CN109652454B - 一种基于gp120/CCR5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gp120/CCR5阻断功能及其生物效应的药物快速筛选方法 Download PDFInfo
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Abstract
本发明建立了一种基于gp120/CCR5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gp120蛋白羧基末端(C端)，构建真核表达载体，并制备gp120‑tagRFP融合蛋白；将人源CD4蛋白基因构建在CMV启动子下；将人源CCR5基因构建在CMV启动子下游，将绿色荧光蛋白基因d2EGFP构建在NF‑κB RE‑TATA‑like人工复合调控元件的下游，两者构建于同一个慢病毒载体内；并建立新型的稳转细胞系CD4.CCR5.CCR5.NF‑κBR‑d2EGFP/Jurkat；以gp120‑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CCR5药的效应，并可同时获得阻断功能与CC趋化因子/CCR5信号通路的生物学效应数据，构建了快速筛选抗CCR5药物和评估其生物学效应的实验模型。
Description
技术领域
本发明涉及一种基于gp120/CCR5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
目前已有几十种抗HIV-1药物得到美国食品与药品监督管理局(FDA)批准，其类型包括逆转录酶抑制剂、蛋白酶抑制剂、CCR5受体抑制剂、整合酶抑制剂、以及融合抑制剂等。然而，已被批准的药物中没有一种是可以完全抑制病毒感染的，而且由于HIV-1突变株的产生，大部分均对不同类型的拮抗剂具有耐药性。此外，随着对病毒入侵过程的深入了解，研究者发现，大多数的HIV都是通过与CCR5或CXCR4结合来感染宿主细胞。通过病毒表面的包膜糖蛋白gp120与T细胞表面受体CD4和趋化因子受体CCR5或CXCR4相互作用进入宿主细胞，是人类免疫缺陷病毒(HIV)致病的关键。因而，辅助受体CCR5是目前抗HIV-1感染的首选靶点，CCR5拮抗剂做为抗HIV-1感染药物的研发成为热门。CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等4类。因此，建立一个高效、快速、简便、精确的高通量筛选系统非常重要，但目前为止尚未见有关报道。本发明在于建立一个基于gp120/CCR5阻断功能及其生物效应的抗HIV-1感染药物的高效、快速、简便、精确的高通量筛选系统。
发明内容
本发明的目的是建立了一种基于gp120/CCR5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gp120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，制备融合蛋自；将人源CD4基因克隆到真核表达载体CMV启动子下，构建表达质粒；将人源CCR5基因构建在CMV启动子下，将绿色荧光蛋白基因d2EGFP构建在NF-κB RE-TATA-like人工复合调控元件的下游，两者构建于同一个慢病毒载体内，获得高表达CCR5和调控表达报告基因质粒；二个基因共转染Jurkat细胞，建立稳转细胞系。将CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等新药与CD4.CCR5.NF-κ B RE-TATA-like-d2EGFP/Jurkat细胞共孵育后再与gp120-tagRFP荧光蛋白融合蛋白一起共孵育；同时设立稳转细胞与gp120-tagRFP荧光蛋白融合蛋白直接共孵育的对照组，清洗非特异性结合后检测荧光值。将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂和筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育为实验组；同时设立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞为空白组，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CCR5药是否具有gp120/CCR5阻断功能；通过检测绿色荧光蛋白d2EGFP荧光值并与对照组比较，判断抗CCR5药是否影响CC趋化因子/CCR5的正常生物学效应。
为了实现本发明目的，第一方面，本发明提供一种基于gp120/CCR5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gp120羧基末端连接构建重组gp120-第一荧光蛋白融合蛋白。
2)将CD4(NM_000616.5)基因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，构建CMV-CD4表达质粒；将CCR5(U54994.1)构建在CMV启动子下，将第二荧光蛋白构建在NF-κB RE-TATA-like人工复合调控元件的下游，两者构建于同一个慢病毒载体内，插入EcoR I/SexA I位点，获得高表达CCR5和调控表达报告基因的CMV-CCR5.NF-κB RE-TATA-like-第二荧光蛋白质粒。
3)将CMV-CCR5.NF-κB RE-TATA-like-第二荧光蛋白、CMV-CD4质粒共转染到Jurkat细胞中，建立稳转细胞系。
4)将CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等新药与稳转细胞共孵育后再与gp120-第一荧光蛋白融合蛋白一起共孵育；同时设立稳转细胞与gp120-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。
5)检测第一荧光蛋白荧光值，并判断新药对gp120/CCR5具有阻断功能。
6)将稳转细胞与CC趋化因子试剂和筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育；同时设立gp120/CCR5细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的稳转细胞为空白组，清洗非特异性结合。
7)检测第二荧光蛋白荧光值，并判断新药对CC趋化因子/CCR5信号通路的影响。
进一步地，第一荧光蛋白是tagRFP红色荧光蛋白；第二荧光蛋白是绿色荧光蛋白d2EGFP。
进一步地，步骤1)中，将红色荧光蛋白tagRFP基因与HIV-1的gp120蛋白基因构建在同一个读码框使红色荧光蛋白tagRFP接gp120蛋白C端形成所述gp120-tagRFP融合蛋白，同时在tagRFP的C端接上6个组氨酸His标签。
进一步地，步骤1)中，将gp120-tagRFP基因克隆至慢病毒表达载体pLV-Puro的CMV启动子下游，插入BamH I/SexA I位点，去掉了原质粒中的PGK启动子和Puro抗性基因，获得pCMV-gp120-tagRFP质粒，并转染至人胚肾细胞293，筛选克隆获得高表达gp120-tagRFP融合蛋白的gp120-tagRFP/293细胞系；扩增该gp120-tagRFP/293细胞，裂解后用His亲和法制备纯化含重组gp120-tagRFP/293融合蛋白。
进一步地，步骤2)中，将CD4构建在慢病毒表达载体pLV-Puro的CMV启动子下游，构建表达质粒pCMV-CD4；将CCR5构建在CMV启动子下，将绿色荧光蛋白d2EGFP构建在NF-κBRE-TATA-like人工复合调控元件的下游，两者构建于同一个慢病毒载体内，插入EcoR I/SexA I位点，获得高表达CCR5和调控表达报告基因质粒pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP。
进一步地，步骤3)中，将pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP、pCMW-CD4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-CD4、CCR5.NF-κB RE-TATA-like-d2EGFP慢病毒，并共转染到Jurkat细胞中，建立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞系。
进一步地，步骤4)中，将CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等新药与CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞共孵育后再与gp120-tagRFP融合蛋白一起共孵育；同时设立稳转细胞与gp120-tagRFP融合蛋白直接共孵育的对照组。
进一步地，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，并判断新药对gp120/CCR5是否具有阻断功能。
进一步地，步骤6)中，将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞与CC趋化因子试剂和筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育；同时设立稳转细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的稳转细胞为空白组。
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绿色荧光蛋白d2EGFP荧光值，并判断新药对CC趋化因子/CCR5信号通路的影响。
另一方面，本发明提供一种稳转细胞系，其能够筛选对gp120/CCR5阻断功能具有阻断功能但不影响CC趋化因子/CCR5信号通路的药物。
进一步地，该稳转细胞系中包含由CMV启动高表达的人源CD4和CCR5二个基因以及一个由人工复合调控元件NF-κB RE-TATA-like调控的荧光蛋白，当CC趋化因子/CCR5信号通路正常连接或被阻断时，能够激发或不激发荧光信号。
另一方面，本发明提供一种基于gp120/CCR5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gp120蛋白羧基末端(C端)，制备gp120-tagRFP融合蛋白。
2)将人源CD4基因克隆到真核表达载体CMV启动子下，获得质粒pCMV-CD4；将CCR5构建在CMV启动子下，将绿色荧光蛋白d2EGFP基因构建在NF-κB RE-TATA-like人工复合调控元件的下游，两者构建于同一个慢病毒载体内，获得质粒pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP；将pCMV-CD4、pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP共转染到Jurkat细胞中，建立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞系。
3)将CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等新药与CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞共孵育后再与gp120-tagRFP融合蛋白一起共孵育；同时设立稳转细胞与gp120-tagRFP融合蛋白直接共孵育的对照组。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，并与对照组比较。
4)依据荧光值判断抗CCR5的新药对gp120/CCR5的阻断作用。
5)将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞与CC趋化因子试剂和筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育；同时设立稳转细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的稳转细胞为空白组。清洗去除非特异性结合后，检测细胞内的绿色荧光蛋白d2EGFP荧光值，并判断新药对CC趋化因子/CCR5信号通路的影响。
进一步地，编码gp120-tagRFP融合蛋白的核酸序列如SEQ ID NO：1所示，表达后gp120-tagRFP融合蛋白的氨基酸序列如SEQ ID NO：2所示；CCR5.NF-κB RE-TATA-like-d2EGFP的DNA基因序列如SEQ ID NO：3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CCR5药的效应。
(4)使用活细胞检测，一个细胞系可同时获得gp120/CCR5的阻断功能与CC趋化因子/CCR5信号通路的生物学效应数据，为快速筛选抗CCR5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-gp120tagRFP质粒图谱
图2pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP质粒图谱
图3使用快筛系统筛选抗CCR5单抗药。检测数据如图所示，空白组的平均荧光强度MFI(PE Texas Red)＝7.3；对照组的MFI(PE Texas Red)＝7929.1，是空白组的1086.2倍；实验组的MFI(PE Texas Red)各不相等，其中筛选出7个阻断性能优良(MFI(PE Texas Red)≤500)的抗CD47单抗药，即anti-CD47-5MFI(PE Texas Red)＝462.9；anti-CD47-12MFI(PE Texas Red)＝36.9；anti-CD47-19MFI(PE Texas Red)＝293.5；anti-CD47-29MFI(PE Texas Red)＝82.6；anti-CD47-33MFI(PE Texas Red)＝7.9；anti-CD47-48MFI(PE Texas Red)＝342.8；anti-CD47-54MFI(PE Texas Red)＝73.9。
图4使用快速筛选系统进一步筛选不影响CC趋化因子生物学效应的CCR5拮抗剂，检测数据如图所示，空白组MFI(Green)＝8.5，对照组MFI(Green)＝4892.6，为空白组的575.6倍，提示该CC趋化因子MIP-1α具有生物学效应。anti-CCR5-40MFI(Green)＝4891.6，说明该CCR5拮抗剂对CCR5的结合不影响CC趋化因子的生物学效应，具有进一步开发价值。而其余5种CCR5拮抗剂的MFI均明显地不同程度低于对照组，证明这几种CCR5拮抗剂和CC趋化因子MIP-1α有不用程度的抑制或竞争作用，应予以淘汰。
图5Elisa法筛选CCR5拮抗剂，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16个阻断性能较好(OD≤0.5)的CCR5拮抗剂，即anti-CCR5-5OD＝0.2；anti-CCR5-8OD＝0.3；anti-CCR5-9OD＝0.2；anti-CCR5-10OD＝0.5；anti-CCR5-23OD＝0.4；anti-CCR5-24OD＝0.3；anti-CCR5-25OD＝0.4；anti-CCR5-29OD＝0.4；anti-CCR5-33OD＝0.3；anti-CCR5-35OD＝0.2；anti-CCR5-37OD＝0.4；anti-CCR5-40OD＝0.5；anti-CCR5-41OD＝0.4；anti-CCR5-48OD＝0.4；anti-CCR5-57OD＝0.4；anti-CCR5-60OD＝0.2。
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gp120-tagRFP融合蛋白的制各
将红色荧光蛋白tagRFP基因与gp120基因构建在同一个读码框使红色荧光蛋白tagRFP接于gp120蛋白C端形成gp120-tagRFP融合蛋白，同时在tagRFP的C端接上6个组氨酸His标签。前后两端的限制性内切酶BamHI(GGATCC)和SexAI(ACCTGGT)用于基因合成后连接于慢病毒表达载体的CMV启动子下游，获得质粒pCMV-gp120-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。将pCMV-gp120-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gp120-tagRFP融合蛋白的gp120-tagRFP/293细胞克隆。扩增收集gp120-tagRFP/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gp120-tagRFP融合蛋白。
gp120-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gp120-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gp120-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、及gp120抗体验证。
如上述步骤中，获得gp120-tagRFP融合蛋白的DNA基因序列如SEQ ID NO：1所示，表达后gp120-tagRFP融合蛋白序列如SEQ ID NO：2所示。
实施例2质粒pCMV-CD4的构建
将人源CD4基因(NM_000616.5)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-CD4。
实施例3 pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP质粒的构建
将人源CCR5基因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-CCR5；再将绿色荧光蛋白d2EGFP构建在NF-κB RE-TATA-like人工复合调控元件下，通过Not I/Sex A I双酶切克隆至上述质粒pCMV-CCR5，去掉了原质粒中的PGK启动子和Puro抗性基因，构建双功能质粒pCMV-CCR5·NF-κB RE-TATA-like-d2EGFP(如图2所示)；其中CCR5基因由CMV启动子高效启动表达，d2EGFP报告基因由NF-κBRE-TATA-like调控表达。
CCR5_NF-κB RE-TATA-like_d2EGFP的DNA基因序列如SEQ ID NO：3所示；其中7-1064bp为CCR5基因、1075-1104bp为3个重复的NF-κB RE调控元件、1105-1253bp为TATA-like框、1254-2099bp为d2EGFP基因。
实施例4建立CD4.CCR5.NF-κB RE-TATA-Iike-d2EGFP/Jurkat稳转细胞系
将pCMV-CD4、pCMV-CCR5·NF-κB RE-TATA-like--d2EGFP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-CD4、CMV-CCR5·NF-κB RE-TATA-like-d2EGFP慢病毒，并共转染Jurkat细胞，筛选克隆建立新型的CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞系。
实施例5：使用快速筛选系统筛选CCR5拮抗剂
先将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与1μg/mL的61种待筛选CCR5拮抗剂(anti-CCR5-1～anti-CCR5-61)分别孵育20分钟。洗去非特异性结合后，再与2μg/mL的gp120-tagRFP融合蛋白共孵育为实验组。同时设立不做任何处理的CD4.CCR5.NF-κBRE-TATA-like-d2EGFP/Jurkat细胞为空白组；设立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与2μg/mL的gp120-tagRFP融合蛋白直接共孵育为对照组。共孵育至20分钟时，洗去非特异性结合，用流式细胞仪分析细胞表面tagRFP荧光值(如以guavaeasyCyte HT流式分析仪Red通道进行数据收集，下同)；
检测数据如图3所示，本实施例5中空白组的平均荧光强度MFI(Red)＝8.5；对照组的MFI(Red)＝34896.3，是空白组的4105.4倍；实验组的MFI(Red)各不相等，其中筛选出6个阻断性能优秀(MFI(Red)≤1000)的CCR5拮抗剂，即anti-CCR5-9MFI(Red)＝181.3；anti-CCR5-25MFI(Red)＝51.2；anti-CCR5-29MFI(Red)＝573.2；anti-CCR5-35MFI(Red)＝655.7；anti-CCR5-40MFI(Red)＝152.9；anti-CCR5-60MFI(Red)＝385.9；
如上所述，使用本文所述之系统从61种待筛选CCR5拮抗剂中筛选到6种具有较好阻断能力的CCR5拮抗剂。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CCR5拮抗剂阻断CCR5和gp120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。
实施例6：使用快速筛选系统进一步筛选不影响CC趋化因子生物学效应的CCR5拮抗剂
将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂(如MIP-1α)和上述实施例5筛到的6种具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育为实验组。同时设立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂(如MIP-1α)共孵育为对照组，设立不做任何处理的CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞为空白组。孵育完成后洗去非特异性结合，再用流式细胞仪分析细胞内的绿色荧光蛋白d2EGFP荧光值(如以guava easyCyte HT流式分析仪Green通道进行数据收集，下同)。
检测数据如图4所示，本实施例中的空白组MFI(Green)＝8.5，对照组MFI(Green)＝4892.6，为空白组的575.6倍，提示该CC趋化因子MIP-1α具有生物学效应。anti-CCR5-40MFI(Green)＝4891.6，证明该CCR5拮抗剂对CCR5的结合不影响CC趋化因子的生物学效应，具有进一步开发价值。而其余5种CCR5拮抗剂的MFI均明显地不同程度低于对照组，证明这几种CCR5拮抗剂和CC趋化因子MIP-1α有不用程度的抑制或竞争作用，应予以淘汰。
如上所述，使用本文所述之系统从61种待筛选CCR5拮抗剂中筛选到6种具有较好阻断能力的CCR5拮抗剂。再从这6种CCR5拮抗剂中进一步筛选到1种既具较好阻断力，又不影响CC趋化因子-CCR5的正常生物学效应的CCR5拮抗剂。进一步结合对比例1的数据，证明本系统在反映CCR5拮抗剂阻断CCR5-gp120结合上，其特异性、精确性和识别度是高度优于其他系统的，且同时能进一步体现其生物学效应，即不影响CC趋化因子对CCR5的功能性。
对比例1：Elisa法筛选CCR5拮抗剂。
先以250ng/mL浓度CCR5胞外端蛋白过夜包被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61种待筛选CCR5拮抗剂(anti-CCR5-1～anti-CCR5-61)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gp120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gp120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(0D)值。
检测数据如图5所示，本对比例1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16个阻断性能较好(OD≤0.5)的CCR5拮抗剂，即anti-CCR5-5OD＝0.2；anti-CCR5-8OD＝0.3；anti-CCR5-9OD＝0.2；anti-CCR5-10OD＝0.5；anti-CCR5-23OD＝0.4；anti-CCR5-24OD＝0.3；anti-CCR5-25OD＝0.4；anti-CCR5-29OD＝0.4；anti-CCR5-33OD＝0.3；anti-CCR5-35OD＝0.2；anti-CCR5-37OD＝0.4；anti-CCR5-40OD＝0.5；anti-CCR5-41OD＝0.4；anti-CCR5-48OD＝0.4；anti-CCR5-57OD＝0.4；anti-CCR5-60OD＝0.2。
对比实施例5和实施例6，不难发现，以Elisa法筛选CCR5拮抗剂，其特异性、精确性和识别度不如本文所述之系统，且难以直观的反映CCR5拮抗剂对于CC趋化因子生物学效应的影响。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CCR5拮抗剂的效应，稳定性好，精确度高，特异性极高。4)一个系统可同时进行CCR5拮抗剂阻断功能和NF-κB信号通路的生物学效应数据，简便多用。为快速筛选CCR5拮抗剂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> 杭州迈尔德生物科技有限公司
<120> 一种基于gp120 CCR5阻断功能及其生物效应的药物快速筛选方法
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 2299
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 1
ggatccatgc gcgtgatggg cacccagagg aactacagcc tgctgtggcg ctggggcacc 60
atcatcttct ggatgatcat gatctgcagc gcctccaacc tgtgggtgac cgtgtactac 120
ggcgtgccta tctggaggga cgccgacacc accctgttct gcgcatccga cgcaaaggca 180
tacgacaccg aggcacacaa cgtgtgggca acccacgcat gcgtgccaac cgacccaaac 240
ccacaggagg tgcctctgga gaacgtgacc gagaacttca acatgtggaa gaacaacatg 300
gtggagcaga tgcacgccga catcatctcc ctgtgggacc agagcctgaa gccatgcgtg 360
aagctgaccc ccctgtgcgt gaccctgaac tgttccggcg tgtacaacag ctccctgaag 420
aacgtgagcg aggagatgaa cggcgagatc aagaactgtt ccttcaacat gaccaccgag 480
ctgcgcgacc agaagcagaa ggtgcacgcc ctgttctaca ggatcgacgt ggtgccaatc 540
aacgactccg acagctccca gatcaccaac gtgagccaga acaacagcaa ctcccagcag 600
agctactccc gctacaggct gatcaactgc aacacctccg ccatcaccca gacctgtcca 660
aaggtgagct tcgagcctat cccaatccac tactgcgcac cagcaggatt cgcaatcctg 720
aagtgtaggg acaaggagtt caacggcacc ggcccttgca agaacgtgag caccgtgcag 780
tgtacccacg gcatccggcc agtggtgtcc acccagctgc tgctgaacgg cagcctggca 840
gagggcaagg tggtcatcag aaccgagaac atcaccgaca acgccaagac catcatcgtg 900
cagctgagcg agcctgtgcg gatcaactgc accagaccaa acaacaacac caggaagtcc 960
gtgcacatcg gaccaggaca gacctactac gcaaccggcg acatcatcgg cgacatccgc 1020
caggcccact gtgacgtgaa caagactgag tggaacaacg ccctgaggaa ggtggccgag 1080
cagctgaaca agacctaccc tatcaacggc accatcaact tcagaaacag ctccggaggc 1140
gacctggaga tcaccaccca ctccttcaac tgcggcggcg agttcttcta ctgtaacacc 1200
tccaagctgt tcaacagcac ctggaacgcc accgacatca acagcaacgt gtccaccgac 1260
agcggcaaga acgacaccat caccctgcca tgccggatca agcagatcat cagaatgtgg 1320
cagagcgtgg gacaggcaat gtacgcacca cctatccagg gcatcatcaa gtgtgagagc 1380
aacatcaccg gactgctgct gacccgggac ggaggcaagg agaacaactc caccaacgag 1440
acctacagac caggaggagg cgacatgagg gacaactgga ggtccgagct gtacaagtac 1500
aaggtggtga agatcgagcc cttcggcatc gcacctaccc tggcaaggcg gagagtggtg 1560
gagagggaga agagactcga gagcgagctg attaaggaga acatgcacat gaagctgtac 1620
atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc 1680
tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc 1740
ttcgacatcc tggctaccag cttcatgtac ggcagcagaa ccttcatcaa ccacacccag 1800
ggcatccccg acttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc 1860
acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc 1920
ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag 1980
aagaaaacac tcggctggga ggccaacacc gagatgctgt accccgctga cggcggcctg 2040
gaaggcagaa gcgacatggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttc 2100
aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat 2160
gtggaccaca gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac 2220
gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacaa gcatcaccat 2280
caccatcact agcggccgc 2299
<210> 2
<211> 761
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 2
Met Arg Val Met Gly Thr Gln Arg Asn Tyr Ser Leu Leu Trp Arg Trp
1 5 10 15
Gly Thr Ile Ile Phe Trp Met Ile Met Ile Cys Ser Ala Ser Asn Leu
20 25 30
Trp Val Thr Val Tyr Tyr Gly Val Pro Ile Trp Arg Asp Ala Asp Thr
35 40 45
Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Ala His
50 55 60
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln
65 70 75 80
Glu Val Pro Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn
85 90 95
Asn Met Val Glu Gln Met His Ala Asp Ile Ile Ser Leu Trp Asp Gln
100 105 110
Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn
115 120 125
Cys Ser Gly Val Tyr Asn Ser Ser Leu Lys Asn Val Ser Glu Glu Met
130 135 140
Asn Gly Glu Ile Lys Asn Cys Ser Phe Asn Met Thr Thr Glu Leu Arg
145 150 155 160
Asp Gln Lys Gln Lys Val His Ala Leu Phe Tyr Arg Ile Asp Val Val
165 170 175
Pro Ile Asn Asp Ser Asp Ser Ser Gln Ile Thr Asn Val Ser Gln Asn
180 185 190
Asn Ser Asn Ser Gln Gln Ser Tyr Ser Arg Tyr Arg Leu Ile Asn Cys
195 200 205
Asn Thr Ser Ala Ile Thr Gln Thr Cys Pro Lys Val Ser Phe Glu Pro
210 215 220
Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys
225 230 235 240
Arg Asp Lys Glu Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr
245 250 255
Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu
260 265 270
Leu Asn Gly Ser Leu Ala Glu Gly Lys Val Val Ile Arg Thr Glu Asn
275 280 285
Ile Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu Ser Glu Pro Val
290 295 300
Arg Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Val His
305 310 315 320
Ile Gly Pro Gly Gln Thr Tyr Tyr Ala Thr Gly Asp Ile Ile Gly Asp
325 330 335
Ile Arg Gln Ala His Cys Asp Val Asn Lys Thr Glu Trp Asn Asn Ala
340 345 350
Leu Arg Lys Val Ala Glu Gln Leu Asn Lys Thr Tyr Pro Ile Asn Gly
355 360 365
Thr Ile Asn Phe Arg Asn Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr
370 375 380
His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Ser Lys
385 390 395 400
Leu Phe Asn Ser Thr Trp Asn Ala Thr Asp Ile Asn Ser Asn Val Ser
405 410 415
Thr Asp Ser Gly Lys Asn Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys
420 425 430
Gln Ile Ile Arg Met Trp Gln Ser Val Gly Gln Ala Met Tyr Ala Pro
435 440 445
Pro Ile Gln Gly Ile Ile Lys Cys Glu Ser Asn Ile Thr Gly Leu Leu
450 455 460
Leu Thr Arg Asp Gly Gly Lys Glu Asn Asn Ser Thr Asn Glu Thr Tyr
465 470 475 480
Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr
485 490 495
Lys Tyr Lys Val Val Lys Ile Glu Pro Phe Gly Ile Ala Pro Thr Leu
500 505 510
Ala Arg Arg Arg Val Val Glu Arg Glu Lys Arg Leu Glu Ser Glu Leu
515 520 525
Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu Gly Thr Val Asn
530 535 540
Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu
545 550 555 560
Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly Gly Pro Leu Pro
565 570 575
Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Arg Thr
580 585 590
Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe
595 600 605
Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly
610 615 620
Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile
625 630 635 640
Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val
645 650 655
Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Asn Thr Glu Met Leu Tyr
660 665 670
Pro Ala Asp Gly Gly Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu
675 680 685
Val Gly Gly Gly His Leu Ile Cys Asn Phe Lys Thr Thr Tyr Arg Ser
690 695 700
Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp
705 710 715 720
His Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu
725 730 735
Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu
740 745 750
Gly His Lys His His His His His His
755 760
<210> 3
<211> 2106
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 3
gaattcatgg actaccaggt gagctcccca atctatgata tcaactacta tacctccgag 60
ccctgccaga agatcaatgt gaagcagatc gcagcaaggc tgctgccacc tctgtattcc 120
ctggtgttca tctttggctt cgtgggcaac atgctggtca tcctgatcct gatcaattgt 180
aagcgcctga agtccatgac cgacatctac ctgctgaacc tggccatctc tgatctgttc 240
tttctgctga cagtgccttt ttgggcacac tacgcagcag cacagtggga cttcggcaat 300
accatgtgcc agctgctgac accggcctgt acttcatcgg cttcttttcc ggcatcttct 360
ttatcatcct gctgaccatc gataggtacc tggccgtggt gcacgccgtg tttgccctga 420
aggcccgcac cgtgacattc ggcgtggtga cctctgtgat cacatgggtg gtggccgtgt 480
ttgcctctct gcctggcatc atcttcaccc ggagccagaa ggagggcctg cactatacat 540
gttctagcca cttcccatac tctcagtatc agttttggaa gaacttccag acactgaaga 600
tcgtgatcct gggcctggtg ctgccactgc tggtcatggt catctgctac agcggcatcc 660
tgaagaccct gctgcggtgt agaaatgaga agaagaggca ccgcgccgtg cggctgatct 720
ttacaatcat gatcgtgtac tttctgttct gggcccccta taacatcgtg ctgctgctga 780
ataccttcca ggagttcttt ggcctgaaca attgctcctc tagcaacaga ctggaccagg 840
ccatgcaggt gaccgagaca ctgggcatga cacactgctg tatcaacccc atcatctacg 900
cctttgtggg cgagaagttc cggaattatc tgctggtgtt ctttcagaag cacatcgcca 960
agagattttg taagtgctgt agcatcttcc agcaggaggc acctgagagg gccagcagcg 1020
tgtacaccag aagcacaggc gagcaggaga tctccgtggg cctgtagcgg ccgcgggact 1080
ttccgggact ttccgggact ttccgccgcc ccgactgcat ctgcgtgttc gaattcgcca 1140
atgacaagac gctgggcggg gtttgtgtca tcatagaact aaagacatgc aaatatattt 1200
cttccgggga caccgccagc aaacgcgagc aacgggccac ggggatgaag cagatggtga 1260
gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 1320
taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 1380
tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 1440
ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 1500
acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 1560
acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 1620
gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 1680
agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 1740
aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 1800
accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 1860
gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 1920
agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag aagcttagcc 1980
atggcttccc gccggaggtg gaggagcagg atgatggcac gctgcccatg tcttgtgccc 2040
aggagagcgg gatggaccgt caccctgcag cctgtgcttc tgctaggatc aatgtgtaga 2100
cctggt 2106
Claims (10)
1.一种基于gp120/CCR5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HIV-1的gp120蛋白羧基末端连接构建重组gp120-第一荧光蛋白融合蛋白质粒；将gp120-tagRFP基因克隆至真核高表达载体CMV启动子下游，在第一荧光蛋白的C端连接6个组氨酸His标签；并转入细胞表达纯化，获得gp120-第一荧光蛋白融合蛋白；
2)将人全长CD4基因克隆到真核表达载体CMV启动子下，构建表达质粒CMV-CD4；将人全长CCR5基因克隆到CMV启动子下，同时将第二荧光蛋白基因构建在核因子κB应答元件NF-κBRE和TATA-like启动子人工复合调控元件的下游，两者构建于同一个慢病毒载体内，构建高表达CCR5和调控表达报告基因质粒CMV-CCR5.NF-κB RE-TATA-like-第二荧光蛋白质粒；将CMV-CCR5.NF-κB RE-TATA-like-第二荧光蛋白基因和CMV-CD4基因共转染到Jurkat细胞，建立稳转细胞系；
3)将待筛选的CCR5拮抗剂与稳转细胞共孵育后再与gp120-第一荧光蛋白融合蛋白一起共孵育；同时设立稳转细胞与gp120-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；
4)检测第一荧光蛋白荧光值，并判断新药对gp120/CCR5具有阻断功能；
5)将稳转细胞与CC趋化因子试剂和上述4)筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育为实验组；同时设立稳转细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的稳转细胞为空白组；清洗非特异性结合；
6)检测第二荧光蛋白荧光值，并判断新药对CC趋化因子/CCR5信号通路的影响，筛选出对gp120/CCR5具有阻断功能但不影响CC趋化因子/CCR5信号通路的药物。
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；第二荧光蛋白是绿色荧光蛋白d2EGFP。
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gp120基因构建在同一个读码框使红色荧光蛋白tagRFP接于gp120蛋白C端形成所述的gp120-tagRFP融合蛋白质粒，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gp120-tagRFP基因克隆至真核高表达载体转染至人胚肾细胞293，筛选克隆获得高表达gp1203-tagRFP融合蛋白的gp120-tagRFP/293细胞系；扩增该gp120-tagRFP/293细胞，裂解后用His亲和法制备纯化含重组gp120-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，人全长CD4基因克隆到真核表达载体CMV启动子下，获得质粒pCMV-CD4；将人全长CCR5基因克隆到CMV启动子下，同时将绿色荧光蛋白d2EGFP基因构建在核因子κB应答元件NF-κB RE和TATA-like启动子人工复合调控元件的下游，两者构建于同一个慢病毒载体内，构建新型的高表达CCR5和调控表达报告基因质粒pCMV-CCR5.NF-κB RE-TATA-like-d2EGFP；将pCMV-CD4、pCMV-CCR5.pNF-κB RE-TATA-like-d2EGFP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-CD4和CMV-CCR5.pNF-κB RE-TATA-like-d2EGFP慢病毒，并共转染到Jurkat细胞，建立稳转细胞系CD4.CCR5.pNF-κB RE-TATA-like-d2EGFP/Jurkat；其中编码gp120-tagRFP融合蛋白的核酸序列如SEQ ID NO：1所示，表达后gp120-tagRFP融合蛋白的氨基酸序列如SEQID NO：2所示；CCR5.NF-κB RE-TATA-Iike-d2EGFP的DNA基因序列如SEQ ID NO：3所示。
6.根据权利要求5的药物快速筛选方法，其中步骤3)中，将待筛选的CCR5拮抗剂与CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞共孵育后再与gp120-tagRFP荧光蛋白融合蛋白一起共孵育；同时设立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与gp120-tagRFP荧光蛋白融合蛋白直接共孵育的对照组。
7.根据权利要求6的药物快速筛选方法，其中步骤5)中，将CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂和筛到的具有gp120/CCR5阻断功能的CCR5拮抗剂共孵育为实验组；同时设立CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞与CC趋化因子试剂共孵育为对照组，设立不做任何处理的CD4.CCR5.NF-κB RE-TATA-like-d2EGFP/Jurkat细胞为空白组。
8.根据权利要求7的药物快速筛选方法，其中步骤4)和6)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；以及细胞内的绿色荧光蛋白d2EGFP荧光值；通过药物组荧光值与对照组比较，判断抗CCR5药对gp120/CCR5阻断功能，以及对CC趋化因子/CCR5信号通路的影响。
9.根据权利要求1-8任一所述的药物快速筛选方法，其特征在于，所述待筛选的CCR5拮抗剂包括趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物。
10.一种如权利要求5的药物快速筛选方法中构建的稳转细胞系，其能够筛选对gp120/CCR5相互作用具有阻断功能但不影响CC趋化因子/CCR5信号通路的药物，其是CD4.CCR5.pNF-κB RE-TATA-like-d2EGFP/Jurkat稳转细胞系。
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