CN111269321B - 一种glp-1类似物融合蛋白质 - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
本发明通过对特定融合蛋白质在分子的GLP‑1部分和Fc部分进行多个位置的替换。其中Fc第11位的Ser、85位的Val、第86位的Val、第87位的Ser和第91位的Val进行了替换,末端Lys缺失,从而在很大程度上克服了施用GLP‑1‑Fc融合物相关的潜在免疫原性的问题,而且体内外活性实验结果还证明,本发明公开的GLP‑1类似物活性明显高于现有GLP‑1类似物。因此,所述的类似物可用于更好地预防、防治或减轻糖尿病和肥胖症和/或并发症。
Description
技术领域
本发明属于遗传工程技术领域,具体涉及一种GLP-1类似物融合蛋白质。
背景技术
糖尿病(diabetes mellitus,DM)是由于体内胰岛素分泌不足或合成受阻导致内分泌代谢紊乱而引起血糖升高的一种复杂的代谢性疾病,主要分为1型糖尿病(type1diabetes mellitus,T1DM)、2型糖尿病(type2 diabetes mellitus,T2DM)以及其他类型的糖尿病。据统计,2型糖尿病所占比例较大,约占糖尿病患者总数的90%以上,其发病期多在35-40岁以后,发病后期会出现较严重的并发症。目前,糖尿病的治疗主要集中在以降低血糖浓度,减少并发症的发生等为目的,治疗方法主要是药物治疗。治疗2型糖尿病的药物主要有磺酰脲类、双胍类、α葡萄糖激酶抑制剂、抗IL-1β抗体和肠促胰岛素类药物,其中肠促胰岛素类药物具有多方面降糖功能而作为新型糖尿病治疗药物备受关注,根据作用机制可分为二肽基肽酶IV(dipeptidylpeptidase 4,DPP-4)抑制剂和胰高血糖素样肽(glucagonlikepeptide-1,GLP-1)受体激动剂两类。研究表明,这两类药物除具有降血糖、极少导致低血糖、安全性和耐受性较好等优点外,还对消化、中枢神经、心血管等多系统发挥保护作用。
胰高血糖素类似肽(Glucagon-like peptide,GLP)是人体分泌的一种肠道激素,由胰高血糖素原分子经肠道蛋白水解酶裂解而成,产生二种胰高血糖素类似肽GLP-1和GLP-2。其中GLP-1有二种活性形成,即GLP-1(7-37)和GLP-1(7-36)酰胺形式,在体内具有相同的促进胰岛素分泌的作用,故又称促胰岛素分泌肽(Incretin或Insulinotropicpeptide)(NegarSadrzadeh et.al.,Pharmaceutical Sciences,Vol.96,1925-1954(2007))。
GLP-1(7-37):
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly。
GLP-1激动剂作为一类多肽药物,目前还只能注射给药,为了提高患者顺应性,缓释注射剂、非注射给药成为这一类药物研发的主要方向。通过结构修饰,掩盖DPP-Ⅳ酶位点,延长生物半衰期,是目前最主流的做法。这种结构修饰包括链接高分子蛋白和聚乙二醇修饰两种,一周一次的Abiglutide和Dulaglutide就属于典型的前一种,尽管已有多个聚乙二醇修饰蛋白或多肽药物上市,但聚乙二醇修饰GLP-1类似物起步较晚,仅索玛鲁肽进入三期临床。当然也有的公司通过制剂的手段,制成缓释微球,实现一周一次,典型的产品就是Bydureon。也有的公司直接避开了注射给药的弊端,开发植入、口服、透皮和吸入之类的产品,其中口服给药是最理想的给药途径,但是需要解决GLP-1类似物在肠胃中被胃酸、酶破坏和吸收问题,以提高生物利用度,降低个体差异,目前口服GLP-1类似物走在最前沿的公司为Oramed公司和Emisphere公司,其中诺和诺德的NN9924就是一种口服索玛鲁肽,国内方面,上海蓝星申报了艾塞那肽肠溶片。
Exendin-4:
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser。
Exendin-4是从蜥蜴唾液中分离出的GLP-1类似物,由39个氨基酸组成,其氨基酸序列与GLP家族中数个成员有序列的相似性,与人GLP-1有53%的同源性。因为N端第二位是Ala(GLP-1为Gly),所以不易被DPP-Ⅳ酶降解,而具有较长的半衰期和较强的生物活性。Exendin-4的化学合成品命名为Exenatide(商品名Byetta),由Amylin和Lilly公司于1995年开始联合研发,2005年4月获得FDA的批准上市。
人GLP-1(7~36)在体内的表达和活性受到严格的调控,当其N端的第二位Ala被二肽基肽酶(dipeptidyl peptidase,DPP)水解后,形成无活性的GLP-1(9~36),该代谢产物还是GLP-1R的体内天然拮抗剂。因此,天然人活性型GLP-1在体内的半衰期很短,其新陈代谢的速率为2min;加上在生理状态下,GLP-1主要通过肾脏排泄,限制了人源GLP-1的临床应用。非人源的人工合成Exenatide的第二位氨基酸Gly不同于人GLP-1的Ala,能有效抵抗二肽酰基肽酶的降解;Exenatide的C端的刚性(PSSGAPPPS)氨基酸序列,可增加多肽稳定性,Exenatide在体内的降血糖能力比GLP-1强1000倍左右。
Exenatide肽二级结构:Exenatide的N端为不规则卷曲,中间部分在同一侧面上相互交替排列带相反电荷侧链的氨基酸残基,通过盐桥或极性氢键形成螺旋,C端则为亲水的“Trp-Cage”。Exenatide与GLP-1受体的相互作用机理已研究的比较清楚。
一系列不同结构设计已经开发用于延长GLP-1类似物结构半衰期,提高生物活性。CN1384755A公开了新型Exendin激动剂制剂及其给药方法,公开了Exenatide的化合物结构和制备方法。CN102532303A公开了使用甲氧基聚乙二醇修饰Exenatide中赖氨酸的氨基或N末端组氨酸残基的氨基。CN101980725B公开了脂肪酸-PEG-Exenatide。CN105753963A公开了单点或多点位氨基酸突变的Exenatide类似物。CN102397558A公开了将Exenatide中某些氨基酸用半胱氨酸取代,并使用PEG或末端被甲基取代的PEG修饰。
丹麦的Novo Nordisk公司开发的酰胺化的GLP-1衍生物Liraglutide(T,DiabetesCare,2007,30:1608-1610),与天然的GLP-1相比34位Lys被Arg所替代,同时含有16个碳的脂肪酸侧链通过谷氨酰基连接到GLP-1的26位Lys上。Liraglutide可与血浆中白蛋白形成非共价连接的衍生物,保留了GLP-1的功能,拮抗DPP IV的降解,其半衰期延长到11-15小时,适合每天一次的给药方式(Elbrond B,Diabetes Care,2002,25:1398-1404)。正在开发的另一个GLP-1类似物CJC-1131中C末端37位赖氨酸经体外修饰后,可以与血浆中白蛋白中34位半胱氨酸残基形成共价连接,同样可以实现一天给药一次的目标(Kim,JG,Diabetes,2003,52:751-759)。由此可见,通过化学或基因工程手段,将GLP-1和exendin-4经突变(1个或几个氨基酸)、缺失或添加氨基酸于C末端的GLP-1类似物,可以较好地保留生物活性。
虽然在GLP-1类似物长效药物开发做了诸多努力,但是目前市场上的GLP-1类似物稳定性较差和药效较低,作为长效药物研发的结构和活性基础,GLP-1类似物的创新性改造和研究仍是一个非常重要的课题。
已经采取多种途径在保持生物学活性的同时延长GLP-1肽的清除半寿期或降低该肽从机体的清除。一条途径涉及将GLP-1肽与免疫球蛋白Fc部分融合。免疫球蛋白一般在体内具有长循环半寿期。例如,IgG分子在人体具有高达23天的半寿期。免疫球蛋白Fc部分是这种体内稳定性的部分原因。在保留GLP-1分子生物学活性的同时,GLP-1-Fc融合蛋白质具有由免疫球蛋白Fc部分提供的稳定性而保留GLP-1分子的生物活性的优点。
然而多种融合蛋白质长时期反复施用时的免疫原性问题还是普遍存在的。通常蛋白质,包括治疗性蛋白,都具有免疫原性,这部分是因为蛋白质通过抗原提呈细胞内吞和蛋白水解后产物肽结合到称为主要组织相容性复合体(MHC)的分子上然后这些分子把肽提呈给T细胞。抗原提呈细胞(APC)表面的抗原肽-MHC复合体刺激T细胞增殖,分化并释放细胞因子。同时,B-细胞分化和抗体产生被诱导,这可以进一步通过清除治疗性蛋白而限制治疗性蛋白的效用。这样,来源于治疗性蛋白的抗原肽能够引起一系列不想要的免疫应答。治疗性蛋白的效用由于抗体的中和而受限,而且因为T-细胞和B-细胞应答会在患者中引起炎性和变态反应,其诱导经常是有害的。
发明内容
本发明提供一种GLP-1类似物融合蛋白质,其包含GLP-1类似物治疗肽和免疫球蛋白Fc部分,所述GLP-1类似物融合蛋白质在Fc部分进行多个位置的替换,从而在很大程度上克服了施用Fc融合物存在的潜在免疫原性问题,提高了稳定性。所述融合蛋白质结合的GLP-1类似物可以是能够与Fc融合的一些治疗肽,比如艾塞那肽、利拉鲁肽,利司那肽、索玛鲁肽、Ideglira、阿必鲁肽、度拉糖肽、ITCA650、LAEx4、Lixilan等。
本发明还提供一种改进的GLP-1类似物,体内外活性实验结果证明,本发明所述GLP-1类似物活性明显高于现有GLP-1类似物。因此,本发明所述的类似物可用于更好地预防、防治或减轻糖尿病和肥胖症和/或并发症。
本发明的GLP-1类似物与免疫球蛋白Fc通过接头形成融合蛋白质,所述免疫球蛋白Fc部分是对人IgG4的Fc片段修饰所得,其序列编号SEQ ID NO:1,具体序列为:
Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Xaa11-Cys-Pro-Ala-Pro-
Glu-Phe-Leu-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr-Arg-Xaa85-Xaa86-Xaa87-Val-Leu-Thr-
Xaa91-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly,
其中第11位的Ser、85位的Val、第86位的Val、第87位的Ser和第91位的Val进行了替换,末端Lys缺失,具体为:
11位的Xaa为Pro;
85位的Xaa为Ala或Phe;
86位的Xaa为Phe或Glu;
87位的Xaa为Glu、Phe或Leu;
91位的Xaa为Gly。
序列1简写为:
在一个优选实施方案中,优选为SEQ ID NO:2,即序列2:
11位替换为Pro;
85位的Xaa为Ala;
86位的Xaa为Phe;
87位的Xaa为Glu;
91位的Xaa为Gly。
序列2简写:
在一个优选实施方案中,优选为SEQ ID NO:3,即序列3:
11位替换为Pro;
85位的Xaa为Phe;
86位的Xaa为Glu;
87位的Xaa为Phe;
91位的Xaa为Gly。
序列3简写:
所述的融合蛋白质,其GLP-1类似物为治疗肽,GLP-1类似物的C末端甘氨酸残基通过肽接头与Fc部分的N末端丙氨酸残基融合,其中肽接头是SEQ ID NO:4的序列,具体为Gly-Ala-Ala-Ala-Ala-Lys-Ser-Ala-Ala-Ala-Ala-Ser。
肽接头还可以选自如下的序列:
a)Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys-Ser-Gly-Gly-Gly-Gly-Ser,如SEQID NO:5所示;
b)Gly-Ala-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser,如SEQ ID NO:6所示;
c)Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys--Ser-Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys-Ser-Gly-Gly-Gly-Gly-Ser,如SEQ ID NO:7所示;
d)Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:8)。
所述融合蛋白质,其中GLP-1类似物是对GLP-1(7-37)修饰后所得,为方便表述对其重新编号,将原7位His编号为1位,序列为如下的SEQ ID NO:9即序列9:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Xaa-Leu-Val-Lys-Gly-Xaa-Xaa;
第25位Xaa为Trp或Phe或His;
第30位Xaa为Val或Glu或Ala;
第31位Xaa为Val-Val-Lys-Leu-Lys-NH2;Ala-Ala-Lys-Gly-Gly-Lys-NH2;Leu-Leu-Leu-Lys-Lys-NH2;Leu-Lys-Leu-Leu-Leu-Arg-NH2。
在一个优选实施方案中,优选SEQ ID NO:10,即序列10:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Ala-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
在一个优选实施方案中,优选SEQ ID NO:11,即序列11:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Ala-Val-Val-Lys-Leu-Lys-NH2。
在一个优选实施方案中,优选SEQ ID NO:12,即序列12:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Glu-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
在一个优选实施方案中,优选SEQ ID NO:13,即序列13:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Val-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
在一个优选实施方案中,优选SEQ ID NO:14,即序列14:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Val-Leu-Leu-Leu-Lys-Lys-NH2。
在一个优选实施方案中,优选SEQ ID NO:15,即序列15:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-His-Leu-Val-Lys-Gly-Ala-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
在一个优选实施方案中,优选SEQ ID NO:16,即序列16:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Ala-Val-Val-Lys-Leu-Lys-NH2。
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:10。
所述的融合蛋白质为序列10-接头序列4-序列2,命名为HIP925-1,序列编号为SEQID NO:17具体蛋白序列为:
所述的融合蛋白质为序列10-接头序列4-序列3,命名为HIP925-2,序列编号为SEQID NO:18具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:11。
所述的融合蛋白质为序列11-接头序列4-序列2,命名为HIP925-3,序列编号为SEQID NO:19具体蛋白序列为:
所述的融合蛋白质为序列11-接头序列4-序列3,命名为HIP925-4,序列编号为SEQID NO:20具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:12。
所述的融合蛋白质为序列12-接头序列4-序列2,命名为HIP925-5,序列编号为SEQID NO:21具体蛋白序列为:
所述的融合蛋白质为序列12-接头序列4-序列3,命名为HIP925-6,序列编号为SEQID NO:22具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:13。
所述的融合蛋白质为序列13-接头序列4-序列2,命名为HIP925-7,序列编号为SEQID NO:23具体蛋白序列为:
所述的融合蛋白质为序列13-接头序列4-序列3,命名为HIP925-8,序列编号为SEQID NO:24具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:14。
所述的融合蛋白质为序列14-接头序列4-序列2,命名为HIP925-9,序列编号为SEQID NO:25具体蛋白序列为:
所述的融合蛋白质为序列14-接头序列4-序列3,命名为HIP925-10,序列编号为SEQ ID NO:26具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:15。
所述的融合蛋白质为序列15-接头序列4-序列2,命名为HIP925-11,序列编号为SEQ ID NO:27具体蛋白序列为:
在一个优选实施方案中,所述的融合蛋白质,其中GLP-1类似物的序列是SEQ IDNO:16。
所述的融合蛋白质为序列16-接头序列4-序列2,命名为HIP925-12,序列编号为SEQ ID NO:28具体蛋白序列为:
上述序列中,与Fc融合前的Glp-1类似物序列10-16表现出比其他Glp-1类似物更优的稳定性,其中序列10和14更优;与Fc融合后能够进一步延长半衰期,其中Fc序列2优于序列3。
所述的融合蛋白质,其中GLP-1类似物还可以是艾塞那肽、利拉鲁肽,利司那肽、索玛鲁肽、Ideglira、阿必鲁肽、度拉糖肽、ITCA650、LAEx4、Lixilan等。
所述融合蛋白可以通过固相合成方法得到,也可以通过真核细胞表达得到,也可以两种方法结合。
固相合成法:
当C末端为羧基时,选择王树脂进行二种多肽的化学固相合成。合成结束后,将得到的侧链保护基的多肽树脂进行裂解,C末端从树脂上断裂下来形成羧基。当多肽C末端为酰胺,选择Fmoc-PAL-PEG-PS树脂进行多肽的化学固相合成。合成结束后,将得到的侧链保护基的多肽树脂进行裂解,C末端从树脂上断裂下来形成酰胺。以上固相合成是在多肽合成仪上进行。
首先将C末端的半胱氨酸连接树脂,然后一个一个的氨基酸从C端向N端进行。待合成结束后,脱保护基和从树脂上断裂后,用反向HPLC层析(Waters公司,C18制备柱)对Glp-1类似物多肽进行纯化。纯化条件为A相含0.5%(V/V)乙酸的水溶液,B相为含80%乙腈和0.5%(V/V)乙酸的水溶液,0-100%B液梯度洗脱。收集目的多肽,经冻干成干粉。经质谱分析,序列10的GLP-1的分子量为4542.06,与理论值一致。
在一个参考实施方案中,本发明所修饰的GLP-1类似物的制备方法如下:
第31位Xaa为Val-Val-Lys-Leu-Lys-NH2时:选择固相合成法,进一步地,采用Fmoc方法。由于多肽C端为酰胺基,对于Fmoc方法,可以使用的树脂为Fmoc-Rink树脂,其原理为利用Fmoc-Lys(Trt)-OH的羧基与载体氨基反应形成稳定的酰胺健,接完肽后可经裂解获得粗品。
第31位为Leu-Lys-Leu-Leu-Leu-Arg-NH2时,选择固相合成法,进一步地,采用Fmoc方法。由于多肽C端为酰胺基,对于Fmoc方法,可以使用的树脂为Fmoc-Rink树脂,其原理为利用Fmoc-Arg(Trt)-OH的羧基与载体氨基反应形成稳定的酰胺健,接完肽后可经裂解获得粗品。
选定以Fmoc-Rink树脂(100~200目,交联度为1-2%,取代值为0.3~0.6mmol NH/g)为起始原料,保护氨基酸与Fmoc-Rink树脂的摩尔比为2:1(或3:1)。取起始Fmoc-Rink树脂(取代值=0.436mmol/g),用20%哌啶(PIP/DMF)为去保护剂,室温反应25分钟,抽滤,用DMF(或DCM)洗涤树脂6次。取等摩尔量的保护氨基酸Fmoc-AAn和HOBt,用DMF溶解,置于-5℃的环境中冷却30分钟以上,另取N,N-二异丙基碳二亚胺的二氯甲烷溶液,慢慢加入至保护氨基酸DMF溶液中,于-5-5℃的环境中搅拌反应30分钟后加入到去保护的树脂中。
在去保护后的树脂与经活化的保护氨基酸反应3小时后,用茚三酮检测监控缩合反应的情况:缩合反应不完全时树脂为蓝色或深蓝色;缩合反应完全时树脂为无色或淡黄色。监测缩合完全缩合后,用DMF洗涤树脂6次。按多肽序列,依次进行活化氨基酸的缩合。完成接肽反应后,用DMF洗涤树脂再用甲醇收缩,减压干燥树脂。
按13mL/g肽树脂用量,将干燥的肽树脂加入到TFA裂解试剂(以ml:ml:ml:g计,TFA:H2O:TIS:DTT=90:2.5:2.5:5),0-5℃反应反应30min后,室温搅拌反应2-3h。反应混合物过滤,收集滤液,35℃减压浓缩至初始体积的25%。将浓缩液加到在-10℃以下冷冻的无水乙醚中,摇匀后在-10℃以下沉淀60分钟,过滤收集滤饼,再用无水乙醚洗涤滤饼3-5次,滤饼减压干燥至恒重,得多肽粗品。
取多肽粗品,用10%醋酸水溶液溶解,备纯化用。使用高效液相制备色谱纯化,流动相系统为0.1%TFA/水溶液-0.1%TFA/乙腈溶液。纯化用色谱填料为10μm的反相C18,紫外检测波长为215nm,50mm*300mm的色谱柱,流速为60mL/min,每次进样2.0-3.0g,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃-37℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥,得到GLP-1类似物的纯品。
细胞表达方法可以参照CN 104277112 A,首先构建融合蛋白质的编码基因;然后将所述基因克隆到真核表达载体,得到可表达所述融合蛋白的真核表达载体;再用所述表达载体转染宿主细胞,使其表达所述重组融合蛋白,然后分离纯化即得。真核表达载体可以为PET32等。所用宿主细胞可以为FreeStyle 293F、酵母细胞、CHO细胞、NSO细胞等。
其中,野生型人类IgG4蛋白质可以从多种来源获得。例如,可以从以可检测水平表达目的mRNA的细胞制备cDNA文库以获得这些蛋白质。使用特定目的蛋白质的公开的DNA或蛋白质序列设计的探针可以对文库进行筛选。例如,在Adams等人,(1980)Biochemistry19:2711-2719;Goughet等人,(1980)Biochemistry 19:2702-2710;Dolby等人,(1980)Proc.Natl.Acad.Sci.USA 77:6027-6031;Rice等人,(1982)Proc.Natl.Acad.Sci.USA79:7862-7862;Falkner等人,(1982)Nature 298:286-288;以及Morrison等人,(1984)Ann.Rev.Immunol.2:239-256中描述了免疫球蛋白轻或重链恒定区。
可以使用标准程序以所选探针筛选cDNA或基因组文库,例如在Sambrook等人,《Molecular Cloning:A Laboratory Manual》,Cold SpringHarbor Laboratory Press,NY(1989)中所述。分离编码免疫球蛋白蛋白质的基因的备选方法是使用PCR方法[Sambrook等人,如上;Dieffenbach等人,《PCR Primer:A Laboratory Manual》,Cold SpringHarborLaboratory Press,NY(1995)]。可以基于已发表的序列设计PCR引物。
从特定文库克隆的全长野生型序列通常可以作为产生本发明IgG4 Fc类似物片段的模板,其中IgG4 Fc类似物片段保留了赋予GLP-1类似物较长血浆半寿期的能力,GLP-1类似物是融合蛋白质的一部分。可以使用PCR技术,用经设计与对应于IgG4 Fc类似物片段所需末端的序列杂交的引物产生该片段。也可以设计PCR引物产生限制性酶位点以便于克隆到表达载体中。
可以通过多种不同的方法产生编码本发明的GLP-1类似物的DNA,所述方法包括如上所述的那些克隆方法和化学合成的DNA。
融合蛋白序列编号SEQ ID NO:17是序列SEQ ID NO:2通过肽接头SEQ ID NO:4与SEQ ID NO:10连接所得融合蛋白质,其优选地多核苷酸序列编号为SEQ ID NO:29,序列为:
在一个优选实施方案中,本发明采用的信号肽氨基酸序列如SEQ ID NO:30所示,具体序列如下:
MGKLIFWLVF WLTIFWLGSA 20。
所述信号肽的多核苷酸序列如SEQ ID NO:31所示,具体序列如下:
ATGGGCAAAC TCATATTCTG GCTTGTGTTT TGGCTGACTA TTTTTTGGCT TGGATCAGCT 60。
以此处所述用于融合蛋白质产生的表达或克隆载体转染或转化宿主细胞,并在经改良适于诱导启动子、选择转化子或扩增编码所希望的序列的基因的常规营养培养基中培养。技术人员不用过多实验方法即可以选择例如培养基、温度、pH等培养条件。一般而言,可以在《Mammalian CellBiotechnology:APractical Approach》,M.Butler编(IRL Press,1991)和如上之Sambrook等人著作中找到将细胞培养生产力最大化的原理、方法和实践技术。普通技术人员已知转染方法,例如CaPO4和电穿孔。在美国专利号4,399,216中描述了哺乳动物细胞宿主系统转化的一般方面。通常根据van Solingen等人,J Bact.130(2):946-7(1977)和Hsiao等人,Proc.Natl.Acad.Sci.USA 76(8):3829-33(1979)的方法进行酵母转化。然而,也可以使用将DNA引入细胞的其他方法,例如核微注射、电穿孔、细菌与完整细胞的原生质体融合、或聚阳离子,例如1,5-二甲基-1,5-二氮十一亚甲基聚甲溴化物(polybrene)或polyomithine。关于转化哺乳动物细胞的多种技术,见Keown等人,Methodsin Enzymology 185:527-37(1990)和Mansour等人,Nature 336(6197):348-52(1988)。
克隆或表达此处载体中核酸(例如DNA)的合适的宿主细胞包括FreeStyle 293F、酵母细胞、CHO细胞、NSO细胞等。
真核微生物例如丝状真菌或酵母是融合蛋白质载体的合适的克隆或表达宿主。酿酒酵母(Saccharomyces cerevisiae)是常用的低等真核宿主微生物。其他包括粟酒裂殖酵母(Schizosaccharomyces pombe)[Beach和Nurse,Nature 290:140-3(1981);1995年5月2日公布的EP 139,383];Muyveromyces宿主[美国专利号4,943,529;Fleer等人,Bio/Technology9(10):968-75(1991)]例如乳酸克鲁维酵母(K.lactis)(MW98-8C、CBS683、CBS4574)[de Louvencourt等人,J.Bacteriol.154(2):737-42(1983)];脆壁克鲁维酵母(K.fiagilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16,045)、威克克鲁维酵母(K wickeramii)(ATCC24,178)、K waltii(ATCC 56,500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC 36.906)[Van den Berg等人,Bio/Technology 8(2):135-9(1990)];K.thermotoierans和马克斯克鲁维酵母(K.marxianus);yarrowia(EP402,226);巴斯德毕赤酵母(Pichia pastoris)(EP 183,070)[Sreekrishna等人,J.BasicMicrobiol.28(4):265-78(1988)];假丝酵母属(Candida);Trichoderma reesia(EP 244,234);粗糙脉孢霉(Neurospora crassa)[Case等人,Proc.Natl.Acad Sci.USA76(10):5259-63(1979)];许旺酵母属(Schwanniomyces),例如许旺酵母(Schwanniomycesoccidentulis)(EP394,538,1990年10月31日发表);以及丝状真菌例如脉孢霉属(Neurospora)、青霉属(Penicillium)、Tolypocladium(WO 91/00357,1991年1月10日发表)以及曲霉属(Aspergillus)宿主,例如构巢曲霉(A.nidulans)[Balance等人,Biochem.Biophys.Res.Comm.112(1):284-9(1983)];Tilburn等人,Gene 26(2-3):205-21(1983);Yelton等人,Proc.Natl.Acad.Sci.USA 81(5):1470-4(1984)]和黑曲菌(A.niger)[Kelly和Hynes,EMBO J.4(2):475-9(1985)]。甲基营养酵母选自汉逊酵母属(Hansenula)、假丝酵母属(Candida)、克勒克酵母属(Kloeckera)、毕赤酵母属(Pichia)、酵母属、球拟酵母属(Torulopsis)和红酵母属(Rhodotorula)。可以在C.Antony,《The Biochemistry ofMethylotrophs》,269(1982)中找到这类酵母示范性的特定种的名单。
表达本发明融合蛋白质的合适的宿主细胞来自多细胞生物。无脊椎动物细胞的实例包括昆虫细胞,例如果蝇S2和灰赤夜蛾(Spodoptera)Sp、灰赤夜蛾high5及植物细胞。有用的哺乳动物宿主细胞系的实例包括NSO骨髓瘤细胞、中国仓鼠卵巢(CHO)细胞、SP2和COS细胞。更特定的实例包括以SV40转化的猴肾脏CVl系(COS-7,ATCC CRL 1651);人胚胎肾系[293或在悬浮培养中生长的亚克隆293细胞,Graham等人,J.Gen Virol.,36(1):59-74(1977)];中国仓鼠卵巢细胞/-DHFR[CHO,Urlaub和Chasin,Proc.Natl.Acad.Sci.USA,77(7):4216-20(1980)];小鼠支持细胞[TM4,Mather,Biol.Reprod.23(1):243-52(1980)];人类肺细胞(W138.ATCCCCL 75);人类肝细胞(Hep G2、HB 8065);以及小鼠乳腺肿瘤(MMT060562,ATCC CCL51)。
表达和克隆载体都含有使载体能够在一种或多种所选宿主细胞中复制的核酸序列。表达和克隆载体一般将含有选择基因,也称为选择标记。典型的选择基因编码(a)赋予抗生素或其他毒素(例如新霉素、氨甲蝶呤或四环素)抗性、(b)弥补自养缺陷、或(c)补充不能从复杂培养基获得的关键营养物(例如编码芽孢杆菌D-丙氨酸消旋酶的基因)的蛋白质。
哺乳动物细胞合适的选择标记的实例是能够鉴定有能力吸收融合蛋白质编码核酸的细胞的那些选择标记,例如DHFR或胸苷激酶。当使用野生型DHFR时,恰当的宿主细胞是如[Urlaub和Chasin,Proc.Natl.Acad.Sci.USA,77(7):4216-20(1980)]所描述的制备并增殖的DHFR活性缺陷的CHO细胞系。在酵母中使用的合适的选择基因是存在于酵母质粒YRp7中的trpl基因[Stinchcomb等人,Nature 282(5734):39-43(1979);Kingsman等人,Gene 7(2):141-52(1979);Tschumper等人,Gene 10(2):157-66(1980)]。Trpl基因为缺乏在色氨酸中生长的能力的酵母突变株例如ATCCNo.44076或PEPC1提供选择标记[Jones,Genetics85:23-33(1977)]。
表达和克隆载体通常含有与融合蛋白质编码核酸序列有效连接以指导mRNA合成的启动子。由多种可能的宿主细胞识别的启动子广为人知。与酵母宿主使用的合适的启动子序列实例包括3-磷酸甘油酸激酶[Hitzeman等人,J.Biol.Chem.255(24):12073-80(1980)]或其他糖酵解酶[Hess等人,J.Adv.Enzyme Reg.7:149(1968);Holland,Biochemistry 17(23):4900-7(1978)],例如烯醇酶、甘油醛-3-磷酸脱氢酶、己糖激酶、丙酮酸脱羧酶、磷酸果糖激酶、葡萄糖-6-磷酸异构酶、3-磷酸甘油酸变位酶、丙酮酸激酶、丙糖磷酸异构酶、磷酸葡萄糖异构酶和葡糖激酶。其他酵母启动子是醇脱氢酶2、异细胞色素C、酸性磷酸酶、与氮素代谢有关的降解酶、金属硫蛋白、3-磷酸甘油醛脱氢酶和负责麦芽糖和半乳糖利用的酶的启动子区,所述启动子是可诱导的启动子,具有由生长条件控制转录的额外的优点。在EP 73,657中进一步描述了用于酵母表达的合适的载体和启动子。例如,通过启动子可以控制融合蛋白质编码mRNA从哺乳动物宿主细胞中载体的转录,所述启动子可以来自病毒基因组,例如多瘤病毒、禽痘病毒、腺病毒(例如腺病毒2)、牛乳头瘤病毒、鸟肉瘤病毒、巨细胞病毒、逆转录病毒、乙型肝炎病毒和猿病毒40(SV 40);异源哺乳动物启动子,例如肌动蛋白启动子或免疫球蛋白启动子,以及热休克启动子,条件是这些启动子与宿主细胞系统相容。
可以通过向载体中插入增强子序列增加高等真核细胞对编码融合蛋白质的多核苷酸的转录。增强子是作用于启动子以增加其转录的DNA顺式作用元件,通常约10至300bp。已知许多来自哺乳动物基因(珠蛋白、弹性蛋白酶、白蛋白、a-酮蛋白(ketoprotein)和胰岛素)的增强子序列。然而,人们一般将使用真核细胞病毒启动子。实例包括复制起点后侧的SV40增强子(bp 100-270)、巨细胞病毒早期启动子增强子、复制起点后侧的多瘤病毒增强子、以及腺病毒增强子。可以将增强子在融合蛋白质编码序列的5′或3′位剪接在载体中,但是优选位于启动子5′位。
用于真核宿主细胞(酵母、真菌、昆虫、植物、动物、人类或其他多细胞生物的有核细胞)的表达载体也将含有转录终止和稳定mRNA所必需的序列。通常可以从真核或病毒DNA或cDNA的5′以及偶然从3′非翻译区获得这些序列。这些区域含有转录为编码融合蛋白质的mRNA中非翻译部分中聚腺苷酸化片段的核苷酸片段。
可以从培养基或宿主细胞裂解物中回收多种形式的融合蛋白质。如果是膜结合的,可以使用合适的去污剂溶液(例如Triton-X 100)或酶切割将其从膜上释放。可以通过多种物理或化学方法(例如循环冻融、超声处理、机械破碎或细胞裂解试剂)来破碎融合蛋白质表达中使用的细胞。
一旦在合适的宿主细胞中表达本发明的融合蛋白质,就可以分离并纯化类似物。下列步骤是适宜的纯化步骤的代表:羧甲基纤维素分级分离;凝胶过滤例如Sephadex G-75;阴离子交换树脂例如DEAE或Mono-Q;阳离子交换例如CM或Mono-S;金属鳌合柱以结合多肽的表位标签形式;反相HPLC;层析聚焦;硅胶;乙醇沉淀;以及硫酸铵沉淀。
可以使用多种蛋白质纯化方法,这类方法为本领域所公知并在例如Deutscher,Methods in Enzymology 182:83-9(1990)和《Scopes,ProteinPurification:Principlesand Practice》,Springer-Verlag,NY(1982)中得到描述。所选的纯化步骤取决于使用的生产方法以及产生的特定融合蛋白质的性质。例如,使用蛋白质A或蛋白质G亲合基质可以有效纯化包含Fc片段的融合蛋白质。可以使用低或高pH缓冲液从亲合基质洗脱融合蛋白质。温和的洗脱条件将有助于防止融合蛋白质的不可逆变性。
Fc部分包含第11、85、86、87、91位的替换,末端lys缺失。替换后的类似物降低了融合蛋白质的免疫原性。
本发明的融合蛋白质含有来自人类IgG4,但是与野生型人类序列相比包括多个替换的Fc部分。Fc部分由通过非共价相互作用和二硫键结合的抗体的两个重链恒定区组成。Fc部分可以包含铰链区,并经CH2和CH3结构域延伸到抗体C末端。Fc部分还可以包含一个或多个糖基化位点。
天然Glp-1(7-37):
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly
本发明融合蛋白质的GLP-1类似物部分与天然Glp-1(7-37)相比包含原第31、36、37位的替换(即本发明新编码序列第25、30和31位的替换)。
本发明所述的GLP-1类似物可采用Fmoc法人工化学合成,其C末端被酰胺化。
本发明的GLP-1类似物可以成盐,包括多种无机或有机盐,如盐酸盐、磷酸盐、硫酸盐、马来酸盐、草酸盐、柠檬酸盐和乳酸盐;与某些无机、有机碱成盐,如氢氧化钠和N-甲基-葡萄糖胺成盐。
本发明所述GLP-1类似物可以以单一药物形式给药或可以与其它药物联合给药,或作为药学上可接受的载体,或为长效多肽药物开发提供可修饰的活性前体。其应用可以使用GLP-1类似物或其可药用盐及药学上可接受的载体。“药学上可接受的载体”不会破坏本发明的化合物和其可用盐的药学活性,同时其有效用量对人体无毒。“药学上可接受的载体”可使用但不限于:离子交换材料、硬脂酸铝、卵磷脂、药物制剂用血清蛋白、饱和植物脂肪酸、纤维素物质、乙烯-聚氧乙烯-嵌段聚合物、环糊精或其经化学修饰的衍生物或其他可溶性衍生物等。
其他可药用辅料如填充剂如无水乳糖、淀粉、乳糖珠粒和葡萄糖,粘合剂如微晶纤维素、崩解剂如交联羧甲基纤维素钠、交联羧甲基淀粉、低取代羟丙基纤维素,润滑剂如硬脂酸镁,吸收促进剂、赋形剂、增溶剂和着色剂等也可加入本发明的药物组合物中。
上述本发明的GLP-1类似物或其可药用盐以及药物组合物可通过肠道或者非肠道途径给药。非肠道给药途径包括皮下、皮内、肌内、经鼻、粘膜给药或吸入。制剂可开发注射剂、霜剂、软膏剂、贴剂和气雾剂等。
本发明的GLP-1类似物或其融合蛋白或其可药用盐以及药物组合物可用于相关疾病的单一用药或联合用药治疗,为本领域技术人员能够理解的范围。
本发明所述融合蛋白质可以用于生产治疗非胰岛素依赖性糖尿病的药物,也可以用于生产在超重受试者中治疗肥胖或诱导体重减轻的药物。
本发明提供了一些术语的解释以便于更好地理解本发明技术方案。
“多肽”是由天然产生或合成的肽键连接的氨基酸残基的聚合物。少于大约10个氨基酸残基的多肽通常被称为“肽”。
“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“蛋白质”是包括一个或多个多肽链的大分子。蛋白质也可以包含非肽的成分,如碳水化合物基团。可以在生产蛋白质的细胞中将碳水化合物和其它非肽取代基团加入到蛋白质中,并且该基团将因细胞的类型不同而不同。本文给蛋白质下定义是依据它们的氨基酸主链结构;对象碳水化合物基团之类的取代基一般不作规定,但其仍然可以存在。
“异源肽”用非宿主DNA分子编码的肽或多肽是“异源”肽或多肽。
“克隆载体”是一种在宿主细胞中具有自我复制能力的核酸分子,例如质粒、粘粒或噬菌体。克隆载体一般含有一个或少量允许在不丧失载体基本生物功能的情况下可以以特定方式插入核酸分子的限制性核酸内切酶识别位点,以及编码适合供克隆载体转化细胞的鉴定和选择之用的标记基因的核苷酸序列。标记基因一般包括提供四环素抗性或氨苄青霉素抗性的基因。克隆载体都有一个松弛的复制子,能带动外源基因,在宿主细胞中复制扩增,它是用来克隆和扩增DNA片段(基因)的载体。
“表达载体”具有克隆载体的基本元件(ori,Ampr,Mcs等)还具有转录/翻译所必需的DNA顺序的载体;是一些用于工程生产的细菌,被导入的目标基因会在此类细菌中得到表达,生产出我们需要的产物,导入的基因是由克隆载体产出的,表达载体具有较高的蛋白质表达效率,一般因为具有强的启动子。
“重组宿主”是一种含有异源核酸分子,如克隆载体或表达载体的细胞。
“整合的转化体”是重组宿主细胞,在其中异源DNA被整合进入到细胞的基因组DNA中。
“融合蛋白质”是由包含至少两个基因的核苷酸序列的核酸分子表达的杂合蛋白质。
“免疫球蛋白部分”是指包含免疫球蛋白恒定区的多肽。例如:免疫球蛋白部分可以包含重链恒定区。
“表达”是指基因产物的生物合成。例如,就结构基因而论,表达包括将结构基因转录到mRNA中和将mRNA翻译为一个或多个多肽。
具体实施方式
以下通过实施例对本发明进行更加详细的说明。根据本发明的要旨,这些实施例仅用于更加具体地说明本发明,不意味着限制本发明的范围,这对于本发明所属技术领域的技术人员而言显而易见。实施例以HIP925-1~12为例进行例证。所用材料中未特殊说明的均为市售可得。
本发明中一些常见缩写具有以下含义:
缩写 | 含义 |
Resin | 树脂 |
Fmoc | 芴甲氧羰基 |
HOBt | 1-羟基苯并三唑 |
DCM | 二氯甲烷 |
DMF | N,N-二甲基甲酰胺 |
DIC | N,N'-二异丙基碳二亚胺 |
Rink Amide-MBHA Resin | Rink酰胺-MBHA |
MeOH | 甲醇 |
TFA | 三氟乙酸 |
TIS | 三异丙基硅烷 |
DTT | 二硫苏糖醇 |
-Trt | 三苯甲基 |
-tBu | 叔丁基 |
-Pbf | 2,2,4,6,7-五甲基苯并呋喃-5-磺酰基 |
-Boc | 叔丁氧羰基 |
-OtBu | 叔丁酯基 |
参考实施例1.序列10的制备
序列10:分子量3910.45;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Ala-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
本实施例提供序列10多肽的制备方法。
序列10多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列10多肽肽树脂,脱除树脂及侧链保护基获得序列10多肽粗品,序列10多肽粗品经制备纯化得序列10多肽精制品。
(1)序列10多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将4.1g Fmoc-Arg(pbf)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列10多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列10多肽粗品的制备:
将以上制得的氨基树脂负载的序列10多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列10多肽粗品7.3g,收率:89.0%,HPLC纯度:53.2%。
(3)序列10多肽精制品的制备:
将7.3g序列10多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样1.8~2.2g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥得序列10多肽精制品3.2g,总收率39%,HPLC纯度99.3%。
参考实施例2.序列11的制备
序列11:分子量3741.2;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Ala-Val-Val-Lys-Leu-Lys-NH2。
本实施例提供序列11多肽的制备方法。
序列11多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列11多肽肽树脂,脱除树脂及侧链保护基获得序列11多肽粗品,序列11多肽粗品经制备纯化得序列11多肽精制品。
(1)序列11多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将3.0g Fmoc-Lys(Boc)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列11多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列11多肽粗品的制备:
将以上制得的氨基树脂负载的序列11多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列11多肽粗品7.1g,收率:90.3%,HPLC纯度:55.1%。
(3)序列11多肽精制品的制备:
将7.1g序列11多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样2~2.5g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,洗脱流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥,得序列11多肽精制品3.1g,总收率39.6%,HPLC纯度99.5%。
参考实施例3.序列12的制备
序列12:分子量3968.5;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Glu-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
本实施例提供序列12多肽的制备方法。
序列12多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列12多肽肽树脂,脱除树脂及侧链保护基获得序列12多肽粗品,序列12多肽粗品经制备纯化得序列12多肽精制品。
(1)序列12多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将4.1g Fmoc-Arg(pbf)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列12多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列12多肽粗品的制备:
将以上制得的氨基树脂负载的序列12多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列12多肽粗品7.2g,收率:86.5%,HPLC纯度:52.0%。
(3)序列12多肽精制品的制备:
将7.2g序列12多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样1.8~2.2g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥得序列12多肽精制品3.2g,总收率38.1%,HPLC纯度98.7%。
参考实施例4.序列13的制备
序列13:分子量3938.5;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Val-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
本实施例提供序列13多肽的制备方法。
序列13多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列13多肽肽树脂,脱除树脂及侧链保护基获得序列13多肽粗品,序列13多肽粗品经制备纯化得序列13多肽精制品。
(1)序列13多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将4.1g Fmoc-Arg(pbf)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列13多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列13多肽粗品的制备:
将以上制得的氨基树脂负载的序列13多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列13多肽粗品7.1g,收率:85.7%,HPLC纯度:56.9%。
(3)序列13多肽精制品的制备:
将7.1g序列13多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样1.8~2.2g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥得序列13多肽精制品3.2g,总收率38.6%,HPLC纯度99.1%。
参考实施例5.序列14的制备
序列14:分子量3797.3;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Phe-Leu-Val-Lys-Gly-Val-Leu-Leu-Leu-Lys-Lys-NH2。
本实施例提供序列14多肽的制备方法。
序列14多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列14多肽肽树脂,脱除树脂及侧链保护基获得序列14多肽粗品,序列14多肽粗品经制备纯化得序列14多肽精制品。
(1)序列14多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将3.0g Fmoc-Lys(Boc)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列14多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列14多肽粗品的制备:
将以上制得的氨基树脂负载的序列14多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列14多肽粗品6.4g,收率:84.1%,HPLC纯度:50.3%。
(3)序列14多肽精制品的制备:
将6.4g序列14多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样1.8~2.2g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,洗脱流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥,得序列14多肽精制品2.8g,总收率36.6%,HPLC纯度98.5%。
参考实施例6.序列15的制备
序列15:分子量3900.4;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-His-Leu-Val-Lys-Gly-Ala-Leu-Lys-Leu-Leu-Leu-Arg-NH2。
本实施例提供序列15多肽的制备方法。
序列15多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列15多肽肽树脂,脱除树脂及侧链保护基获得序列15多肽粗品,序列15多肽粗品经制备纯化得序列15多肽精制品。
(1)序列15多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将4.1g Fmoc-Arg(pbf)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列15多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列15多肽粗品的制备:
将以上制得的氨基树脂负载的序列15多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列15多肽粗品7.0g,收率:85.8%,HPLC纯度:53.7%。
(3)序列15多肽精制品的制备:
将7.0g序列15多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样1.8~2.2g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥得序列15多肽精制品3.1g,总收率37.6%,HPLC纯度98.9%。
参考实施例7.序列16的制备
序列16:分子量3780.3;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Ala-Val-Val-Lys-Leu-Lys-NH2。
本实施例提供序列16多肽的制备方法。
序列16多肽的制备方法:采用提供Fmoc保护的氨基树脂,脱除氨基树脂上的Fmoc保护基后,合成序列16多肽肽树脂,脱除树脂及侧链保护基获得序列16多肽粗品,序列16多肽粗品经制备纯化得序列16多肽精制品。
(1)序列16多肽肽树脂的制备:
在100ml的固相反应器中加入Rink Amide-MBHA Resin树脂(5.0g,0.42mmol/g,2.1mmol)及40ml CH2Cl2,将树脂溶胀30min,抽除CH2Cl2,用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用30ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(6×40ml)洗涤树脂,备用;同时将3.0g Fmoc-Lys(Boc)-OH(6.3mmol)和0.9g HOBt(6.6mmol)置于100ml烧瓶中,加入40ml DMF溶解,冰浴降温至0~5℃,滴加1.0ml DIC(6.6mmol),加完后反应5min,将该反应液转移至上述所得脱除Fmoc的氨基树脂中,N2鼓泡混匀,缩合反应3h后,抽除反应液,用DMF(6×40ml)洗涤树脂。
重复上述脱保护及缩合步骤,采用逐一偶联的方式,继续依次偶联Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Lys(Boc)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、、Fmoc-Ala-OH、Fmoc-His(Trt)-OH直至完成氨基树脂负载的序列16多肽肽树脂的合成,其中脱保护与缩合的步骤基本相同。合成完毕,用DMF(6×40ml)洗涤树脂,向固相反应器中加入40ml 20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,再次用40ml20%哌啶/DMF溶液脱除树脂上的Fmoc保护基,10min后,抽除反应液,采用DMF(4×40ml)洗涤树脂,再用MeOH(2×40ml)洗涤树脂,35℃真空烘干燥肽树脂。
(2)序列16多肽粗品的制备:
将以上制得的氨基树脂负载的序列16多肽肽树脂加入150ml TFA/TIS/H2O(v/v/v=95:2.5:2.5)混合溶液脱除树脂和侧链保护基,2.5h后抽滤,收集滤液,再用TFA/TIS/H2O(体积比为95:2.5:2.5)混合溶液(10ml)洗涤树脂,抽滤,合并滤液,35℃以下,减压浓缩至馏分不再流出,将浓缩液缓慢倒入3L冷乙醚中,搅拌,析出白色沉淀,过滤,滤饼碾碎,再用冷乙醚洗涤3次,35℃真空干燥5小时,得序列16多肽粗品7.1g,收率:89.7%,HPLC纯度:56.8%。
(3)序列16多肽精制品的制备:
将7.1g序列16多肽粗品溶于20ml 10%醋酸水溶液中,0.45μm膜过滤,用制备型HPLC纯化:选用C18柱子上样,每次上样2~2.5g,波长214nm,(10%乙腈—H2O(含1%的TFA)等梯度洗脱5min,10%至80%的乙腈—H2O(含1%的TFA)梯度洗脱35min,洗脱流速50ml/min,采用梯度系统洗脱,循环进样纯化,收集主峰并用分析液相检测纯度,纯化主峰纯度应大于98%。制备洗脱液在30℃~35℃水浴条件下减压浓缩,得到一次纯化物料浓缩液。
将一次纯化收集到的物料浓缩液进行二次除盐纯化,条件为:流动相A相:纯化水;B相:乙腈溶液,检测波长:215nm,流速为30~60ml·min-1。收集目标馏分,将收集到的馏分于水温低于35℃减压浓缩至馏分不再流出,将剩余馏分冻干,得到精制品,并真空冷冻干燥,得序列16多肽精制品3.1g,总收率39.4%,HPLC纯度99.6%。
验证实施例1.测定本发明GLP-1化合物融合蛋白的体外活性
如下所述测定GLP-1的类似物的效价,即在含有表达人GLP-1受体的膜的培养基中刺激环AMP(cAMP)的形成。
用目的GLP-1类似物或衍生物刺激表达人GLP-1受体的来自稳定转染的细胞系BHK467-12A(tk-ts13)的纯化的质膜,用来自PerkinElmer Life Sciences的AlphaScreenTM cAMP测定试剂盒测量产生cAMP的效价。AlphaScreen测定的基本原理是内源cAMP和外源加入的生物素-cAMP之间的竞争。通过用与受体珠缀合的特异性抗体实现cAMP捕获。
细胞培养和膜的制备:选择稳定转染的细胞系和高表达克隆用于筛选。让细胞在DMEM、5%FCS、1%Pen/Strep(青霉素/链霉素)和0.5mg/ml的选择标记G418中于5%CO2生长。
用PBS洗涤2X大约80%汇合的细胞,用Versene(乙二胺四乙酸四钠盐的水溶液)收获,以1000rpm离心5分钟,去掉上清液。另外的步骤都在冰上进行。通过Ultrathurax让细胞沉淀在10ml缓冲液1(20mM Na-HEPES、10mM EDTA、pH=7.4)中均质化20-30秒,以20,000rpm离心15分钟,将沉淀重悬于10ml缓冲液2(20mMNa-HEPES、0.1mM EDTA、pH=7.4)中。让悬浮液均质化20-30秒,以20,000rpm离心15分钟。让缓冲液2中的悬浮液再次重复均质化和离心,将膜重悬于缓冲液2中。测定蛋白质浓度,将膜储存在-80℃直到使用。
在平底的1/2-面积的96-孔板中实施测定。最终体积为每孔50μl。
溶液和反应试剂如下:
来自Perkin Elmer Life Sciences的AlphaScreen cAMP测定试剂盒;含有抗-cAMP受体珠(10U/μl)、链霉抗生物素供体珠(10U/μl)和生物素化的-cAMP(133U/μl)。
AlphaScreen缓冲液,pH=7.4:50mM TRIS-HCl;5mM HEPES;10mM MgCl2,6H2O;150mM NaCl;0.01%吐温。使用前将以下加入到AlphaScreen缓冲液中(指最终浓度):BSA:0.1%;IBMX:0.5mM;ATP:1mM;GTP:1uM。
cAMP标准(测定中稀释系数=5):cAMP溶液:5μL的5mMcAMP-储液+495μLAlphaScreen缓冲液。
在AlphaScreen缓冲液中制备cAMP标准以及待测GLP-1类似物或衍生物的合适的稀释系列,例如以下8种浓度的GLP-1化合物:10-7、10-8、10-9、10-10、10-11、10-12、10-13和10-14M和例如cAMP的系列10-6至3×10-11。
膜/受体珠:使用hGLP-1/BHK 467-12A膜;6μg/孔对应于0.6mg/ml(每孔所用膜量可变化)。
“无膜”:AlphaScreen缓冲液中的受体珠(最终15μg/ml)
“6μg/孔膜”:AlphaScreen缓冲液中的膜+受体珠(最终15μg/ml)。
加10μl“无膜”到cAMP标准(每孔一式二份)、阳性和阴性对照中。
加10μl“6μg/孔膜”到GLP-1和类似物(每孔一式二份/一式三份)
阳性对照:10μl“无膜”+10μl AlphaScreen缓冲液
阴性对照:10μl“无膜”+10μl cAMP储液(50μM)
因为珠对直接光照敏感,所以任何处理都在黑暗(尽可能黑)或在绿光中。在冰上进行所有稀释。
操作方法:
制备AlphaScreen缓冲液;
在AlphaScreen缓冲液中溶解并稀释GLP-1/类似物/cAMP标准;
制备供体珠溶液并在R.T孵育30分钟;
将cAMP/GLP-1/类似物加到板中:每孔10μl;
制备膜/受体珠溶液,并将其加到板中:每孔10μl;
加入供体珠:每孔30μl;
用铝箔包裹板,于RT在振荡器中孵育3小时(极为缓慢);
在AlphaScreen上计数-在AlphaScreen中计数前让每一板预孵育3分钟;
用Graph-Pad Prism软件(版本5)计算EC50[pM]值,结果见表1。
杜拉糖肽EC50值为13.712nM,HIP925-1的EC50值为0.124nM。HIP925-1~12效果接近。
表1.各融合蛋白的体外活性EC50值比较
融合蛋白 | EC50(nM) |
HIP925-1 | 0.124 |
HIP925-2 | 0.266 |
HIP925-3 | 0.422 |
HIP925-4 | 1.425 |
HIP925-5 | 1.057 |
HIP925-6 | 2.589 |
HIP925-7 | 2.874 |
HIP925-8 | 2.771 |
HIP925-9 | 1.732 |
HIP925-10 | 2.225 |
HIP925-11 | 2.527 |
HIP925-12 | 2.449 |
杜拉鲁肽 | 15.712 |
验证实施例2.接头对融合的GLP-1类似物肽的活性的影响
比较肽接头SEQ ID NO:4-8对活性的影响。按照上述实施例中所述的cAMP测定方法测试不同接头的融合肽对人GLP-1受体的活性。所测试的融合分子能够在cAMP细胞基测定中活化人GLP-1受体。在该测定中,接头序列4、6和7稍优于5和8。
验证实施例3.db/db糖尿病小鼠单次注射的血糖变化情况
雄性糖尿病db/db小鼠,8周龄,体重42±2g,按体重随机分为5组,每组6只。受试药组按1.5mg/kg剂量皮下注射HIP925-1~12,阳性组注射1.5mg/kg的杜拉鲁肽,模型组注射等体积剂量(10mL/kg)的PBS缓冲液。各组动物分别于给药前(0h)、给药后1h、2h、4h、6h、24h、48h、72h、96h、120h、144h、168h、180h、192h、204h、216h、228h、240h和252h,再分别腹腔注射40%的葡萄糖溶液200μl,给予葡萄糖后30、60分钟尾静脉取血测定血糖值RBG(血糖仪测定)。血糖数据以均数±标准差(means±SD)形式表示,采用SPSS18.0统计软件分析数据。正态分布,多组间均数差异采用单因素方差分析,方差齐性采用LSD检验,方差不齐采用Dunnett-T3检验;非正态性分布采用非参数检验,P<0.05表示具有显著性统计学差异。
在相同剂量下,实验组和阳性对照药杜拉鲁肽均有降血糖作用。经t-test检验,在0h~6h,HIP925-1~12组与杜拉鲁肽组动物的随机血糖值相对模型组均出现显著性降低,P<0.01,说明二者在体内的起效时间相近,短期内降糖效果相似;另外从给药后9天内小鼠RBG值变化中得知,杜拉鲁肽降血糖作用能维持至第3.5天,在给药后第100小时,其血糖值与模型组相比已不具有统计学差异,而HIP925-1~12组能维持至给药后228-252小时,即第10.5天小鼠的血糖水平与模型组相比,仍具有统计学差异(P<0.05),其中HIP925-1组能维持至给药后252h,HIP925-9组能维持至给药后228h。本发明其他融合蛋白稳定性也高于杜拉糖肽。因此,本发明所述融合蛋白稳定性高,有望开发成每周或更长周期给药一次的长效GLP-1受体激动剂。
表2.各融合蛋白维持降糖效果的持续时间比较
融合蛋白 | 维持降糖效果的持续时间(h) |
HIP925-1 | 252 |
HIP925-2 | 248 |
HIP925-3 | 246 |
HIP925-4 | 248 |
HIP925-5 | 243 |
HIP925-6 | 242 |
HIP925-7 | 242 |
HIP925-8 | 238 |
HIP925-9 | 228 |
HIP925-10 | 232 |
HIP925-11 | 235 |
HIP925-12 | 229 |
杜拉鲁肽 | 100 |
验证实施例4.db/db糖尿病小鼠连续10周给予HIP925-1~12的随机血糖及HbA1c含量变化
SPF级雄性db/db小鼠(购自上海斯莱克实验动物有限公司),8周龄,适应性饲养1周后,将30只db/db小鼠按随机血糖值(random blood glucose,RBG)随机分为5组(n=6):模型组、杜拉鲁肽组、HIP925-1~12按低(0.75mg/kg)、中(1.5mg/kg)、高(3mg/kg)剂量给药。各给药组皮下注射给予相应剂量的药物溶液,模型组皮下注射PBS缓冲液,给药体积均为10ml/kg。各组动物每周给药一次,连续给药10周,分别用血糖仪(安准血糖仪,长沙三诺生物传感股份有限公司产品)检测给药后不同采血时间点各组小鼠RBG值,并记录数据。采血时间点设定:第一次给药前(0d)、给药后第7d、14d、21d、28d、35d、42d、49d、56d、63d和70d。第70d,各组小鼠禁食14h后眼眶取血,然后立即用糖化血红蛋白测定试剂盒(免疫比浊法)及其配套仪器H700特定蛋白分析仪检测全血中糖化血红蛋白(HbA1c)含量,结果以HbA1c占总血红蛋白的百分比(%)表示。
数据以均数±标准差(means±SD)形式表示,采用SPSS18.0统计软件分析数据。正态分布,多组间均数差异采用单因素方差分析,方差齐性采用Dunnett-t检验,方差不齐采用Dunnett-t3检验;非正态性分布采用非参数检验,P<0.05表示具有显著性统计学差异。
对各组小鼠连续给药10周随机血糖值的变化趋势研究发现,HIP925-1~12高、中、低剂量组的小鼠血糖值相对模型组都有一定程度的降低,并且其降血糖活性呈现剂量依赖性。提示HIP925-1~12能有效、持续地控制db/db糖尿病小鼠的血糖水平。而且,首次给药和末次给药HIP925-1~12的降糖效果相似,说明未出现HIP925-1~12长期给药导致的受体敏感性降低的作用。
糖化血红蛋白(HbA1c)是血中葡萄糖与红细胞的血红蛋白相结合的产物,它与血中的葡萄糖水平呈正比的关系。由于红细胞在血循环中的寿命约为120天,因此糖化血红蛋白可反映取血前4-12周血糖的总水平,弥补了空腹血糖只反映瞬时血糖的不足。因而,HbA1c是长期控制血糖最重要的评估指标,也是临床决定是否要更换治疗方案的重要依据。本实施例中HbA1c检查结果可以稳定、可靠的反映出取血前2~3个月小鼠的血糖控制情况。连续给药10周后各组小鼠HbA1c含量测定结果见下表3。
表3.HIP925-1对db/db小鼠随机血糖的影响(means±SD,n=6)
注:各剂量组与模型组相比*P<0.05;**P<0.01。单位mmol/L。
而且,HIP925-1每周一次0.75mg治疗组受试者HbAlc降至正常水平(HbAlc<7%)的比例较每周一次0.75mg杜拉糖肽高12%。HIP925-1~12效果接近。
SEQUENCE LISTING
<110>鲁南制药集团股份有限公司3
<120> 一种GLP-1类似物融合蛋白质 GLP-1
<160> 31
<170> PatentIn version 3.5
<210> 1
<211> 229
<212> PRT
<213> synthetic construct
<220>
<221> UNSURE
<222> (11)..(11)
<223> Xaa at position 11 is Pro
<220>
<221> UNSURE
<222> (85)..(85)
<223> Xaa at position 85 is Ala or Phe
<220>
<221> UNSURE
<222> (86)..(86)
<223> Xaa at position 86 is Phe or Glu
<220>
<221> UNSURE
<222> (87)..(87)
<223> Xaa at position 87 is Glu, Phe or Leu
<220>
<221> UNSURE
<222> (91)..(91)
<223> Xaa at position 91 is Gly
<400> 1
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Xaa Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Xaa Xaa Xaa Val Leu Thr Xaa Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 2
<211> 229
<212> PRT
<213> synthetic construct
<400> 2
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 3
<211> 229
<212> PRT
<213> synthetic construct
<400> 3
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 4
<211> 12
<212> PRT
<213> synthetic construct
<400> 4
Gly Ala Ala Ala Ala Leu Ser Ala Ala Ala Ala Ser
1 5 10
<210> 5
<211> 15
<212> PRT
<213> synthetic construct
<400> 5
Gly Gly Gly Gly Ser Lys Lys Lys Lys Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 6
<211> 22
<212> PRT
<213> synthetic construct
<400> 6
Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Gly Ser
20
<210> 7
<211> 25
<212> PRT
<213> synthetic construct
<400> 7
Gly Gly Gly Gly Ser Lys Lys Lys Lys Ser Gly Gly Gly Gly Ser Lys
1 5 10 15
Lys Lys Lys Ser Gly Gly Gly Gly Ser
20 25
<210> 8
<211> 15
<212> PRT
<213> synthetic construct
<400> 8
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 9
<211> 31
<212> PRT
<213> synthetic construct
<220>
<221> UNSURE
<222> (25)..(25)
<223> Xaa at position 25 is Trp, Phe or His
<220>
<221> UNSURE
<222> (30)..(30)
<223> Xaa at position 30 is Val, Glu or Ala
<220>
<221> UNSURE
<222> (31)..(31)
<223> Xaa at position 31 is Val-Val-Lys-Leu-Lys-NH2,
Ala-Ala-Lys-Gly-Gly-Lys-NH2, Leu-Leu-Leu-Lys-Lys-NH2 or
Leu-Leu-Leu-Trp-Leu-Leu-Leu-Arg-NH2
<400> 9
His Ala 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 Xaa Leu Val Lys Gly Xaa Xaa
20 25 30
<210> 10
<211> 36
<212> PRT
<213> synthetic construct
<400> 10
His Ala 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 Phe Leu Val Lys Gly Ala Leu Lys
20 25 30
Leu Leu Leu Arg
35
<210> 11
<211> 35
<212> PRT
<213> synthetic construct
<400> 11
His Ala 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 Phe Leu Val Lys Gly Ala Val Val
20 25 30
Lys Leu Lys
35
<210> 12
<211> 36
<212> PRT
<213> synthetic construct
<400> 12
His Ala 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 Phe Leu Val Lys Gly Glu Leu Lys
20 25 30
Leu Leu Leu Arg
35
<210> 13
<211> 36
<212> PRT
<213> synthetic construct
<400> 13
His Ala 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 Phe Leu Val Lys Gly Val Leu Lys
20 25 30
Leu Leu Leu Arg
35
<210> 14
<211> 35
<212> PRT
<213> synthetic construct
<400> 14
His Ala 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 Phe Leu Val Lys Gly Val Leu Leu
20 25 30
Leu Lys Lys
35
<210> 15
<211> 36
<212> PRT
<213> synthetic construct
<400> 15
His Ala 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 His Leu Val Lys Gly Ala Leu Lys
20 25 30
Leu Leu Leu Arg
35
<210> 16
<211> 35
<212> PRT
<213> synthetic construct
<400> 16
His Ala 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 Ala Val Val
20 25 30
Lys Leu Lys
35
<210> 17
<211> 277
<212> PRT
<213> synthetic construct
<400> 17
His Ala 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 Phe Leu Val Lys Gly Ala Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 18
<211> 277
<212> PRT
<213> synthetic construct
<400> 18
His Ala 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 Phe Leu Val Lys Gly Ala Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 19
<211> 276
<212> PRT
<213> synthetic construct
<400> 19
His Ala 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 Phe Leu Val Lys Gly Ala Val Val
20 25 30
Lys Leu Lys Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser Ala
35 40 45
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
50 55 60
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
65 70 75 80
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
85 90 95
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
100 105 110
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
115 120 125
Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
145 150 155 160
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
180 185 190
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
245 250 255
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
260 265 270
Leu Ser Leu Gly
275
<210> 20
<211> 276
<212> PRT
<213> synthetic construct
<400> 20
His Ala 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 Phe Leu Val Lys Gly Ala Val Val
20 25 30
Lys Leu Lys Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser Ala
35 40 45
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
50 55 60
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
65 70 75 80
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
85 90 95
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
100 105 110
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
115 120 125
Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
145 150 155 160
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
180 185 190
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
245 250 255
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
260 265 270
Leu Ser Leu Gly
275
<210> 21
<211> 277
<212> PRT
<213> synthetic construct
<400> 21
His Ala 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 Phe Leu Val Lys Gly Glu Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 22
<211> 277
<212> PRT
<213> synthetic construct
<400> 22
His Ala 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 Phe Leu Val Lys Gly Glu Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 23
<211> 277
<212> PRT
<213> synthetic construct
<400> 23
His Ala 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 Phe Leu Val Lys Gly Val Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 24
<211> 277
<212> PRT
<213> synthetic construct
<400> 24
His Ala 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 Phe Leu Val Lys Gly Val Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 25
<211> 276
<212> PRT
<213> synthetic construct
<400> 25
His Ala 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 Phe Leu Val Lys Gly Val Leu Leu
20 25 30
Leu Lys Lys Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser Ala
35 40 45
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
50 55 60
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
65 70 75 80
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
85 90 95
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
100 105 110
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
115 120 125
Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
145 150 155 160
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
180 185 190
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
245 250 255
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
260 265 270
Leu Ser Leu Gly
275
<210> 26
<211> 276
<212> PRT
<213> synthetic construct
<400> 26
His Ala 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 Phe Leu Val Lys Gly Val Leu Leu
20 25 30
Leu Lys Lys Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser Ala
35 40 45
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
50 55 60
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
65 70 75 80
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
85 90 95
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
100 105 110
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
115 120 125
Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
145 150 155 160
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
180 185 190
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
245 250 255
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
260 265 270
Leu Ser Leu Gly
275
<210> 27
<211> 277
<212> PRT
<213> synthetic construct
<400> 27
His Ala 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 His Leu Val Lys Gly Ala Leu Lys
20 25 30
Leu Leu Leu Arg Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser
35 40 45
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
50 55 60
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
65 70 75 80
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
85 90 95
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
100 105 110
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
115 120 125
Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp
130 135 140
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
145 150 155 160
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
165 170 175
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
180 185 190
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
195 200 205
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
210 215 220
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
225 230 235 240
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
245 250 255
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
260 265 270
Ser Leu Ser Leu Gly
275
<210> 28
<211> 276
<212> PRT
<213> synthetic construct
<400> 28
His Ala 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 Ala Val Val
20 25 30
Lys Leu Lys Gly Ala Ala Ala Ala Lys Ser Ala Ala Ala Ala Ser Ala
35 40 45
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
50 55 60
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
65 70 75 80
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
85 90 95
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
100 105 110
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
115 120 125
Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
145 150 155 160
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
180 185 190
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
245 250 255
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
260 265 270
Leu Ser Leu Gly
275
<210> 29
<211> 831
<212> DNA
<213> synthetic construct
<400> 29
cacgccgaag gaaccttcac ctcagatgtg tcatcatacc tcgaaggcga agccgccaag 60
gaattcatcg cattcctcgt gaaaggagca ctcaaactcc tcctccgtgg cgcggcggcg 120
gcgaaaagcg cggcggcggc gagcgccgaa tcgaaatatg gtcccccctg tccaccatgt 180
cccgctcccg aatttcttgg tggtccttct gtttttcttt ttcccccaaa accgaaagat 240
actcttatga tttccggcac tccagaggtt acttgtgttg ttgtggacgc ctcacaggag 300
gatccagagg cccaattcaa ttggtatgtc gatggcgtcg aagtccacaa cgccaaaacc 360
aaaccccgcg aagagcagtt caattctact tatcgtgcct tcgaagtcct tactggtctc 420
catcaagatt ggcttaatgg caaggaatac aaatgcaagg tctccaacaa aggcctcccc 480
tcatcaatcg aaaaaaccat ctcgaaagcc aaaggccaac cccgcgagcc ccaggtctat 540
actctccccc cctctcaaga agagatgacc aaaaaccagg cctcactcac gtgcctcgtc 600
aaaggattct acccgtccga tattgccgtc gagtgggaat caaacggaga gccagagaac 660
aactacaaaa ccaccccacc ggtgctcgat tcagatggct catttttcct ctactcacgt 720
ctcactgtcg acaaatcccg ctggcaggaa ggaaacgtct tttcatgttc agtcatgcac 780
gaagccctcc acaatcatta tacccaaaaa tcgctgtcac tcatgttggg a 831
<210> 30
<211> 20
<212> PRT
<213> synthetic construct
<400> 30
Met Gly Lys Leu Ile Phe Trp Leu Val Phe Trp Leu Thr Ile Phe Trp
1 5 10 15
Leu Gly Ser Ala
20
<210> 31
<211> 60
<212> DNA
<213> synthetic construct
<400> 31
atgggcaaac tcatattctg gcttgtgttt tggctgacta ttttttggct tggatcagct 60
Claims (5)
1.一种GLP-1类似物融合蛋白质,其由GLP-1类似物和免疫球蛋白Fc部分组成,两者通过肽接头融合,该融合蛋白质序列选自SEQ ID NO:17-28。
2.一种多核苷酸,其编码权利要求1所述的融合蛋白质。
3.一种载体,其包含权利要求2的多核苷酸。
4.一种宿主细胞,其包含权利要求3的载体,为CHO细胞或NSO细胞。
5.权利要求1所述的融合蛋白质用于生产治疗非胰岛素依赖性糖尿病的药物的用途。
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CN1703424A (zh) * | 2002-10-11 | 2005-11-30 | 株式会社三和化学研究所 | Glp-1衍生物及其经粘膜吸收的制剂 |
CN101328221A (zh) * | 2008-04-14 | 2008-12-24 | 中国药科大学 | 一种降糖多肽融合蛋白及其衍生物的结构及用途 |
WO2009058734A4 (en) * | 2007-10-30 | 2009-07-23 | Univ Indiana Res & Tech Corp | Compounds exhibiting glucagon antagonist and glp-1 agonist activity |
CN106397569A (zh) * | 2015-08-01 | 2017-02-15 | 深圳红石科创生物科技发展有限公司 | 一种治疗代谢疾病的突变细胞因子融合蛋白 |
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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CN1703424A (zh) * | 2002-10-11 | 2005-11-30 | 株式会社三和化学研究所 | Glp-1衍生物及其经粘膜吸收的制剂 |
WO2009058734A4 (en) * | 2007-10-30 | 2009-07-23 | Univ Indiana Res & Tech Corp | Compounds exhibiting glucagon antagonist and glp-1 agonist activity |
CN101328221A (zh) * | 2008-04-14 | 2008-12-24 | 中国药科大学 | 一种降糖多肽融合蛋白及其衍生物的结构及用途 |
CN106397569A (zh) * | 2015-08-01 | 2017-02-15 | 深圳红石科创生物科技发展有限公司 | 一种治疗代谢疾病的突变细胞因子融合蛋白 |
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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