CN109666698B - 一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法 - Google Patents

一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法 Download PDF

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CN109666698B
CN109666698B CN201811653268.XA CN201811653268A CN109666698B CN 109666698 B CN109666698 B CN 109666698B CN 201811653268 A CN201811653268 A CN 201811653268A CN 109666698 B CN109666698 B CN 109666698B
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范春雷
吴王亲
武虎
匡红
刘美星
莫一平
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Abstract

本发明建立了一种基于Tim‑3/Galectin‑9阻断功能及其生物效应的药物快速筛选方法：将红色荧光蛋白tagRFP偶联于Galectin‑9蛋白羧基末端(C端)，构建真核表达载体，并制备Galectin‑9‑tagRFP融合蛋白；将Ceacam‑1构建在真核表达载体的CMV启动子下游，黄色荧光蛋白基因Ypet构建在Tim‑3蛋白的下游，以及青色荧光蛋白基因CyPet通过一个连接肽(G4S)3构建在Bat3基因的下游，分别构建真核表达载体，并获得新型的稳转细胞系Ceacam‑1.Tim‑3Ypet.Bat3CyPet/Jurkat，以Galectin‑9‑tagRFP与细胞系的亲和进行荧光检测筛选阻断剂。本方法可精确反映抗Tim‑3和抗Galectin‑9药的效应，可同时获得阻断功能与Tim‑3/Galectin‑9信号通路的生物学效应数据，构建快速筛选抗Tim‑3和抗Galectin‑9药物和评估其生物学效应的实验模型。

Description

一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快 速筛选方法
技术领域
本发明涉及一种基于Tim-3/Galectin-9靶点及其生物效应的药物快速筛选方法，属于生物技术领域。
背景技术
膜型Tim-3分子是一种单跨膜分子，c端位于胞内，胞内域富含酪氨酸；N端位于胞外，胞外域包括IgV亚单位、黏蛋白样结构域，它们共同参与配体的识别。Ceacam-1又称为CD66a(分化簇66a)，人糖蛋白，是癌胚抗原(CEA)基因家族一员。有研究表明T细胞中TIM-3的表达与Ceacam-1的表达具有非常强的相关性。半乳糖凝集素-9(Galectin-9，Gal-9)是Tim-3的配体，也是一种膜蛋白分子，在肿瘤细胞表面往有高表达。Galectin-9与T细胞表面的Tim-3结合，触发胞内的信号通路。Tim-3在非激活状态时，胞内段与Bat3结合而被抑制；当Tim-3胞外域与Galectin-9结合时，Bat3从Tim-3胞内段解离而使Tim-3向胞内转导抑制信号。最终可导致T细胞功能的抑制，如促进性细胞因子产生的减少，T细胞增殖力降低，促进T细胞衰竭和凋亡。Tim-3/Galectin-9信号通路在T细胞活性和数量的调节上起着重要作用。因此，Tim-3和Galectin-9分子与机体的抗肿瘤免疫、抗病毒免疫和自身免疫性疾病关系密切，已成为抗肿瘤药物研究的热门靶点之一。
基于Tim-3/Galectin-9阻断功能的抗癌新药，包括抗Tim-3、抗Galectin-9单抗药、小分子阻断剂、多肽阻断剂等的研发是目前医药领域热点之一。但目前为止尚未见有关基于Tim-3/Galectin-9阻断功能及其生物学效应的新药快速、高效而精准的筛选系统。
发明内容
本发明的目的是建立了一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法。
本发明构思如下：
采用红色荧光蛋白tagRFP偶联于Galectin-9蛋白羧基末端，将该荧光蛋白标记基因构建载体并在细胞内表达，制备融合蛋白。将黄色荧光蛋白基因Ypet构建在Tim-3蛋白的下游，并克隆到真核表达载体CMV启动子下；将青色荧光蛋白基因CyPet通过一个连接肽(G4S)3构建在Bat3基因的下游，并克隆到真核表达载体CMV启动子下；将三个基因质粒共转染到细胞，建立稳转细胞系。将抗Tim-3的单抗药、小分子、或多肽阻断剂等新药与稳转细胞一起共孵育后再与Galectin-9-tagRFP融合蛋白共孵育；或将抗Galectin-9的单抗药、小分子、或多肽阻断剂等新药与Galectin-9-tagRFP融合蛋白共孵育后再与稳转细胞一起共孵育；清洗非特异性结合后检测荧光值。通过检测红色荧光蛋白tagRFP荧光值判断该抗Tim-3药和抗Galectin-9药是否具有Tim-3/Galectin-9阻断功能；通过检测CyPet/Ypet的FRET荧光值并与对照组比较，判断抗Tim-3药和抗Galectin-9药是否具有Tim-3.Bat3信号通路的生物学效应。
为了实现本发明目的，第一方面，本发明提供一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法，包括以下步骤：
1)将第一荧光蛋白与Galectin-9蛋白羧基末端构建重组Galectin-9-第一荧光蛋白融合蛋白；
2)将第二荧光蛋白连与Tim-3蛋白羧基末端，并克隆在真核表达载体的CMV启动子下，构建表达质粒；将Ceacam-1克隆到CMV启动子下，构建表达质粒；
3)将第三荧光蛋白与Bat3蛋白羧基末端，并克隆在真核表达载体的CMV启动子下，构建表达质粒。
4)将CMV-Ceacam-1、Tim-3-第二荧光蛋白和Bat3-第三荧光蛋白质粒共转染到人源T淋巴细胞中，建立稳转细胞系；
5)抗Tim-3的单抗药、小分子、或多肽阻断剂等新药与稳转细胞孵育后再与重组Galectin-9-第一荧光蛋白融合蛋白共孵育，清洗非特异性结合；
6)抗Galectin-9的单抗药、小分子、或多肽阻断剂等新药与重组Galectin-9-第一荧光蛋白融合蛋白共孵育后，与稳转细胞孵育，清洗非特异性结合；
7)分别检测第一荧光蛋白荧光值、和第二荧光蛋白/第三荧光蛋白的FRET荧光值，并判断新药对Tim-3/Galectin-9的阻断功能及对Tim-3.Bat3信号通路产生的生物学效应。
进一步地，第一荧光蛋白是tagRFP红色荧光蛋白；第二荧光蛋白是黄色荧光蛋白Ypet；第三荧光蛋白是青色荧光蛋白CyPet。
进一步地，步骤1)中，将红色荧光蛋白tagRFP基因与人全长Galectin-9基因构建在同一个读码框使红色荧光蛋白tagRFP融合在Galectin-9蛋白C端形成所述Galectin-9-tagRFP融合蛋白，同时在tagRFP的C端接上6个组氨酸His标签，获得表达质粒pCMV-Gal-9tagRFP。
进一步地，步骤1)中，将Galectin-9-tagRFP基因克隆至真核高表达载体CMV启动子下游，获得质粒pCMV-Galectin-9-tagRFP，并转染至人胚肾细胞293，筛选克隆获得高表达Galectin-9-tagRFP融合蛋白的Galectin-9-tagRFP/293细胞系；扩增该Galectin-9-tagRFP/293细胞，裂解后用His亲和法制备纯化重组Galectin-9-tagRFP融合蛋白。
进一步地，步骤2)中，将黄色荧光蛋白Ypet基因与人全长Tim-3基因构建在同一个读码框使黄色荧光蛋白Ypet融合在Tim-3蛋白C端，并克隆在真核表达载体的CMV启动子下，构建表达质粒pCMV-Tim-3Ypet；将Ceacam-1克隆到CMV启动子下，构建表达质粒pCMV-Ceacam-1。
进一步地，步骤3)中，将青色荧光蛋白CyPet基因与Bat3基因构建在同一个读码框使青色荧光蛋白CyPet连于Bat3蛋白C端，并克隆在真核表达载体的CMV启动子下，构建表达质粒pCMV-Bat3CyPet。
进一步地，步骤4)中，将pCMV-Ceacam-1、pCMV-Tim-3Ypet和pCMV-Bat3CyPet质粒分别与慢病毒包装质粒pH1、pH2共转染到慢病毒包装系细胞293V，制备CMV-Tim-3Ypet、CMV-Bat3(G4S)3CyPet、CMV-Ceacam-1慢病毒，并共转染到人急性T淋巴细胞白血病细胞Jurkat中，建立稳转细胞系Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat。
进一步地，步骤5)中，抗Tim-3的单抗药、小分子、或多肽阻断剂等新药与稳转细胞Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat孵育后再与重组Galectin-9-tagRFP融合蛋白共孵育；同时设立重组Galectin-9-tagRFP融合蛋白与稳转细胞直接共孵育的对照组。
进一步地，步骤6)中，抗Galectin-9的单抗药、小分子、或多肽阻断剂等新药与重组Galectin-9-tagRFP融合蛋白共孵育后，与稳转细胞Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat共孵育；同时设立重组Galectin-9-tagRFP融合蛋白与稳转细胞直接共孵育的对照组。
进一步地，步骤7)中，经缓冲液清洗去除非特异性结合后用流式细胞仪、或荧光酶标仪分析细胞表面的红色荧光蛋白tagRFP荧光值以及细胞内的CyPet/Ypet的FRET荧光值。通过药物组荧光值与对照组比较，判断抗Tim-3和抗Galectin-9对Tim-3/Galectin-9的阻断功能。
另一方面，本发明提供一种稳转细胞系，其能够筛选对Tim-3/Galectin-9(Tim-3/Bat3)信号通路具有阻断功能的药物。
进一步地，该稳转细胞系中包含三个独立的均由CMV启动高表达基因，即Ceacam-1、Tim-3-Ypet融合蛋白基因和Bat3-CyPet融合蛋白基因；当Tim-3/Galectin-9(Tim-3/Bat3)信号通路正常连接或被阻断时，能够激发或不激发CyPet/Ypet的FRET荧光信号。
另一方面，本发明提供一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法，包括以下步骤：
1)将红色荧光蛋白tagRFP偶联于Galectin-9蛋白羧基末端(C端)，制备Galectin-9-tagRFP融合蛋白。
2)将人源Ceacam-1基因克隆到真核表达载体CMV启动子下，获得质粒pCMV-Ceacam-1；将黄色荧光蛋白Ypet基因与人全长Tim-3基因构建在同一个读码框使Ypet融合在Tim-3蛋白C端，并克隆在真核表达载体的CMV启动子下，获表达质粒pCMV-Tim-3Ypet；将青色荧光蛋白CyPet基因与Bat3基因构建在同一个读码框使CyPet融合在Bat3蛋白C端，并克隆在真核表达载体的CMV启动子下，获表达质粒pCMV-Bat3CyPet；将pCMV-Ceacam-1、pCMV-Tim-3Ypet和pCMV-Bat3CyPet分别与慢病毒包装质粒pH1、pH2共转染到慢病毒包装系细胞293V，制备CMV-Ceacam-1、CMV-Tim-3Ypet和CMV-Bat3CyPet慢病毒，并共转染到Jurkat细胞，建立Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat稳转细胞系。
3)将抗Tim-3的单抗药、小分子、或多肽阻断剂等新药与稳转细胞Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat孵育后再与重组Galectin-9-tagRFP融合蛋白共孵育；同时设立重组Galectin-9-tagRFP融合蛋白与稳转细胞直接共孵育的对照组。
4)将抗Galectin-9的单抗药、小分子、或多肽阻断剂等新药与重组Galectin-9-tagRFP融合蛋白共孵育后，与稳转细胞Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat共孵育；同时设立重组Galectin-9-tagRFP融合蛋白与稳转细胞直接共孵育的对照组。
5)清洗去除非特异性结合后，检测细胞表面的红色荧光蛋白tagRFP荧光值；以及细胞内CyPet/Ypet的FRET荧光值，并与对照组比较。
6)依据荧光值判断抗Tim-3和抗Galectin-9新药对Tim-3/Galectin-9信号通路的阻断作用。
进一步地，编码Galectin-9-tagRFP融合蛋白的核酸序列如SEQ ID NO：1所示，表达后Galectin-9-tagRFP融合蛋白的氨基酸序列如SEQ ID NO：2所示；
编码Tim-3-Ypet融合蛋白的DNA基因序列如SEQ ID NO：3所示，表达后Tim-3-Ypet融合蛋白的氨基酸序列如SEQ ID NO：4所示；
编码Bat3-CyPet融合蛋白的DNA基因序列如SEQ ID NO：5所示，表达后Bat3-CyPet融合蛋白的氨基酸序列如SEQ ID NO：6所示。
借由上述技术方案，本发明至少具有下列优点及有益效果：
(1)快速简便、低成本，系统除了必要的阻断效应实验步骤外无需另外处理，如用荧光素酶的话需裂解细胞并要用昂贵的荧光素酶试剂盒。本发明的方法可直接用活细胞，即测即得，并可进行动态监测。
(2)可用通用的流式细胞仪、荧光酶标仪等方法测定，适用绝大多数普通实验室。
(3)系统可精确反映抗Tim-3和抗Galectin-9药的效应。
(4)使用活细胞检测，一个样本可同时获得Tim-3/Galectin-9的阻断功能与Tim-3/Galectin-9(Tim-3/Bat3)信号通路的生物学效应数据，为快速筛选抗Tim-3和抗Galectin-9药物和评估其生物学效应提供了一个新型强有力的系统和实验模型。
附图说明
图1pCMV-Gal-9tagRFP质粒图谱
图2pCMV-Tim-3Ypet质粒图谱
图3pCMV-Bat3CyPet质粒图谱
图4使用快速筛选系统筛选抗Tim-3单抗药。检测数据如上图所示，空白组的平均荧光强度MFI(PE Texas Red)＝6.6；对照组的MFI(PE Texas Red)＝7432.1，是空白组的1126.1倍；实验组的MFI(PE Texas Red)各不相等，其中筛选出4个阻断性能优良(MFI(PETexas Red)≤500)的抗Tim-3单抗药，即anti-Tim-3-6MFI(PE Texas Red)＝414.9；anti-Tim-3-11MFI(PE Texas Red)＝108.9；anti-Tim-3-19MFI(PE Texas Red)＝7.8；anti-Tim-3-3IMFI(PE Texas Red)＝122.7。
图5使用基于快筛系统检测抗Tim-3单抗药的生物学效应。检测数据如上图所示，空白组的平均荧光强度MFI(FITC)＝6293.7；对照组的MFI(FITC)＝126.3，为空白组的49.8倍：实验组的MFI(FITC)各不相等，其中可筛选出4个生物效应优良(MFI(FITC)≥6000)的抗Tim-3单抗药，即anti-Tim-3-6MFI(FITC)＝6009.5；anti-Tim-3-11MFI(FITC)＝6203.4；anti-Tim-3-19MFI(FITC)＝6287.3；anti-Tim-3-31MFI(FITC)＝6191.9。
图6使用快速筛选系统筛选抗Gal-9单抗药。检测数据如上图所示，空白组的平均荧光强度MFI(PE Texas Red)＝6.6；对照组的MFI(PE Texas Red)＝7430.9，是空白组的1125.9倍；实验组的MFI(PE Texas Red)各不相等，其中筛选出9个阻断性能优良(MFI(PETexas Red)≤500)的抗Gal-9单抗药，即anti-Gal-9-4MFI(PE Texas Red)＝472.7；anti-Gal-9-10MFI(PE Texas Red)＝37.7；anti-Gal-9-21MFI(PE Texas Red)＝299.7；anti-Gal-9-28MFI(PE Texas Red)＝84.3；anti-Gal-9-41MFI(PE Texas Red)＝8.1；anti-Gal-9-47MFI(PE Texas Red)＝350.0；anti-Gal-9-53MFI(PE Texas Red)＝75.5；anti-Gal-9-59MFI(PE Texas Red)＝49.0；anti-Gal-9-66MFI(PE Texas Red)＝305.6。
图7使用基于快筛系统检测抗Gal-9单抗药的生物学效应。检测数据如上图所示，空白组的平均荧光强度MFI(FITC)＝6295.2；对照组的MFI(FITC)＝126.3，为空白组的49.8倍；实验组的MFI(FITC)各不相等，其中可筛选出9个生物效应优良(MFI(FITC)≥6000)的抗Tim-3单抗药，即anti-Gal-9-4MFI(Frrc)＝6000.1；anti-Gal-9-10MFI(FITC)＝6263.9；anti-Gal-9-21MFI(FITC)＝6046.6；anti-Gal-9-28MFI(FITC)＝6225.2；anti-Gal-9-41MFI(FITC)＝6228.5；anti-Gal-9-47MFI(FITC)＝6004.8；anti-Gal-9-53MFI(FITC)＝6232.6；anti-Gal-9-59MFI(FITC)＝6254.5；anti-Gal-9-66MFI(FITC)＝6041.7。
图8Elisa法筛选抗Tim-3单抗药，检测数据如上所示，空白组的OD(450nm，下同)＝0.2；对照组的OD＝1.9，是空白组的9.5倍；实验组的OD值各不相等，其中筛选出22个阻断性能较好(OD≤0.5)的抗TIM-3单抗药，即anti-Tim-3-1OD＝0.4；anti-Tim-3-3OD＝0.2；anti-Tim-3-5OD＝0.3；anti-Tim-3-6OD＝0.3；anti-Tim-3-7OD＝0.3；anti-Tim-3-8OD＝0.4；anti-Tim-3-11OD＝0.2；anti-Tim-3-12OD＝0.3；anti-Tim-3-15OD＝0.4；anti-Tim-3-16OD＝0.2；anti-Tim-3-17OD＝0.5；anti-Tim-3-18OD＝0.5；anti-Tim-3-19OD＝0.2；anti-Tim-3-21OD＝0.3；anti-Tim-3-23OD＝0.2；anti-Tim-3-25OD＝0.2；anti-Tim-3-26OD＝0.4；anti-Tim-3-28OD＝0.4；anti-Tim-3-31OD＝0.2；anti-Tim-3-33OD＝0.4；anti-Tim-3-34OD＝0.3；anti-Tim-3-36OD＝0.2。
图9Elisa法筛选抗Gal-9单抗药，检测数据如上所示，空白组的OD(450nm，下同)＝0.2；对照组的OD＝1.8，是空白组的9倍；实验组的OD值各不相等，其中筛选出28个阻断性能较好(OD≤0.5)的抗TIM-3单抗药，即anti-Gal-9-3OD＝0.5；anti-Gal-9-4OD＝0.3；anti-Gal-9-7OD＝0.5；anti-Gal-9-10OD＝0.2；anti-Gal-9-12OD＝0.5；anti-Gal-9-18OD＝0.3；anti-Gal-9-19OD＝0.3；anti-Gal-9-21OD＝0.3；anti-Gal-9-22OD＝0.3；anti-Gal-9-23OD＝0.3；anti-Gal-9-26OD＝0.4；anti-Gal-9-28OD＝0.2；anti-Gal-9-31OD＝0.2；anti-Gal-9-35OD＝0.3；anti-Gal-9-36OD＝0.3；anti-Gal-9-35OD＝0.5；anti-Gal-9-41OD＝0.2；anti-Gal-9-42OD＝0.4；anti-Gal-9-47OD＝0.3；anti-Gal-9-48OD＝0.4；anti-Gal-9-49OD＝0.4；anti-Gal-9-53OD＝0.2；anti-Gal-9-58OD＝0.4；anti-Gal-9-59OD＝0.2；anti-Gal-9-65OD＝0.3；anti-Gal-9-66OD＝0.3；anti-Gal-9-68OD＝0.3；anti-Gal-9-69OD＝0.3。
具体实施方式
以下是本发明的具体实施例，对本发明的技术方案作进一步的描述，但本发明并不限于这些实施例。
实施例1重组Galectin-9-tagRFP融合蛋白的制备
将红色荧光蛋白tagRFP基因与人全长Galectin-9基因构建在同一个读码框使红色荧光蛋白tagRFP融合在Galectin-9蛋白C端形成Galectin-9-tagRFP融合蛋白，同时在tagRFP的C端接上6个组氨酸His标签。由于Galectin-9与Tim-3的结合位点在胞外域的N端，故tagRFP接于Galectin-9胞内域C端，不影响Galectin-9与Tim-3的结合。前后两端的限制性内切酶EcoRI(GAATTC)和SexAI(ACCTGGT)用于基因合成后连接于慢病毒表达载体pLV-Puro的CMV启动子下游，获得质粒pCMV-Galectin-9-tagRFP(图1)，去掉了原质粒中的PGK启动子和Puro抗性基因，有利于提高慢病毒的转染效率，而阳性克隆的筛选可利用目标蛋白中tagRFP荧光蛋白直接用分选型流式细胞仪筛选或荧光显微镜下挑取。末端的6个His标签用于蛋白His亲和柱纯化以及用His6抗体鉴定目标蛋白。Flag标签(DYKDDDDK)前面为Galectin-9蛋白，后面为tagRFP蛋白；可用Flag抗体鉴定目标蛋白，蛋白纯化后还可以用肠激酶(特异性识别序列DDDDK)将Galectin-9蛋白与tagRFP蛋白切开。将pCMV-Galectin-9-tagRFP与质粒pH1、pH2共转染至慢病毒包装系细胞293V，经5代或20天以上培养获得稳定表达目的蛋白细胞后，荧光显微镜下，用50μl的移液枪挑取高表达Galectin-9-tagRFP融合蛋白的Galectin-9tagRFP/293细胞克隆。扩增收集Galectin-9tagRFP/293细胞，可用匀浆、超声、或冷冻高压细胞破碎仪裂解细胞，15000g高速离心后取沉淀加His结合缓冲液，上Ni2+或Co2+His亲和法柱制备纯化重组Galectin-9-tagRFP融合蛋白。
Galectin-9-tagRFP融合蛋白的制备纯化和鉴定的具体过程包括：
(1)将扩增的Galectin-9-tagRFP/293细胞离心收集，800g、4℃、20min；并用结合缓冲液(20mM Tris-HCl(PH8.0)、150mM NaCl、2μg/ml Aprotinin、2μg/ml Leupeptin、1mM的PMSF、0.1％DNA酶和0.05％RNA酶)、10％(V/V)Glycerol清洗三遍。
(2)沉淀细胞加入裂解结合缓冲液(按1g细胞湿重/mL缓冲液)，裂解结合缓冲液即为结合缓冲液中加有0.5％NP-40；冰浴研磨50次，并超声裂解10min(300w、工作3s、间隔10s)。
(3)高速(8,000g、4℃)离心20min，取上清过0.22μm滤膜，加入Ni2+-NTA-Sepharose亲和柱材料，混匀后，摇床4℃、140rpm/min震荡1-2小时。
(4)放入重力柱，先用结合缓冲液洗3遍，弃洗脱液；用清洗缓冲液(含20-60mM咪唑的结合缓冲液)清洗三遍。
(5)用洗脱液(含200mM咪唑的结合缓冲液)洗脱，收集洗脱液。
(6)用10KD蛋白分子量截留的超滤管离心(6000g、4℃、30min)，浓缩蛋白；用PBSpH7.4置换3遍，收集蛋白液，因为Galectin-9-tagRFP融合蛋白有红色荧光蛋白标签，纯化后的蛋白液显红色，红色越深目标蛋白浓度越高；蛋白液加15％甘油，分装于4℃保存。
(7)取2.5μL用BCA法测蛋白质浓度。
(8)取4μg蛋白质样品进行SDS-PAGE电泳，考马斯亮蓝染色，脱色后观察蛋白质纯度。
(9)Western-blot验证目标蛋白质特异性，包括用His6抗体、Flag抗体、及Galectin-9抗体验证。
如上述步骤中，获得的Galectin-9-tagRFP融合蛋白的DNA基因序列如SEQ ID NO：1所示，表达后Galectin-9-tagRFP融合蛋白序列如SEQ ID NO：2所示。
实施例2质粒pCMV-Ceacam-1的构建
将人源Ceacam-1(NM_001712.5)构建在慢病毒表达载体pLV-Puro的CMV启动子下游，插入EcoR I/Not I位点，获得质粒pCMV-Ceacam-1。
实施例3pCMV-Tim-3Ypet质粒的构建
将黄色荧光蛋白基因Ypet构建在Tim-3蛋白基因的下游，并克隆(EcoRI/SexAI)到慢病毒表达载体pLV-Puro的CMV启动子下游，获得质粒pCMV-Tim-3Ypet(如图2所示)，去掉了原质粒中的PGK启动子和Puro抗性基因。Tim-3Ypet融合蛋白的DNA基因序列如SEQ IDNO：3所示，表达后融合蛋白的序列如SEQ ID NO：4所示。
实施例4质粒pCMV-Bat3CyPet的构建
将青色荧光蛋白CyPet基因构建在Bat3蛋白基因的下游，并克隆(EcoRI/SexAI)到慢病毒表达载体pLV-Puro的CMV启动子下游，获得质粒pCMV-Bat3CyPet(如图3所示)，去掉了原质粒中的PGK启动子和Puro抗性基因。Bat3CyPet融合蛋白的DNA基因序列如SEQ IDNO：5所示，表达后Bat3CyPet融合蛋白的氨基酸序列如SEQ ID NO：6所示。
实施例5建立Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat稳转细胞系
将pCMV-Ceacam-1、pCMV-Tim-3Ypet和pCMV-Bat3CyPet分别与病毒蛋白pH1、pH2共转染到慢病毒包装系细胞293V，制备CMV-Ceacam-1、CMV-Tim-3Ypet和CMV-Bat3CyPet慢病毒，并共转染Jurkat细胞，筛选克隆建立新型的Ceacam-1.Tim-3Ypet.Bat3CyPet/Jurkat稳转细胞系。
实施例6快速筛选系统筛选抗Tim-3单抗药
先将Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞与各1μg/mL的36种待筛选抗Tim-3单抗药(anti-Tim-3-1～anti-Tim-3-36)分别孵育20分钟。洗去非特异性结合后，再与2μg/mL的Gal-9-RFP融合蛋白共孵育为实验组。同时设立不做任何处理的Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞为空白组；设立Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞与2μg/mL的Gal-9-RFP融合蛋白直接共孵育为对照组。共孵育至20分钟时，从上述组别中取部分细胞，洗去非特异性结合，用流式细胞仪分析细胞表面RFP荧光值(如以BDFACSVerseTM流式细胞仪PE Texas Red通道进行数据收集，下同)；共孵育至1小时时，取上述组别的剩余细胞。洗去非特异性结合后，按CyPet/Ypet荧光蛋白FRET法用流式细胞仪分析细胞表面Ypet荧光值(如以BD FACSVerseTM流式细胞仪FITC通道进行数据收集，下同)。
检测数据如图4所示，本实施例6中空白组的平均荧光强度MFI(PE Texas Red)＝6.6；对照组的MFI(PE Texas Red)＝7432.1，是空白组的1126.1倍；实验组的MFI(PE TexasRed)各不相等，其中筛选出4个阻断性能优良(MFI(PE Texas Red)≤500)的抗Tim-3单抗药，即anti-Tim-3-6MFI(PE Texas Red)＝414.9；anti-Tim-3-11MFI(PE Texas Red)＝108.9；anti-Tim-3-19MFI(PE Texas Red)＝7.8；anti-Tim-3-31MFI(PE Texas Red)＝122.7。
检测数据又如图5所示，本实施例6中空白组的平均荧光强度MFI(FITC)＝6293.7；对照组的MFI(FITC)＝126.3，为空白组的49.8倍；实验组的MFI(FITC)各不相等，其中可筛选出4个生物效应优良(MFI(FITC)≥6000)的抗Tim-3单抗药，即anti-Tim-3-6MFI(FITC)＝6009.5；anti-Tim-3-11MFI(FITC)＝6203.4；anti-Tim-3-19MFI(FITC)＝6287.3；anti-Tim-3-31MFI(FITC)＝6191.9；抗Tim-3单抗阻断Tim-3/Gal-9结合的能力和生物学效应在本文所述之体系中表现出很好的一致性。
如上所述，使用本文所述之系统从36种待筛选抗Tim-3单抗药中筛选到4种具有较好阻断能力和生物学效应的抗Tim-3单抗药。进一步结合对比例1(具体实施见下文)的数据，证明本系统在反映抗Tim-3单抗药阻断Tim-3和Gal-9的结合上，其特异性、精确性和识别度是高度优于其他系统的。且同时还能体现其生物学效应，即Tim3与Gal-9的结合使Bat3自Tim3胞内段解离而使Tim-3向胞内转导抑制信号，最终导致T细胞功能的抑制，而抗Tim3单抗药通过对Tim-3/6al-9的阻断作用使Bat3和Tim-3保持正常结合状态，维持了T细胞的活性、增殖和对肿瘤细胞的免疫杀伤作用。
实施例7：快筛系统筛选抗Gal-9单抗药
实验组先将2μg/mL的Gal-9-RFP融合蛋白分别与各1μg/mL的69种待筛选的抗Gal-9单抗药(anti-Gal-9-1～anti-Gal-9-69)分别孵育20分钟。洗去非特异性结合后，再同Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞共孵育为实验组。同时设立不做任何处理的Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞为空白组；设立Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat细胞与各2μg/mL的Gsl-9-RFP融合蛋白直接共孵育为对照组。共孵育至20分钟时，从上述组别中取部分细胞，洗去非特异性结合，用流式细胞仪分析细胞表面RFP荧光值；共孵育至1小时时，取上述组别的剩余细胞。洗去非特异性结合后，按CyPet/Ypet荧光蛋白FRET法用流式细胞仪分析细胞表面Ypet荧光值。
检测数据如图6所示，本实施例7中空白组的平均荧光强度MFI(PE TexasRed)＝6.6；对照组的MFI(PE Texas Red)＝7430.9，是空白组的1125.9倍；实验组的MFI(PE TexasRed)各不相等，其中筛选出9个阻断性能优良(MFI(PE Texas Red)≤500)的抗Gal-9单抗药，即anti-Gal-9-4MFI(PE Texas Red)＝472.7；anti-Gal-9-10MFI(PE Texas Red)＝37.7；anti-Gal-9-21MFI(PE Texas Red)＝299.7；anti-Gal-9-28MFI(PE Texas Red)＝84.3；anti-Gal-9-41MFI(PE Texas Red)＝8.1；anti-Gal-9-47MFI(PE Texas Red)＝350.0；anti-Gal-9-53MFI(PE Texas Red)＝75.5；anti-Gal-9-59MFI(PE Texas Red)＝49.0；anti-Gal-9-66MFI(PE Texas Red)＝305.6。
检测数据又如图7所示，本实施例7中空白组的平均荧光强度MFI(FITC)＝6295.2；对照组的MFI(FITC)＝126.3，为空白组的49.8倍；实验组的MFI(FITC)各不相等，其中可筛选出9个生物效应优良(MFI(FITC)≥6000)的抗Tim-3单抗药，即anti-Gal-9-4MFI(FITC)＝6000.1；anti-Gal-9-10MFI(FITC)＝6263.9；anti-Gal-9-21MFI(FITC)＝6046.6；anti-Gal-9-28MFI(FITC)＝6225.2；anti-Gal-9-41MFI(FITC)＝6228.5；anti-Gal-9-47MFI(FITC)＝6004.8；anti-Gal-9-53MFI(FITC)＝6232.6；anti-Gal-9-59MFI(FITC)＝6254.5；anti-Gal-9-66MFI(FITC)＝6041.7。抗Gal-9单抗阻断Tim-3-GAL-9结合的能力和生物学效应在本文所述之体系中表现出很好的一致性。
如上所述，使用本文所述之系统从69种待筛选抗Gal-9单抗药中筛选到9种具有较好阻断能力和生物学效应的抗Gal-9单抗药。进一步结合对比例2(具体实施见下文)的数据，证明本系统在反映抗Gal-9单抗药阻断Tim-3和Gal-9的结合上，其特异性、精确性和识别度是高度优于其他系统的。且同时还能体现其生物学效应，即Tim3与Gal-9的结合使Bat3自Tim3胞内段解离而使Tim-3向胞内转导抑制信号，最终导致T细胞功能的抑制，而抗Gal单抗药通过对Tim-3/Gal-9的阻断作用使Bat3和Tim-3保持正常结合状态，维持了T细胞的活性、增殖和对肿瘤细胞的免疫杀伤作用。
对比例1：ELISA法筛选抗Tim-3单抗药。
先以250ng/mL浓度Tim-3蛋白过夜包被96孔板。洗板5次尽去非特异性结合后，实验组加入各1μg/mL的36种待筛选抗Tim-3单抗药(anti-Tim-3-1～anti-Tim-3-36)分别孵育30分钟。洗板5次尽去非特异性结合后，加入生物素标记的Gal-9蛋白，孵育30分钟；洗板5次尽去非特异性结合。加入辣根过氧化物酶(HRP)标记的亲合素，孵育30分钟。洗板5次尽去非特异性结合后，加底物TMB显色10分钟，随后加酸终止。同时设立不加任何单抗药和生物素标记的Gal-9蛋白，仅显色终止的为空白组；设立仅加辣根过氧化物酶(HRP)标记的亲合素，随后显色、终止为对照组。最后以上各组用酶标仪在450nm波长下测定吸光度(OD)值。
检测数据如图8所示，本对比例1中空白组的OD＝0.2；对照组的OD＝1.9，是空白组的9.5倍；实验组的OD值各不相等，其中筛选出22个阻断性能较好(OD≤0.5)的抗TIM-3单抗药，即anti-Tim-3-1OD＝0.4；anti-Tim-3-3OD＝0.2；anti-Tim-3-5OD＝0.3；anti-Tim-3-6OD＝0.3；anti-Tim-3-7OD＝0.3；anti-Tim-3-8OD＝0.4；anti-Tim-3-11OD＝0.2；anti-Tim-3-12OD＝0.3；anti-Tim-3-15OD＝0.4；anti-Tim-3-16OD＝0.2；anti-Tim-3-17OD＝0.5；anti-Tim-3-18OD＝0.5；anti-Tim-3-19OD＝0.2；anti-Tim-3-21OD＝0.3；anti-Tim-3-23OD＝0.2；anti-Tim-3-25OD＝0.2；anti-Tim-3-26OD＝0.4；anti-Tim-3-28OD＝0.4；anti-Tim-3-31OD＝0.2；anti-Tim-3-33OD＝0.4；anti-Tim-3-34OD＝0.3；anti-Tim-3-36OD＝0.2。
对比实施例6，不难发现，以ELISA法筛选抗Tim-3单抗药，其特异性、精确性和识别度不如本文所述之系统，且仅能反映抗Tim-3单抗药对于Tim-3-Gal-9结合的阻断作用，不能获得生物学效应的数据。而且，以ELISA法进行筛选还有操作繁琐、特异性低、误差较大、试剂盒成本较高等缺点。
对比例2：ELISA法筛选抗Gal-9单抗药。
先以250ng/mL浓度Gal-9蛋白过夜包被96孔板。洗板5次尽去非特异性结合后，实验组加入各1μg/mL的69种待筛选抗Gal-9单抗药(anti-Gal-9-1～anti-Gal-9-69)分别孵育30分钟。洗板5次尽去非特异性结合后，加入生物素标记的Tim-3蛋白，孵育30分钟；洗板5次尽去非特异性结合。加入辣根过氧化物酶(HRP)标记的亲合素，孵育30分钟。洗板5次尽去非特异性结合后，加底物TMB显色10分钟，随后加酸终止。同时设立不加任何单抗药和生物素标记的Tim-3蛋白，仅显色终止的为空白组；设立仅加辣根过氧化物酶(HRP)标记的亲合素，随后显色、终止为对照组。最后以上各组用酶标仪在450nm波长下测定吸光度(OD)值。
检测数据如图9所示，本对比例2中空白组的OD＝0.2；对照组的OD＝1.8，是空白组的9倍；实验组的OD值各不相等，其中筛选出28个阻断性能较好(OD≤0.5)的抗TIM-3单抗药，即anti-Gal-9-3OD＝0.5；anti-Gal-9-4OD＝0.3；anti-Gal-9-7OD＝0.5；anti-Gal-9-10OD＝0.2；anti-Gal-9-12OD＝0.5；anti-Gal-9-18OD＝0.3；anti-Gal-9-19OD＝0.3；anti-Gal-9-21OD＝0.3；anti-Gal-9-22OD＝0.3；anti-Gal-9-23OD＝0.3；anti-Gal-9-26OD＝0.4；anti-Gal-9-28OD＝0.2；anti-Gal-9-31OD＝0.2；anti-Gal-9-35OD＝0.3；anti-Ga1-9-36OD＝0.3；anti-Gal-9-35OD＝0.5；anti-Gal-9-41OD＝0.2；anti-Gal-9-42OD＝0.4；anti-Gal-9-47OD＝0.3；anti-Gal-9-48OD＝0.4；anti-Gal-9-49OD＝0.4；anti-Gal-9-53OD＝0.2；anti-Gal-9-58OD＝0.4；anti-Gal-9-59OD＝0.2；anti-Gal-9-65OD＝0.3；anti-Gal-9-66OD＝0.3；anti-Gal-9-68OD＝0.3；anti-Gal-9-69OD＝0.3。
对比实施例6，不难发现，以ELISA法筛选抗Gal-9单抗药，其特异性、精确性和识别度不如本文所述之系统，且仅能反映抗Gal-9单抗药对于Tim-3-Gal-9结合的阻断作用，不能获得生物学效应的数据。而且，以ELISA法进行筛选还有操作繁琐、特异性低、误差较大、试剂盒成本较高等缺点。
综上所述，本系统1)快速简便、低成本，系统除了必要的阻断效应实验步骤外无需另外处理，即测即得，并可进行动态监测。2)可用通用的流式细胞仪、荧光酶标仪等多种方法测定，普适性好。3)系统可精确反映抗Tim-3和抗Gal-9药的效应，稳定性好，精确度高，特异性极高。4)一个系统可同时进行抗Tim-3药物和抗Gal-9药物的筛选，简便多用。5)一个样本可同时获得Tim-3/Gal-9的阻断功能与Tim-3/Gal-9信号通路的生物学效应数据，为快速筛选抗Tim-3/Gal-9药物和评估其生物学效应提供了一个新型强有力的系统和实验模型。
以上所述，仅为本发明的优选实施例，应当指出，对本技术领域的普通技术人员来说，在不脱离本发明的核心技术特点的前体下，还可以做出变化和改进。这些变化和改进均属于本发明的专利保护范围。与本发明的权利要求书相当的含义和范围内的任何改变，都应认为是包括在权利要求书的范围内。
SEQUENCE LISTING
<110> 杭州科兴生物科技有限公司
<120> 一种基于Tim3/Galectin-9阻断功能及其生物效应的药物快速筛选方法
<130> 1
<160> 6
<170> PatentIn version 3.5
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gccttccact tcaaccctcg gtttgaagat ggagggtacg tggtgtgcaa cacgaggcag 240
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caaaaacctc ccggcgtgtg gcctgccaac ccggctccca ttacccagac agtcatccac 600
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ccccaccccg cctatccgat gcctttcatc accaccattc tgggagggct gtacccatcc 720
aagtccatcc tcctgtcagg cactgtcctg cccagtgctc agaggttcca catcaacctg 780
tgctctggga accacatcgc cttccacctg aacccccgtt ttgatgagaa tgctgtggtc 840
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Arg Asp Ser Gly Ala Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly
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Gly Ile Arg Ser Glu Glu Asn Ile Tyr Thr Ile Glu Glu Asn Val Tyr
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Glu Val Glu Glu Pro Asn Glu Tyr Tyr Cys Tyr Val Ser Ser Arg Gln
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Gln Pro Ser Gln Pro Leu Gly Cys Arg Phe Ala Met Pro Asp Tyr Lys
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Asp Asp Asp Asp Lys Leu Glu Lys Gly Glu Glu Leu Phe Thr Gly Val
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Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe
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Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
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Leu Val Thr Thr Leu Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro
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Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
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<210> 5
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<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 5
gaattcatgg agcctaatga tagtaccagt accgctgtgg aggagcctga cagcttggag 60
gtgttggtga agaccttgga ctctcaaact cgtaccttta ttgtgggggc ccagatgaat 120
gtaaaagagt ttaaggagca cattgctgcc tctgtcagca tcccatctga aaaacaacgg 180
ctcatttacc agggacgagt tctgcaagat gataagaagc ttcaggaata caatgttggg 240
ggaaaggtta tccacctggt ggaacgggct cctcctcaga ctcacctccc ttctggggca 300
tcttctggga cggggtctgc ctcagccact catggtgggg gatctccccc tggtactcgg 360
gggcctgggg cctctgttca tgaccggaat gccaacagct atgtcatggt tggaaccttc 420
aatcttccta gtgacggctc tgctgtggat gttcacatca acatggaaca ggccccgatt 480
cagagtgagc cccgggtacg gctggtgatg gctcagcaca tgatcaggga tatacagacc 540
ttactatccc ggatggagtg tcgaggaggg ccccaaccgc agcacagtca gccgcccccg 600
cagccaccgg ctgtgacccc ggagccagta gccttgagct ctcaaacatc agaaccagtt 660
gaaagtgaag cacctccccg ggagcccatg gaggcagaag aagtggagga gcgtgcccca 720
gcccagaacc cggagctcac tcctggccca gccccagcgg gcccaacacc tgccccggaa 780
acaaatgcac ccaaccatcc ttcccctgcg gagtatgtcg aggtgctcca ggagctacag 840
cggctggaga gtcgcctcca gcccttcttg cagcgctact acgaggttct gggtgctgct 900
gccaccacgg actacaataa caatcacgag ggccgggagg aggatcagcg gttgatcaac 960
ttggtagggg agagcctgcg actgctgggc aacacctttg ttgcactgtc tgacctgcgc 1020
tgcaatctgg cctgcacgcc cccacgacac ctgcatgtgg tccggcctat gtctcactac 1080
accaccccca tggtgctcca gcaggcagcc attcccatac agatcaatgt gggaaccact 1140
gtgaccatga caggaaatgg gactcggccc cccccaactc ccaatgcaga ggcacctccc 1200
cctggtcctg ggcaggcctc atccgtggct ccgtcttcta ccaatgtcga gtcctcagct 1260
gagggggctc ccccgccagg tccagctccc ccgccagcca ccagccaccc gagggtcatc 1320
cggatttccc accagagtgt ggaacccgtg gtcatgatgc acatgaacat tcaagattct 1380
ggcacacagc ctggtggtgt tccgagtgct cccactggcc ccctgggacc ccctggtcat 1440
ggccaaaccc tgggacagca ggtgccaggc ttcccaacag ctccaacccg ggtggtgatt 1500
gcccggccca ctcctccaca ggctcggcct tcccatcctg gagggccccc agtctctggg 1560
acactgcagg gcgccggtct gggtaccaat gcctcgttgg cccagatggt gagcggcctt 1620
gtggggcagc ttcttatgca gccagtcctt gtggctcagg ggaccccagg tatggctcca 1680
ccgccagccc ctgccactgc ttctgccagt gctggcacca ccaacacagc taccacagct 1740
ggccccgctc ctggggggcc tgcccagcct ccacccaccc ctcaaccctc catggctgat 1800
cttcagttct ctcagcttct ggggaacctg ctagggcctg cagggccagg ggctggaggg 1860
tctggtgtgg cttctcccac catcactgtg gcgatgcctg gtgtccctgc ctttctccaa 1920
ggcatgactg acttcttgca ggcaacacag acagcccctc caccaccccc acctcctcca 1980
cccccaccac ctgccccaga gcagcagacc atgcccccac caggctcccc ttctggtggc 2040
gcagggagtc ctggaggcct gggtcttgag agcctgtcac cggagttttt tacctcagtg 2100
gtgcagggtg tgctcagctc cctgctgggc tccctggggg ctcgggctgg cagcagtgaa 2160
agtattgctg ccttcataca acgcctcagt ggatctagca acatctttga gcctggagct 2220
gatggggccc ttggattctt tggggccttg ctttctcttc tgtgccagaa cttctctatg 2280
gtggacgtag tgatgcttct ccatgggcat ttccagccac tacaacggct ccagccccag 2340
ctgcgatcct tcttccacca gcactacctg ggtggtcagg agcccacacc cagtaacatc 2400
cggatggcaa cccacacatt gatcacgggg ctagaagagt atgtgcggga gagtttttcc 2460
ttggtgcagg ttcagccagg tgtggacatc atccggacaa acctggaatt tctccaagag 2520
cagtttaata gcattgctgc gcatgtgctg cattgcacag atagtggatt tggggcccgg 2580
ttgctggagt tgtgtaacca aggcctgttt gaatgcctgg ccctaaacct gcactgcttg 2640
gggggacagc agatggagct tgctgctgtt atcaatggcc gaatacgtcg tatgtctcgt 2700
ggggtgaatc cctccttggt gagctggctg accactatga tgggactgag gcttcaggtg 2760
gtactggagc acatgcctgt aggccctgat gccattctca gatacgttcg cagggttggt 2820
gatccccccc agccacttcc tgaggagcca atggaagttc agggagcaga aagagcttcc 2880
cctgagcctc agcgggagaa tgcttcccca gcccctggaa caacagcaga agaggccatg 2940
tcccgaggtc cacctcctgc tcctgagggg ggctcccggg atgaacagga tggagcttca 3000
gctgagacag aaccttgggc agctgcagtc cccccagaat gggtccctat tatccagcag 3060
gacattcaga gccagcggaa ggtgaaaccg cagccccctc tgagtgatgc ctacctcagt 3120
ggtatgcctg ccaagagacg caagacgatg cagggtgagg gcccccagct gcttctctca 3180
gaggctgtga gccgggcagc taaggcagcc ggagctcggc ccctgacgag ccccgagagc 3240
ctgagccggg acctggaggc accagaggtt caggagagct acaggcagca gctccggtct 3300
gatatacaaa aacgactgca ggaagacccc aactacagtc cccagcgctt ccccaatgcc 3360
cagcgggcct ttgctgatga tcctgactac aaggacgacg acgacaaggg aggaggaggc 3420
agcggcggag gaggctccgg cggcggcggc agcggcgccg tgagcaaggg agaggaactg 3480
ttcggcggca tcgtgcccat cctggtggag ctggagggcg acgtgaacgg ccacaagttc 3540
agcgtgagcg gcgagggcga gggcgacgcc acctacggca agctgaccct gaagttcatc 3600
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctgg tgaccaccct gacctggggc 3660
gtgcagtgct tcagccggta ccccgaccac atgaagcagc acgacttctt caagagcgtg 3720
atgcccgagg gctacgtgca ggagcggacc atcttcttca aggacgacgg caactacaag 3780
acccgggccg aggtgaagtt cgagggcgac accctggtga accggatcga gctgaagggc 3840
atcgacttca aggaggacgg caacatcctg ggccacaagc tggagtacaa ctacatcagc 3900
cacaacgtgt acatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaaggcc 3960
cggcacaaca tcaccgacgg cagcgtgcag ctggccgacc actaccagca gaacaccccc 4020
atcggcgacg gccccgtgat cctgcccgac aaccactacc tgagcaccca gagcgccctg 4080
agcaaggacc ccaacgagaa gcgggaccac atggtgctgc tggagttcgt gaccgccgcc 4140
ggcatcaccc acggcatgga cgaactgtac aaacatgatg agcttcatca ccatcaccat 4200
cactagcggc cgc 4213
<210> 6
<211> 1399
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 6
Met Glu Pro Asn Asp Ser Thr Ser Thr Ala Val Glu Glu Pro Asp Ser
1 5 10 15
Leu Glu Val Leu Val Lys Thr Leu Asp Ser Gln Thr Arg Thr Phe Ile
20 25 30
Val Gly Ala Gln Met Asn Val Lys Glu Phe Lys Glu His Ile Ala Ala
35 40 45
Ser Val Ser Ile Pro Ser Glu Lys Gln Arg Leu Ile Tyr Gln Gly Arg
50 55 60
Val Leu Gln Asp Asp Lys Lys Leu Gln Glu Tyr Asn Val Gly Gly Lys
65 70 75 80
Val Ile His Leu Val Glu Arg Ala Pro Pro Gln Thr His Leu Pro Ser
85 90 95
Gly Ala Ser Ser Gly Thr Gly Ser Ala Ser Ala Thr His Gly Gly Gly
100 105 110
Ser Pro Pro Gly Thr Arg Gly Pro Gly Ala Ser Val His Asp Arg Asn
115 120 125
Ala Asn Ser Tyr Val Met Val Gly Thr Phe Asn Leu Pro Ser Asp Gly
130 135 140
Ser Ala Val Asp Val His Ile Asn Met Glu Gln Ala Pro Ile Gln Ser
145 150 155 160
Glu Pro Arg Val Arg Leu Val Met Ala Gln His Met Ile Arg Asp Ile
165 170 175
Gln Thr Leu Leu Ser Arg Met Glu Cys Arg Gly Gly Pro Gln Pro Gln
180 185 190
His Ser Gln Pro Pro Pro Gln Pro Pro Ala Val Thr Pro Glu Pro Val
195 200 205
Ala Leu Ser Ser Gln Thr Ser Glu Pro Val Glu Ser Glu Ala Pro Pro
210 215 220
Arg Glu Pro Met Glu Ala Glu Glu Val Glu Glu Arg Ala Pro Ala Gln
225 230 235 240
Asn Pro Glu Leu Thr Pro Gly Pro Ala Pro Ala Gly Pro Thr Pro Ala
245 250 255
Pro Glu Thr Asn Ala Pro Asn His Pro Ser Pro Ala Glu Tyr Val Glu
260 265 270
Val Leu Gln Glu Leu Gln Arg Leu Glu Ser Arg Leu Gln Pro Phe Leu
275 280 285
Gln Arg Tyr Tyr Glu Val Leu Gly Ala Ala Ala Thr Thr Asp Tyr Asn
290 295 300
Asn Asn His Glu Gly Arg Glu Glu Asp Gln Arg Leu Ile Asn Leu Val
305 310 315 320
Gly Glu Ser Leu Arg Leu Leu Gly Asn Thr Phe Val Ala Leu Ser Asp
325 330 335
Leu Arg Cys Asn Leu Ala Cys Thr Pro Pro Arg His Leu His Val Val
340 345 350
Arg Pro Met Ser His Tyr Thr Thr Pro Met Val Leu Gln Gln Ala Ala
355 360 365
Ile Pro Ile Gln Ile Asn Val Gly Thr Thr Val Thr Met Thr Gly Asn
370 375 380
Gly Thr Arg Pro Pro Pro Thr Pro Asn Ala Glu Ala Pro Pro Pro Gly
385 390 395 400
Pro Gly Gln Ala Ser Ser Val Ala Pro Ser Ser Thr Asn Val Glu Ser
405 410 415
Ser Ala Glu Gly Ala Pro Pro Pro Gly Pro Ala Pro Pro Pro Ala Thr
420 425 430
Ser His Pro Arg Val Ile Arg Ile Ser His Gln Ser Val Glu Pro Val
435 440 445
Val Met Met His Met Asn Ile Gln Asp Ser Gly Thr Gln Pro Gly Gly
450 455 460
Val Pro Ser Ala Pro Thr Gly Pro Leu Gly Pro Pro Gly His Gly Gln
465 470 475 480
Thr Leu Gly Gln Gln Val Pro Gly Phe Pro Thr Ala Pro Thr Arg Val
485 490 495
Val Ile Ala Arg Pro Thr Pro Pro Gln Ala Arg Pro Ser His Pro Gly
500 505 510
Gly Pro Pro Val Ser Gly Thr Leu Gln Gly Ala Gly Leu Gly Thr Asn
515 520 525
Ala Ser Leu Ala Gln Met Val Ser Gly Leu Val Gly Gln Leu Leu Met
530 535 540
Gln Pro Val Leu Val Ala Gln Gly Thr Pro Gly Met Ala Pro Pro Pro
545 550 555 560
Ala Pro Ala Thr Ala Ser Ala Ser Ala Gly Thr Thr Asn Thr Ala Thr
565 570 575
Thr Ala Gly Pro Ala Pro Gly Gly Pro Ala Gln Pro Pro Pro Thr Pro
580 585 590
Gln Pro Ser Met Ala Asp Leu Gln Phe Ser Gln Leu Leu Gly Asn Leu
595 600 605
Leu Gly Pro Ala Gly Pro Gly Ala Gly Gly Ser Gly Val Ala Ser Pro
610 615 620
Thr Ile Thr Val Ala Met Pro Gly Val Pro Ala Phe Leu Gln Gly Met
625 630 635 640
Thr Asp Phe Leu Gln Ala Thr Gln Thr Ala Pro Pro Pro Pro Pro Pro
645 650 655
Pro Pro Pro Pro Pro Pro Ala Pro Glu Gln Gln Thr Met Pro Pro Pro
660 665 670
Gly Ser Pro Ser Gly Gly Ala Gly Ser Pro Gly Gly Leu Gly Leu Glu
675 680 685
Ser Leu Ser Pro Glu Phe Phe Thr Ser Val Val Gln Gly Val Leu Ser
690 695 700
Ser Leu Leu Gly Ser Leu Gly Ala Arg Ala Gly Ser Ser Glu Ser Ile
705 710 715 720
Ala Ala Phe Ile Gln Arg Leu Ser Gly Ser Ser Asn Ile Phe Glu Pro
725 730 735
Gly Ala Asp Gly Ala Leu Gly Phe Phe Gly Ala Leu Leu Ser Leu Leu
740 745 750
Cys Gln Asn Phe Ser Met Val Asp Val Val Met Leu Leu His Gly His
755 760 765
Phe Gln Pro Leu Gln Arg Leu Gln Pro Gln Leu Arg Ser Phe Phe His
770 775 780
Gln His Tyr Leu Gly Gly Gln Glu Pro Thr Pro Ser Asn Ile Arg Met
785 790 795 800
Ala Thr His Thr Leu Ile Thr Gly Leu Glu Glu Tyr Val Arg Glu Ser
805 810 815
Phe Ser Leu Val Gln Val Gln Pro Gly Val Asp Ile Ile Arg Thr Asn
820 825 830
Leu Glu Phe Leu Gln Glu Gln Phe Asn Ser Ile Ala Ala His Val Leu
835 840 845
His Cys Thr Asp Ser Gly Phe Gly Ala Arg Leu Leu Glu Leu Cys Asn
850 855 860
Gln Gly Leu Phe Glu Cys Leu Ala Leu Asn Leu His Cys Leu Gly Gly
865 870 875 880
Gln Gln Met Glu Leu Ala Ala Val Ile Asn Gly Arg Ile Arg Arg Met
885 890 895
Ser Arg Gly Val Asn Pro Ser Leu Val Ser Trp Leu Thr Thr Met Met
900 905 910
Gly Leu Arg Leu Gln Val Val Leu Glu His Met Pro Val Gly Pro Asp
915 920 925
Ala Ile Leu Arg Tyr Val Arg Arg Val Gly Asp Pro Pro Gln Pro Leu
930 935 940
Pro Glu Glu Pro Met Glu Val Gln Gly Ala Glu Arg Ala Ser Pro Glu
945 950 955 960
Pro Gln Arg Glu Asn Ala Ser Pro Ala Pro Gly Thr Thr Ala Glu Glu
965 970 975
Ala Met Ser Arg Gly Pro Pro Pro Ala Pro Glu Gly Gly Ser Arg Asp
980 985 990
Glu Gln Asp Gly Ala Ser Ala Glu Thr Glu Pro Trp Ala Ala Ala Val
995 1000 1005
Pro Pro Glu Trp Val Pro Ile Ile Gln Gln Asp Ile Gln Ser Gln
1010 1015 1020
Arg Lys Val Lys Pro Gln Pro Pro Leu Ser Asp Ala Tyr Leu Ser
1025 1030 1035
Gly Met Pro Ala Lys Arg Arg Lys Thr Met Gln Gly Glu Gly Pro
1040 1045 1050
Gln Leu Leu Leu Ser Glu Ala Val Ser Arg Ala Ala Lys Ala Ala
1055 1060 1065
Gly Ala Arg Pro Leu Thr Ser Pro Glu Ser Leu Ser Arg Asp Leu
1070 1075 1080
Glu Ala Pro Glu Val Gln Glu Ser Tyr Arg Gln Gln Leu Arg Ser
1085 1090 1095
Asp Ile Gln Lys Arg Leu Gln Glu Asp Pro Asn Tyr Ser Pro Gln
1100 1105 1110
Arg Phe Pro Asn Ala Gln Arg Ala Phe Ala Asp Asp Pro Asp Tyr
1115 1120 1125
Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
1130 1135 1140
Ser Gly Gly Gly Gly Ser Gly Ala Val Ser Lys Gly Glu Glu Leu
1145 1150 1155
Phe Gly Gly Ile Val Pro Ile Leu Val Glu Leu Glu Gly Asp Val
1160 1165 1170
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
1175 1180 1185
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys
1190 1195 1200
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly
1205 1210 1215
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp
1220 1225 1230
Phe Phe Lys Ser Val Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
1235 1240 1245
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
1250 1255 1260
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
1265 1270 1275
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
1280 1285 1290
Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln
1295 1300 1305
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ala Arg His Asn Ile Thr
1310 1315 1320
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
1325 1330 1335
Ile Gly Asp Gly Pro Val Ile Leu Pro Asp Asn His Tyr Leu Ser
1340 1345 1350
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
1355 1360 1365
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His Gly
1370 1375 1380
Met Asp Glu Leu Tyr Lys His Asp Glu Leu His His His His His
1385 1390 1395
His

Claims (10)

1.一种基于Tim-3/Galectin-9阻断功能及其生物效应的药物快速筛选方法，包括以下步骤：
1）将第一荧光蛋白与Galectin-9蛋白羧基末端连接构建重组Galectin-9-第一荧光蛋白融合蛋白；在第一荧光蛋白的C端连接6个组氨酸His标签；
2）将第二荧光蛋白连接在Tim-３蛋白的羧基末端，并克隆到真核表达载体CMV启动子下，构建表达质粒；将第三荧光蛋白通过一个连接肽(G4S)3连在Bat3蛋白的羧基末端，并克隆到真核表达载体CMV启动子下，构建表达质粒；将Ceacam-1克隆到CMV启动子下，构建表达质粒；
将上述三个表达质粒与慢病毒包装质粒pH1、pH2共转染到慢病毒包装系细胞293V，制备三种慢病毒，并共转染到Jurkat细胞，并建立新型的稳转细胞系Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat；
将抗Tim-3的单抗药、小分子、或多肽阻断剂与步骤2）获得的稳转细胞系共孵育后加入重组Galectin-9-第一荧光蛋白融合蛋白共孵育；同时设立步骤2）的稳转细胞与重组Galectin-9-第一荧光蛋白融合蛋白直接共孵育的对照组；清洗非特异性结合；
将抗Galectin-9的单抗药、小分子、或多肽阻断剂与Galectin-9-第一荧光蛋白融合蛋白共孵育后再与步骤2）获得的稳转细胞系一起共孵育；同时设立步骤2）的稳转细胞与Galectin-9-第一荧光蛋白融合蛋白直接共孵育的对照组；清洗非特异性结合；
分别检测第一荧光蛋白荧光值、第二荧光蛋白/第三荧光蛋白FRET荧光值，并判断新药对Tim-3/Galectin-9信号通路的阻断功能。
2.根据权利要求1的药物快速筛选方法，其中第一荧光蛋白是tagRFP红色荧光蛋白；第二荧光蛋白是黄色荧光蛋白Ypet；第三荧光蛋白是青色荧光蛋白CyPet。
3.根据权利要求2的药物快速筛选方法，其中步骤1）中，将红色荧光蛋白tagRFP基因与人全长Galectin-9基因构建在同一个读码框使红色荧光蛋白tagRFP融合在Galectin-9蛋白C端形成所述Galectin-9-tagRFP融合蛋白，同时在tagRFP的C端接上6个组氨酸His标签。
4.根据权利要求3的药物快速筛选方法，其中步骤1）中，将Galectin-9-tagRFP基因克隆至真核高表达载体CMV启动子下游，并转染至人胚肾细胞293，筛选克隆获得高表达Galectin-9-tagRFP融合蛋白的Galectin-9-tagRFP/293细胞系；扩增该Galectin-9-tagRFP/293细胞，裂解后用His亲和法制备纯化重组Galectin-9-tagRFP融合蛋白。
5.根据权利要求4的药物快速筛选方法，其中步骤2）中，将黄色荧光蛋白Ypet基因与人全长Tim-3基因构建在同一个读码框使黄色荧光蛋白Ypet融合在Tim-3蛋白C端形成所述Tim-3-Ypet融合蛋白，并克隆到真核表达载体CMV启动子下，构建表达质粒pCMV-Tim-3Ypet。
6.根据权利要求5的药物快速筛选方法，其中步骤2）中，将青色荧光蛋白CyPet基因通过一个连接肽(G4S)3连在Bat3蛋白的羧基末端，并克隆到真核表达载体CMV启动子下，构建表达质粒pCMV-Bat3(G4S)3CyPet；将Ceacam-1克隆到CMV启动子下，构建表达质粒pCMV-Ceacam-1。
7.根据权利要求6的药物快速筛选方法，其中步骤2）中，将pCMV-Tim-3Ypet、pCMV-Bat3(G4S)3CyPet、pCMV-Ceacam-1分别与慢病毒包装质粒pH1、pH2共转染到慢病毒包装系细胞293V，制备CMV-Tim-3Ypet、CMV-Bat3(G4S)3CyPet、CMV-Ceacam-1慢病毒，并共转染到Jurkat细胞，建立新型的稳转细胞系Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat；
其中Galectin-9-tagRFP融合蛋白的氨基酸序列如SEQ ID NO:2所示；
Tim-3-Ypet融合蛋白的氨基酸序列如SEQ ID NO：4所示；
Bat3(G4S)3CyPet融合蛋白的氨基酸序列如SEQ ID NO：6所示。
8.根据权利要求7的药物快速筛选方法，其中步骤3）中，将抗Tim-3的单抗药、小分子、或多肽阻断剂与Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat稳转细胞共孵育后加入重组Galectin-9-tagRFP融合蛋白共孵育；同时设立Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat稳转细胞与重组Galectin-9-tagRFP融合蛋白直接共孵育的对照组。
9.根据权利要求8的药物快速筛选方法，其中步骤4）中，将抗Galectin-9的单抗药、小分子、或多肽阻断剂与Galectin-9-tagRFP融合蛋白共孵育后再与Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat稳转细胞共孵育；同时设立Ceacam-1.Tim-3Ypet.Bat3_CyPet/Jurkat稳转细胞与重组Galectin-9-tagRFP融合蛋白直接共孵育的对照组。
10.根据权利要求9的药物快速筛选方法，其中步骤5）中，经缓冲液清洗去除非特异性结合后用流式细胞仪、或荧光酶标仪分析细胞表面的红色荧光蛋白tagRFP荧光值；以及细胞内的CyPet/Ypet的FRET荧光值；通过药物组荧光值与对照组比较，判断抗Tim-3药和抗Galectin-9对Tim-3/Galectin-9阻断功能，以及对Tim-3.Bat3信号通路产生的生物学效应。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1902227A (zh) * 2003-10-03 2007-01-24 布赖汉姆妇女医院 Tim－3配体及其方法
CN101671653A (zh) * 2009-09-22 2010-03-17 浙江大学 一种hiv侵染细胞的筛选系统及其应用
CN108611395A (zh) * 2016-12-12 2018-10-02 中山大学 一种抗人乙肝病毒药物的筛选方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1902227A (zh) * 2003-10-03 2007-01-24 布赖汉姆妇女医院 Tim－3配体及其方法
CN101671653A (zh) * 2009-09-22 2010-03-17 浙江大学 一种hiv侵染细胞的筛选系统及其应用
CN108611395A (zh) * 2016-12-12 2018-10-02 中山大学 一种抗人乙肝病毒药物的筛选方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy;Catherine A Sabatos-Peyton等;《Oncoimmunology》;20171231;第7卷(第2期);全文 *
Soluble Tim-3 and Gal-9 are associated with renal allograft dysfunction in kidney transplant recipients: A cross-sectional study;Ya Mei Li等;《Int Immunopharmacol》;20180106;第55卷;全文 *

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