CN114106194A - 一种用于治疗糖尿病和/或肥胖症的融合蛋白 - Google Patents
一种用于治疗糖尿病和/或肥胖症的融合蛋白 Download PDFInfo
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- CN114106194A CN114106194A CN202010900880.3A CN202010900880A CN114106194A CN 114106194 A CN114106194 A CN 114106194A CN 202010900880 A CN202010900880 A CN 202010900880A CN 114106194 A CN114106194 A CN 114106194A
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Abstract
本发明涉及一种用于治疗糖尿病和/或肥胖症的融合蛋白，该融合蛋白的结构为：GLP‑1或其类似物、衍生物—连接肽—白蛋白结合蛋白；所述白蛋白结合蛋白包括ABD结构域(GA3、ABD035、ABDCon)或可以靶向HSA的DARPin。本发明通过与ABD结构域或DARPin蛋白的连接，解决了GLP‑1或Ex半衰期较短的瓶颈问题，使其能够更好的应用于临床上。通过本发明试验可以看出白蛋白结合蛋白显著延长了GLP‑1或Ex的半衰期，并提高了在小鼠体内的降血糖活性。改造后的GLP‑1或Ex具有一定的减轻体重的作用，可以用来治疗或辅助治疗肥胖。
Description
技术领域
本发明属于生物技术领域，具体涉及一系列新型的半衰期延长且活性提高的GLP-1受体激动剂，其主要应用于糖尿病治疗领域或肥胖治疗领域。
背景技术
随着人们生活水平的提高、饮食结构的变化以及体力劳动的减少等，糖尿病患者的数量呈现逐渐增长的态势。
糖尿病是一种以高血糖为主要特征的慢性代谢疾病，目前已成为严重威胁人类健康的疾病之一，且发病率呈逐年上升的趋势。2017年全世界糖尿病患者为4.2亿，预计到2045年这一数字将达到6.3亿。我国糖尿病人口总数已经超过了1.14亿，2型糖尿病患者超过90％以上，而其中肥胖人群占约80％以上。2型糖尿病的主要病因是胰岛素抵抗(Insulinresistance)，并伴有不同程度的胰岛素分泌不足。
胰高血糖素样肽-1(Glucagon like peptide 1，GLP-1)是由小肠L-细胞在饮食后分泌，具有葡萄糖依赖的肠促胰岛素效应(Glucose dependent incretin effect)的多肽类激素。GLP-1在体内有两种结构GLP-1(7-37)和N端酰胺化的GLP-1(7-36)，分别含有31个和30个氨基酸。这两种结构都能与细胞表面受体(GLP-1R，一种G蛋白偶联受体)结合，发挥促进胰岛素分泌、促进胰岛β细胞的增殖及分化、抑制胰高血糖素分泌、产生饱腹感、抑制进食、抑制胃排空等作用，从而能够显著降低血糖水平，并能有效减轻体重。因此，GLP-1可以被用来治疗糖尿病和肥胖。
艾塞那肽(Exenatide，Ex)是GLP-1的类似物，含有39个氨基酸，是从毒蜥蜴的唾液中分离出来的多肽类激素；与哺乳动物GLP-1具有53％的同源性，其在体内也能与GLP-1受体结合，发挥与GLP-1类似的作用；GLP-1与Ex也被称为GLP-1受体激动剂。GLP-1和Ex被认为是治疗2型糖尿病和肥胖的理想药物。因此，鉴于GLP-1受体激动剂具有治疗2型糖尿病和肥胖的双重功能，2010年，美国FDA先批准了诺和诺德的GLP-1类似物利拉鲁肽(商标名Victoza)用来治疗2型糖尿病；然后，在2014年，又批准了利拉鲁肽(商标名Saxenda)用于治疗肥胖或超重。
然而，GLP-1由于在体内受到二肽基肽酶-4(Dipeptidyl Peptidase 4，DPP-4)对其N端的降解，以及肾脏的快速过滤排出作用，其半衰期仅有1-2分钟，严重影响其作为药物的应用潜力。而艾塞那肽的分子量也较小，容易受到肾脏的过滤排出作用，在人体内的半衰期仅有2.4h，需要每日注射两次，较高的注射频率严重降低了患者的依从性。因此，解决GLP-1和艾塞那肽半衰期较短这一关键技术瓶颈将推动GLP-1和艾塞那肽在临床上的应用，具有重要意义。
目前用于延长GLP-1或艾塞那肽的半衰期的方法有：
1)氨基酸替换。天然的GLP-1的前两个氨基酸是组氨酸和丙氨酸，容易被体内的DPP-4所识别而切割，从而失去活性。因此，丙氨酸可以被替换成丝氨酸、甘氨酸或其他的非天然氨基酸，这样DPP-4不能识别该位点，从而延长半衰期；且突变后其活性不受损失。如艾塞那肽在这个位置的氨基酸为甘氨酸，半衰期显著延长。
2)PEG修饰。聚乙二醇(PEG)是美国FDA批准的可体内注射的药用成分之一。将PEG与GLP-1序列上的组氨酸和赖氨酸反应，可以将不同分子量的PEG与GLP-1连接，从而增加GLP-1的分子大小，减缓肾脏的过滤排出作用，也能降低蛋白酶的降解，从而延长半衰期。
3)构建融合蛋白。融合蛋白是将2个或2个以上的基因序列连接，并由一个宿主细胞进行表达后的产物。如2个GLP-1分子串联后，在与人血清白蛋白(HSA)进行结合，研发出了阿必鲁肽，是葛兰素史克(GSK)与2014年EMA批准上市的一个长效GLP-1受体激动剂。该药物是在GLP-1氨基酸序列改造(甘氨酸取代丙氨酸)的基础上，在GLP-1的C-末端融合了人血清白蛋白(585个氨基酸)，这样不仅可以避免了DPP-4的降解，还增加了GLP-1的分子量，增加了其流体力学体积，从而减少了肾脏对GLP-1的过滤排出作用，从而显著延长了半衰期，在人体内的半衰期达到了6-8天。
美国礼来公司研发了一种GLP-1与IgG Fc的融合蛋白(杜拉鲁肽)，杜拉鲁肽对GLP-1的序列进行了一些改造，将第8位丙氨酸突变为甘氨酸，将26位的甘氨酸替换为谷氨酸，将第36位的精氨酸替换为甘氨酸，然后通过一个连接肽与Fc片段连接，在人体内的半衰期达到了4天。
4)FcRn(新生儿Fc受体)介导的循环再生再生作用来延长半衰期。阿必鲁肽和杜拉鲁肽具有显著延长的半衰期，除了依靠人血清白蛋白和Fc显著增加了GLP-1的分子大小外，还能与FcRn进行结合，依靠FcRn介导的循环再生作用，而延长半衰期。FcRn是一种膜蛋白受体，主要存在于上皮细胞和内皮细胞，但在肝脏、肾脏和肠细胞也有分布。在血液中，人血清白蛋白和IgG能与FcRn以一种pH依赖的方式结合；与FcRn结合后，经胞吞作用被细胞吸收后，在内涵体的酸性环境(pH<6.5)中与FcRn结合，可以免受溶酶体的降解；在生理pH(7.4)时，HSA和IgG重新释放到血液中，从而明显延长了人血清白蛋白和IgG的半衰期，使其在体内的半衰期可达19天和21天。利用FcRn介导的循环再生机制延长其在血液中的半衰期。
虽然以上技术能够延长GLP-1或者艾塞那肽的半衰期，但是在实际生产和应用过程中也存在一些问题。如PEG方法，生产成本较高，会导致药物活性降低；近年来又发现PEG修饰的蛋白和其降解物容易在肾脏中累积，干扰了正常肾脏的过滤作用；且人会对PEG产生一定的抗体，表明其具有一定的免疫原性。
Fc的细胞毒作用和补体激活作用有时会对机体造成一定的伤害；Fc融合蛋白多为二聚体形式，过大的分子质量会影响药物分子通过粘膜的速率；Albumin融合蛋白在其发酵、纯化和储存过程中易发生降解和聚合现象，会导致活性损失，并有可能引起毒副作用；Albumin和Fc需要通过酵母或者哺乳动物细胞进行表达，周期较长，成本较高。
因此，有必要开发一种免疫原性低、成本较低、表达纯化更简单的载体，来延长GLP-1或艾塞那肽(Ex)的半衰期，进一步降低生产成本，减轻患者的经济负担，提高患者的依从性。
发明内容
本发明主要是将糖尿病治疗多肽GLP-1或艾塞那肽(Ex)以基因融合的方式通过连接肽与白蛋白结合蛋白(各种ABD结构域或DARPin蛋白)连接，并在表达系统中进行表达，从而获得GLP-1、Ex与ABD或DARPin的融合蛋白，获得的融合蛋白具有延长的半衰期和延长的降血糖活性，可以应用于治疗2型糖尿病或减轻体重。
本发明采用的技术方案为：
一种融合蛋白，该融合蛋白的结构为：GLP-1或其类似物(如Ex)、衍生物—连接肽—白蛋白结合蛋白。
所述白蛋白结合蛋白包括ABD结构域(GA3、ABD035、ABDCon)或可以靶向HSA的DARPin。
所述的白蛋白结合蛋白的序列包括(为)如下序列的一种：
GA3序列(SEQ ID NO.3)
LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
ABD035序列(SEQ ID NO.4)
LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
ABDCon序列(SEQ ID NO.5)
LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA
DARPin序列：(SEQ ID NO.30)
DLGKKLLEAARAGQDDEVRELLKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKAGADVNAQDIFGKTPADIAADAGHEDIAEVLQKAA。
所述GLP-1或其类似物、衍生物为具有SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.37、SEQ ID NO.24所述的序列或与该序列具有至少60％相同性的序列(更优选地，至少具有80％的相同性；更优选地，至少具有95％的相同性)。
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO.1)
HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO.2)
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG(SEQ ID NO.37)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO.24)
所述连接肽是含有氨基酸GS的柔性肽或者含有氨基酸EA的刚性肽。在GLP-1、Ex与白蛋白结合蛋白(ABD结构域、DARPin)的中间，起到连接与空间分离的作用。
更优选地，所述的连接肽为如下的一种GS(GGGGS)x或GS(EAAAK)y或(PAPA)n；其中x,y,n是1-6的整数；更优选地，x,y,n是1-3的整数。
本发明还提供一种多核苷酸，其编码上述的融合蛋白。
本发明还提供了上述融合蛋白的制备方法，构建含有所述融合蛋白的基因序列的表达载体，然后将所述的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的融合蛋白。
所述宿主细胞为大肠杆菌、酵母菌、哺乳动物细胞。
本发明还提供了上述融合蛋白在制备治疗糖尿病和/或肥胖的药物中的应用。
本发明还提供了一种用于治疗糖尿病和/或肥胖的药物组合物，包括上述的融合蛋白及至少一种药学上可接受的载体或赋形剂。
所述“GLP-1类似物”指的是：与GLP-1相比，具有一个或多个氨基酸取代、删除、倒置或添加的分子。
本发明中所述的“GLP-1衍生物”指的是具有GLP-1或GLP-1类似物的氨基酸序列；但是在一个或多个氨基酸侧链基团、α-碳原子、末端氨基基团或末端羧酸基团上的具有其它化学修饰的分子。
本发明所具有的有益效果：
本发明通过与ABD结构域或DARPin蛋白的连接，解决了GLP-1或Ex半衰期较短的瓶颈问题，使其能够更好的应用于临床上。通过本发明试验可以看出白蛋白结合蛋白显著延长了GLP-1或Ex的半衰期，并提高了在小鼠体内的降血糖活性。改造后的GLP-1或Ex具有一定的减轻体重的作用，可以用来治疗或辅助治疗肥胖。
本发明利用ABD或DARPin蛋白来延长GLP-1或Ex半衰期的半衰期还具有如下优点：不需要额外的化学偶联，化学偶联常常面临产物不均一、活性损失较严重等问题；其分子量较低，可以采用大肠杆菌表达系统进行表达，成本低，效率高；溶解度高，可以实现可溶表达，对于后期的纯化十分有利。
附图说明
图1为用15％SDS-PAGE分析各个融合蛋白的电泳图。1，GLP-GA3-L0；2，GLP-GA3-GS-L1；3，GLP-GA3-GS-L2；4，GLP-GA3-GS-L3；5，GLP-ABDCon-GS-L3；6，GLP-ABD035-GS-L3；7，GLP-ABD035-EA-L3；8，Ex-GA3-GS-L3；9，Ex-ABD035-GS-L3；10，Ex-GA3-EA-L3；11，Ex-ABDCon-EA-L3。
图2为GLP-ABD融合蛋白的降血糖活性。小鼠先过夜禁食(14-16h)，然后皮下注射各个融合蛋白30nmol/kg；血糖变化使用血糖仪进行检测，进食量的变化使用减少量与体重的比值表示。A，将不同连接肽长度的GLP-GA3注射入小鼠体内，小鼠的血糖变化值与曲线下面积(AUC)；B，天然GLP-1与GLP-GA3-GS-L3的降血糖活性和AUC对比；C和D，GLP-GA3-GS-L3,GLP-ABD035-GS-L3,GLP-ABDCon-GS-L3和GLP-ABD035-EA-L3的对小鼠的降血糖活性、AUC变化(C)以及进食量的变化(D)。数据表示为mean±SEM，每组有5-6只小鼠。*p<0.05，**p<0.01，***p<0.001表示实验组与PBS组相互比较；#p<0.05，##p<0.01表示GLP-GA3-GS-L3与其他实验组比较；ns表示没有显著性差异。
图3GLP-ABD融合蛋白的口服葡萄糖耐受性实验。将小鼠先过夜禁食(14-16h)，然后皮下注射各个融合蛋白15nmol/kg；30min后灌胃2g/kg葡萄糖(0min)血糖变化使用血糖仪进行检测。A，小鼠的血糖变化值；B，曲线下面积(AUC)。数据表示为mean±SEM，每组有5-6只小鼠。*p<0.05，**p<0.01，***p<0.001表示实验组与PBS组相互比较；ns表示没有显著性差异。
图4多次注射GLP-ABD融合蛋白能够降低小鼠的体重。将30nmol/kg的各个GLP-ABD融合蛋白注射入小鼠体内，连续注射5天，在第1天和第5天检测其非空腹体重。数据表示为mean±SEM，每组有4只小鼠。*p<0.05表示实验组与PBS组相互比较
图5融合蛋白在小鼠血液中的浓度变化。将FITC标记的融合蛋白注射入BALB/C小鼠体内，在不同的时间从小鼠的尾部取血，检测血药浓度。
图6Ex-ABD融合蛋白的降血糖活性研究。小鼠先过夜禁食(14-16h)，然后皮下注射各个融合蛋白30nmol/kg；血糖变化使用血糖仪进行检测，进食量的变化使用减少量与体重的比值表示。A，小鼠的血糖变化值；B，曲线下面积(AUC)。数据表示为mean±SEM，每组有5-6只小鼠。***p<0.001表示实验组与PBS组相互比较；ns表示没有显著性差异。
图7GLP-1或Ex与DARPin的融合蛋白的口服葡萄糖耐受性实验。将小鼠先过夜禁食(14-16h)，然后皮下注射各个融合蛋白30nmol/kg；30min后灌胃2g/kg葡萄糖(0min)血糖变化使用血糖仪进行检测。A，GLP-DARPin-GS-L3与GLP-DARPin-DARPin-GS-L3降低小鼠的血糖水平；B，Ex-DARPin-GS-3降低小鼠的血糖水平。数据表示为mean±SEM，每组有4-5只小鼠。*p<0.05，**p<0.01，***p<0.001表示实验组与PBS组相互比较。
图8多次注射GLP-DARPin-GS-L3融合蛋白显著降低小鼠的体重。将90nmol/kg的GLP-DARPin-GS-L3融合蛋白每隔1天注射入小鼠体内，连续注射13天；中间每隔一天检测小鼠的进食量和体重。数据表示为mean±SEM，每组有4只小鼠。**p<0.01表示实验组与PBS组相互比较。
图9DARPin融合蛋白在小鼠血液中的浓度变化。将FITC标记的融合蛋白注射入Balb/c小鼠体内，在不同的时间从小鼠的尾部取血，检测血药浓度。
具体实施方式：
在以下的实施例中，我们将GLP-1或Ex与不同的ABD结构域(GA3、ABD035、ABDCon)及DARPin蛋白通过不同的连接肽进行连接，经表达获得一系列的融合蛋白，并对这些融合蛋白的活性及半衰期在小鼠体内进行研究。研究结果表明，白蛋白结合蛋白显著延长了GLP-1或Ex的半衰期，并提高了在小鼠体内的降血糖活性。改造后的GLP-1或Ex具有一定的减轻体重的作用，可以用来治疗或辅助治疗肥胖。
实施例一：将GLP-1与不同的ABD结构域连接
1.载体构建
采用全基因合成的方式获得ABD与GLP-1的融合蛋白基因，其中GLP-1与ABD的N端或C端连接，并与pET-24d连接，构建pET-24d-GLP-ABD-X-Ln或pET-24d-ABD-GLP-X-Ln(X表示GS连接肽或EA连接肽；n＝0、1、2、3个GGGGS或EAAAK重复)；在所有的目的蛋白的N端均含有His-tag以及TEV蛋白酶酶切位点，MKHHHHHHPMSDYDIPTTENLYFQ(SEQ ID NO.34)。
GLP-1序列(31个氨基酸)：
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO.1)
或HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO.2)
GA3序列(SEQ ID NO.3)
LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
ABD035序列(SEQ ID NO.4)
LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
ABDCon序列(SEQ ID NO.5)
LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA
柔性连接肽
GS-L1：GSGGGGS(SEQ ID NO.6)
GS-L2：GSGGGGSGGGGS(SEQ ID NO.7)
GS-L3：GSGGGGSGGGGSGGGGS(SEQ ID NO.8)
刚性连接肽
EA-L1：GSEAAAK(SEQ ID NO.9)
EA-L2：GSEAAAKEAAAK(SEQ ID NO.10)
EA-L3：GSEAAAKEAAAKEAAAK(SEQ ID NO.11)
GLP-GA3-L0序列：(SEQ ID NO.12)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-GS-L1序列(SEQ ID NO.13)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-GS-L2序列(SEQ ID NO.14)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-GS-L3序列(SEQ ID NO.15)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-EA-L1序列(SEQ ID NO.16)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSEAAAKLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-EA-L2序列(SEQ ID NO.17)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSEAAAKEAAAKLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-GA3-EA-L3序列(SEQ ID NO.18)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSEAAAKEAAAKEAAAKLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
GLP-ABD035-GS-L3序列(SEQ ID NO.19)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
GLP-ABD035-EA-L3序列(SEQ ID NO.20)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSEAAAKEAAAKEAAAKLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
GLP-ABDCon-GS-L3序列(SEQ ID NO.21)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSGGGGSLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA
GLP-ABDCon-EA-L3序列(SEQ ID NO.22)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSEAAAKEAAAKEAAAKLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA
GA3-GLP-GS-L3序列(SEQ ID NO.23)
LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPSGSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
2.表达
将各个表达载体分别转化E.coli BL21(DE3)感受态细胞中，挑取单菌落于含50μg/mL卡那霉素的LB培养基中，37℃摇床过夜培养后，以1％接种量转接下一级，37℃继续培养至菌液OD600约为0.4-0.6时加入终浓度为1.0mM的IPTG，并于30℃诱导表达12h。表达结束后在4℃，5000g条件下离心10min，收集菌体。
收集菌体后，用平衡缓冲液(25mM Tris/HCl,300mM NaCl,10mM咪唑，pH 8.0)进行重悬，然后，用细胞高压破碎仪(广州聚能生物科技有限公司)对菌体进行破碎，破碎结束后，14000rpm离心30min。
3.纯化
吸取上清到Ni2+-NTA柱子(预先用平衡缓冲液进行平衡)中；利用AKTA purifier10蛋白纯化仪对蛋白分别用漂洗缓冲液(25mM Tris/HCl,300mM NaCl,30mM咪唑，pH 8.0)进行漂洗，然后用洗脱缓冲液(25mM Tris/HCl,300mM NaCl,400mM咪唑，pH 8.0)进行洗脱，收集洗脱液。
融合蛋白经Ni2+-NTA纯化后，切除His-tag，并用HiTrapTM Q离子交换柱对各个融合蛋白进行进一步的纯化。用AKTA Purifier 10蛋白纯化系统进行线性洗脱，得到纯度较高的融合蛋白。经15％SDS-PAGE检测发现(图1)，各个纯化的融合蛋白纯度较高。
4.活性检测
为了确定将GLP-1与ABD(GA3、ABD035及ABDCon)连接后的融合蛋白对小鼠血糖水平的控制作用，我们进行了降血糖实验。将C57BL/6小鼠随机组，每组5-6只小鼠，分为PBS组和各个实验组(GLP-GA3-L0、GLP-GA3-GS-L1、GLP-GA3-GS-L2、GLP-GA3-GS-L3、GLP-ABO035-GS-L3、GLP-ABDCon-GS-L3、GLP-ABDCon-EA-L3)。小鼠先过夜禁食(14-16h)，实验组分别皮下注射30nmol/kg药物，PBS组给予相同体积的PBS，并每隔一定的时间用血糖仪检测血糖水平。实验结果显示(图2)，随着连接肽的增加，GLP-GA3融合蛋白的降血糖活性越高，GLP-GA3-GS-L3有最高的降血糖活性。GLP-GA3-GS-L3能够在8-10h内显著降低血糖水平，而天然GLP-1降血糖效果不明显，有研究表明GLP-1的降血糖活性最多仅能维持0.5h。因此，GA3显著延长了GLP-1的半衰期，也显著增加了控制血糖的时间。而GLP-ABD035-GS-L3的活性比GLP-ABDCon-GS-L3和GLP-GA3-GS-L3高，而GLP-ABD035-GS-L3的活性与GLP-ABD035-EA-L3之间没有显著性差异。这几种融合蛋白均具有显著的抑制进食的作用，注射后能在很长一段时间内显著减少了食物的摄入量，表明可以通过注射融合蛋白来达到减少进食、减轻体重的作用。这也表明三种ABD结构域(GA3、ABDCon和ABD035)在延长半衰期方面存在一定的差异，可能与Albumin的亲和力有关。而在一定长度下，EA连接肽和GS连接肽对活性的影响较小。
急性降血糖作用。采用口服葡萄糖耐受性方法检测融合蛋白的降血糖活性。小鼠先过夜禁食(14-16h)，然后皮下注射C57BL/6小鼠各个融合蛋白，剂量15nmol/kg；30min后，灌胃2g/kg葡萄糖，马上在0、15、30、60、120min时检测血糖变化。PBS组给予相同体积的PBS。结果表明(图3)，注射葡萄糖后，PBS组血糖水平迅速增加，而GLP-ABD035-GS-L3、GLP-ABDCon-GS-L3和GLP-GA3-GS-L3组则一直维持一个比较低的血糖水平，表明GLP-ABD融合蛋白显著提高了小鼠的葡萄糖耐受性。
减轻体重作用。将C57BL/6小鼠随机组，每组4只小鼠，分为PBS组和各个实验组。实验组每天一次皮下注射30nmol/kg药物(GLP-GA3-GS-L3、GLP-ABD035-GS-L3、GLP-ABDCon-GS-L3、GLP-ABD035-EA-L3)，PBS组给予相同体积的PBS。在第1天和第5天分别称量体重。实验结果表明，在第5天时，实验组小鼠的体重均发生了显著的下降(图4)。这可能是由于连续注射药物，导致小鼠持续的进食量降低从而使体重减轻。这说明，这些融合蛋白具有减轻体重、治疗肥胖的作用。
5.体内稳定性检测
将融合蛋白使用FITC(Sigma，上海)进行标记。将FITC标记的融合蛋白通过皮下注射入BALB/C小鼠体内，每隔一定时间从尾部取血，滴入预先用EDTA-Na2处理过的Ep管中，取血后立即(30s以内)加入蛋白酶抑制剂Aprotinin(索来宝，北京)，5000rpm离心10min后收集血浆，并使用酶标仪在激发波长485nm，发射波长535nm条件下检测。样品中融合蛋白的浓度使用标准曲线进行计算。经计算如图5和表1所示，GLP-GA3-GS-L3、GLP-ABD035-GS-L3及GLP-ABDCon-GS-L3在小鼠体内的半衰期分别为36.3±7.8h、31.3±1.0h和38.3±2.7h，相互之间没有显著性差异。而有研究表明GLP-1(A8G)在小鼠体内的半衰期仅有16min(Fremaux J,et al.Nature Communications,2019,10:924)，所以ABD结构域显著延长了GLP-1的半衰期。
实施例二：将艾塞那肽(Ex)与不同的ABD结构域连接
1.载体构建
采用全基因合成的方式获得各种ABD与Ex的融合蛋白基因，将艾塞那肽(Ex)与ABD的N端或C端连接，并与表达载体pET-24d等连接，构建pET-24d-Ex-ABD-X-Ln或pET-24d-ABD-Ex-X-Ln(X表示GS连接肽或EA连接肽；n＝0、1、2、3个GGGGS或EAAAK重复)；在所有的目的蛋白的N端均含有His-tag以及TEV蛋白酶酶切位点，MKHHHHHHPMSDYDIPTTENLYFQ(SEQ IDNO.34)。
艾塞那肽(Ex)的序列(39个氨基酸)：(SEQ ID NO.24)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
Ex-GA3-GS-L2序列(SEQ ID NO.25)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
Ex-GA3-GS-L3序列(SEQ ID NO.26)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
Ex-GA3-EA-L3序列(SEQ ID NO.27)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSEAAAKEAAAKEAAAKLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP
Ex-ABD035-GS-L3序列(SEQ ID NO.28)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
Ex-ABDCon-GS-L3序列(SEQ ID NO.29)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSGGGGSGGGGSGGGGSLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA
2.融合蛋白的表达、纯化、降血糖活性检测以及体内稳定性检测等参照实施例一。
实验结果表明，Ex与各个ABD结构域(GA3、ABD035、ABCon)连接后，经纯化可以获得纯度较高的融合蛋白(图1)。降血糖活性显示，Ex与ABD结构域融合后，能够显著增加Ex的降血糖活性(图6)。Ex只能在2h内降低小鼠的血糖水平，而Ex-ABD融合蛋白能够在24h内降低血糖，而GA3、ABD035或ABDCon对Ex的活性的提高没有显著差别。柔性的GS连接肽或刚性的EA连接肽之间没有显著差别。与之前的研究发现Ex在小鼠体内的半衰期只有0.17h(Schellenberger V,Nature Biotechnology,2009,27(12):1186-1192)，而连接ABD结构域后，Ex的半衰期被延长到40h左右，不同的ABD结构域之间没有显著差异(表1)。以上实验结果表明，ABD结构域显著延长了Ex的半衰期，且延长了Ex在体内发挥降血糖作用的时间。因Ex与GLP-1的受体是相同的，都是GLP-1R，因此Ex在体内也能发挥抑制进食和减轻体重的作用。
实施例三：将GLP-1或艾塞那肽Ex与DARPin蛋白连接
1.载体构建
通过基因融合的方式将GLP-1或Ex与DARPin的N端连接在一起，采用全基因合成的方式合成所有的基因，并与表达载体pET-28a连接，构建表达载体pET-28a-GLP-DARPin-GS-L3或pET-28a-Ex-DARPin-GS-L3等；
将两个DARPin串联起来，并将GLP-1连接在其N端，构建表达载体pET-28a-GLP-DARPin-DARPin-GS-L3。两个DARPin中用GTGPTPTPTGSGPTPTPTGGS(SEQ ID NO.35)序列连接。
在所有的目的蛋白的N端均含有His-tag以及TEV蛋白酶酶切位点，MGSSHHHHHHSSGENLYFQ(SEQ ID NO.36)。
DARPin序列：(SEQ ID NO.30)
DLGKKLLEAARAGQDDEVRELLKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKAGADVNAQDIFGKTPADIAADAGHEDIAEVLQKAA
GLP-DARPin-GS-L3序列；(SEQ ID NO.31)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSGGGGSDLGKKLLEAARAGQDDEVREL
LKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKAGADVNAQD
IFGKTPADIAADAGHEDIAEVLQKAA
Ex-DARPin-GS-L3序列；(SEQ ID NO.32)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGSGGGGSGGGGSGGGGSDLGKKLLEAARAG QDDEVRELLKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKA GADVNAQDIFGKTPADIAADAGHEDIAEVLQKAA
GLP-DARPin-DARPin-GS-L3序列：(SEQ ID NO.33)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGGSGGGGSGGGGSDLGKKLLEAARAGQDDEVRELLKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKAGADVNAQDIFGKTPADIAADAGHEDIAEVLQKAAGTGPTPTPTGSGPTPTPTGGSDLGKKLLEAARAGQDDEVRELLKAGADVNAKDYFSHTPLHLAARNGHLKIVEVLLKAGADVNAKDFAGKTPLHLAANEGHLEIVEVLLKAGADVNAQDIFGKTPADIAADAGHEDIAEVLQKAA
2.融合蛋白的表达、纯化、降血糖活性检测、进食量检查以及体内稳定性检测等参照实施例一。
血糖检测。口服葡萄糖耐受性实验结果表明(图7)，将GLP-1与1个或2个DARPin蛋白融合表达后，GLP-1仍然能发挥显著的降血糖功能。GLP-DARPin-GS-L3的降血糖活性更高，可能是由于两个DARPin分子之间存在一定的竞争关系和空间位阻效应，阻碍了GLP-1结合并激活受体。将艾塞那肽Ex与DARPin连接后(Ex-DARPin-GS-L3)同样保持了显著的降血糖活性。表明DARPin不影响GLP-1或Ex结合并激活受体的功能。同样，柔性的GS连接肽或刚性的EA连接肽之间没有显著差别。
进食量与体重检测。将90nmol/kg的GLP-DARPin-GS-L3融合蛋白每隔1天皮下注射入小鼠体内，连续注射13天；中间每隔1天检测小鼠的进食量和体重。实验结果表明(图8)，多次注射GLP-DARPin-GS-L3融合蛋白能显著降低小鼠的进食量和体重。表明，GLP-DARPin-GS-L3具有减轻体重和治疗肥胖的潜力。
半衰期检测。连接DARPin蛋白后，DARPin可以与HSA结合，从而可以利用FcRn介导的循环再生作用延长GLP-1或Ex的半衰期。GLP-DARPin-GS-L3、GLP-DARPin-DARPin-GS-L3以及Ex-DARPin-GS-L3在小鼠体内的半衰期分别为18.0、52.3和26.8h(图9，表2)。以上实验结果表明，DARPin蛋白可以通过HSA显著延长了GLP-1或Ex的半衰期，且显著增强了GLP-1或Ex的降血糖功能。
表1各个ABD融合蛋白在小鼠体内的半衰期
表2各个DARPin融合蛋白在小鼠体内的浓度变化
| 融合蛋白 | GLP-DARPin-GS-L3 | GLP-DARPin-DARPin-GS-L3 | Ex-DARPin-GS-L3 |
| 半衰期 | 18.0±2.8h | 52.3±3.3h | 26.8±4.1h |
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种用于治疗糖尿病和/或肥胖症的融合蛋白
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Val Ser Asp Phe Tyr Lys Arg Leu Ile Asn Lys Ala Lys Thr Val Glu
65 70 75 80
Gly Val Glu Ala Leu Lys Leu His Ile Leu Ala Ala Leu Pro
85 90
<210> 20
<211> 94
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
35 40 45
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
50 55 60
Val Ser Asp Phe Tyr Lys Arg Leu Ile Asn Lys Ala Lys Thr Val Glu
65 70 75 80
Gly Val Glu Ala Leu Lys Leu His Ile Leu Ala Ala Leu Pro
85 90
<210> 21
<211> 93
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Leu Lys Glu Ala Lys Glu Lys Ala Ile Glu Glu Leu Lys Lys Ala Gly
50 55 60
Ile Thr Ser Asp Tyr Tyr Phe Asp Leu Ile Asn Lys Ala Lys Thr Val
65 70 75 80
Glu Gly Val Asn Ala Leu Lys Asp Glu Ile Leu Lys Ala
85 90
<210> 22
<211> 93
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
35 40 45
Leu Lys Glu Ala Lys Glu Lys Ala Ile Glu Glu Leu Lys Lys Ala Gly
50 55 60
Ile Thr Ser Asp Tyr Tyr Phe Asp Leu Ile Asn Lys Ala Lys Thr Val
65 70 75 80
Glu Gly Val Asn Ala Leu Lys Asp Glu Ile Leu Lys Ala
85 90
<210> 23
<211> 95
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
1 5 10 15
Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu
20 25 30
Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Ser Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
65 70 75 80
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
85 90 95
<210> 24
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 25
<211> 97
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp
50 55 60
Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys
65 70 75 80
Thr Val Glu Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu
85 90 95
Pro
<210> 26
<211> 102
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys Val Leu Ala
50 55 60
Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu
65 70 75 80
Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp Glu
85 90 95
Ile Leu Ala Ala Leu Pro
100
<210> 27
<211> 102
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Glu Ala Ala Ala Lys Glu Ala
35 40 45
Ala Ala Lys Glu Ala Ala Ala Lys Leu Ala Glu Ala Lys Val Leu Ala
50 55 60
Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu
65 70 75 80
Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp Glu
85 90 95
Ile Leu Ala Ala Leu Pro
100
<210> 28
<211> 102
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys Val Leu Ala
50 55 60
Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Phe Tyr Lys Arg Leu
65 70 75 80
Ile Asn Lys Ala Lys Thr Val Glu Gly Val Glu Ala Leu Lys Leu His
85 90 95
Ile Leu Ala Ala Leu Pro
100
<210> 29
<211> 101
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Leu Lys Glu Ala Lys Glu Lys Ala
50 55 60
Ile Glu Glu Leu Lys Lys Ala Gly Ile Thr Ser Asp Tyr Tyr Phe Asp
65 70 75 80
Leu Ile Asn Lys Ala Lys Thr Val Glu Gly Val Asn Ala Leu Lys Asp
85 90 95
Glu Ile Leu Lys Ala
100
<210> 30
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
20 25 30
Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu
35 40 45
Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
50 55 60
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His
65 70 75 80
Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
85 90 95
Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly
100 105 110
His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
115 120
<210> 31
<211> 172
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
50 55 60
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
65 70 75 80
Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu
85 90 95
Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
100 105 110
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His
115 120 125
Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
130 135 140
Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly
145 150 155 160
His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
165 170
<210> 32
<211> 180
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu
50 55 60
Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala
65 70 75 80
Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His
85 90 95
Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
100 105 110
Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu
115 120 125
His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
130 135 140
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
145 150 155 160
Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu
165 170 175
Gln Lys Ala Ala
180
<210> 33
<211> 317
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
50 55 60
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
65 70 75 80
Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu
85 90 95
Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
100 105 110
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His
115 120 125
Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
130 135 140
Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly
145 150 155 160
His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Thr Gly Pro
165 170 175
Thr Pro Thr Pro Thr Gly Ser Gly Pro Thr Pro Thr Pro Thr Gly Gly
180 185 190
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp
195 200 205
Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
210 215 220
Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His
225 230 235 240
Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
245 250 255
Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly
260 265 270
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn
275 280 285
Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala
290 295 300
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
305 310 315
<210> 34
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro
1 5 10 15
Thr Thr Glu Asn Leu Tyr Phe Gln
20
<210> 35
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Gly Thr Gly Pro Thr Pro Thr Pro Thr Gly Ser Gly Pro Thr Pro Thr
1 5 10 15
Pro Thr Gly Gly Ser
20
<210> 36
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Met Gly Ser Ser His His His His His His Ser Ser Gly Glu Asn Leu
1 5 10 15
Tyr Phe Gln
<210> 37
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly
20 25 30
Claims (10)
1.一种融合蛋白，其特征在于：该融合蛋白的结构为：GLP-1或其类似物、衍生物—连接肽—白蛋白结合蛋白。
2.根据权利要求1所述的一种融合蛋白，其特征在于：所述白蛋白结合蛋白包括ABD结构域(GA3、ABD035、ABDCon)或可以靶向HSA的DARPin。
3.根据权利要求2所述的一种融合蛋白，其特征在于：所述的白蛋白结合蛋白的序列包括如下序列的一种：SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.30。
4.根据权利要求1所述的一种融合蛋白，其特征在于：所述GLP-1或其类似物、衍生物为具有SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.37、SEQ ID NO.24所述的序列或与该序列具有至少60％相同性的序列；更优选地，至少具有80％的相同性；更优选地，至少具有95％的相同性。
5.根据权利要求1所述的一种融合蛋白，其特征在于：所述连接肽是含有氨基酸GS的柔性肽或者含有氨基酸EA或P的刚性肽。
6.根据权利要求5所述的一种融合蛋白，其特征在于：所述的连接肽为如下的一种GS(GGGGS)x或GS(EAAAK)y或(PAPA)n；其中x,y，n是1-6的整数；更优选地，x,y，n是1-3的整数。
7.一种多核苷酸，其特征在于：其编码权利要求1-6所述的融合蛋白。
8.权利要求1-6任一项所述融合蛋白的制备方法，其特征在于：构建含有所述融合蛋白的基因序列的表达载体，然后将所述的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的融合蛋白。
9.根据权利要求8所述的融合蛋白的制备方法，其特征在于：所述宿主细胞为大肠杆菌、酵母菌或哺乳动物细胞。
10.权利要求1-6任一项所述融合蛋白在制备治疗糖尿病和/或肥胖的药物中的应用。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802386A (zh) * | 2003-06-12 | 2006-07-12 | 伊莱利利公司 | Glp－1类似物融合蛋白质 |
| CN103459415A (zh) * | 2010-11-26 | 2013-12-18 | 分子组合公司 | 设计的与血清白蛋白结合的重复蛋白 |
| CN105367664A (zh) * | 2015-11-04 | 2016-03-02 | 成都贝爱特生物科技有限公司 | 激活GLP-1受体和Amylin受体双功能作用的融合蛋白制备及其用途 |
| CN108424460A (zh) * | 2017-02-13 | 2018-08-21 | 成都贝爱特生物科技有限公司 | GLP-1类似物和davalintide类似物的融合蛋白制备及其用途 |
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- 2020-08-31 CN CN202010900880.3A patent/CN114106194B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802386A (zh) * | 2003-06-12 | 2006-07-12 | 伊莱利利公司 | Glp－1类似物融合蛋白质 |
| CN103459415A (zh) * | 2010-11-26 | 2013-12-18 | 分子组合公司 | 设计的与血清白蛋白结合的重复蛋白 |
| CN105367664A (zh) * | 2015-11-04 | 2016-03-02 | 成都贝爱特生物科技有限公司 | 激活GLP-1受体和Amylin受体双功能作用的融合蛋白制备及其用途 |
| CN108424460A (zh) * | 2017-02-13 | 2018-08-21 | 成都贝爱特生物科技有限公司 | GLP-1类似物和davalintide类似物的融合蛋白制备及其用途 |
Non-Patent Citations (1)
| Title |
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| 徐龙福: "PEG定点修饰及白蛋白结合肽融合的长效策略", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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