat 7560
tgggttttgc ccagtacaaa aggtcaatag ggggtgagtc aatgggtttt tcccattatt 7620
ggcacataca taaggtcaat aggggtgact agtcagtggg cagagcgcac atcgcccccg 7680
agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 7740
actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt 7800
atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 7860
agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 7920
gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 7980
cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 8040
cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 8100
tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 8160
ctacaaacag tagttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 8220
tcactatagg agggccatca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg 8280
cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt 8340
ggaggacgac ttcgctggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca 8400
ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct 8460
gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat 8520
gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa 8580
ctgcgtgcac tttgtggcag aggagcagga ctaaatctag aattatccct aatacctgcc 8640
accccactct taatcagtgg tggaagaacg gtctcagaac tgtttgtttc aattggccat 8700
ttaagtttag tagtaaaaga ctggttaatg ataacaatgc atcgtaaaac cttcagaagg 8760
aaaggagaat gttttgtgga ccactttggt tttctttttt gcgtgtggca gttttaagtt 8820
attagttttt aaaatcagta ctttttaatg gaaacaactt gaccaaaaat ttgtcacaga 8880
attttgagac ccattaaaaa agttaaatga gaaacctgtg tgttcctttg gtcaacaccg 8940
agacatttag gtgaaagaca tctaattctg gttttacgaa tctggaaact tcttgaaaat 9000
gtaattcttg agttaacact tctgggtgga gaatagggtt gttttccccc cacataattg 9060
gaaggggaag gaatatcatt taaagctatg ggagggttgc tttgattaca acactggaga 9120
gaaatgcagc atgttgctga ttgcctgtca ctaaaacagg ccaaaaactg agtccttggg 9180
ttgcatagaa agctg 9195
<210> 2
<211> 10018
<212> DNA
<213> 人工
<223> 质粒pTrioz-CD45 Ab-Furin-DTA-6 X His
<400> 2
cctgcaggcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 60
cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 120
tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 180
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 240
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 300
gctattacca tgatgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 360
tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttgactagtc 420
agggccccaa cccccccaag cccccatttc acaacacgct ggcgctacag gcgcgtgact 480
tccccttgct ttggggcggg gggctgagac tcctatgtgc tccggattgg tcaggcacgg 540
ccttcggccc cgcctcctgc caccgcagat tggccgctag gcctccccga gcgccctgcc 600
tccgagggcc ggcgcaccat aaaagaagcc gccctagcca cgtcccctcg cagttcggcg 660
gtcccgcggg tctgtctcaa gcttgccgcc agaacacagg taagtgccgt gtgtggttcc 720
cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccatgccc 780
ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 840
gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcttgggcg 900
ctggggccgc cgcgtgctaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgctaa 960
gtctctagcc atttaaaatt tttgataacc agctgcgacg ctttttttct ggcgagatag 1020
tcttgtaaat gcgggccagg atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 1080
cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 1140
accgagaatc ggacgggggt agtctcaaac tggccggcct gctctggtgc ctggcctcgc 1200
gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 1260
agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 1320
cccgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cttcctcatc 1380
cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttgtc 1440
gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 1500
ccacactgag tgggtggaga ctgaagagtt aggccagctt ggcacttgat gtaattctcc 1560
ttggaatttg ccctttttga gtttggatct tgcctcattc tcaagcctca gacagtggtt 1620
caaagttttt ttcttccatt tcaggtgtcg tgaaaactac ccctaaaagc caccggcgag 1680
gcgcgccaag tttaaacacc atggcaatga tgtcctctgc tcagttcctt ggtctcctgt 1740
tgctctgttt tcaaggtaaa attgactata atgggaattt tgctgttgca cggtgtgata 1800
tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga gtcaccatca 1860
gttgcagtgc aagtcagggc attagcaatt atttaaactg gtatcagcag aaaccagatg 1920
gaactgttaa actcctgatc tattacacat caagtttaca ctcaggagtc ccatcaaggt 1980
tcagtggcag tgggtctggg acagattatt ctctcaccat cagcaacctg gaacctgaag 2040
atattgccac ttactattgt cagcagtata gtaagcttcc tccgtggacg ttcggtggag 2100
gcaccaagct ggaaatcaaa cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat 2160
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat aacttctatc 2220
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg 2280
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc accctgacgc 2340
tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc catcagggcc 2400
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttagagggac ctagctggcc 2460
agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2520
atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2580
taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2640
ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggaaatgt taattaacta 2700
gccatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 2760
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2820
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2880
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 2940
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 3000
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 3060
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 3120
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 3180
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 3240
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 3300
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 3360
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 3420
cacatgttct taattaacct gcaggcgaaa ctcagcttcc ttcgtttccg acttttccat 3480
ccgcgtcctc cacttccccg ttccgccctc ccccattgcc aacattctgg ctgagtcacg 3540
gcgccccaga gcgcgccagg ctgggggaaa ggagcagaag ggagggccct agcgacccgc 3600
gggatgtggt ccgagtcacg tccgaggggg gtggggaggg atcgtgttct cggcgcccgc 3660
cccttcctag cgcggcctct gggctgcgcc tctcgggggc ggcccgtagc ccagtccgtc 3720
gactagttcc gccagagcgc gcgagggcct ccagcggccg cccctccccc acagcagggg 3780
cggggtcccg cgctcaccgg aaggagcggg ctcggggcgg gcggcgctga ttggccgggg 3840
cgggcctgac gccgacgcgg ctataagaga ccacaagcga cccgcagggc cagacgttct 3900
tcgccgaagc ttgccgtcag aacgcaggtg aggggcgggt gtggcttccg cgggccgccg 3960
agctggaggt cctgctccga gcgggccggg ccccgctgtc gtcggcgggg attagctgcg 4020
agcattcccg cttcgagttg cgggcggcgc gggaggcaga gtgcgaggcc tagcggcaac 4080
cccgtagcct cgcctcgtgt ccggcttgag gcctagcgtg gtgtccgcgc cgccgccgcg 4140
tgctactccg gccgcactct ggtctttttt ttttttgttg ttgttgccct gctgccttcg 4200
attgccgttc agcaataggg gctaacaaag ggagggtgcg gggcttgctc gcccggagcc 4260
cggagaggtc atggttgggg aggaatggag ggacaggagt ggcggctggg gcccgcccgc 4320
cttcggagca catgtccgac gccacctgga tggggcgagg cctggggttt ttcccgaagc 4380
aaccaggctg gggttagcgt gccgaggcca tgtggcccca gcacccggca cgatctggct 4440
tggcggcgcc gcgttgccct gcctccctaa ctagggtgag gccatcccgt ccggcaccag 4500
ttgcgtgcgt ggaaagatgg ccgctcccgg gccctgttgc aaggagctca aaatggagga 4560
cgcggcagcc cggtggagcg ggcgggtgag tcacccacac aaaggaagag ggcctggtcc 4620
ctcaccggct gctgcttcct gtgaccccgt ggtcctatcg gccgcaatag tcacctcggg 4680
cttttgagca cggctagtcg cggcgggggg aggggatgta atggcgttgg agtttgttca 4740
catttggtgg gtggagacta gtcaggccag cctggcgctg gaagtcattt ttggaatttg 4800
tccccttgag ttttgagcgg agctaattct cgggcttctt agcggttcaa aggtatcttt 4860
taaacccttt tttaggtgtt gtgaaaacca ccgctaattc aaagcaaccg gtcgcacgcg 4920
tagatatcat gagcgtgctg attcttttgt ggctgttcac agcctttcct ggtgagtgta 4980
tcctgtctga tgtgcagctt caggagtcag gacctggcct ggtgaaacct tctcagtcac 5040
tttccctcac ctgcactgtc actggctact ccatcaccag tggttatgac tggaactgga 5100
tccggcagtt tccaggaaac aaactggagt ggatgggcta cataagctac agtgtagcaa 5160
gtactacaac ccatctctca aaagtcgaat ctctatcact cgagacacat ccaagaacca 5220
gttctccctg gaatattctg ctactgagga cacagccaca tattactgtg caagaggtag 5280
ctactaccgt agtaataaac ggagacggtg accgtggtcc ctgtgcccca gacatcgaag 5340
taccagtagc tagcaccaag ggcccatcgg tcttccccct ggcaccctcc tccaagagca 5400
cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc gagccggtga 5460
cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg gctgtcctac 5520
agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc agcttgggca 5580
cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg gacaagaaag 5640
ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca cctgaactcc 5700
tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc 5760
ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt 5820
tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc 5880
agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga 5940
atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa 6000
ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc 6060
gggacgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca 6120
gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc 6180
ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga 6240
gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc 6300
actacacgca gaagagcctc tccctgtctc cgggtaaagc aggaaatcgt gtcaggcgat 6360
cagtaggtag ctcattgtca ggcgctgatg atgttgttga ttcttctaaa tcttttgtga 6420
tggaaaactt ttcttcgtac cacgggacta aacctggtta tgtagattcc attcaaaaag 6480
gtatacaaaa gccaaaatct ggtacacaag gaaattatga cgatgattgg aaagggtttt 6540
atagtaccga caataaatac gacgctgcgg gatactctgt agataatgaa aacccgctct 6600
ctggaaaagc tggaggcgtg gtcaaagtga cgtatccagg actgacgaag gttctcgcac 6660
taaaagtgga taatgccgaa actattaaga aagagttagg tttaagtctc actgaaccgt 6720
tgatggagca agtcggaacg gaagagttta tcaaaaggtt cggtgatggt gcttcgcgtg 6780
tagtgctcag ccttcccttc gctgagggga gttctagcgt ttcctatatt aataactggg 6840
aacaggcgaa agcgttaagc gtagaacttg agattaattt tgaaacccgt ggaaaacgtg 6900
gccaagatgc gatgtatgag tatatggctc aagcctgtgc aggaaatcgt gtcaggcgat 6960
cagtaggtag ctcattgtca tgcataaatc ttgattggga tgtcataagg gataaaacta 7020
agacaaagat agagtctttg aaagagcatg gccctatcaa aaataaaatg agcgaaagtc 7080
ccaataaaac agtatctgag gaaaaagcta aacaatacct agaagaattt catcaaacgg 7140
cattagagca tcctgaattg tcagaactta aaaccgttac tgggaccaat cctgtattcg 7200
ctggggctaa ctatgcggcg tgggcagtaa acgttgcgca agttatagat ctcgaaacag 7260
ctgataattt ggaaaagaca actgctgctc tttcgatact tcctggtatc ggtagcgtaa 7320
tgggcattgc agacggtgcc gttcaccaca atacagaaga gatagtggca caatcaatag 7380
ctttatcgtc tttaatggtt gctcaagcta ttccattggt aggagagcta gttgatattg 7440
gtttcgctgc atataatttt gtagagagta ttatcaattt atttcaagta gttcataatt 7500
cgtataatcg tcccgcgtat tctccggggc ataaaacgca accatttctt catcaccatc 7560
atcaccaccc taggagcagg tttccccaat gacacaaaac gtgcaacttg aaactccgcc 7620
tggtctttcc aggtctagaa gctcgctttc ttgctgtcca atttctatta aaggttcctt 7680
tgttccctaa gtccaactac taaactgggg gatattatga agggccttga gcatctggat 7740
tctgcctaat aaaaaacatt tattttcatt gcaatgatgt atttaaatta tttctgaata 7800
ttttactaaa aagggaatgt gggaggtcag tgcatttaaa acataaagaa atgaagagct 7860
agttcaaacc ttgggaaaat acactatatc ttaaactcca tgaaagaagg tgaggctgca 7920
aacagctaat gcacattggc aacagcccct gatgcctatg ccttattcat ccctcagaaa 7980
aggattcaag tagaggcttg atttggaggt taaagttttg ccatgctgta ttttagaatt 8040
cctgcaggag tcaatgggaa aaacccattg gagccaagta cactgactca atagggactt 8100
tccattgggt tttgcccagt acataaggtc aatagggggt gagtcaacag gaaagtccca 8160
ttggagccaa gtacattgag tcaataggga ctttccaatg ggttttgccc agtacataag 8220
gtcaatggga ggtaagccaa tgggtttttc ccattactga catgtatact gagtcattag 8280
ggactttcca atgggttttg cccagtacat aaggtcaata ggggtgaatc aacaggaaag 8340
tcccattgga gccaagtaca ctgagtcaat agggactttc cattgggttt tgcccagtac 8400
aaaaggtcaa tagggggtga gtcaatgggt ttttcccatt attggcacat acataaggtc 8460
aataggggtg actagtcagt gggcagagcg cacatcgccc ccgagaagtt ggggggaggg 8520
gtcggcaatt gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc 8580
gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc 8640
gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg 8700
gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg 8760
agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta 8820
agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga 8880
ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg 8940
ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctacaaa cagtagttga 9000
caattaatca tcggcatagt atatcggcat agtataatac gactcactat aggagggcca 9060
tcatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg 9120
tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgctg 9180
gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg 9240
acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg 9300
aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc 9360
agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cactttgtgg 9420
cagaggagca ggactaaatc tagaattatc cctaatacct gccaccccac tcttaatcag 9480
tggtggaaga acggtctcag aactgtttgt ttcaattggc catttaagtt tagtagtaaa 9540
agactggtta atgataacaa tgcatcgtaa aaccttcaga aggaaaggag aatgttttgt 9600
ggaccacttt ggttttcttt tttgcgtgtg gcagttttaa gttattagtt tttaaaatca 9660
gtacttttta atggaaacaa cttgaccaaa aatttgtcac agaattttga gacccattaa 9720
aaaagttaaa tgagaaacct gtgtgttcct ttggtcaaca ccgagacatt taggtgaaag 9780
acatctaatt ctggttttac gaatctggaa acttcttgaa aatgtaattc ttgagttaac 9840
acttctgggt ggagaatagg gttgttttcc ccccacataa ttggaagggg aaggaatatc 9900
atttaaagct atgggagggt tgctttgatt acaacactgg agagaaatgc agcatgttgc 9960
tgattgcctg tcactaaaac aggccaaaaa ctgagtcctt gggttgcata gaaagctg 10018
<210> 3
<211> 1127
<212> PRT
<213> 人工
<223> 靶向CD45抗体药物偶联物蛋白序列
<400> 3
Met Ala Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys
1 5 10 15
Phe Gln Gly Lys Ile Asp Tyr Asn Gly Asn Phe Ala Val Ala Arg Cys
20 25 30
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
35 40 45
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr
50 55 60
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
65 70 75 80
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
85 90 95
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
100 105 110
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Pro
115 120 125
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
130 135 140
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
145 150 155 160
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
165 170 175
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
180 185 190
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
195 200 205
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
210 215 220
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
225 230 235 240
Ser Phe Asn Arg Gly Glu Cys Met Ser Val Leu Ile Leu Leu Trp Leu
245 250 255
Phe Thr Ala Phe Pro Gly Glu Cys Ile Leu Ser Asp Val Gln Leu Gln
260 265 270
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr
275 280 285
Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr Asp Trp Asn Trp
290 295 300
Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser
305 310 315 320
Tyr Ser Val Ala Ser Thr Thr Thr His Leu Ser Lys Val Glu Ser Leu
325 330 335
Ser Leu Glu Thr His Pro Arg Thr Ser Ser Pro Trp Asn Ile Leu Leu
340 345 350
Leu Arg Thr Gln Pro His Ile Thr Val Gln Glu Val Ala Thr Thr Val
355 360 365
Val Ile Asn Gly Asp Gly Asp Arg Gly Pro Cys Ala Pro Asp Ile Glu
370 375 380
Val Pro Val Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
385 390 395 400
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
405 410 415
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
420 425 430
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
435 440 445
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
450 455 460
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
465 470 475 480
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
485 490 495
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
500 505 510
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
515 520 525
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
530 535 540
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
545 550 555 560
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
565 570 575
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
580 585 590
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
595 600 605
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
610 615 620
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
625 630 635 640
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
645 650 655
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
660 665 670
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
675 680 685
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
690 695 700
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ala Gly Asn
705 710 715 720
Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser Gly Ala Asp Asp Val
725 730 735
Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His
740 745 750
Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln Lys
755 760 765
Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Gly Phe
770 775 780
Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn
785 790 795 800
Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val Val Lys Val Thr Tyr
805 810 815
Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val Asp Asn Ala Glu Thr
820 825 830
Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln
835 840 845
Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly Asp Gly Ala Ser Arg
850 855 860
Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser Ser Ser Val Ser Tyr
865 870 875 880
Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser Val Glu Leu Glu Ile
885 890 895
Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu Tyr
900 905 910
Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser
915 920 925
Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr
930 935 940
Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly Pro Ile Lys Asn Lys
945 950 955 960
Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys Gln
965 970 975
Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu Leu Ser
980 985 990
Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn
995 1000 1005
Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Leu Glu
1010 1015 1020
Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu Ser Ile Leu
1025 1030 1035
Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val His
1040 1045 1050
His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser
1055 1060 1065
Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp
1070 1075 1080
Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu
1085 1090 1095
Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro
1100 1105 1110
Gly His Lys Thr Gln Pro Phe Leu His His His His His His
1115 1120 1125
<210> 4
<211> 31
<212> DNA
<213> 人工
<223> 引物IgHC-F-NheI
<400> 4
gctagcacca agggcccatc ggtcttcccc c 31
<210> 5
<211> 65
<212> DNA
<213> 人工
<223> 引物IgHC-R-Furin
<400> 5
tgacaatgag ctacctactg atcgcctgac acgatttcct gctttacccg gagacaggga 60
gaggc 65
<210> 6
<211> 1032
<212> DNA
<213> 人工
<223> 基因片段IgHC
<400> 6
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgagccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggacgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa gcaggaaatc gtgtcaggcg atcagtaggt 1020
agctcattgt ca 1032
<210> 7
<211> 65
<212> DNA
<213> 人工
<223> 引物DTA-F-Furin
<400> 7
gcaggaaatc gtgtcaggcg atcagtaggt agctcattgt caggcgctga tgatgttgtt 60
gattc 65
<210> 8
<211> 48
<212> DNA
<213> 人工
<223> 引物DTA-R-6XHis-AvrII
<400> 8
cctagggtgg tgatgatggt gatgaagaaa tggttgcgtt ttatgccc 48
<210> 9
<211> 1212
<212> DNA
<213> 人工
<223> 基因片段DTA
<400> 9
gcaggaaatc gtgtcaggcg atcagtaggt agctcattgt caggcgctga tgatgttgtt 60
gattcttcta aatcttttgt gatggaaaac ttttcttcgt accacgggac taaacctggt 120
tatgtagatt ccattcaaaa aggtatacaa aagccaaaat ctggtacaca aggaaattat 180
gacgatgatt ggaaagggtt ttatagtacc gacaataaat acgacgctgc gggatactct 240
gtagataatg aaaacccgct ctctggaaaa gctggaggcg tggtcaaagt gacgtatcca 300
ggactgacga aggttctcgc actaaaagtg gataatgccg aaactattaa gaaagagtta 360
ggtttaagtc tcactgaacc gttgatggag caagtcggaa cggaagagtt tatcaaaagg 420
ttcggtgatg gtgcttcgcg tgtagtgctc agccttccct tcgctgaggg gagttctagc 480
gtttcctata ttaataactg ggaacaggcg aaagcgttaa gcgtagaact tgagattaat 540
tttgaaaccc gtggaaaacg tggccaagat gcgatgtatg agtatatggc tcaagcctgt 600
gcaggaaatc gtgtcaggcg atcagtaggt agctcattgt catgcataaa tcttgattgg 660
gatgtcataa gggataaaac taagacaaag atagagtctt tgaaagagca tggccctatc 720
aaaaataaaa tgagcgaaag tcccaataaa acagtatctg aggaaaaagc taaacaatac 780
ctagaagaat ttcatcaaac ggcattagag catcctgaat tgtcagaact taaaaccgtt 840
actgggacca atcctgtatt cgctggggct aactatgcgg cgtgggcagt aaacgttgcg 900
caagttatag atctcgaaac agctgataat ttggaaaaga caactgctgc tctttcgata 960
cttcctggta tcggtagcgt aatgggcatt gcagacggtg ccgttcacca caatacagaa 1020
gagatagtgg cacaatcaat agctttatcg tctttaatgg ttgctcaagc tattccattg 1080
gtaggagagc tagttgatat tggtttcgct gcatataatt ttgtagagag tattatcaat 1140
ttatttcaag tagttcataa ttcgtataat cgtcccgcgt attctccggg gcataaaacg 1200
caaccatttc tt 1212
<210> 10
<211> 2220
<212> DNA
<213> 人工
<223> 基因片段IgHC-DTA
<400> 10
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgagccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggacgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa gcaggaaatc gtgtcaggcg atcagtaggt 1020
agctcattgt caggcgctga tgatgttgtt gattcttcta aatcttttgt gatggaaaac 1080
ttttcttcgt accacgggac taaacctggt tatgtagatt ccattcaaaa aggtatacaa 1140
aagccaaaat ctggtacaca aggaaattat gacgatgatt ggaaagggtt ttatagtacc 1200
gacaataaat acgacgctgc gggatactct gtagataatg aaaacccgct ctctggaaaa 1260
gctggaggcg tggtcaaagt gacgtatcca ggactgacga aggttctcgc actaaaagtg 1320
gataatgccg aaactattaa gaaagagtta ggtttaagtc tcactgaacc gttgatggag 1380
caagtcggaa cggaagagtt tatcaaaagg ttcggtgatg gtgcttcgcg tgtagtgctc 1440
agccttccct tcgctgaggg gagttctagc gtttcctata ttaataactg ggaacaggcg 1500
aaagcgttaa gcgtagaact tgagattaat tttgaaaccc gtggaaaacg tggccaagat 1560
gcgatgtatg agtatatggc tcaagcctgt gcaggaaatc gtgtcaggcg atcagtaggt 1620
agctcattgt catgcataaa tcttgattgg gatgtcataa gggataaaac taagacaaag 1680
atagagtctt tgaaagagca tggccctatc aaaaataaaa tgagcgaaag tcccaataaa 1740
acagtatctg aggaaaaagc taaacaatac ctagaagaat ttcatcaaac ggcattagag 1800
catcctgaat tgtcagaact taaaaccgtt actgggacca atcctgtatt cgctggggct 1860
aactatgcgg cgtgggcagt aaacgttgcg caagttatag atctcgaaac agctgataat 1920
ttggaaaaga caactgctgc tctttcgata cttcctggta tcggtagcgt aatgggcatt 1980
gcagacggtg ccgttcacca caatacagaa gagatagtgg cacaatcaat agctttatcg 2040
tctttaatgg ttgctcaagc tattccattg gtaggagagc tagttgatat tggtttcgct 2100
gcatataatt ttgtagagag tattatcaat ttatttcaag tagttcataa ttcgtataat 2160
cgtcccgcgt attctccggg gcataaaacg caaccatttc ttcatcacca tcatcaccac 2220
<210> 11
<211> 420
<212> DNA
<213> 人工
<223> 基因片段CD45 Ab轻链
<400> 11
atgagcgtgc tgattctttt gtggctgttc acagcctttc ctggtgagtg tatcctgtct 60
gatgtgcagc ttcaggagtc aggacctggc ctggtgaaac cttctcagtc actttccctc 120
acctgcactg tcactggcta ctccatcacc agtggttatg actggaactg gatccggcag 180
tttccaggaa acaaactgga gtggatgggc tacataagct acagtgtagc aagtactaca 240
acccatctct caaaagtcga atctctatca ctcgagacac atccaagaac cagttctccc 300
tggaatattc tgctactgag gacacagcca catattactg tgcaagaggt agctactacc 360
gtagtaataa acggagacgg tgaccgtggt ccctgtgccc cagacatcga agtaccagta 420
<210> 12
<211> 26
<212> DNA
<213> 人工
<223> 引物CD45 Ab-L-F-NcoI
<400> 12
ccatggcaat gatgtcctct gctcag 26
<210> 13
<211> 26
<212> DNA
<213> 人工
<223> 引物CD45 Ab-L-R-BsiWI
<400> 13
cgtacgtttg atttccagct tggtgc 26
<210> 14
<211> 428
<212> DNA
<213> 人工
<223> 基因片段CD45 Ab L
<400> 14
ccatggcaat gatgtcctct gctcagttcc ttggtctcct gttgctctgt tttcaaggta 60
aaattgacta taatgggaat tttgctgttg cacggtgtga tatccagatg acacagacta 120
catcctccct gtctgcctct ctgggagaca gagtcaccat cagttgcagt gcaagtcagg 180
gcattagcaa ttatttaaac tggtatcagc agaaaccaga tggaactgtt aaactcctga 240
tctattacac atcaagttta cactcaggag tcccatcaag gttcagtggc agtgggtctg 300
ggacagatta ttctctcacc atcagcaacc tggaacctga agatattgcc acttactatt 360
gtcagcagta tagtaagctt cctccgtgga cgttcggtgg aggcaccaag ctggaaatca 420
aacgtacg 428
<210> 15
<211> 9608
<212> DNA
<213> 人工
<223> 中间载体pTrioz-L-Furin-DTA-6 X His
<400> 15
cctgcaggcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 60
cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 120
tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 180
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 240
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 300
gctattacca tgatgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 360
tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttgactagtc 420
agggccccaa cccccccaag cccccatttc acaacacgct ggcgctacag gcgcgtgact 480
tccccttgct ttggggcggg gggctgagac tcctatgtgc tccggattgg tcaggcacgg 540
ccttcggccc cgcctcctgc caccgcagat tggccgctag gcctccccga gcgccctgcc 600
tccgagggcc ggcgcaccat aaaagaagcc gccctagcca cgtcccctcg cagttcggcg 660
gtcccgcggg tctgtctcaa gcttgccgcc agaacacagg taagtgccgt gtgtggttcc 720
cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccatgccc 780
ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 840
gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcttgggcg 900
ctggggccgc cgcgtgctaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgctaa 960
gtctctagcc atttaaaatt tttgataacc agctgcgacg ctttttttct ggcgagatag 1020
tcttgtaaat gcgggccagg atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 1080
cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 1140
accgagaatc ggacgggggt agtctcaaac tggccggcct gctctggtgc ctggcctcgc 1200
gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 1260
agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 1320
cccgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cttcctcatc 1380
cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttgtc 1440
gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 1500
ccacactgag tgggtggaga ctgaagagtt aggccagctt ggcacttgat gtaattctcc 1560
ttggaatttg ccctttttga gtttggatct tgcctcattc tcaagcctca gacagtggtt 1620
caaagttttt ttcttccatt tcaggtgtcg tgaaaactac ccctaaaagc caccggcgag 1680
gcgcgccaag tttaaacacc atggcaatga tgtcctctgc tcagttcctt ggtctcctgt 1740
tgctctgttt tcaaggtaaa attgactata atgggaattt tgctgttgca cggtgtgata 1800
tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga gtcaccatca 1860
gttgcagtgc aagtcagggc attagcaatt atttaaactg gtatcagcag aaaccagatg 1920
gaactgttaa actcctgatc tattacacat caagtttaca ctcaggagtc ccatcaaggt 1980
tcagtggcag tgggtctggg acagattatt ctctcaccat cagcaacctg gaacctgaag 2040
atattgccac ttactattgt cagcagtata gtaagcttcc tccgtggacg ttcggtggag 2100
gcaccaagct ggaaatcaaa cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat 2160
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat aacttctatc 2220
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg 2280
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc accctgacgc 2340
tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc catcagggcc 2400
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttagagggac ctagctggcc 2460
agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2520
atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2580
taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2640
ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggaaatgt taattaacta 2700
gccatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 2760
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2820
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2880
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 2940
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 3000
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 3060
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 3120
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 3180
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 3240
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 3300
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 3360
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 3420
cacatgttct taattaacct gcaggcgaaa ctcagcttcc ttcgtttccg acttttccat 3480
ccgcgtcctc cacttccccg ttccgccctc ccccattgcc aacattctgg ctgagtcacg 3540
gcgccccaga gcgcgccagg ctgggggaaa ggagcagaag ggagggccct agcgacccgc 3600
gggatgtggt ccgagtcacg tccgaggggg gtggggaggg atcgtgttct cggcgcccgc 3660
cccttcctag cgcggcctct gggctgcgcc tctcgggggc ggcccgtagc ccagtccgtc 3720
gactagttcc gccagagcgc gcgagggcct ccagcggccg cccctccccc acagcagggg 3780
cggggtcccg cgctcaccgg aaggagcggg ctcggggcgg gcggcgctga ttggccgggg 3840
cgggcctgac gccgacgcgg ctataagaga ccacaagcga cccgcagggc cagacgttct 3900
tcgccgaagc ttgccgtcag aacgcaggtg aggggcgggt gtggcttccg cgggccgccg 3960
agctggaggt cctgctccga gcgggccggg ccccgctgtc gtcggcgggg attagctgcg 4020
agcattcccg cttcgagttg cgggcggcgc gggaggcaga gtgcgaggcc tagcggcaac 4080
cccgtagcct cgcctcgtgt ccggcttgag gcctagcgtg gtgtccgcgc cgccgccgcg 4140
tgctactccg gccgcactct ggtctttttt ttttttgttg ttgttgccct gctgccttcg 4200
attgccgttc agcaataggg gctaacaaag ggagggtgcg gggcttgctc gcccggagcc 4260
cggagaggtc atggttgggg aggaatggag ggacaggagt ggcggctggg gcccgcccgc 4320
cttcggagca catgtccgac gccacctgga tggggcgagg cctggggttt ttcccgaagc 4380
aaccaggctg gggttagcgt gccgaggcca tgtggcccca gcacccggca cgatctggct 4440
tggcggcgcc gcgttgccct gcctccctaa ctagggtgag gccatcccgt ccggcaccag 4500
ttgcgtgcgt ggaaagatgg ccgctcccgg gccctgttgc aaggagctca aaatggagga 4560
cgcggcagcc cggtggagcg ggcgggtgag tcacccacac aaaggaagag ggcctggtcc 4620
ctcaccggct gctgcttcct gtgaccccgt ggtcctatcg gccgcaatag tcacctcggg 4680
cttttgagca cggctagtcg cggcgggggg aggggatgta atggcgttgg agtttgttca 4740
catttggtgg gtggagacta gtcaggccag cctggcgctg gaagtcattt ttggaatttg 4800
tccccttgag ttttgagcgg agctaattct cgggcttctt agcggttcaa aggtatcttt 4860
taaacccttt tttaggtgtt gtgaaaacca ccgctaattc aaagcaaccg gtcgcacgcg 4920
tagatatccg tcatgaaagc tagcaccaag ggcccatcgg tcttccccct ggcaccctcc 4980
tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 5040
gagccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 5100
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 5160
agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 5220
gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 5280
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 5340
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 5400
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 5460
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 5520
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 5580
atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 5640
cccccatccc gggacgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 5700
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 5760
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 5820
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 5880
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaagc aggaaatcgt 5940
gtcaggcgat cagtaggtag ctcattgtca ggcgctgatg atgttgttga ttcttctaaa 6000
tcttttgtga tggaaaactt ttcttcgtac cacgggacta aacctggtta tgtagattcc 6060
attcaaaaag gtatacaaaa gccaaaatct ggtacacaag gaaattatga cgatgattgg 6120
aaagggtttt atagtaccga caataaatac gacgctgcgg gatactctgt agataatgaa 6180
aacccgctct ctggaaaagc tggaggcgtg gtcaaagtga cgtatccagg actgacgaag 6240
gttctcgcac taaaagtgga taatgccgaa actattaaga aagagttagg tttaagtctc 6300
actgaaccgt tgatggagca agtcggaacg gaagagttta tcaaaaggtt cggtgatggt 6360
gcttcgcgtg tagtgctcag ccttcccttc gctgagggga gttctagcgt ttcctatatt 6420
aataactggg aacaggcgaa agcgttaagc gtagaacttg agattaattt tgaaacccgt 6480
ggaaaacgtg gccaagatgc gatgtatgag tatatggctc aagcctgtgc aggaaatcgt 6540
gtcaggcgat cagtaggtag ctcattgtca tgcataaatc ttgattggga tgtcataagg 6600
gataaaacta agacaaagat agagtctttg aaagagcatg gccctatcaa aaataaaatg 6660
agcgaaagtc ccaataaaac agtatctgag gaaaaagcta aacaatacct agaagaattt 6720
catcaaacgg cattagagca tcctgaattg tcagaactta aaaccgttac tgggaccaat 6780
cctgtattcg ctggggctaa ctatgcggcg tgggcagtaa acgttgcgca agttatagat 6840
ctcgaaacag ctgataattt ggaaaagaca actgctgctc tttcgatact tcctggtatc 6900
ggtagcgtaa tgggcattgc agacggtgcc gttcaccaca atacagaaga gatagtggca 6960
caatcaatag ctttatcgtc tttaatggtt gctcaagcta ttccattggt aggagagcta 7020
gttgatattg gtttcgctgc atataatttt gtagagagta ttatcaattt atttcaagta 7080
gttcataatt cgtataatcg tcccgcgtat tctccggggc ataaaacgca accatttctt 7140
catcaccatc atcaccaccc taggagcagg tttccccaat gacacaaaac gtgcaacttg 7200
aaactccgcc tggtctttcc aggtctagaa gctcgctttc ttgctgtcca atttctatta 7260
aaggttcctt tgttccctaa gtccaactac taaactgggg gatattatga agggccttga 7320
gcatctggat tctgcctaat aaaaaacatt tattttcatt gcaatgatgt atttaaatta 7380
tttctgaata ttttactaaa aagggaatgt gggaggtcag tgcatttaaa acataaagaa 7440
atgaagagct agttcaaacc ttgggaaaat acactatatc ttaaactcca tgaaagaagg 7500
tgaggctgca aacagctaat gcacattggc aacagcccct gatgcctatg ccttattcat 7560
ccctcagaaa aggattcaag tagaggcttg atttggaggt taaagttttg ccatgctgta 7620
ttttagaatt cctgcaggag tcaatgggaa aaacccattg gagccaagta cactgactca 7680
atagggactt tccattgggt tttgcccagt acataaggtc aatagggggt gagtcaacag 7740
gaaagtccca ttggagccaa gtacattgag tcaataggga ctttccaatg ggttttgccc 7800
agtacataag gtcaatggga ggtaagccaa tgggtttttc ccattactga catgtatact 7860
gagtcattag ggactttcca atgggttttg cccagtacat aaggtcaata ggggtgaatc 7920
aacaggaaag tcccattgga gccaagtaca ctgagtcaat agggactttc cattgggttt 7980
tgcccagtac aaaaggtcaa tagggggtga gtcaatgggt ttttcccatt attggcacat 8040
acataaggtc aataggggtg actagtcagt gggcagagcg cacatcgccc ccgagaagtt 8100
ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg tggcgcgggg taaactggga 8160
aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag 8220
tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa 8280
gcttcgaggg gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc 8340
acgccggttg agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc 8400
cgtctaggta agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag 8460
cctacctaga ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt 8520
ctttgtttcg ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctacaaa 8580
cagtagttga caattaatca tcggcatagt atatcggcat agtataatac gactcactat 8640
aggagggcca tcatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 8700
gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 8760
gacttcgctg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 8820
gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 8880
gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 8940
atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 9000
cactttgtgg cagaggagca ggactaaatc tagaattatc cctaatacct gccaccccac 9060
tcttaatcag tggtggaaga acggtctcag aactgtttgt ttcaattggc catttaagtt 9120
tagtagtaaa agactggtta atgataacaa tgcatcgtaa aaccttcaga aggaaaggag 9180
aatgttttgt ggaccacttt ggttttcttt tttgcgtgtg gcagttttaa gttattagtt 9240
tttaaaatca gtacttttta atggaaacaa cttgaccaaa aatttgtcac agaattttga 9300
gacccattaa aaaagttaaa tgagaaacct gtgtgttcct ttggtcaaca ccgagacatt 9360
taggtgaaag acatctaatt ctggttttac gaatctggaa acttcttgaa aatgtaattc 9420
ttgagttaac acttctgggt ggagaatagg gttgttttcc ccccacataa ttggaagggg 9480
aaggaatatc atttaaagct atgggagggt tgctttgatt acaacactgg agagaaatgc 9540
agcatgttgc tgattgcctg tcactaaaac aggccaaaaa ctgagtcctt gggttgcata 9600
gaaagctg 9608
<210> 16
<211> 377
<212> DNA
<213> 人工
<223> 基因片段CD45 Ab重链
<400> 16
atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taaaattgac 60
tataatggga attttgctgt tgcacggtgt gatatccaga tgacacagac tacatcctcc 120
ctgtctgcct ctctgggaga cagagtcacc atcagttgca gtgcaagtca gggcattagc 180
aattatttaa actggtatca gcagaaacca gatggaactg ttaaactcct gatctattac 240
acatcaagtt tacactcagg agtcccatca aggttcagtg gcagtgggtc tgggacagat 300
tattctctca ccatcagcaa cctggaacct gaagatattg ccacttacta ttgtcagcag 360
tatagtaagc ttcctcc 377
<210> 17
<211> 31
<212> DNA
<213> 人工
<223> CD45 Ab-H-F-EcoRV
<400> 17
gatatcatga gcgtgctgat tcttttgtgg c 31
<210> 18
<211> 26
<212> DNA
<213> 人工
<223> 引物CD45 Ab-H-R-NheI
<400> 18
gctagctact ggtacttcga tgtctg 26
<210> 19
<211> 432
<212> DNA
<213> 人工
<223> 基因片段CD45 Ab H
<400> 19
gatatcatga gcgtgctgat tcttttgtgg ctgttcacag cctttcctgg tgagtgtatc 60
ctgtctgatg tgcagcttca ggagtcagga cctggcctgg tgaaaccttc tcagtcactt 120
tccctcacct gcactgtcac tggctactcc atcaccagtg gttatgactg gaactggatc 180
cggcagtttc caggaaacaa actggagtgg atgggctaca taagctacag tgtagcaagt 240
actacaaccc atctctcaaa agtcgaatct ctatcactcg agacacatcc aagaaccagt 300
tctccctgga atattctgct actgaggaca cagccacata ttactgtgca agaggtagct 360
actaccgtag taataaacgg agacggtgac cgtggtccct gtgccccaga catcgaagta 420
ccagtagcta gc 432
<210> 20
<211> 22
<212> DNA
<213> 人工
<223> 引物Dph7-gRNA-F
<400> 20
tgttgatgct tctctttctc ca 22
<210> 21
<211> 20
<212> DNA
<213> 人工
<223> 引物Dph7-gRNA-R
<400> 21
tctgaaccag tgtccagcac 20
<210> 22
<211> 10847
<212> DNA
<213> 人工
<223> 基因工程质粒pX260a-GPF-Dph 7
<400> 22
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggttttagag ctatgctgtt ttgaatggtc ccaaaactgt tgatgcttct 300
ctttctccag ttttagagct atgctgtttt gaatggtccc aaaacttttt ctagcgcgtg 360
cgccaattct gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc 420
ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg 480
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 540
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 600
tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 660
ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc 720
tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 780
atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg 840
ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt 900
ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 960
ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 1020
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1080
tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt 1140
attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt 1200
gccaccatgg actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa 1260
gacgatgacg ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca 1320
gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc 1380
gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac 1440
cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc 1500
gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc 1560
tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac 1620
agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc 1680
ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga 1740
aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc 1800
cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc 1860
gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac 1920
cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc 1980
agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga 2040
aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc 2100
gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg 2160
gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc 2220
atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc 2280
tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg 2340
cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac 2400
gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc 2460
ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg 2520
cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg 2580
cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag 2640
atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac 2700
agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag 2760
gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat 2820
aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc 2880
gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc 2940
ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg 3000
accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa 3060
atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa 3120
attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc 3180
gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat 3240
gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg 3300
ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc 3360
ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac 3420
gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc 3480
ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag 3540
acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc 3600
gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag 3660
agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac 3720
cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg 3780
cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac 3840
catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga 3900
agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg 3960
aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat 4020
ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga 4080
cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg 4140
aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag 4200
tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac 4260
aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa 4320
aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg 4380
aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac 4440
agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag 4500
cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat 4560
tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag 4620
gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg 4680
atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg 4740
gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt 4800
gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc 4860
gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct 4920
aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa 4980
ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc 5040
agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg 5100
gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga 5160
gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat 5220
aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga 5280
gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc 5340
aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg 5400
atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag 5460
gcaaaaaaga aaaagtaaga attcgcccct ctccctcccc cccccctaac gttactggcc 5520
gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc 5580
cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta 5640
ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag 5700
ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga 5760
accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg 5820
caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 5880
ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 5940
tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa 6000
acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata 6060
tggccacaac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 6120
tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 6180
atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 6240
cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 6300
accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 6360
gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 6420
gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 6480
tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 6540
agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 6600
tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 6660
ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 6720
atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 6780
tgtacaagta agaattccga tcatattcaa taacccttaa tataacttcg tataatgtat 6840
gctatacgaa gttattaggt ctgaagagga gtttacgtcc agccaagctt aggatctcga 6900
cctcgaaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt 6960
tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca 7020
catccaccgg tagcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac 7080
tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca 7140
aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa 7200
gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca 7260
gcagctggga agggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc 7320
gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg 7380
ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga tctcgagcag 7440
ctgaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 7500
cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 7560
gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 7620
gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 7680
accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 7740
gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 7800
cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 7860
aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 7920
cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 7980
accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 8040
cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc 8100
catgcatcga tgatatcaga tccccgggat gcagaaattg atgatctatt aaacaataaa 8160
gatgtccact aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag 8220
tacctacatt ttgaatggaa ggattggagc ggcgcctgat gcggtatttt ctccttacgc 8280
atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 8340
cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 8400
cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 8460
ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 8520
cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 8580
ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 8640
tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 8700
ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 8760
aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 8820
gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 8880
cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 8940
ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 9000
ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 9060
gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 9120
acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 9180
tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 9240
agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 9300
cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 9360
aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 9420
gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 9480
agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 9540
aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 9600
gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 9660
ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 9720
aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 9780
aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 9840
tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 9900
agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 9960
ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 10020
ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 10080
ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 10140
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 10200
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 10260
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 10320
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 10380
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 10440
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 10500
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 10560
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 10620
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 10680
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 10740
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 10800
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt 10847
<210> 23
<211> 25
<212> DNA
<213> 人工
<223> 引物Dph7-Screen-F
<400> 23
cttcctcaca gacagagtca cccag 25
<210> 24
<211> 22
<212> DNA
<213> 人工
<223> 引物Dph7-Screen-R
<400> 24
cgagaagggc ccgagagcct cc 22
<210> 25
<211> 908
<212> DNA
<213> 人工
<223> 基因片段Dph7 screen
<400> 25
cttcctcaca gacagagtca cccaggggag cttcctcaca gacagagtca cccaggggag 60
cttcctcaca gacagagtca tccaggggag cttcctcaca gacagagtca cccaggggag 120
cttcctcaca gacagagtca cccaggggag cttcctcaca gacagagtca cccaggggag 180
cttcctcaca gacagagtca cccaggggag cttcctcaca gacagagtca cccaggggag 240
cttcctcaca gacagagtca ccccggggag cttcctcaca gacagtcacc caggggagct 300
tcctcacaga cagtcaccca ggggagcttc ctcacagaca gagtcatcca ggggagcttc 360
ctcacagaca gagtcaccca ggggagcttc ctcacagaca gagtcaccca ggggagcttc 420
ctcacagaca gtcaccccgg ggagcttcct cacagacagt cacccagggg agcttcctca 480
cagacagagt caccccgggg agcttcctca cagacagtca ccccggggag cttcctcaca 540
gacatcaacg gcctaactga gaacactcat ttcctgtcat tagtggtgtt gatgcttctc 600
tttctccaca cagggggcga cgatggcctt ctgaggggct gggacaccag ggtacccggc 660
aaatttctct tcaccagcaa aaggtaactg cccaagggcc agcactcggt gcccacacca 720
gcccccgcgt tccctgggtc accagccctc ccgttccctg ggtgggtctg tgtgctggac 780
actggttcag aagccgcctc tgcttgcaga cacaccatgg gtgtgtgcag catccagagc 840
agccctcatc gggagcacat cctggccacg ggaaggtgag tccccaggag gctctcgggc 900
ccttctcg 908
<210> 26
<211> 41
<212> DNA
<213> 人工
<223> 引物DTA-F-BamHI
<400> 26
accaccggat ccatgggcgc tgatgatgtt gttgattctt c 41
<210> 27
<211> 34
<212> DNA
<213> 人工
<223> 引物DTA-R(EcoRI)
<400> 27
accaccgaat tcttagtggg aattagtcat gccc 34
<210> 28
<211> 1200
<212> DNA
<213> 白喉杆菌
<223> 基因片段DTA
<400> 28
accaccggat ccatgggcgc tgatgatgtt gttgattctt ctaaatcttt tgtgatggaa 60
aacttttctt cgtaccacgg gactaaacct ggttatgtag attccattca aaaaggtata 120
caaaagccaa aatctggtac acaaggaaat tatgacgatg attggaaagg gttttatagt 180
accgacaata aatacgacgc tgcgggatac tctgtagata atgaaaaccc gctctctgga 240
aaagctggag gcgtggtcaa agtgacgtat ccaggactga cgaaggttct cgcactaaaa 300
gtggataatg ccgaaactat taagaaagag ttaggtttaa gtctcactga accgttgatg 360
gagcaagtcg gaacggaaga gtttatcaaa aggttcggtg atggtgcttc gcgtgtagtg 420
ctcagccttc ccttcgctga ggggagttct agcgtttcct atattaataa ctgggaacag 480
gcgaaagcgt taagcgtaga acttgagatt aattttgaaa cccgtggaaa acgtggccaa 540
gatgcgatgt atgagtatat ggctcaagcc tgtgcaggaa atcgtgtcag gcgatcagta 600
ggtagctcat tgtcatgcat aaatcttgat tgggatgtca taagggataa aactaagaca 660
aagatagagt ctttgaaaga gcatggccct atcaaaaata aaatgagcga aagtcccaat 720
aaaacagtat ctgaggaaaa agctaaacaa tacctagaag aatttcatca aacggcatta 780
gagcatcctg aattgtcaga acttaaaacc gttactggga ccaatcctgt attcgctggg 840
gctaactatg cggcgtgggc agtaaacgtt gcgcaagtta tagatctcga aacagctgat 900
aatttggaaa agacaactgc tgctctttcg atacttcctg gtatcggtag cgtaatgggc 960
attgcagacg gtgccgttca ccacaataca gaagagatag tggcacaatc aatagcttta 1020
tcgtctttaa tggttgctca agctattcca ttggtaggag agctagttga tattggtttc 1080
gctgcatata attttgtaga gagtattatc aatttatttc aagtagttca taattcgtat 1140
aatcgtcccg cgtattctcc ggggcataaa acgcaaccat ttctttaaga attcggtggt 1200
<210> 29
<211> 7185
<212> DNA
<213> 人工
<223> TR细胞株检测载体pMyc-DTA-IRES-GFP
<400> 29
ctgcataatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 60
caaggcatgg aaaaatacat aactgagaat agaaaagttc agatcaaggt caggaacaga 120
tggaacagct gaatatgggc caaagcggat atctgtggta agcagttcct gccccggctc 180
agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag 240
ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca 300
gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc 360
cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg 420
agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt 480
cgcccgggta cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc 540
gctgatcctt gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt 600
tggaggttcc accgagattt ggagacccca gcccagggac caccgacccc cccgccggga 660
ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc 720
atctaatgtt tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc 780
cgtggtggaa ctgacgagtt cggaacaccc ggccgcaacc ctgggagacg tcccagggac 840
ttcgggggcc gtttttgtgg cccgacctga gtccaaaaat cccgatcgtt ttggactctt 900
tggtgcaccc cccttagagg agggatatgt ggttctggta ggagacgaga acctaaaaca 960
gttcccgcct ccgtctgaat ttttgctttc ggtttgggac cgaagccgcg ccgcgcgtct 1020
tgtctgctgc agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt atttgtctga 1080
aaatatgggc ccgggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa 1140
agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac 1200
cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa 1260
ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc 1320
agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt 1380
caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc 1440
ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc 1500
tctaggcgcc cccatatggc catatgagat cttatatggg gcacccccgc cccttgtaaa 1560
cttccctgac cctgacatga caagagttac taacagcccc tctctccaag ctcacttaca 1620
ggctctctac ttagtccagc acgaagtctg gagacctctg gcggcagcct accaagaaca 1680
actggaccga ccggtggtac ctcaccctta ccgagtcggc gacacagtgt gggtccgccg 1740
acaccagact aagaacctag aacctcgctg gaaaggacct tacacagtcc tgctgaccac 1800
ccccaccgcc ctcaaagtag acggcatcgc agcttggata cacgccgccc acgtgaaggc 1860
tgccgacccc gggggtggac catcctctag actgccggat ccaccaccgg atccatgggc 1920
gctgatgatg ttgttgattc ttctaaatct tttgtgatgg aaaacttttc ttcgtaccac 1980
gggactaaac ctggttatgt agattccatt caaaaaggta tacaaaagcc aaaatctggt 2040
acacaaggaa attatgacga tgattggaaa gggttttata gtaccgacaa taaatacgac 2100
gctgcgggat actctgtaga taatgaaaac ccgctctctg gaaaagctgg aggcgtggtc 2160
aaagtgacgt atccaggact gacgaaggtt ctcgcactaa aagtggataa tgccgaaact 2220
attaagaaag agttaggttt aagtctcact gaaccgttga tggagcaagt cggaacggaa 2280
gagtttatca aaaggttcgg tgatggtgct tcgcgtgtag tgctcagcct tcccttcgct 2340
gaggggagtt ctagcgtttc ctatattaat aactgggaac aggcgaaagc gttaagcgta 2400
gaacttgaga ttaattttga aacccgtgga aaacgtggcc aagatgcgat gtatgagtat 2460
atggctcaag cctgtgcagg aaatcgtgtc aggcgatcag taggtagctc attgtcatgc 2520
ataaatcttg attgggatgt cataagggat aaaactaaga caaagataga gtctttgaaa 2580
gagcatggcc ctatcaaaaa taaaatgagc gaaagtccca ataaaacagt atctgaggaa 2640
aaagctaaac aatacctaga agaatttcat caaacggcat tagagcatcc tgaattgtca 2700
gaacttaaaa ccgttactgg gaccaatcct gtattcgctg gggctaacta tgcggcgtgg 2760
gcagtaaacg ttgcgcaagt tatagatctc gaaacagctg ataatttgga aaagacaact 2820
gctgctcttt cgatacttcc tggtatcggt agcgtaatgg gcattgcaga cggtgccgtt 2880
caccacaata cagaagagat agtggcacaa tcaatagctt tatcgtcttt aatggttgct 2940
caagctattc cattggtagg agagctagtt gatattggtt tcgctgcata taattttgta 3000
gagagtatta tcaatttatt tcaagtagtt cataattcgt ataatcgtcc cgcgtattct 3060
ccggggcata aaacgcaacc atttctttaa gaattcctgc aggcctcgag gcggccgcta 3120
cgtaaattcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa 3180
taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 3240
gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct 3300
ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 3360
tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 3420
gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 3480
ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 3540
gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 3600
gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc 3660
cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg 3720
gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 3780
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 3840
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 3900
gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 3960
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 4020
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 4080
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 4140
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 4200
atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 4260
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 4320
ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 4380
ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc 4440
ggcccagcca cagtggtcga ccgggccgcc agcacagtgg gggggggccc gcgattagtc 4500
caatttgtta aagacaggat atcagtggtc caggctctag ttttgactca acaatatcac 4560
cagctgaagc ctatagagta cgagccatag atagaataaa agattttatt tagtctccag 4620
aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta agtaagccat 4680
tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggttaggaa 4740
cagagagaca ggagaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 4800
ctcagggcca agaacagttg gaacagcaga atatgggcca aacaggatat ctgtggtaag 4860
cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca 4920
gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg 4980
tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc 5040
ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg 5100
cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc 5160
tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc 5220
attctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 5280
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 5340
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 5400
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 5460
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 5520
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 5580
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 5640
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 5700
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 5760
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 5820
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 5880
aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 5940
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 6000
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 6060
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6120
atgagattat caaaaaggat cttcacctag atccttttgc ggccggccgc aaatcaatct 6180
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 6240
tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 6300
ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 6360
gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 6420
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 6480
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 6540
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 6600
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 6660
tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 6720
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 6780
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata 6840
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 6900
aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 6960
actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 7020
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 7080
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 7140
aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttc 7185
<210> 30
<211> 22
<212> DNA
<213> 人工
<223> Dph7 gRNA序列
<400> 30
tgttgatgct tctctttctc ca 22

Claims (6)

1. 靶向CD45的抗体药物偶联物，其特征在于，所述抗体药物偶联物为CD45 Ab-DTA重组蛋白，其氨基酸序列如SEQ ID NO.3所示。
2.包括如权利要求1所述的靶向CD45的抗体药物偶联物的药物组合物。
3. 表达如权利要求1所述的靶向CD45的抗体药物偶联物的生产载体，其特征在于，所述生产载体包括编码以下部分氨基酸的核酸序列：靶向CD45抗体轻链、靶向CD45抗体重链、白喉毒素、白喉毒素与重链之间由Furin蛋白剪切酶识别位点的氨基酸以及6 X His；
所述生产载体为pTrioz-CD45 Ab-Furin-DTA-6 X His，其序列为SEQ ID NO. 2。
4.构建如权利要求3所述的生产载体的方法，其特征在于，包括以下步骤：
(1)以pTrioz空白质粒为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQID NO.5所示的反向引物进行PCR，扩增出序列如SEQ ID NO.6所示的基因片段IgHC；
(2) 以pIRES-proHB EGF WT质粒为模板，利用序列如SEQ ID NO.7所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行PCR，扩增出序列如SEQ ID NO.9所示的基因片段DTA；
(3) 以步骤（1）和（2）得到的基因片段IgHC与DTA为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行Overlap PCR得到序列如SEQ IDNO.10所示的融合基因片段IgHC-Furin-DTA-6Xhis；
(4) 将目的基因片段IgHC-Furin-DTA-6XHis利用酶切位点NheI、AvrII克隆至载体pTrioz，得到载体pTrioz-Furin-DTA-6 X His，如 SEQ ID NO.1所示；
(5) 合成如SEQ ID NO.11所示序列CD45 Ab轻链；以合成产物为模板，利用序列如SEQID NO.12所示的正向引物和序列如SEQ ID NO.13所示的反向引物进行PCR，扩增出序列如SEQ ID NO.14所示的基因片段CD45 Ab L；将PCR产物利用酶切位点NcoI、BsiWI克隆至载体pTrioz-Furin-DTA-6 X His，得到载体pTrioz-L-Furin-DTA-6 X His,如 SEQ ID NO.15所示；
(6) 合成如SEQ ID NO.16所示序列CD45 Ab重链；以合成产物为模板，利用序列如SEQID NO.17所示的正向引物和序列如SEQ ID NO.18所示的反向引物进行PCR，扩增出序列如SEQ ID NO.19所示的基因片段CD45 Ab H；将PCR产物利用酶切位点NheI、EcoRV克隆至载体pTrioz-L-Furin-DTA-6 X His，得到生产载体pTrioz-CD45 Ab-Furin-DTA-6 X His,如SEQ ID NO. 2所示。
5. 利用如权利要求4所述的生产载体来制备CD45 Ab-DTA重组蛋白的方法，其特征在于，所述CD45 Ab-DTA重组蛋白的氨基酸序列如SEQ ID NO.3所示，所述方法包括以下步骤：
(1) 培养TR 细胞；
(2) 将pTrioz-CD45 Ab-Furin-DTA-6 X His生产载体转染至TR 细胞；
(3) 培养转染后的TR 细胞并收取CD45 Ab DTA重组蛋白。
6.如权利要求1所述的靶向CD45的抗体药物偶联物在制备用于清除正常、病变、癌变的免疫细胞方面的药物、制备治疗血液系统疾病方面的药物、制备辅助自体、异体骨髓移植方面的药物、制备治疗免疫系统疾病方面的药物中的应用；所述免疫系统疾病包括但不限于艾滋病。
CN202010438614.3A 2020-05-22 2020-05-22 靶向cd45的抗体药物偶联物及其制备和应用 Active CN111617261B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010438614.3A CN111617261B (zh) 2020-05-22 2020-05-22 靶向cd45的抗体药物偶联物及其制备和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010438614.3A CN111617261B (zh) 2020-05-22 2020-05-22 靶向cd45的抗体药物偶联物及其制备和应用

Publications (2)

Publication Number Publication Date
CN111617261A CN111617261A (zh) 2020-09-04
CN111617261B true CN111617261B (zh) 2023-08-29

Family

ID=72267879

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010438614.3A Active CN111617261B (zh) 2020-05-22 2020-05-22 靶向cd45的抗体药物偶联物及其制备和应用

Country Status (1)

Country Link
CN (1) CN111617261B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113144214A (zh) * 2021-03-30 2021-07-23 乾元康安(苏州)生物科技有限公司 一种通过化学偶联构建的靶向cd45的新型抗体药物的合成与应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108137648A (zh) * 2015-04-06 2018-06-08 哈佛学院校长同事会 用于非清髓性预处理的组合物和方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108137648A (zh) * 2015-04-06 2018-06-08 哈佛学院校长同事会 用于非清髓性预处理的组合物和方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
唐晓文等.急性髓细胞白血病的治疗进展.《国外医学输血及血液学分册》.2002,第25卷(第25期),第17-21页. *

Also Published As

Publication number Publication date
CN111617261A (zh) 2020-09-04

Similar Documents

Publication Publication Date Title
EP2707025B1 (en) Hendra and nipah virus g glycoprotein immunogenic compositions
KR102272932B1 (ko) 이종 유전자로 무장된 종양살상 아데노바이러스
Milich et al. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero?
AU2014217569B2 (en) Cetuximab with modified glycosylation and uses thereof
KR20140095556A (ko) 사이토메갈로바이러스의 치료를 위한 조성물 및 방법
HU229222B1 (en) Hbv core antigen particles with multiple immunogenic components attached via peptide ligands
CN101107005A (zh) 用于预防和治疗补体相关紊乱的CRIg多肽
KR20110084280A (ko) 종양 항원의 생물 활성을 특이적으로 차단하는 항체
KR20200115525A (ko) 그룹 b 아데노바이러스-함유 제형
CN111617261B (zh) 靶向cd45的抗体药物偶联物及其制备和应用
KR102140984B1 (ko) 신장 결합 항원 １에 대한 항체 및 이의 항원 결합 단편
JPS6269991A (ja) 牛下痢症ウイルス由来のワクチンと診断薬
Enjuanes et al. Antigenic specificities of the cellular immune response of C57BL/6 mice to the Moloney leukemia/sarcoma virus complex
KR102102239B1 (ko) 유방암을 치료하기 위한 방법
CN111770767B (zh) 用于从生物流体中去除纤溶蛋白的体外设备和基质、其方法和用途
KR102230593B1 (ko) 코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물
US20230082323A1 (en) METHODS, COMPLEXES AND KITS FOR DETECTING OR DETERMINING AN AMOUNT OF A ß CORONAVIRUS ANTIBODY IN A SAMPLE
Liu et al. Protective mcmv immunity by vaccination of the salivary gland via wharton's duct: Replication-deficient recombinant adenovirus expressing individual mcmv genes elicits protection similar to that of mcmv
AU2013262426B2 (en) Cellular vaccine and method of inducing an immune response in a subject
CN115335390A (zh) 基于SARS-CoV-2的S蛋白的疫苗和组合物
JP2003192616A (ja) 歯周病用ｄｎａワクチン
EP4000633A1 (en) Neutralizing antibody for tooth regeneration treatment targeting usag-1 molecule
Hillman et al. A polymer containing a repeating peptide sequence can stimulate T-cell-independent IgG antibody production in vivo
CN110317264B (zh) 一种抗肿瘤蛋白内皮抑素的衍生物
US20140199344A1 (en) Anti-measles cancer immunotherapy

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant