CN113150172B - Glp-1r/gipr双靶点激动剂融合蛋白及其制备方法与应用 - Google Patents
Glp-1r/gipr双靶点激动剂融合蛋白及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种对胰高血糖素样肽‑1(GLP‑1)受体(GLP‑1R)和葡萄糖依赖性促胰岛素多肽(GIP)受体(GIPR)具有双重激活活性的长效融合蛋白,及其制备方法与用途。所述融合蛋白从N‑端至C端依次由艾塞那肽(exendin‑4)或其突变体、人葡萄糖依赖性促胰岛素多肽(GIP)或其突变体、人免疫球蛋白IgG的Fc片段三个结构功能域通过连接肽(linker)或直接连接而成。本发明还提供了在大肠杆菌中可溶性表达上述融合蛋白的基因工程制备方法，该方法工艺简单，便于直接从细胞破壁液中纯化获得具有生物活性的蛋白产物。本发明所述融合蛋白对GLP‑1R和GIPR具有显著的双重激动剂活性，可应用于制备治疗糖尿病、肥胖、高血脂及其它相关疾病的药物。
Description
技术领域
本发明属于生物制药技术，具体涉及一种GLP-1R/GIPR双靶点激动剂融合蛋白及其制备方法，以及其在制备治疗糖尿病、肥胖、高血脂及其它相关疾病药物中的应用。
背景技术
糖尿病(Diabetes Mellitus,DM)是一种以高血糖为特征的内分泌代谢性疾病，主要由胰岛素分泌缺陷和/或胰岛素作用不足所致。糖尿病可分为1型(Type 1 diabetesmellitus，T1DM)和2型(Type 2 diabetes mellitus，T2DM)。其中，T1DM不产胰岛素，属胰岛素依赖性疾病，而T2DM主要是由于机体对胰岛素反应不敏感，产生胰岛素抵抗，占糖尿病患者90％以上。糖尿病会导致多种致命性并发症，如肥胖、心血管疾病、肾病等，严重危害人类健康。
治疗2型糖尿病的药物主要有磺脲类、双胍类、格列奈类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、二肽基肽酶IV(DPP-4)抑制剂、胰高血糖素样肽-1(GLP-1)受体(GLP-1R)激动剂、和钠–葡萄糖协同转运蛋白2(SGLT2)抑制剂等。其中，DPP-4抑制剂和GLP-1受体激动剂除具有降血糖、极少导致低血糖、安全性和耐受性较好等优点外，还对心血管、中枢神经、消化等多系统具有保护作用。
艾塞那肽(exendin-4)是一种从北美洲西北部钝尾毒蜥唾液中发现的外源性GLP-1R激动剂(GLP-1RAs)，由39个氨基酸组成，与人肠促胰岛素GLP-1氨基酸序列有53％的同源性，在哺乳动物体内的生理功能与GLP-1相似。由于艾塞那肽对DPP-4不敏感，与GLP-1相比，其体内半衰期显著延长，达3.3～4小时。艾塞那肽于2005年4月由美国Amylin与Eli Lilly公司开发上市，并于2009年8月在中国上市，成为首个上市的GLP-1受体激动剂药物。
GLP-1受体激动剂药物已在临床上广泛应用于2型糖尿病治疗，具体机制是其与GLP-1受体结合后，可刺激胰岛β细胞分泌胰岛素，同时抑制胰岛α细胞分泌胰高血糖素，并增强组织对胰岛素的敏感性，从而达到降低血糖的功效。这类药物最显著优点是，刺激胰岛素分泌具有葡萄糖依赖性，即只在高血糖浓度时发挥作用，而在血糖正常或偏低时不发挥作用，这样就有效避免了低血糖现象的发生，从而保证了临床用药安全性。葡萄糖依赖性促胰岛素多肽(Glucose-dependent insulinotropic polypeptide，GIP)也是一种肠促胰岛素，包含42个氨基酸残基，由十二指肠和空肠上段K细胞分泌。GIP可与胰岛β细胞表面的GIP受体(GIPR)结合，通过葡萄糖依赖的方式促进胰岛素的合成和分泌，即高血糖条件下刺激胰岛素分泌，降低血糖。然而，在低血糖条件下，GIP则可诱导胰高血糖素产生，由此维持血糖平衡(Christensen et al.(2011)Diabetes 60:3103-9)。P.K.等报道，GIP类似物ZP4165可增强GLP-1激动剂降血糖作用及减肥效果( et al.(2018)Diabetes Obes Metab 20:60-68)。Finan,B.等报道了一种对GLP-1R和GIPR具有共激活作用的单分子双促胰岛素(unimolecular dual incretins)多肽，其在db/db小鼠、ZDF大鼠、猴及人中均表现出较好的促进胰岛素分泌和降血糖作用(Finan et al.(2013)Sci TranslMed 5:209ra151)。
免疫球蛋白IgG是血液中丰度最高的蛋白之一，其体内半衰期可长达21天。因此，人的IgGFc片段(即人IgG的铰链区和恒定区CH2-CH3)可与其它活性蛋白或多肽进行融合以获得体内半衰期显著延长的长效融合蛋白药物。如，2008年美国Amgen公司研发上市的Romiplostim(罗米司亭)，就是一种由血小板生成素受体(TPO)结合肽与人IgG1Fc连接而成的融合蛋白，该融合蛋白既保留了TPO结合肽激活促血小板生成素(TPO)受体的功能，同时其体内半衰期大大延长，在临床上用于治疗慢性免疫性血小板减少性紫癜(ITP)。该融合蛋白在E.coli中表达时形成包涵体，下游处理工艺复杂，需经过变性和复性处理才能形成具有生物活性的可溶性蛋白。
目前未见GLP-1R/GIPR双靶点激动剂融合蛋白相关报道。
发明内容
发明目的：针对上述现有技术，本申请提供了一种新型的对人GLP-1R和GIPR具有双重激活活性的激动剂融合蛋白及其制备方法，还提供了融合蛋白在制备治疗糖尿病、肥胖、高血脂及其它相关疾病的药物中的应用。
技术方案：本申请所述的GLP-1R/GIPR双靶点激动剂融合蛋白从N-端至C端依次由艾塞那肽(exendin-4)或其突变体、人葡萄糖依赖性促胰岛素多肽(GIP)或其突变体、人免疫球蛋白IgG的Fc片段三个结构功能域通过连接肽(linker)或直接连接而成。
其中，所述野生型艾塞那肽序列见SEQ ID NO:1，其突变体由野生型艾塞那肽第21位Leu突变为Lys或Arg或His而成，具有SEQ ID NO:2～4所示的氨基酸序列或与该序列有90％以上同源性的序列。
所述天然人GIP(1-30)序列见SEQ ID NO:5，其突变体由第2位Ala突变为Gly或Ser而成，具有SEQ ID NO:6～7所示的氨基酸序列或与该序列有90％以上同源性。
所述人免疫球蛋白IgG的Fc片段是人IgG1Fc、IgG2Fc、IgG3Fc或IgG4Fc中的任意一种。
所述连接肽为富含Gly和/或Ala和/或Ser的柔性肽，长度在1～100个氨基酸残基之间，优选连接肽为(Gly-Gly-Gly-Gly-Ser)n，其中n是2～10的整数。
所述人免疫球蛋白IgG的Fc片段包括铰链区及恒定区CH2-CH3。所述铰链区可以是天然的或优化突变的人IgG的铰链区。所述恒定区CH2-CH3可以是天然的人IgG恒定区CH2-CH3或与其有90％以上同源性的序列。
优选的人IgG1铰链区具有SEQ ID NO:8所示序列，该序列由天然的人IgG1铰链区原形序列-VEPKSCDKTHTCPPCP-(SEQ ID NO:9)突变而成。
优选的人IgG1恒定区CH2-CH3：具有SEQ ID NO:10所示序列，或与该序列有90％以上同源性的序列。
进一步优选的，本申请所述GLP-1R/GIPR双靶点激动剂长效融合蛋白如EX-GIP-Fc、EX-L21K-GIP-Fc、EX-L21R-GIP-Fc、EX-L21H-GIP-Fc，分别具有SEQ ID NO:11～14所示氨基酸序列或与该序列有90％以上同源性。
本申请还公开了上述GLP-1R/GIPR双靶点激动剂长效融合蛋白的制备方法，包括以下步骤：
(1)设计合成并克隆所述融合蛋白的编码基因；
(2)构建成表达质粒后转化大肠杆菌或酵母，或转染哺乳动物细胞进行表达；
(3)收集菌体破壁液或培养液上清，分离纯化得到可溶性目的融合蛋白。
本申请还公开了上述GLP-1R/GIPR双靶点激动剂融合蛋白在制备治疗糖尿病、肥胖、高血脂及其它相关疾病的药物中的应用。
有益效果：本申请提供的GLP-1R/GIPR双靶点激动剂融合蛋白可同时激活人GLP-1R和GIPR，具有降糖活性高、起效快和降糖作用持续时间长的显著优点，有利于降低获得治疗效果的用药剂量，减少给药频率，提高药物治疗的依从性，改善治疗效果，为治疗糖尿病和、肥胖高血脂及其它相关等相关疾病提供新的药物。并且本申请所述GLP-1R/GIPR双靶点激动剂融合蛋白可在大肠杆菌E.coli中进行可溶性表达，且以可溶的二聚体形式存在，因此可直接从大肠杆菌E.coli破壁液上清中直接分离纯化得到具有生物活性的表达产物，避免了由于形成包涵体而带来的复杂的下游变性和复性处理过程。
附图说明
图1是GLP-1R/GIPR双靶点激动剂融合蛋白EX-L21K-GIP-Fc结构示意图,主要包括：起始甲硫氨酸Met、艾塞那肽突变体EX-L21K、连接肽(GGGGS)3、GIP-A2G、连接肽(GGGGS)2、突变的人IgG1铰链区(-DKTHTCPPCP-)、人IgG1恒定区CH2-CH3。编码基因5’端为起始密码子ATG，3’末端为两个终止密码子TAATAG；
图2是GLP-1R/GIPR双靶点激动剂融合蛋白EX-L21K-GIP-Fc表达质粒图谱，其中，T7promoter：T7启动子；EX-L21K-GIP-Fc：融合蛋白EX-L21K-GIP-Fc编码基因，克隆于NdeI与HindⅢ酶切位点之间；f1 origin：f1噬菌体基因组DNA的复制区序列；Kan：卡那霉素抗性基因；Ori：质粒复制起始原点；lacI：乳糖操纵子阻遏蛋白编码基因；
图3是Westernblot检测GLP-1R/GIPR双靶点激动剂长效融合蛋白EX-L21K-GIP-Fc的表达，检测抗体为HRP标记的抗人IgG抗体；
图4是FPLC凝胶色谱层析洗脱图谱；
图5是12％(w/v)还原型SDS-PAGE(A)和western blot(B)检测纯化产物EX-L21K-GIP-Fc结果图；
图6是分析型SEC-HPLC检测标准蛋白(BSA、鸡卵清蛋白、胰凝乳蛋白酶原和溶菌酶)结果图；
图7是分析型SEC-HPLC检测融合蛋白EX-L21K-GIP-Fc纯度及分子量结果图；
图8是分析型RP-HPLC检测融合蛋白EX-L21K-GIP-Fc纯度结果图；
图9是融合蛋白EX-L21K-GIP-Fc对HEK293/GLP-1R/CRE4细胞GLP-1R的激活作用结果图；
图10是融合蛋白EX-L21K-GIP-Fc对HEK293/GIPR/CRE4细胞GIPR的激活作用结果图；
图11是各组动物D36时经口葡萄糖糖负荷后的血糖-时间曲线下面积(AUC)结果图；“+++”表示相比正常对照组，P<0.001；“***”表示相比模型对照组，P<0.001；
图12是胰腺HE染色结果图。
具体实施方式
下面结合具体实施例对本申请作出详细说明。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下述实例中所用的材料、试剂、装置、仪器、设备等，如无特殊说明，均可从商业途径获得。
实施例1 GLP-1R/GIPR双靶点激动剂长效融合蛋白的设计
GLP-1R/GIPR双靶点激动剂长效融合蛋白结构如图1所示，从N端至C端依次由以下多肽结构域串联组成：高活性艾塞那肽突变体(EX-L21K)、富含Gly的连接肽(GGGGS)3、人葡萄糖依赖性促胰岛素多肽(GIP)突变体(GIP-A2G)、富含Gly的连接肽(GGGGS)3、优化突变的人IgG1的铰链区DKTHTCPPCP(下划线表示)、人IgG1恒定区CH2-CH3。
其中，高活性艾塞那肽突变体EX-L21K由野生型艾塞那肽的第21位Leu突变为Lys得到。
GIP突变体GIP-A2G由天然GIP第2位Ala突变为Gly得到。
优化突变的人IgG1的铰链区：具有-DKTHTCPPCP-(SEQ ID NO:8)所示氨基酸序列。
人IgG1恒定区CH2-CH3：具有SEQ ID NO:10所示序列。
上述GLP-1R/GIPR双靶点激动剂融合蛋白EX-L21K-GIP-Fc氨基酸序列如SEQ IDNO:12所示，其编码基因序列如SEQ ID NO:15所示。
实施例2 GLP-1R/GIPR双靶点激动剂长效融合蛋白的克隆与表达
(1)重组质粒pET27b-IgG1Fc的构建
利用实验室已合成的含人IgG1Fc编码基因(SEQ ID NO:16)的质粒为模板，设计以下正、反向引物(下划线分别表示BamH I和HindIII酶切位点)：
BamHI-IgG1Fc-F：AATTGGATCCGGTGGTGGTGGTTCTGACAAAACCCACACCTGC(SEQ ID NO:17)；
HindIII-IgG1Fc-R：GGCCGCAAGCTTCTATTATTTACCCGGA(SEQ ID NO:18)。
上述引物由南京GenScript生物技术公司合成。
采用PCR技术扩增得到IgG1Fc基因片段，BamH I和Hind III酶切后插入表达载体pET27b相应酶切位点，得到含人IgG1Fc编码基因的重组质粒pET27b-IgG1Fc。
(2)重组质粒pET27b-EX-L21K-GIP-Fc的构建
编码EX-L21K-GIP融合蛋白的基因(SEQ ID NO:19)由上海生工合成并克隆，Nde I和BamH I双酶切后亚克隆到上述含人IgG1Fc基因的质粒pET27b-IgG1Fc中，从而构建成表达GLP-1R/GIPR双靶点激动剂长效融合蛋白EX-L21K-GIP-Fc的质粒pET27b-EX-L21K-GIP-Fc(图2)，DNA测序正确后，CaCl2转化E.coliBL21(DE3)宿主菌，37℃于LBK(含50μg/mL卡那霉素的LB培养基)平板培养后，于-70℃甘油管保存。
(3)融合蛋白EX-L21K-GIP-Fc的摇瓶表达及产物鉴定
将上述pET27b-EX-L21K-GIP-Fc/E.coliBL21(DE3)工程菌于37℃LBK平板划线培养过夜，挑单菌落至30mlLBK液体培养基，于37℃，220rpm振荡培养8h。按2％(v/v)接种量转至50mL TB培养基(胰蛋白胨1.2％(w/v)，酵母粉2.4％(w/v)，甘油0.4％(v/v)，17mMKH2PO4，72mM K2HPO4·3H2O，50μg/mL卡那霉素)，37℃，220rpm振荡培养至OD600为1左右，加入乳糖至1g/L，于25℃，220rpm诱导表达12～16h。同时设立pET27b/E.coliBL21(DE3)对照，相同条件下对之进行摇瓶培养和乳糖诱导表达。
表达完成后，取2mL发酵液4℃离心(6000rpm，10min)，收集菌泥，加入1mL 100mMTris-HCl溶液(pH 8.0)重悬，冰浴条件下，3s间隙超声破碎1min。细胞破碎液4℃离心(12000rpm，30min)。取pET27b-EX-L21K-GIP-Fc/E.coliBL21(DE3)破壁液上清、破壁液沉淀重悬液，以及pET27b/E.coliBL21(DE3)对照组全菌破壁液进行12％SDS-PAGE电泳。然后，于4℃、100V恒压转印100min，将蛋白转印到PVDF膜(Roche,cat#03010040001)上。将膜置于5％封闭液(含5％脱脂牛奶和0.1％吐温20的TBS)中，室温封闭2h。于5％封闭液按1：5000稀释的HRP-conjugated Rabbit anti-human IgG抗体(cat#D110149，上海生工)中4℃孵育过夜。TBST(含0.1％吐温20的TBS)洗膜5次，每次5min，ECL显色。结果如图3所示，泳道1：pET27b-EX-L21K-GIP-Fc/BL21(DE3)菌体破壁液上清；泳道2：pET27b-EX-L21K-GIP-Fc/BL21(DE3)菌体破壁液沉淀；泳道3：pET27b/BL21(DE3)空质粒对照菌体破壁液。根据图示可见，表达EX-L21K-GIP-Fc菌体破壁液上清在分子量35kDa处有明显条带(泳道1)，而菌体破壁液沉淀在35kDa处只有微量蛋白(泳道2)。此外，空载体pET27b/BL21(DE3)对照组在35kDa处无蛋白条带。由此说明，目的蛋白EX-L21K-GIP-Fc在该表达条件下为可溶性表达。
实施例3融合蛋白EX-L21K-GIP-Fc工程菌发酵及分离纯化
(1)种子液制备
甘油管挑取菌液，在LBK培养板上进行2次三区划线培养。从LBK平板上挑取单菌落，接入30mL LBK液体培养基，37℃，220rpm培养12h左右，此为一级种子液。将一级种子液以2％(v/v)的接种量转入两个装有100mL LBK液体培养基的500mL锥形瓶中于37℃，220rpm培养12h，此为高密度发酵二级种子液，
(2)补料分批发酵
将二级种子液以4％(v/v)的接种量接入装有4.5L发酵培养基(酵母粉2.4％(w/v)，胰蛋白胨1.2％(w/v)，甘油0.4％(v/v)，17mM KH2PO4，72mM K2HPO4·3H2O，消泡剂0.1％(v/v)，121℃灭菌20min，灭菌降温后加入终浓度为100μg/mL的卡那霉素)的7L发酵罐中。37℃培养，搅拌速度230～400rpm/min，每隔一小时取样检测OD600。培养至对数生长期时(约3h)，将发酵罐温度降至25℃，通过蠕动泵向发酵罐中流加约10g乳糖，持续诱导蛋白表达9h，总发酵时长约12h。其间，控制pH在7.0左右，并通过调节转速和通气量将溶氧控制在10％。当溶氧和pH明显上升时，说明培养基中葡萄糖耗尽，菌体生长减慢，开始利用含氮有机物作为碳源，通过溶氧及pH反馈的方式进行补料。
(3)菌体破碎
发酵液离心收集菌泥，用100mM Tris-HCl溶液(pH 8.0)按10％(w/v)比例重悬，并加入终浓度为0.2M的精氨酸溶液(pH 8.0)。用ATS均质机(AH100B，ATS Engineering Inc.，Canada)破碎细胞至破碎液澄清，破碎过程中维持均质机压力在600bar左右。收集菌体破碎液，4℃离心(12000rpm，30min)，收集上清，0.45μm滤膜过滤，再用0.22μm滤膜过滤。
(4)Protein A柱亲和层析
取Protein A-Agarose填料(Cat#11134515001，Roche)装柱，水洗去除20％的乙醇保存液，然后用100mM Tris-HCl溶液(pH 8.0)充分平衡色谱柱。将过滤后的上清液上样至平衡好的色谱柱，调节上样流速为1mL/min。上样后用10mM Tris-HCl溶液(pH 8.0)洗色谱柱，除去未结合的杂蛋白，再用100mM Glycine(pH 3.0)洗脱目的蛋白，分管收集蛋白洗脱峰。洗脱收集液中即刻加入10％(v/v)的中和缓冲液1M Tris缓冲液(pH 8.0)进行混匀，将样品pH调至7.5-8.0。为保持蛋白活性，亲和层析于4℃层析柜中进行操作。
(5)超滤浓缩
Protein A亲和层析洗脱收集液于4℃离心(5500rpm，30min)，收集上清。然后，采用Amicon Ultra-15 10K超滤管(Cat#UFC901096，Millipore)，对其于4℃，5500rpm离心超滤，直至浓缩至1mL左右。向超滤管内加入10mM柠檬酸盐缓冲液(pH 5.8)，4℃，5500rpm离心超滤至管内溶液体积约1mL。柠檬酸盐缓冲液置换溶液3次。收集超滤管内蛋白，0.22μm滤膜过滤后，于4℃暂存。
(6)凝胶色谱层析
采用凝胶色谱层析在FPLC系统(Biologic Duo-Flow，BIO-RAD)上进行分离纯化，色谱柱为Superdex 200Increase 10/300(Cat#17-5715-01，GE)。用平衡缓冲液(10mM柠檬酸盐缓冲液，pH5.8)将色谱柱平衡后，上样500μL样品进行分离，流速为0.4mL/min，检测波长为280nm。洗脱峰图如图4，峰1为微量目的蛋白多聚体；峰2为目的蛋白二聚体。跟据峰图分管收集不同的蛋白洗脱峰。收集目的蛋白洗脱峰，于超净台内用0.22μm滤膜对样品进行无菌推滤，分装后于-80℃冰箱长期保存。
采用12％SDS-PAGE和western blot对分离纯化过程中收集的样品进行分析，结果分别见图5A和图5B，其中，M：标准蛋白marker，泳道1：菌体破壁液上清，泳道2-5：Protein A柱穿过液、10mM Tris-HCl洗杂液、100mM Gly洗脱液(管1)、100mM Gly洗脱液(管2)，泳道6-7：Superdex 200Increase 10/300分子筛柱层析洗脱峰1及峰2。
实施例4融合蛋白EX-L21K-GIP-Fc分子量检测及纯度分析
(1)融合蛋白EX-L21K-GIP-Fc分子量检测
采用分子排阻法(Size Exclusion Chromatography，SEC)在HPLC系统(LC-2010AHT，SHIMADZU Corp.)上进行分子量检测，色谱柱为Shodex PROTEIN KW-802.5(cat#F6989000，SHOWA DENKO K.K.，Japan)，流动相为20mM磷酸缓冲液(pH 7.4)，含0.1M Na2SO4(按层析柱说明书要求添加)，流速为0.7mL/min，检测波长为280nm，上样量为20μL，按面积归一化法计算样品的纯度。
以上海源叶生物科技公司购得的BSA(MW 66.4kDa)、鸡卵清蛋白(MW 45kDa)、胰凝乳蛋白酶原(MW 25.6kDa)和溶菌酶(MW 14.3kDa)为标准品进行分析，其保留时间分别是10.509min，11.216min，12.434min，13.656min(图6A)，所对应的保留体积分别为7.63mL，7.85mL，8.70mL，9.56mL。根据各标准蛋白的分子量和保留体积做出标准曲线(图6B)，得到分子量计算公式为lgMW＝-0.299Ve+4.019。
融合蛋白EX-L21K-GIP-Fc三次进样的平均保留时间为10.489min，对应的保留体积为7.34mL。根据上述公式推算出的目的蛋白EX-L21K-GIP-Fc分子量为66.64kDa，为ExPASy网站(http：//web.expasy.org/compute_pi/)预测的单体分子量34.81kDa的2倍。由此说明，纯化得到的目的蛋白EX-L21K-GIP-Fc以可溶的二聚体形式存在。
(2)融合蛋白EX-L21K-GIP-Fc纯度检测
采用上述SEC-HPLC系统(HPLC系统为LC-2010A HT,SHIMADZU Corp.，层析柱为ShodexPROTEIN KW-802.5。洗脱液为20mM磷酸缓冲液(pH 7.4)+0.1M Na2SO4，流速为0.7mL/min，检测波长为280nm，上样量为20μL)检测融合蛋白EX-L21K-GIP-Fc纯度(图7A)，在保留时间约14.5min时出现溶剂峰(图7B)，扣除溶剂峰，按面积归一化法计算样品EX-L21K-GIP-Fc的纯度为97.24％。三次进样平均保留时间为10.489min，对应的保留体积为7.34mL，由此推算出目的蛋白EX-L21K-GIP-Fc子量为66.64kDa。
采用反相高效液相色谱法(RP-HPLC)在HPLC系统(LC-2010A HT，SHIMADZU Corp.)上检测EX-L21K-GIP-Fc纯度，反相柱为Symmetry 300 C4(4.6mm×250mm，5μm)(cat#186000289,Waters)。A相(0.05％三氟乙酸水溶液)和B相(0.05％三氟乙酸乙腈溶液)。室温梯度洗脱14min，B相由30％升高至60％。流速：0.5mL/min；检测波长：280nm；上样量：10μL。A.检测融合蛋白EX-L21K-GIP-Fc；B.检测溶剂。结果如图8A所示。在保留时间约6min时出现溶剂峰，如图8B所示，扣除溶剂峰按面积归一化法计算样品EX-L21K-GIP-Fc的纯度为99.65％。
实施例5融合蛋白EX-L21K-GIP-Fc对胰高血糖素样肽-1受体(GLP-1R)的激活作用
(1)构建HEK293/CRE4细胞
为了克隆含4个非回文CRE(cAMP响应元件)序列(Chepurny et al.(2007)JBiomol Screen 12:740-6)的DNA片段，合成以下2条互补寡核苷酸链(下划线所示为CRE序列)：
CRE4-F：5-CTAGCAGCCTGACGTCCGAGAGCCTGACGTCCGAGAGCCTGACGTCCGAGAGCCTGACG TCCGAGA-3(SEQ ID NO:20)
CRE4-R：5-GATCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGT CAGGCTG-3(SEQ ID NO:21)
取上述正、负链(100μM)各10μl，5×退火缓冲液(cat#D0251，beyotime公司)8μl，ddH2O 2μl，混合后于95℃变性5min，缓慢降至室温(2h内)进行退火，获得含4个CRE的双链DNA片段。另外，用限制性内切酶NheI和Bgl-II对pGL4.26质粒(cat#E8441，Promega)进行双酶切，并用琼脂糖电泳回收载体片段。然后，用T4DNA连接酶(cat#M0202S，NEB)将含4个CRE的DNA片段与pGL4.26载体于16℃连接2h后，转化E.coliDH 5α宿主菌，并涂布于氨苄抗性LB平板，筛选阳性克隆，送上海生工进行DNA测序验证，重组质粒命名为pGL4.26-CRE。
人胚胎肾HEK293细胞(购自中科院上海生命科学研究院细胞库)，培养于DMEM(cat#12800-058，Gibco)完全培养基。6孔板铺细胞，106个细胞/孔，培养18～24小时。PBS洗细胞后，每孔加入1mL无血清且无双抗的opti-MEM(cat#31985-062，Gibco)，继续培养1小时。用250μl opti-MEM稀释2.5μg质粒pGL4.26-CRE，混匀后加入7μl转染试剂lipo3000转染试剂(cat#L3000001，Thermofisher)(质粒w：转染试剂v＝1:3)，混匀8s，静置10min。将质粒-转染试剂混合物加入6孔板细胞中，6h后更换成DMEM完全培养基继续培养48h。用含50μg/ml潮霉素B(cat#31282-04-9，beyotime公司)的选择培养基进行加压筛选，每3天更换一次培养基，筛选2周左右，并设未转染质粒的孔作为对照。消化并稀释细胞至每毫升5细胞/ml，铺96孔板，1个细胞/200μl/孔。培养2-3周,传至24孔板，长满后再传至6孔板。提取基因组，用PCR方法扩增荧光素酶基因(Luc)基因的方法，筛选出pGL4.26-CRE质粒高拷贝整合到基因组的HEK293/CRE4细胞阳性克隆。
(2)构建HEK293/GLP-1R/CRE4细胞
人GLP-1R编码基因(NM-002062.2)由金唯智生物科技公司合成。采用TAKARA公司软件设计In-Fusion PCR引物：
GLP-1R-F：5-ACCCAAGCTGGCTAGCATGGCCGGCGCCCCCGGC-3(SEQ ID NO:22)
GLP-1R-R：5-GCTGATCAGCGGTTTAAACTCAGCTGCAGGAGGCCTGG-3(SEQ ID NO:23)
以合成的人GLP-1R编码基因为模板，用保真DNA聚合酶PCR扩增人GLP-1R编码基因，并用凝胶回收试剂盒回收。另外，用NheI和PmeI内切酶对pcDNA3.1(-)质粒(cat#V79520，Invitrogen)进行双酶切，并回收载体片段。然后，用In-Fusion HD Cloning kit(cat#638910，Takara)将人GLP-1R编码基因克隆到pcDNA3.1中，DNA测序验证，得到pcDNA3.1-GLP1R重组质粒。
用Lipo3000转染试剂将pcDNA3.1-GLP1R质粒转染实施例5(1)构建的HEK293-pGL4.26-CRE4细胞。经300μg/ml G418(cat#108321-42-2，Biofroxx)加压筛选后，96孔板铺单克隆，提取基因组DNA，并以其为模板采用PCR扩增G418抗性基因的方法，筛选出pcDNA3.1-GLP1R质粒已整合到基因组中的阳性单克隆。G418抗性基因PCR所需引物序列如下：
G418-F：5-CTATTCGGCTATGACTGGGC-3(SEQ ID NO:24)
G418-R：5-AATATCACGGGTAGCCAACG-3(SEQ ID NO:25)
对筛选出的pcDNA3.1-GLP1R质粒已整合到基因组中的阳性单克隆，提取蛋白，用Anti-GLP-1R抗体(cat#26196-1-AP，Proteintech)进行western blot检测，筛选出GLP1R蛋白高表达的克隆。然后，用300μg/ml G418和50μg/ml潮霉素加压筛选4周，再通过稀释法在96孔板中进行单克隆分离培养，得到HEK293/GLP-1R/CRE4单克隆。
在此基础上，用BeyoGold全黑96孔细胞培养板(CAT#FCP966，beyotime公司)对上述筛选得到的EK293/GLP-1R/CRE4细胞进行铺板，细胞密度为10000细胞/100μl/孔，用10倍梯度稀释的(0.0001～10000nM)的艾塞那肽(cat#052143，HPLC纯度99.41，吉尔生化)处理细胞6小时，同时设置HEK293/CRE4细胞阴性对照组。采用Bright-LumiTM萤火虫萤光素酶报告基因检测试剂盒(cat#D7170M，beyotime公司)方法，在多功能酶标仪上进行化学发光检测，筛选出对艾塞那肽激动剂反应性最强的HEK293/GLP-1R/CRE4克隆，用于后续GLP-1R激动剂活性检测。
(3)检测融合蛋白EX-L21K-GIP-Fc对GLP-1R的激动剂活性
用含10％FBS的DMEM完全培养基，配制10倍梯度稀释的(0.0001～10000nM)艾塞那肽及EX-L21K-GIP-Fc融合蛋白药物溶液(为进行活性比较，EX-L21K-GIP-Fc融合蛋白按单体分子量计算其摩尔浓度)。
按实施例5(2)所述药物处理方法，将艾塞那肽及EX-L21K-GIP-Fc融合蛋白(纯度≥95％)处理HEK293/GLP-1R/CRE4细胞，并采用Varioskan flash全波长荧光酶标仪检测萤光素酶活性。读取化学发光的信号，每孔读取时间为1500ms。实验重复3次。结果以平均值±SEM表示。用GraphPad Prism.6软件分析剂量-反应关系，并采用四参数方程拟合剂量反应曲线并计算EC50。结果如图9及表1所示，说明融合蛋白EX-L21K-GIP-Fc对GLP-1R具有显著激活作用，且活性优于阳性对照药艾塞那肽。
表1 融合蛋白EX-L21K-GIP-Fc对GLP-1R的激动剂活性
实施例6融合蛋白EX-L21K-GIP-Fc对葡萄糖依赖性促胰岛素多肽受体(GIPR)的激活作用
(1)构建HEK293/GIPR/CRE4细胞
人GIPR编码基因(NM_000164.2)由金唯智生物科技公司合成。采用TAKARA公司软件设计In-Fusion PCR引物：
GIPR-F：5-ACCCAAGCTGGCTAGCGCCACCATGACTACCTCTCCG-3(SEQ ID NO:26)
GIPR-R：5-GCTGATCAGCGGTTTAAACCTAGCAGTAACTTTCCAACTCCCGG-3(SEQ ID NO:27)
以合成的人GIPR编码基因为模板，用高保真DNA聚合酶PCR扩增人GIPR编码基因。另外，用NheI和PmeI双酶切pcDNA3.1(-)质粒(cat#V79520，Invitrogen)进行，并回收载体片段。然后，用In-Fusion HD Cloning kit(cat#638910，Takara)将人GIPR编码基因克隆到pcDNA3.1中得到pcDNA3.1-GIPR重组质粒。
按实施例5(2)中所述pcDNA3.1-GLP1R质粒转染方法，将pcDNA3.1-GIPR质粒转染实施例5(1)构建的HEK293-pGL4.26-CRE4细胞，并通过筛选获得对GIP-A2G多肽激动剂反应性最强的HEK293/GIPR/CRE4克隆，用于后续GIPR激动剂活性检测。
(2)检测融合蛋白EX-L21K-GIP-Fc对GIPR的激动剂活性
按实施例5(2)所述药物处理方法，将GIP-A2G及EX-L21K-GIP-Fc融合蛋白(纯度≥95％)处理HEK293/GLP-1R/CRE4细胞，并采用Varioskan flash全波长荧光酶标仪检测萤光素酶活性。读取化学发光的信号，每孔读取时间为1500ms。实验重复3次。结果以平均值±SEM表示。用GraphPad Prism.6软件分析剂量-反应关系，并采用四参数方程拟合剂量反应曲线并计算EC50。结果见图10及表2，说明融合蛋白EX-L21K-GIP-Fc对GIPR具有显著激活作用，且活性优于GIP-A2G阳性对照。
表2 融合蛋白EX-L21K-GIP-Fc对GIPR的激动剂活性
实施例7融合蛋白EX-L21K-GIP-Fc动物体内药效试验
(1)实验动物及分组
5周龄雄性dbdb小鼠及C57BL/6背景鼠(江苏集萃药康生物科技股份有限公司)，C57BL/6鼠正常饲料饲养，dbdb鼠高脂饲料饲养。适应性喂养1周，给药时为6周龄。dbdb鼠尾尖采血测血糖。不禁食血糖≥11.6即为合格模型鼠。
按表3进行实验动物分组(n＝7)。正常对照组为C57BL/6小鼠，正常饲料饲养。其余各组为dbdb小鼠，高脂饲料饲养。
表3实验动物分组给药剂量
(2)动物给药
模型对照组：D1、D8、D11、D15、D18、D22、D25、D29给药一次；
阳性药(Exendin-4)组：D1单次给药；D8～D29每日给药两次；
EX-L21K-GIP-Fc组：D1、D8、D11、D15、D18、D22、D25、D29给药一次；
(3)体重
所有动物分组前称重1次；D8、D15、D22、D29、D35各称重1次，结果(表4)表明，EX-L21K-GIP-Fc具有显著减肥效果。
表4体重(g)统计数据
与模型对照组比较，*P＜0.05，**P＜0.01
(4)血糖检测
所有动物D1和D29给药前、药后30min、1h、2h、4h、8h、12h、24h、36h、48h、60h、72h、96h、120h、144h各测定1次血糖，结果分别见表5～6。D9、D16、D23各测定1次非空腹血糖(阳性对照组为当日首次给药后1h测定),结果见表7。D12、D19、D26各测定1次空腹血糖(禁食16h；阳性对照组为当日首次药后1h),结果见表8。取血方法：用手术刀片从小鼠尾尖处取血，第一滴舍弃不用，收集第二滴用于检测血糖。上述结果表明，EX-L21K-GIP-Fc降血糖效果显著，具有显著长效性。
表5 D1单次给药血糖值统计数据
与模型对照组比较，*P＜0.05，**P＜0.01
与模型对照组比较，*P＜0.05，**P＜0.01
与模型对照组比较，*P＜0.05，**P＜0.01
表6 D29血糖值统计数据
与模型对照组比较，*P＜0.05，**P＜0.01
与模型对照组比较，*P＜0.05，**P＜0.01
与模型对照组比较，*P＜0.05，**P＜0.01
表7非空腹血糖值统计数据
与模型对照组比较，*P＜0.05，**P＜0.01
表8空腹血糖值统计数据
与模型对照组比较，*P＜0.05，**P＜0.01
(5)口服糖耐量(OGTT)检测
所有动物D36测定空腹血糖后，灌胃给予1g/kg的葡萄糖溶液，测定葡萄糖负荷后15min、30min、60min、90min、120min的血糖。
计算血糖-时间曲线下面积(AUC)：AUC＝1/2(0min+15min)×0.25h+1/2(15min+30min)×0.25h+1/2(30min+60min)×0.5h……。结果(图11)表明，相比正常对照组，模型组的糖耐量明显受损。相比模型组，阳性对照药Exendin-4和双靶点融合蛋白EX-L21K-GIP-Fc对db/db小鼠糖耐量均有明显改善，但EX-L21K-GIP-Fc的改善效果更为显著。
(6)糖化血红蛋白(HbA1c)
所有动物D36(正常对照组为D37)实验结束后,异氟烷麻醉下腹主动脉,取血0.3～0.5mL,EDTA-2K抗凝,采用德国罗氏P800全自动生化分析仪测定糖化血红蛋白(HbA1c)。结果显示，与模型组相比，双靶点融合蛋白EX-L21K-GIP-Fc与阳性药Exendin-4均具有显著降低HbA1c效果，且EX-L21K-GIP-Fc明显优于阳性药Exendin-4(表9)。
(7)血脂测定
所有动物D36(正常对照组为D37)实验结束后,异氟烷麻醉下腹主动脉,取血0.3～0.5mL,置促凝管，3000rpm离心10min分离血清，采用德国罗氏P800全自动生化分析仪测定总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇。结果显示，双靶点融合蛋白EX-L21K-GIP-Fc与阳性药Exendin-4均具有显著降LDL-C和TC的功效，且EX-L21K-GIP-Fc优于阳性药Exendin-4。此外，EX-L21K-GIP-Fc与阳性药Exendin-4均有明显降TG功效(表9)。
表9糖化血红蛋白及血脂统计数据
与模型对照组比较，*P＜0.05，**P＜0.01；与Exendin-4组比较，#P＜0.05，##P＜0.01
(8)胰腺HE染色
实验终点时，小鼠胰腺组织经HE染色后，结果如图12所示，正常组C57BL/6小鼠，胰岛结构完整，细胞排列整齐。模型组db/db小鼠胰岛损伤严重，胰岛边缘萎缩变形。与模型组相比，EX-L21K-GIP-Fc及Exendin-4给药后均对db/db糖尿病小鼠胰岛损伤具有明显保护作用，胰岛结构及细胞形态明显改善。
序列表
<110> 中国药科大学
<120> GLP-1R/GIPR双靶点激动剂融合蛋白及其制备方法与应用
<141> 2021-04-28
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<212> DNA
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atgcacggtg aaggtacctt cacctctgac ctgtctaaac agatggaaga agaagcggtt 60
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aaagcgctgc cggcgccgat cgaaaaaacc atctctaaag cgaaaggtca gccgcgtgaa 660
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<211> 837
<212> DNA
<213> 人工序列(Artificial Sequence)
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atgcacggtg aaggtacctt cacctctgac ctgtctaaac agatggaaga agaagcggtt 60
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gcgccggaac tgctgggtgg tccgtctgtt ttcctgttcc cgccgaaacc gaaagacacc 240
ctgatgatct ctcgtacccc ggaagttacc tgcgttgttg ttgacgtttc tcacgaagac 300
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tacaaaacca ccccgccggt tctggactct gacggttctt tcttcctgta ctctaaactg 720
accgttgaca aatctcgttg gcagcagggt aacgttttct cttgctctgt tatgcacgaa 780
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<210> 17
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aattggatcc ggtggtggtg gttctgacaa aacccacacc tgc 43
<210> 18
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ggccgcaagc ttctattatt tacccgga 28
<210> 19
<211> 277
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tacatatgca cggtgaaggt accttcacct ctgacctgtc taaacagatg gaagaagaag 60
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cgtctggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct tacggtgaag 180
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<210> 20
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ctagcagcct gacgtccgag agcctgacgt ccgagagcct gacgtccgag agcctgacgt 60
ccgaga 66
<210> 21
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gatctctcgg acgtcaggct ctcggacgtc aggctctcgg acgtcaggct ctcggacgtc 60
aggctg 66
<210> 22
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
acccaagctg gctagcatgg ccggcgcccc cggc 34
<210> 23
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gctgatcagc ggtttaaact cagctgcagg aggcctgg 38
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ctattcggct atgactgggc 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
aatatcacgg gtagccaacg 20
<210> 26
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
acccaagctg gctagcgcca ccatgactac ctctccg 37
<210> 27
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gctgatcagc ggtttaaacc tagcagtaac tttccaactc ccgg 44
Claims (4)
1.一种GLP-1R/GIPR双靶点激动剂融合蛋白，其特征在于，所述融合蛋白从N-端至C端依次由艾塞那肽exendin-4突变体、人葡萄糖依赖性促胰岛素多肽GIP突变体、人免疫球蛋白IgG的Fc片段三个结构功能域通过连接肽linker连接而成，其具体的氨基酸序列如SEQID NO.12所示。
2.根据权利要求1所述的GLP-1R/GIPR双靶点激动剂融合蛋白，其特征在于，所述融合蛋白，其编码基因序列如SEQ ID NO:15所示。
3.权利要求1所述GLP-1R/GIPR双靶点激动剂融合蛋白的制备方法，其特征在于，包括以下步骤：
（1）设计合成并克隆权利要求1所述融合蛋白的编码基因；
（2）构建权利要求1所述融合蛋白的表达质粒，将其转化大肠杆菌进行表达；
（3）对已表达的权利要求1所述融合蛋白进行分离纯化。
4.权利要求1所述GLP-1R/GIPR双靶点激动剂融合蛋白在制备治疗糖尿病、肥胖、高血脂的药物中的应用。
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