CN100540565C - 修饰的Exendins及其应用 - Google Patents
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Abstract
提供了在一个或多个下列位置：2、14、27、28修饰的新的Exendin-4及其聚乙二醇衍生物。这些化合物作为GLP-1受体激动剂用于治疗II型糖尿病。
Description
技术领域
本发明涉及一类长效促胰岛素分泌肽衍生物及其可药用盐，具体地说涉及一类经用聚乙二醇修饰的促胰岛素分泌肽化合物及其可药用盐，本发明还涉及其制备方法，和该化合物通过作用于胰高血糖素类似肽-1(Glucagon-like Peptide 1，GLP-1)受体而刺激β-细胞分泌胰岛素来调整血糖而在预防、治疗II型糖尿病中的应用。
技术背景
近年来，随着生活水平的提高，生活模式的现代化和社会的老龄化，世界各地糖尿病的患病率正在逐年增加，尤其是那些由贫变富的发展中国家上升更为明显。糖尿病继肿瘤、心脑血管疾病之后已成为第三位严重的慢性非传染疾病，是造成致死、致残的主要原因之一。1997年WHO报告，全世界已有糖尿病人1.35亿，到2000年估计将达1.75亿人。中国最新的调查报告显示20岁以上自然人群中糖尿病的患病率为3.21％，初步估计，中国糖尿病患者至少在2000万以上，其中95％以上为II型糖尿病病人。据统计美国自1987年至1992年每年直接或间接用于糖尿病的经费开支从10亿美元增加至920亿美元。中国近年来糖尿病的费用也以惊人的速度增加，从1993年的有关资料统计分析，当年直接用于糖尿病的治疗费用高达22.16亿元，而且这一费用尚不包括糖尿病所致并发症的治疗费用，医院外的治疗和保健开支以及间接的社会经济损失。
目前控制II型糖尿病的方法有节制饮食，注意锻炼，以及用药物来调节血糖浓度。最常用的药物包括胰岛素，磺酰脲类，双胍类以及格列酮类的化合物。这些药物强调促使体内血糖趋于正常，无法更正由糖尿病而引起的并发症，尤其是对肾脏，心血管***，视觉及神经***产生损伤。这些并发症与糖尿病所致死亡率的增加有直接关系。第一代治疗糖尿病药物的主要副作用包括低血糖，体重增加以及水肿。这些药物其作用机理可能有所不同，但是没有一个机理有保护具有分泌胰岛素功能的β-细胞的作用，从而没有可能维持体内正常的血糖代谢和内分泌调节。在许多情况下，使用单一药物会慢慢失去作用，迫使采用复方治疗方法，往往病人同时还使用降血压，降胆固醇药，所以这样的方案的长期效果不一。因此，研究开发控制血糖新药，协同目前现在的药物，强调保护及修复β-细胞的功能，调节内分泌***对食物摄入的反应，将会对治疗糖尿病有革命性的推进。
针对胰高血糖素类似肽-1(GLP-1)受体的激动剂方面的研究前景可观，这一领域的研究和开发有可能掀开治疗II型糖尿病领域的新篇章。胰高血糖素类似肽-1发现于1984年，它是一种肠内分泌激素。如果给予II型糖尿病人输入此激素，其血糖浓度可调节至正常水平(Nathan，DM，et al.Diabetes Care 1992；15：270-6；Zander，M，etal.Lancet 2002；359：824-30)。研究表明，胰高血糖素类似肽及其受体激动剂的作用主要是激活胰腺β-细胞表面的胰高血糖素类似物-1受体而分泌胰岛素所致。因为这种效应是由体内本身血糖浓度的高低来决定的，所以这样就不会产生会象传统药物一样，即使在胰高血糖素类似肽-1及其受体激动剂存在的情况下也导致严重低血糖而造成具有生命危险的低血糖休克。具体地说，当体内血糖浓度高于6mmol/L时，胰高血糖素类似肽-1能具有显著的促进胰岛素分泌的作用，当体内血糖趋于正常的水平时，则不再继续作用。另外，这类激动剂还具有刺激啮齿动物(大鼠)胰岛β-细胞的生长，增加β-细胞组织的作用。这种修复胰岛β-细胞的功能为治愈II型糖尿病提供了前景，至少可以推迟从II型发展成I型的时间。再者，胰高血糖素类似肽-1及其受体激动剂同时能抑制胰高血糖素的分泌从而减少肝脏血糖输出的可能。更有意义的是，这类激动剂能有效地抑制胃肠道能动性及胃排空从而导致减少食物的摄入，使体重减少。这样能帮助控制好II型糖尿病人的体重。
发明内容
本发明的目的在于提供一类长效的用聚乙二醇修饰的促胰岛素分泌肽化合物及其可药用盐。它们具有激活胰高血糖素类似肽-1(GLP-1)受体而促进胰岛素分泌、降低血糖的作用，因此可以用来治疗II型糖尿病或用来预防II型糖尿病的发生。这类化合物之所以会在体内具有更长的逗留时间而发挥功效作用，不仅是因为经聚乙二醇修饰后，其经肾脏排除时间推迟，而且更重要的是其多肽骨架在体内具有更好的酶介和化学稳定性，从而保证了由聚乙二醇修饰后带来的长效作用，这样会减少病人注射使用次数，真正方便病人而提高治疗质量和效益。
具体地说，本发明涉及包括但不限于列在序列表所有用作聚乙二醇修饰的前体多肽，以及用不同分子量的聚乙二醇修饰后而形成的化合物及其可药用盐。
本发明的另一目的在于提供一种制备长效的用聚乙二醇修饰的促胰岛素分泌肽化合物及其可药用盐的方法。
本发明的又一目的在于提供这类长效促胰岛素分泌肽衍生物和/或可药用盐，作为胰高血糖素类似肽-1(GLP-1)受体激动剂在预防、治疗II型糖尿病中的应用。
本发明的目的是由以下技术方案来达到的，本发明涉及一类促胰岛素分泌肽及其可药用的盐，其多肽骨架具有优化的体内酶介及化学稳定性。特别是，本发明涉及促胰岛素分泌肽，其包括(A)序列号(SEQ ID NO.)4至141所示的氨基酸序列，(B)含有与序列号4至141所示氨基酸序列实质上同一的氨基酸序列。
本发明还涉及含有在序列号4至141所示促胰岛素分泌肽氨基酸序列中特别是在其2位，14位，27位，28位，通过一个或者多于一个经聚乙二醇修饰衍生后所得的促胰岛素分泌肽及其可药用盐，所述的聚乙二醇的分子量为5,000-80,000，优选为20,000-60,000。本发明的这些促胰岛素分泌肽的氨基酸化合物具有关键的修饰部位，其包括促胰岛素分泌肽的氨基酸序列2位，14位，27位，28位。
本发明还涉及提供一种制备上述促胰岛素分泌肽及其可药用盐的方法，其中包括固相和液相合成方法，经过反相高效液相、离子交换和凝胶过滤纯化方法，冷冻干燥。
本发明进一步提供一种经聚乙二醇修饰前或后的促胰岛素分泌肽衍生物及其可药用盐在治疗和/或预防II型糖尿病方面的应用。
临床实验数据表明，当对血糖浓度控制较差的II型糖尿病人使用胰高血糖素类似肽-1(GLP-1)治疗时，其空腹血糖浓度能恢复正常(Gutniak，et al，New Eng.J.Med.326：1316-1322，1992)。长期使用胰高血糖素类似肽-1(GLP-1)能使其β-细胞的功能达到正常人的水平(Rachman，et al.，Diabetes 45：1524-1530，1996)。胰高血糖素类似肽-1(GLP-1)能够对那些对葡萄糖耐受功能不健全病人的β-细胞回应葡萄糖功能有所改善(Byrne，et al.，Diabetes 47：1259-1265，1998)。但是因为胰高血糖素类似肽-1(GLP-1)在体内很容易被一种二肽内切酶(DPP IV)所破坏而失活，同时，胰高血糖素类似肽-1(GLP-1)也存在着被其它多肽内切酶(如NEP24.11)的切点，这样，胰高血糖素类似肽-1(GLP-1)在体内的作用是短暂的。只有通过连续给药的方法，才能取得真正的治疗效果。因此许多实验室都注重开发更稳定的胰高血糖素类似肽-1(GLP-1)受体激动剂，这类化合物主要以修饰胰高血糖素类似肽-1(GLP-1)本身而形成的。有意义的是，80年代末、90年代初，Eng等人从美国西南部的大毒蜥(GilaMonster，Heloderma Sespectrum)的唾液分泌器官中分离出Exendin-4(Eng，J.，et al.，J.Biol.Chem.，265：20259-62，1990，Eng，J.，et al.，J.Biol.Chem.，267：7402-05，1992)。Exendin-4是一个具有39个氨基酸的多肽，与胰高血糖素类似肽-1(GLP-1)享有53％的同源性。它与胰高血糖素类似肽-1(GLP-1)受体亲和，其能力比胰高血糖素类似肽-1(GLP-1)本身强。在体内Exendin-4的调节体内糖代谢的能力比胰高血糖素类似肽-1(GLP-1)更好，其产生促胰岛素分泌的作用所需的浓度比胰高血糖素类似肽-1(GLP-1)要低，而且在体内的半衰期比胰高血糖素类似肽-1(GLP-1)更长(Kudsen，L.B.J.Med.Chem.47：4128-4134，2004)。这主要是Exendin-4具有其独特的酶介稳定性，这些酶介稳定性主要来源于消除了多肽内切酶(如NEP 24。11)的切点。
目前，已有大量文献报道了具有胰高血糖素类似肽-1(GLP-1)受体激动剂功能的化合物如GLP-1(7-36)、GLP-1(7-37)、Exendin-4以及其他GLP-1及Exendin-4的衍生物。这些文献包括WO98/43658、WO00/15224、WO00/66629、WO01/98331、WO01/04156、US Pateat No.5,545,618、US Patent No 5,118、WO03/058203、US PatentApplication Serial No.60/395,738、WO04/022004以及其内引用的文献等。
下面将天然存在的GLP-1受体激动剂对照如下
| 肽名 | 序列 | SEQ ID NO |
| GLP-1(7-36) | HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 | 1 |
| GLP-1(7-37) | HAEGTFTSDV SSYLEGQAAK EFIAWLVKGRG | 2 |
| Exendin-4 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG | 3 |
| PSSGAPPPS-NH2 |
式中英文缩写H(His)组氨酸，A(Ala)丙氨酸，E(Glu)谷氨酸，G(Gly)甘氨酸，T(Thr)苏氨酸，F(Phe)苯丙氨酸，S(Ser)丝氨酸，D(Asp)天冬氨酸，V(Val)缬氨酸，Y(Tyr)酪氨酸，L(Leu)亮氨酸，Q(Gln)谷氨酰酶，K(Lys)赖氨酸，I(Ile)异亮氨酸，R(Arg)精氨酸，M(Met)甲硫氨酸，N(Asn)天冬酰胺，P(Pro)脯氨酸。
尽管许多实验室开发更稳定的胰高血糖素类似肽-1(GLP-1)受体激动剂，但其在体内的作用效果仍是短暂的，因此开发长效的、基于胰高血糖素类似肽-1(GLP-1)受体激动机理的促胰岛素分泌肽衍生物就迫在眉睫。由于此类化合物的治疗作用和引起副作用(呕吐及恶心)的窗户较窄，所以用储藏缓释制剂来解决的方法成功的机率亦较小。解决获得长效的胰高血糖素类似肽-1(GLP-1)受体激动剂的方法，只有通过产生一个在体内具有足够逗留时间，稳定的化合物来实现。
将聚乙二醇引进活性蛋白和多肽会延长活性蛋白和多肽逗留的时间。这一技术已成功地应用在许多以蛋白质为主的生物药物上，如PEG-Intron，PEGASYS，Neulasta，Somavert等等。将聚乙二醇引进多肽或蛋白骨架的方法和化学可参见有关文献，如Veronese的综述(Veronese，FM，Biomaterial 200122：405-417)。鉴于胰高血糖素类似肽-1(GLP-1)和Exendin-4都为胰高血糖素类似肽-1(GLP-1)受体的事实，美国专利USP05424286及世界专利WO98/05351公开了胰高血糖素类似肽-1(GLP-1)和Exendin-4体内促胰岛素分泌作用的对比实验。实验表明Exendin-4体内作用比胰高血糖素类似肽-1(GLP-1)强而且时间较胰高血糖素类似肽-1(GLP-1)长，其原因是Exendin-4对体内的多肽水介酶更稳定(DPP IV，NEP24.11等)。世界专利申请2004/022004公开了有关用聚乙二醇修饰的胰高血糖素类似肽-1(GLP-1)受体激动剂，并提出当使用聚乙二醇的分子量大于30000道尔顿之上时，所获得的衍生物有可能不再有由于脑内胰高血糖素类似肽-1(GLP-1)受体活化而导致的恶心和呕吐副作用。这表明，经聚乙二醇修饰了的胰高血糖素类似肽-1(GLP-1)受体激动剂不仅会延长体内作用时间，而且有可能减少其副作用。但是这些公开的化合物除体内、体外活性有限制之外，其多肽骨架的体内酶介及化学稳定性并未改善，这限制了这些化合物成为长效制剂的可能性。其减弱的体内、体外活性将会使生产成本加高。因此，用酶介稳定性较好的Exendin-4骨架作为用聚乙二醇修饰前体的起点，成功的可能性会更大。虽然，世界专利申请WO00/66629公开了直接用Exendin-4作为聚乙二醇修饰前体所得的化合物和方法，但是这离真正得到一个长效稳定的、低成本的药品仍有距离。其原因如下：虽然Exendin-4在体内逗留时间从原来的几小时延长到几十小时、甚至更长时间，但其N端的His-Gly是仍有被二肽内切酶(如DPP IV)切断的可能性，导致胰高血糖素类似肽-1(GLP-1)受体激动活性功能失活；同样，也因为长效的聚乙二醇修饰的胰高血糖素类似肽-1(GLP-1)受体激动剂还必需具有很好的化学稳定性，尤其是体内37℃的条件下，而Exendin-4骨架中的14位甲硫氨酸易被氧化而导致生物活性的变化，同时亦会给制剂造成麻烦；而28位天冬酰胺的水解则是导致药物失活以及制剂困难的主要原因。其水解机理如下：
从水解机理可以看出，由于天冬酰胺的存在，所产生的五元环水解，不仅会降低修饰后胰高血糖素类似肽-1(GLP-1)受体激动剂的活性，而且会导致聚乙二醇与多肽骨架的分离从而影响长效化合物在体内逗留的时间。因此对2位甘氨酸的修改将提高基于Exendin-4多肽骨架的酶介稳定性和化学稳定性；对14位甲硫氨酸和28位天冬酰胺的修改将提高基于Exendin-4多肽骨架的化学稳定性。WO00/66629强调用聚乙二醇修饰Exendin-4中引进的赖氨酸侧链氨基来进行酰化反应而形成聚乙二醇结合物。因Exendin-4本身同时存在着赖氨酸，这样修饰的反应选择性只有通过使用保护基而实现，从而对生产成本必然会有影响。把修饰连接聚乙二醇的接点和反应专一的基团安排在多肽的羧基端(C-端)不仅不会减少聚乙二醇对多肽与受体的作用的影响，而且反应专一，其生产成本将明显减少。
本发明公开一系列在2位，14位，27位或者28位聚乙二醇修改了的Exendin-4衍生物，以及用这些多肽骨架进行聚乙二醇修饰而得的促胰岛素分泌肽。这些用聚乙二醇修饰的促胰岛素分泌肽在体内具有长效作用，可制成长效注射剂使用。
本发明所公开的促胰岛素分泌肽化合物具有体内和体外活化位于β-细胞表面的GLP-1受体而分泌胰岛素的功能，从而使血糖浓度降低。这些促胰岛素分泌肽的例子包括但不限于表1所列的多肽序列及其用聚乙二醇修饰所得的化合物。作为聚乙二醇修饰点，可以将39位丝氨酸用半胱氨酸或其它含巯基的合成氨基酸取代；同样，多点聚乙二醇修饰可按下列方式完成，这就是在羧基端加上二个或多个含巯基的氨基酸(如半胱氨酸)，利用这些延长了的多肽衍生物作为聚乙二醇修饰的前体，其用作两点修饰的通式为Cys(39)-(Xaa)(n-1)-Cys(n+39)，其中n＝1-10，Xaa为任一氨基酸。
这些多肽可以用化学合成方法制备，这包括液相片断合成，固相合成(见如Merrifield，J.Am.Chem.Soc.1963，85：2149-2154)或者固相、液相相结合的方法；多肽合成包括手工和自动方式，若采用自动方式，可使用Applied Biosystems 431A多肽合成仪、Csbio多肽合成仪等等；另外多肽合成也可采用组合合成的方法。
经化学合成而制备的多肽需经过高效液相制备方法提纯，通常用反相材料作为柱填料(如C4，C8，C18)。所得的多肽经分析鉴定(如高效液相色谱(HPLC)，质谱(MS)，氨基酸分析(AAA))证明后，才能进行体外、体内药效研究。这些化合物经高效液相制备方法提纯后，冻干即得产品。
聚乙二醇可以从不同的商家购买或用已知的方式合成。聚乙二醇的分子量范围为5000-80,000道尔顿，优选范围为20,000-60,000道尔顿，更好为40,000道尔顿左右。
聚乙二醇和多肽应在多肽羧基端连接，这样可以最低限度地减少聚乙二醇链对多肽本身与其受体之间作用的干扰。这就是说聚乙二醇可以连接在从29到39位残基的任何位置。这要求用含巯基的氨基酸(如半胱氨酸)取代任一或同时几个氨基酸。如果是作单一点聚乙二醇修饰，最好是用半胱氨酸去取代位于羧基端39位的丝氨酸；同样，如果作二点修饰，最佳方法是用半胱氨酸去取代39位丝氨酸，然后再另加一半胱氨酸在40位，或者39+n位(n＝1-10)。
利用半胱氨酸或巯基连接聚乙二醇的方法在许多文献中都有描述(见Veronese，Biomaterials 2001，22：405-417)。受过化学训练的技术人员都能把聚乙二醇连接到含巯基的促胰岛素分泌肽上去。
具体地说，利用巯基来实现连接反应可以用下列方式：
1)巯基来自于多肽链，通过引入如下式描述的氨基酸
n＝1-10
这时聚乙二醇应含有麦克尔加成接受体如马来酰亚胺中的双链，卤素及磺酸酯取代的基团，多肽与聚乙二醇形成硫醚链而连接。
2)巯基来自于修改的多肽中氨基酸的侧链，如巯基连接于赖氨酸的侧链氨基。侧链修改的氨基酸如下列通式：
n＝1-10；m＝1-10
这时聚乙二醇应含有麦克尔加成接受体如马来酰亚胺中的双链，卤素及磺酸酯取代的基团，多肽与聚乙二醇形成硫醚链而连接。
3)巯基来自于聚乙二醇，这时多肽中的连接点应含有麦克尔加成接受体如马来酰亚胺中的双链，卤素及磺酸酯取代的基团，多肽与聚乙二醇形成硫醚链面连接。
4)若聚乙二醇和多肽均含巯基，则可以用形成不对称双硫键的方式连接。
优选聚乙二醇与本发明中所公开的多肽通过形成硫醚的方法形成共价连接。聚乙二醇与这些公开了的多肽序列连接并不只限于这一化学连接方法，其它连接方法如通过酰化反应，还原胺化反应，肟形成反应等都包括在本发明之内。
本发明中适合于作为聚乙二醇修饰的前体多肽衍生物见表1，这只是本发明所列举的例子，本发明并非限定于此序列。在序列表中，优选的多肽序列选自SEQ ID NO80至SEQ ID NO 141。
这些经聚乙二醇修饰的促胰岛素分泌肽以及其多肽前体本身是两性化合物，可以与酸性或碱性物质反应形成盐。通常成盐所用的酸选自：盐酸，氨溴酸，氢碘酸，硫酸，磷酸，对甲苯磺酸，甲磺酸，草酸，对溴苯基磺酸，碳酸，琥珀酸，柠檬酸，苯甲酸，乙酸、三氟乙酸等。这类盐的例子包括硫酸盐，焦硫酸盐，硫酸氢盐，亚硫酸盐，亚硫酸氢盐，磷酸盐，磷酸氢盐，磷酸二氢盐，偏磷酸盐，焦磷酸盐，盐酸盐，氢溴酸盐，氢碘酸盐，乙酸盐，丙酸盐，癸酸盐，辛酸盐，丙烯酸盐，甲酸盐，异丁酸盐，己酸盐，庚酸盐，丙炔酸盐，草酸盐，丙二酸盐，丁二酸盐，辛二酸盐，癸二酸盐，富马酸盐，马来酸盐，丁炔-1，4-二酸盐，己炔-1，6-二酸盐，苯甲酸盐，氯苯甲酸盐，对甲基苯甲酸盐，二硝基苯甲酸盐，羟基苯甲酸盐，甲氧基苯甲酸盐，苯乙酸盐，苯丙酸盐，苯丁酸盐，柠檬酸盐，乳酸盐，r-羟基丁酸盐，甘油酸盐，酒石酸盐，甲磺酸盐，丙磺酸盐，萘-1-磺酸盐，萘-2-磺酸盐，扁桃酸盐等。优选的酸加成盐是盐酸盐、硫酸盐、乙酸盐、三氟乙酸盐；通常成盐所用的碱选自氢氧化钠、氢氧化钾、氨水、碳酸钾等。
本发明所公开的促胰岛素分泌肽化合物，特别是用聚乙二醇修饰的促胰岛素分泌肽化合物，由于其具有修复β-细胞功能的潜力，所以可以用于预防、治疗II型糖尿病，特别是对于由体重过重甚至肥胖而引起的分泌不太正常的病人。
因此，本发明还涉及一种治疗和/或预防II型糖尿病的方法，其中向需要治疗和/或预防II型糖尿病II的人使用药物有效剂量的本发明所述的促胰岛素分泌肽。
本发明所公开的促胰岛素分泌肽化合物用来治疗糖尿病时可以单独使用，更适合于与其它抗糖尿病药联合使用(如PPAR激动剂、磺酰脲类药、非磺酰脲类似物(Secretagogues)、α-glucosidase(α-葡萄糖苷酶)抑制制、胰岛素增敏剂、胰岛素Seeretagogues、肝糖释放抑制剂、胰岛素和其它抗肥胖药)。
临床用量应根据具体化合物的药效而定。其范围为0.0001mg/kg到大约200mg/kg体重，优选范围为0.001mg/kg到20mg/kg体重，最优选范围为0.01mg/kg到1mg/kg体重。给药方式可以用注射方式(包括静注、肌注、皮下)或其它连续注射方式。这些化合物亦可以做成各种各样的制剂，以其它常规的途径给药，如口服，皮肤给药，肺、鼻、口腔喷雾剂，栓剂等等。
附图说明
图1是SEQ ID NO.95的LC-MS图。
图2显示db/db小鼠皮下注射PEG-EX-4类似物当天对糖耐量的影响。
图3显示db/db小鼠皮下注射PEG-EX-4类似物后第3天对糖耐量的影响。
图4显示db/db小鼠皮下注射PEG-EX-4类似物后第6天对糖耐量的影响。
图5显示db/db小鼠皮下注射PEG-EX-4类似物后第9天对糖耐量的影响。
图6显示小鼠皮下注射PEG-EX-4类似物1100μg/kg后，其血糖降低作用。
图7小鼠皮下注射PEG-EX-4类似物3300μg/kg后，其血糖降低作用。
具体实施方式
为了更详细地说明本发明，给出下列实例。但本发明的范围并非限定于此。
实施例一
本发明所得化合物SEQ ID NO.95的固相合成制备
(1)合成所采用的氨基酸单体
Fmoc-His(Trt)-OH，Fmoc-dAla-OH，Fmoc-Gly-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Thr(tBu)-OH，Fmoc-Phe-OH，Fmoc-Ser(tBu)-OH，Fmoc-Asp(OtBu)-OH，
Fmoc-Leu-OH，Fmoc-Lys(Boc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Nle-OH，
Fmoc-Ala-OH，Fmoc-Val-OH，Fmoc-Arg(pbf)-OH，Fmoc-Ile-OH，Fmoc-Trp(Boc)-OH，Fmoc-Asn(Trt)-OH，Fmoc-Pro-OH，Fmoc-Cys(Trt)-OH
上面缩写为：Fmoc：9-芴基甲氧羰基；Boc：叔丁氧羰基；Trt：三苯甲基；OtBU：叔丁基氧基；tBu：叔丁基。
(2)所用药品：试剂为：N，N-二异丙基乙胺，二异丙基碳二亚胺(DIC)，N，N-二甲基甲酰胺(DMF)，二氯甲烷、六氢吡啶、1-羟基苯并***、Rinkamide树脂、茚三酮、甲醇、苯甲醚、三异丙基硅烷、三氟乙酸。
(3)操作
A.合成：以0.25mmol规模为例，称取0.5g Rink酰胺树脂置于反应器中，将氨基酸称取1mmol，用DIC/HOBt方法活化，按照多肽序列从C端到N端依次进行合成。反应在25℃室温条件下进行，照下列步骤进行操作：
1.用20％六氢吡啶的DMF溶液脱保护基Fmoc，每次10分钟；
2.用10毫升DMF洗三次，抽干；
3.称取保护的氨基酸(1mmol)，HOBt(1mmol)，溶于10毫升DMF中，加入DIC(1mmol)活化10分钟；
4.将此活化的氨基酸溶液加入反应器内，振摇反应1小时；
5.用DMF洗树脂三次，抽干；
6.如果茚三酮反应显阴性，进行下一循环，重复步骤1到5；
如果茚三酮反应显阳性，重复步骤3到5。
多肽链逐步合成完毕后，用甲醇洗干树脂，凉干。
B.脱保护基及切除多肽
将带有多肽的树脂1克置于反应器中，加入裂解液。比例如下表：
| 溶剂 | 用量(毫升) |
| 苯甲醚 | 2 |
| 甲醇 | 2 |
| 三异丙基硅烷 | 2 |
| 三氟乙酸 | 6 |
室温振摇2小时后，过滤，收集滤液，树脂用少量醋酸洗涤，合并收集液，浓缩后，加入***产生沉淀，过滤沉淀，用少量***洗涤后，得粗品。
C.高效液相分离纯化，冻干
将得到的粗品溶于少量10％醋酸溶液中，上柱，经高效液相制备分离纯化、冻干得产品，所得多肽经色质联用检测证明为所求化合物。
色谱柱：luna C18(2)，5u，
分离波长：λ＝220nm，Waters制备***
梯度：(TFA：三氟乙酸)
| T(分钟) | A：(0.05％TFA)CH<sub>3</sub>CN | B：(0.05TFA)H<sub>2</sub>O |
| 0 | 10％ | 90％ |
| 20 | 45％ | 55％ |
| 30 | 45％ | 55％ |
| 30.1 | 10％ | 90％ |
所得化合物分子量：4212.6g/mol；理论分子量：4213g/mol。
图1：SEQIDNO95LC-MS图
实施例二
聚乙二醇修饰促胰岛素分泌肽的方法
用聚乙二醇修饰多肽可按常规的方法操作。本发明强调基于修饰巯基而成硫醚的方法，把聚乙二醇和多肽结合起来。具体地说在优化了的Exendin-4衍生物羧基端加上一个或多个半胱氨酸，然后采用含马来酸酰亚胺功能基团的聚乙二醇进行聚乙二醇修饰反应，麦克尔加成反应后形成硫醚链而使多肽和聚乙二醇共价结合。通常，将所需的多肽溶于0.1M磷酸盐缓冲液中，在去氧情况下，加入聚乙二醇。聚乙二醇和多肽的摩尔比为1∶1。反应的pH为6-7.5，反应液中加入EDTA可减少巯基的氧化。反应2小时后，反应液用反相高效液相方法分离提纯，过剩、未反应的聚乙二醇可用离子交换方法去除。所得产品用质谱证实分子量，用RP-HPLC及凝胶色谱分析纯度。以修饰SEQ ID NO 95为例，当用一个43KD PEG修饰时，其收率为70-90％。(以聚乙二醇计)
实施例三
多肽稳定性测定
本发明中所公开的Exendin-4衍生物具有优化了的酶介或化学稳定性。下面的方法用来检查本发明中公开的一些多肽的化学稳定性。
将1毫克各供试品溶于150mM氯化钠，20mM磷酸钠盐的缓冲液中，配成浓度为4mg/ml的溶液，其pH为8.0，将供试品溶液于40℃的恒温箱中放置，用液相色谱-质谱联用方法检查多肽的纯度，主峰面积消失的比例与时间的关系表达多肽的化学稳定性。
| 纯度(％) | 0天 | 5天 | 10天 | 15天 |
| 供试品1 | 98.2 | 88.0 | 81.6 | 76.2 |
| 供试品2 | 98 | 93.1 | 90.4 | 88.2 |
| 供试品3 | 98.9 | 98.8 | 98.8 | 98.8 |
| 供试品4 | 99.7 | 99.4 | 99.0 | 99.3 |
表2Exendin-4类化合物稳定性测定表
其中，供试品1：Exendin-4对照。序列为：
His-Gly2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met14-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn28-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser39。
供试品2：将供试品1中的2#Gly改为d-Ala，39#改为Cys。供试品3：将供试品1中的2#Gly改为d-Ala，14#Met改为Nle，28#Asn改为Gln。供试品4：将供试品2与PEG40K通过C末端的Cys共价连接，其中，供试品1-4的C末端均是酰胺。
结论：
供试品1的序列是自然界分离得到的多肽序列，但其N末端的His-Gly是二肽酶合适的底物，而且它含有的Met容易氧化，Asn容易自身反应，都造成了供试品1不太稳定；通过对2位、14位和28位三个点的替换，肽的稳定性有较大提高，而且三个位点都替换，其稳定性提高的程度大大超过了仅His-Gly被替换成His-dAla时稳定性的提高程度；供试品2虽然不是很稳定，但在与PEG40K连接后(即供试品4)变得非常稳定，这说明了PEG在提高多肽稳定性方面有很大的作用。
实施例四
制剂
PEG-EX-4类似物 5g
间甲酚 0.04g
冰醋酸 适量
冰醋酸钠 适量
注射用水 适量
100ml/100支装
无菌制备
实施例一
多肽口服葡萄糖耐量实验
将序号为SEQ ID NO 25的多肽经分子量为40000左右的聚乙二醇修饰后(编号：供试品5)，对正常小鼠进行口服葡萄糖耐量实验，结果如下表：
表1.不同剂量供试品5皮下注射对正常小鼠，可见第一和第三天口服葡萄糖耐量及血糖曲线下面积的影响
(第1天)
v.s Con ***P＜0.001；n＝10
(第3天)
v.s Con，*P＜0.05，**P＜0.01，***P＜0.001；n＝10
实验例二
PEG-EXENDIN-4(PEG-EX-4)类似物对II型糖尿病db/db小鼠的影响
1.受试动物：种属、品系：db/db小鼠，来源：南京大学模式动物中心提供，小鼠体重：35-50g，雌雄各半。动物数：45只，每组5-6个。饲养条件：SPF级动物房饲养，温度：22-24℃，湿度：45-80％，光照：150-300Lx，12小时昼夜交替。
2.试验方法：
剂量设置：设5个给药组：0.03、0.1、0.3、1和3mg/kg；同时设立空白对照组；给药途径：皮下注射；给药容积：0.05ml/kg体重。
(1)对非禁食状态下db/db小鼠血糖的影响
db/db小鼠，按非禁食血糖及体重分组，每组6只，雌雄各半，分别为空白对照和5个PEG-EX-4类似物给药组。各组动物分别单次皮下注射给予受试药物和生理盐水，于给药前及给药后1、2、4、8、24小时测血糖，此后每隔24小时测定一次非禁食血糖，观察受试物降血糖作用的维持时间，同时测定各组动物给药后摄食量及体重的变化。
(2)对禁食状态下db/db小鼠血糖的影响
db/db小鼠，按非禁食、禁食血糖及体重分组，每组6只，雌雄各半，分别为空白对照和5个PEG-EX-4类似物给药组。各组动物于禁食5小时后分别单次皮下注射给予受试药物和生理盐水，于给药前及给药后1、2小时测血糖，此后每隔24小时测定其非禁食和禁食血糖，观察受试物降血糖作用的维持时间，同时测定各组动物给药后摄食量及体重的变化。
(3)对禁食状态下db/db小鼠糖耐量的影响
db/db小鼠，按禁食血糖及体重分组，每组5只，分别为空白对照和5个PEG-EX-4类似物给药组。于禁食5小时后分别单次皮下注射给予受试药物和生理盐水，于给药后15分钟时口服葡萄糖2.5g/kg，于给糖后0、30、60和120分钟时测定血糖值。在给药后第3、6及9天时，再分别进行口服糖耐量试验。观察受试物对db/db糖耐量的影响及其作用的维持时间，并测定各组动物给药后摄食量及体重的变化。
3.试验结果：参见附图2-5以及表1-表6，PEG-EX-4类似物对db/db小鼠血糖的影响
(1)对禁食和非禁食状态下db/db小鼠血糖的影响
表1：db/db小鼠皮下注射PEG-EX-4类似物后对禁食血糖的影响(均值±SD，n＝6)
表2：db/db小鼠皮下注射PEG-EX-4类似物后对每日非禁食血糖的影响(均值±SD，n＝6)
(2)对禁食状态下db/db小鼠糖耐量的影响
表3：db/db小鼠皮下注射PEG-EX-4类似物当天对糖耐量的影响(均值±SD，n＝5)
表4：db/db小鼠皮下注射PEG-EX-4类似物后第3天对糖耐量的影响(均值±SD，n＝5)
表5：db/db小鼠皮下注射PEG-EX-4类似物后第6天对糖耐量的影响(均值±SD，n＝5)
表6：db/db小鼠皮下注射PEG-EX-4类似物后第9天对糖耐量的影响(均值±SD，n＝5)
*，P＜0.05；**，P＜0.01；***，P＜0.001，与空白对照组相比
实验例三
PEG-EXENDIN-4(PEG-EX-4)类似物对KKAy小鼠血糖的影响预初试验结果
1.试验方法：
KKAy小鼠一次皮下注射不同剂量的PEG-EX-4类似物后，在不同时间测定其血糖的变化情况。
2.试验结果：
(1)参见图6，KKAy小鼠一次皮下注射PEG-EX-4类似物1100μg/kg后，其血糖降低作用可维持3-4天左右。
(2)参见图7，KKAy小鼠一次皮下注射PEG-EX-4类似物3300μg/kg后，其血糖降低作用可维持3-4天左右。
以下表7中可见本发明中所述的长效促胰岛素分泌肽的氨基酸序列：
| 编号 | 序列 | SEOIDNO |
| HR1 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS | 4 |
| HR2 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC | 5 |
| HR3 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC | 6 |
| HR4 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC | 7 |
| HR5 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC | 8 |
| HR6 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup> | 9 |
| HR7 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 10 |
| HR8 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC-NH<sub>2</sub> | 11 |
| HR9 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH<sub>2</sub> | 12 |
| HR10 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC-NH<sub>2</sub> | 13 |
| HR11 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH<sub>2</sub> | 14 |
| HR12 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 15 |
| HR13 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 16 |
| HR14 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS | 17 |
| HR15 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC | 18 |
| HR16 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC | 19 |
| HR17 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC | 20 |
| HR18 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC | 21 |
| HR19 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup> | 22 |
| HR20 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 23 |
| HR21 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH<sub>2</sub> | 24 |
| HR22 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC-NH<sub>2</sub> | 25 |
| HR23 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH<sub>2</sub> | 26 |
| HR24 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSS GAPPPhC-NH<sub>2</sub> | 27 |
| HR25 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH<sub>2</sub> | 28 |
| HR26 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 29 |
| HR27 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH2 | 30 |
| HR28 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS | 31 |
| HR29 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC | 32 |
| HR30 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC | 33 |
| HR31 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC | 34 |
| HR32 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhChC | 35 |
| HR33 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup> | 36 |
| HR34 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 37 |
| HR35 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH<sub>2</sub> | 38 |
| HR36 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH<sub>2</sub> | 39 |
| HR37 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PS SGAPPPCC-NH<sub>2</sub> | 40 |
| HR38 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC-NH<sub>2</sub> | 41 |
| HR39 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH<sub>2</sub> | 42 |
| HR40 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 43 |
| HR41 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 44 |
| HR42 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS | 45 |
| HR43 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC | 46 |
| HR44 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC | 47 |
| HR45 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC | 48 |
| HR46 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPhChC | 49 |
| HR47 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup> | 50 |
| HR48 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 51 |
| HR49 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH<sub>2</sub> | 52 |
| HR50 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC-NH<sub>2</sub> | 53 |
| HR51 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPCC-NH<sub>2</sub> | 54 |
| HR52 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPhC-NH<sub>2</sub> | 55 |
| HR53 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPhChC-NH<sub>2</sub> | 56 |
| HR54 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 57 |
| HR55 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 58 |
| HR56 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS | 59 |
| HR57 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC | 60 |
| HR58 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC | 61 |
| HR59 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC | 62 |
| HR60 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhChC | 63 |
| HR61 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup> | 64 |
| HR62 | HdAEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 65 |
| HR63 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH<sub>2</sub> | 66 |
| HR64 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH<sub>2</sub> | 67 |
| HR65 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPS SGAPPPCC-NH<sub>2</sub> | 68 |
| HR66 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC-NH<sub>2</sub> | 69 |
| HR67 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH<sub>2</sub> | 70 |
| HR68 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 71 |
| HR69 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 72 |
| HR70 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS | 73 |
| HR71 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC | 74 |
| HR72 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC | 75 |
| HR73 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPhC | 76 |
| HR74 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhChC | 77 |
| HR75 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup> | 78 |
| HR76 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 79 |
| HR77 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH<sub>2</sub> | 80 |
| HR78 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC-NH<sub>2</sub> | 81 |
| HR79 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC-NH<sub>2</sub> | 82 |
| HR80 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC-NH<sub>2</sub> | 83 |
| HR81 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPS SGAPPPhChC-NH<sub>2</sub> | 84 |
| HR82 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 85 |
| HR83 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 86 |
| HR84 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPS | 87 |
| HR85 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC | 88 |
| HR86 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPCC | 89 |
| HR87 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPhC | 90 |
| HR88 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPhChC | 91 |
| HR89 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup> | 92 |
| HR90 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 93 |
| HR91 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPS-NH<sub>2</sub> | 94 |
| HR92 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH<sub>2</sub> | 95 |
| HR93 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPCC-NH<sub>2</sub> | 96 |
| HR94 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPhChC-NH<sub>2</sub> | 97 |
| HR95 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 98 |
| HR96 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 99 |
| HR97 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPS | 100 |
| HR98 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC | 101 |
| HR99 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPCC | 102 |
| HR100 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC | 103 |
| HR101 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhChC | 104 |
| HR102 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSS GAPPPK<sup>*</sup> | 105 |
| HR103 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 106 |
| HR104 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPS-NH<sub>2</sub> | 107 |
| HR105 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC-NH<sub>2</sub> | 108 |
| HR106 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPCC-NH<sub>2</sub> | 109 |
| HR107 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC-NH<sub>2</sub> | 110 |
| HR108 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPhChC-NH<sub>2</sub> | 111 |
| HR109 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 112 |
| HR110 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 113 |
| HR111 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPS | 114 |
| HR112 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPC | 115 |
| HR113 | HdAEGTFTSDL SKQMEEEAVRLFIEWLVKGG PSSGAPPPCC | 116 |
| HR114 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC | 117 |
| HR115 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhChC | 118 |
| HR116 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK<sup>*</sup> | 119 |
| HR117 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 120 |
| HR118 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPS-NH<sub>2</sub> | 121 |
| HR119 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPC-NH<sub>2</sub> | 122 |
| HR120 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPCC-NH<sub>2</sub> | 123 |
| HR121 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC-NH<sub>2</sub> | 124 |
| HR122 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG | 125 |
| PSSGAPPPhChC-NH<sub>2</sub> | ||
| HR123 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 126 |
| HR124 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 127 |
| HR125 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPS | 128 |
| HR126 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPC | 129 |
| HR127 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPCC | 130 |
| HR128 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhC | 131 |
| HR129 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhChC | 132 |
| HR130 | HdAEGTFTSDL SKQNleEEEAVRLFIEWLKGG PSSGAPPPK<sup>*</sup> | 133 |
| HR131 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPK<sup>*</sup>K<sup>*</sup> | 134 |
| HR132 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPS-NH<sub>2</sub> | 135 |
| HR133 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPC-NH<sub>2</sub> | 136 |
| HR134 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPCC-NH<sub>2</sub> | 137 |
| HR135 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhC-NH<sub>2</sub> | 138 |
| HR136 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPhChC-NH<sub>2</sub> | 139 |
| HR137 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPK<sup>*</sup>-NH<sub>2</sub> | 140 |
| HR138 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPK<sup>*</sup>K<sup>*</sup>-NH<sub>2</sub> | 141 |
表7中，C，hC，K*为聚乙二醇修饰点。其C为半胱氨酸，hC为高级半胱氨酸，K*为侧链修饰了的赖氨酸，如巯基丙酸连接在其侧链氨基上。如果两个CC，hChC，或K*K*出现在序列中，意味着两个聚乙二醇修饰点。Nle为正亮氨酸，dAla为D型丙氨酸，-NH2为C端酰胺。
序列表
<110>无锡宏创医药科技有限公司
<120>长效促胰岛素分泌肽衍生物及其可药用盐和其制备方法及应用
<160>141
<170>PatentIn version 3.3
<210>1
<211>30
<212>PRT
<213>人工序列
<400>1
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 Arg
20 25 30
<210>2
<211>31
<212>PRT
<213>人工序列
<400>2
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 Arg Gly
20 25 30
<210>3
<211>39
<212>PRT
<213>人工序列
<400>3
His Gly Gly 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>4
<211>39
<212>PRT
<213>人工序列
<400>4
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>5
<211>39
<212>PRT
<213>人工序列
<400>5
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 Cys
35
<210>6
<211>40
<212>PRT
<213>人工序列
<400>6
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 Cys Cys
35 40
<210>7
<211>39
<212>PRT
<213>人工序列
<400>7
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 Cys
35
<210>8
<211>40
<212>PRT
<213>人工序列
<400>8
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 Cys Cys
35 40
<210>9
<211>39
<212>PRT
<213>人工序列
<400>9
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 Lys
35
<210>10
<211>40
<212>PRT
<213>人工序列
<400>10
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 Lys Lys
35 40
<210>11
<211>39
<212>PRT
<213>人工序列
<400>11
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 Cys
35
<210>12
<211>40
<212>PRT
<213>人工序列
<400>12
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 Cys Cys
35 40
<210>13
<211>39
<212>PRT
<213>人工序列
<400>13
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 Cys
35
<210>14
<211>40
<212>PRT
<213>人工序列
<400>14
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 Cys Cys
35 40
<210>15
<211>39
<212>PRT
<213>人工序列
<400>15
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 Lys
35
<210>16
<211>40
<212>PRT
<213>人工序列
<400>16
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 Lys Lys
35 40
<210>17
<211>39
<212>PRT
<213>人工序列
<400>17
His Ala 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>18
<211>39
<212>PRT
<213>人工序列
<400>18
His Ala 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 Cys
35
<210>19
<211>40
<212>PRT
<213>人工序列
<400>19
His Ala 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 Cys Cys
35 40
<210>20
<211>39
<212>PRT
<213>人工序列
<400>20
His Ala 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 Cys
35
<210>21
<211>40
<212>PRT
<213>人工序列
<400>21
His Ala 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 Cys Cys
35 40
<210>22
<211>39
<212>PRT
<213>人工序列
<400>22
His Ala 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 Lys
35
<210>23
<211>40
<212>PRT
<213>人工序列
<400>23
His Ala 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 Lys Lys
35 40
<210>24
<211>39
<212>PRT
<213>人工序列
<400>24
His Ala 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>39
<212>PRT
<213>人工序列
<400>25
His Ala 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 Cys
35
<210>26
<211>40
<212>PRT
<213>人工序列
<400>26
His Ala 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 Cys Cys
35 40
<210>27
<211>39
<212>PRT
<213>人工序列
<400>27
His Ala 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 Cys
35
<210>28
<211>40
<212>PRT
<213>人工序列
<400>28
His Ala 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 Cys Cys
35 40
<210>29
<211>39
<212>PRT
<213>人工序列
<400>29
His Ala 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 Lys
35
<210>30
<211>40
<212>PRT
<213>人工序列
<400>30
His Ala 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 Lys Lys
35 40
<210>31
<211>39
<212>PRT
<213>人工序列
<400>31
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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>32
<211>39
<212>PRT
<213>人工序列
<400>32
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>33
<211>40
<212>PRT
<213>人工序列
<400>33
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>34
<211>39
<212>PRT
<213>人工序列
<400>34
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>35
<211>40
<212>PRT
<213>人工序列
<400>35
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>36
<211>39
<212>PRT
<213>人工序列
<400>36
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys
35
<210>37
<211>40
<212>PRT
<213>人工序列
<400>37
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys Lys
35 40
<210>38
<211>39
<212>PRT
<213>人工序列
<400>38
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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>39
<211>39
<212>PRT
<213>人工序列
<400>39
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>40
<211>40
<212>PRT
<213>人工序列
<400>40
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>41
<211>39
<212>PRT
<213>人工序列
<400>41
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>42
<211>40
<212>PRT
<213>人工序列
<400>42
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>43
<211>39
<212>PRT
<213>人工序列
<400>43
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys
35
<210>44
<211>40
<212>PRT
<213>人工序列
<400>44
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys Lys
35 40
<210>45
<211>39
<212>PRT
<213>人工序列
<400>45
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>46
<211>39
<212>PRT
<213>人工序列
<400>46
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>47
<211>40
<212>PRT
<213>人工序列
<400>47
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>48
<211>39
<212>PRT
<213>人工序列
<400>48
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>49
<211>39
<212>PRT
<213>人工序列
<400>49
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Cys Cys
35
<210>50
<211>39
<212>PRT
<213>人工序列
<400>50
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>51
<211>40
<212>PRT
<213>人工序列
<400>51
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>52
<211>39
<212>PRT
<213>人工序列
<400>52
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>53
<211>39
<212>PRT
<213>人工序列
<400>53
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>54
<211>40
<212>PRT
<213>人工序列
<400>54
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>55
<211>39
<212>PRT
<213>人工序列
<400>55
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>56
<211>40
<212>PRT
<213>人工序列
<400>56
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>57
<211>39
<212>PRT
<213>人工序列
<400>57
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>58
<211>40
<212>PRT
<213>人工序列
<400>58
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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>59
<211>39
<212>PRT
<213>人工序列
<400>59
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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>60
<211>39
<212>PRT
<213>人工序列
<400>60
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>61
<211>40
<212>PRT
<213>人工序列
<400>61
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>62
<211>39
<212>PRT
<213>人工序列
<400>62
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>63
<211>40
<212>PRT
<213>人工序列
<400>63
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>64
<211>39
<212>PRT
<213>人工序列
<400>64
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys
35
<210>65
<211>40
<212>PRT
<213>人工序列
<400>65
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys Lys
35 40
<210>66
<211>39
<212>PRT
<213>人工序列
<400>66
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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>67
<211>39
<212>PRT
<213>人工序列
<400>67
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 ProPro Cys
35
<210>68
<211>40
<212>PRT
<213>人工序列
<400>68
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>69
<211>39
<212>PRT
<213>人工序列
<400>69
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys
35
<210>70
<211>40
<212>PRT
<213>人工序列
<400>70
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Cys Cys
35 40
<210>71
<211>39
<212>PRT
<213>人工序列
<400>71
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys
35
<210>72
<211>40
<212>PRT
<213>人工序列
<400>72
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle 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 Lys Lys
35 40
<210>73
<211>39
<212>PRT
<213>人工序列
<400>73
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>74
<211>39
<212>PRT
<213>人工序列
<400>74
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>75
<211>40
<212>PRT
<213>人工序列
<400>75
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>76
<211>39
<212>PRT
<213>人工序列
<400>76
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>77
<211>40
<212>PRT
<213>人工序列
<400>77
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>78
<211>39
<212>PRT
<213>人工序列
<400>78
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>79
<211>40
<212>PRT
<213>人工序列
<400>79
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>80
<211>39
<212>PRT
<213>人工序列
<400>80
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>81
<211>39
<212>PRT
<213>人工序列
<400>81
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>82
<211>40
<212>PRT
<213>人工序列
<400>82
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>83
<211>39
<212>PRT
<213>人工序列
<400>83
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>84
<211>40
<212>PRT
<213>人工序列
<400>84
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>85
<211>39
<212>PRT
<213>人工序列
<400>85
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>86
<211>40
<212>PRT
<213>人工序列
<400>86
His Ala 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 Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>87
<211>39
<212>PRT
<213>人工序列
<400>87
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>88
<211>39
<212>PRT
<213>人工序列
<400>88
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>89
<211>40
<212>PRT
<213>人工序列
<400>89
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>90
<211>39
<212>PRT
<213>人工序列
<400>90
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>91
<211>40
<212>PRT
<213>人工序列
<400>91
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>92
<211>39
<212>PRT
<213>人工序列
<400>92
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>93
<211>40
<212>PRT
<213>人工序列
<400>93
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>94
<211>39
<212>PRT
<213>人工序列
<400>94
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>95
<211>39
<212>PRT
<213>人工序列
<400>95
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>96
<211>40
<212>PRT
<213>人工序列
<400>96
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>97
<211>40
<212>PRT
<213>人工序列
<400>97
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>98
<211>39
<212>PRT
<213>人工序列
<400>98
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>99
<211>40
<212>PRT
<213>人工序列
<400>99
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>100
<211>39
<212>PRT
<213>人工序列
<400>100
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu PheIle Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>101
<211>39
<212>PRT
<213>人工序列
<400>101
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>102
<211>40
<212>PRT
<213>人工序列
<400>102
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>103
<211>39
<212>PRT
<213>人工序列
<400>103
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>104
<211>40
<212>PRT
<213>人工序列
<400>104
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>105
<211>39
<212>PRT
<213>人工序列
<400>105
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>106
<211>40
<212>PRT
<213>人工序列
<400>106
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>107
<211>39
<212>PRT
<213>人工序列
<400>107
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>108
<211>39
<212>PRT
<213>人工序列
<400>108
His Ala Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>109
<211>40
<212>PRT
<213>人工序列
<400>109
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>110
<211>39
<212>PRT
<213>人工序列
<400>110
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>111
<211>40
<212>PRT
<213>人工序列
<400>111
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>112
<211>39
<212>PRT
<213>人工序列
<400>112
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>113
<211>40
<212>PRT
<213>人工序列
<400>113
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>114
<211>39
<212>PRT
<213>人工序列
<400>114
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>115
<211>39
<212>PRT
<213>人工序列
<400>115
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>116
<211>40
<212>PRT
<213>人工序列
<400>116
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>117
<211>39
<212>PRT
<213>人工序列
<400>117
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>118
<211>40
<212>PRT
<213>人工序列
<400>118
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>119
<211>39
<212>PRT
<213>人工序列
<400>119
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>120
<211>40
<212>PRT
<213>人工序列
<400>120
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>121
<211>39
<212>PRT
<213>人工序列
<400>121
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>122
<211>39
<212>PRT
<213>人工序列
<400>122
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>123
<211>40
<212>PRT
<213>人工序列
<400>123
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu PheIle Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>124
<211>39
<212>PRT
<213>人工序列
<400>124
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>125
<211>40
<212>PRT
<213>人工序列
<400>125
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>126
<211>39
<212>PRT
<213>人工序列
<400>126
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>127
<211>40
<212>PRT
<213>人工序列
<400>127
His Ala 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 Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>128
<211>39
<212>PRT
<213>人工序列
<400>128
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>129
<211>39
<212>PRT
<213>人工序列
<400>129
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>130
<211>40
<212>PRT
<213>人工序列
<400>130
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>131
<211>39
<212>PRT
<213>人工序列
<400>131
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>132
<211>40
<212>PRT
<213>人工序列
<400>132
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>133
<211>39
<212>PRT
<213>人工序列
<400>133
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>134
<211>40
<212>PRT
<213>人工序列
<400>134
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>135
<211>39
<212>PRT
<213>人工序列
<400>135
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>136
<211>39
<212>PRT
<213>人工序列
<400>136
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>137
<211>40
<212>PRT
<213>人工序列
<400>137
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>138
<211>39
<212>PRT
<213>人工序列
<400>138
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>139
<211>40
<212>PRT
<213>人工序列
<400>139
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>140
<211>39
<212>PRT
<213>人工序列
<400>140
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>141
<211>40
<212>PRT
<213>人工序列
<400>141
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
Claims (9)
1.促胰岛素分泌肽，其中所述促胰岛素分泌肽的氨基酸序列选自序列号SEQ IDNO.5至141所示的氨基酸序列。
2.根据权利要求1所述的促胰岛素分泌肽，其中所述促胰岛素分泌肽为经聚乙二醇修饰后的序列号5至141所示的氨基酸序列。
3.根据权利要求2所述的促胰岛素分泌肽，其中所述促胰岛素分泌肽为经聚乙二醇修饰后的序列号95所示的氨基酸序列。
4.根据权利要求3所述的促胰岛素分泌肽，其中在经聚乙二醇修饰时将修饰连接聚乙二醇的接点和反应专一基团安排在促胰岛素分泌肽的羧基端。
5.根据权利要求2所述的促胰岛素分泌肽，其中所述的聚乙二醇的分子量范围在5,000-80,000道尔顿。
6.根据权利要求2所述的促胰岛素分泌肽，其中所述的聚乙二醇的分子量范围在20,000-60,000道尔顿。
7.根据上述任何一项权利要求所述的促胰岛素分泌肽，其中包括所述的促胰岛素分泌肽的可药用盐。
8.一种制备权利要求1或2所述的促胰岛素分泌肽的方法，其中包括固相和液相合成方法，反相高效液相、离子交换和凝胶过滤纯化方法以及冷冻干燥。
9.根据权利要求1或2所述的促胰岛素分泌肽在制备治疗和/或预防II型糖尿病药物中的用途。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510038102 | 2005-01-14 | ||
| CN200510038102.3 | 2005-01-14 | ||
| PCT/CN2006/000029 WO2006074600A1 (fr) | 2005-01-14 | 2006-01-10 | Exendines modifiees et utilisations correspondantes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1976948A CN1976948A (zh) | 2007-06-06 |
| CN100540565C true CN100540565C (zh) | 2009-09-16 |
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| US (1) | US8097586B2 (zh) |
| EP (2) | EP1845105A4 (zh) |
| JP (2) | JP4785206B2 (zh) |
| CN (1) | CN100540565C (zh) |
| CA (1) | CA2603630C (zh) |
| ES (1) | ES2541633T3 (zh) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109414469A (zh) * | 2016-03-16 | 2019-03-01 | 普罗林科斯有限责任公司 | 艾塞那肽类似物的缓释偶联物 |
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| AU2008258548B2 (en) * | 2007-06-08 | 2014-07-10 | Sanofi-Aventis Deutschland Gmbh | Long-acting transient polymer conjugates of exendin |
| WO2009035540A2 (en) * | 2007-09-07 | 2009-03-19 | Ipsen Pharma S.A.S. | Analogues of exendin-4 and exendin-3 |
| US20100317827A1 (en) * | 2008-02-06 | 2010-12-16 | Biocon Limited | Method of Purifying a Peptide |
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| CN101870728A (zh) | 2009-04-23 | 2010-10-27 | 派格生物医药(苏州)有限公司 | 新型Exendin变体及其缀合物 |
| PT2498802E (pt) * | 2009-11-13 | 2015-04-13 | Sanofi Aventis Deutschland | Composição farmacêutica que compreende um agonista de glp-1, uma insulina e metionina |
| CN102397558B (zh) * | 2010-09-09 | 2013-08-14 | 中国人民解放军军事医学科学院毒物药物研究所 | Exendin-4类似物的定位聚乙二醇化修饰物及其用途 |
| CN102786590A (zh) * | 2011-05-19 | 2012-11-21 | 江苏豪森药业股份有限公司 | 分枝型peg修饰的glp-1类似物及其可药用盐 |
| WO2013003449A2 (en) * | 2011-06-27 | 2013-01-03 | Phasebio Pharmaceuticals, Inc. | Methods of treatment with glp-1 receptor agonists |
| CN102617728B (zh) * | 2012-04-13 | 2013-07-03 | 上海交通大学医学院附属瑞金医院 | 一种Exendin-4的18F标记产物的制备方法 |
| UA116217C2 (uk) | 2012-10-09 | 2018-02-26 | Санофі | Пептидна сполука як подвійний агоніст рецепторів glp1-1 та глюкагону |
| JP2016503771A (ja) | 2012-12-21 | 2016-02-08 | サノフイ | エキセンジン−４誘導体 |
| CA2907454C (en) | 2013-03-21 | 2021-05-04 | Sanofi-Aventis Deutschland Gmbh | Synthesis of hydantoin containing peptide products |
| EP2976325B1 (en) | 2013-03-21 | 2017-03-01 | Sanofi-Aventis Deutschland GmbH | Synthesis of cyclic imide containing peptide products |
| EP3080149A1 (en) | 2013-12-13 | 2016-10-19 | Sanofi | Dual glp-1/glucagon receptor agonists |
| TW201609796A (zh) | 2013-12-13 | 2016-03-16 | 賽諾菲公司 | 非醯化之艾塞那肽－４（ｅｘｅｎｄｉｎ－４）胜肽類似物 |
| EP3080150B1 (en) | 2013-12-13 | 2018-08-01 | Sanofi | Exendin-4 peptide analogues as dual glp-1/gip receptor agonists |
| WO2015086729A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Dual glp-1/gip receptor agonists |
| RU2016137264A (ru) * | 2014-02-21 | 2018-03-26 | МЕДИММЬЮН, ЭлЭлСи | Слияния антитела к pcsk9~glp-1 и способы применения |
| TW201625670A (zh) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | 衍生自ｅｘｅｎｄｉｎ－４之雙重ｇｌｐ－１／升糖素受體促效劑 |
| TW201625669A (zh) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | 衍生自艾塞那肽－４（Ｅｘｅｎｄｉｎ－４）之肽類雙重ＧＬＰ－１／升糖素受體促效劑 |
| TW201625668A (zh) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | 作為胜肽性雙重ｇｌｐ－１／昇糖素受體激動劑之艾塞那肽－４衍生物 |
| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
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| CN107778361B (zh) * | 2016-08-30 | 2021-07-09 | 上海交通大学 | 多肽hl-39及其制备与应用 |
| CN107952064B (zh) * | 2016-10-14 | 2023-10-20 | 江苏豪森药业集团有限公司 | 含有聚乙二醇洛塞那肽的药物制剂及其制备方法 |
| CN109929025A (zh) * | 2017-12-18 | 2019-06-25 | 江苏豪森药业集团有限公司 | 一种基于反应动力学的聚乙二醇洛塞那肽的制备方法 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109414469A (zh) * | 2016-03-16 | 2019-03-01 | 普罗林科斯有限责任公司 | 艾塞那肽类似物的缓释偶联物 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4785206B2 (ja) | 2011-10-05 |
| CA2603630A1 (en) | 2006-07-20 |
| CN1976948A (zh) | 2007-06-06 |
| US20100009904A1 (en) | 2010-01-14 |
| WO2006074600A1 (fr) | 2006-07-20 |
| JP2008526899A (ja) | 2008-07-24 |
| EP2505207B1 (en) | 2015-04-22 |
| EP2505207A3 (en) | 2012-11-14 |
| CA2603630C (en) | 2015-06-09 |
| EP1845105A1 (en) | 2007-10-17 |
| EP1845105A4 (en) | 2009-02-18 |
| JP2011173886A (ja) | 2011-09-08 |
| JP5528380B2 (ja) | 2014-06-25 |
| ES2541633T3 (es) | 2015-07-22 |
| US8097586B2 (en) | 2012-01-17 |
| EP2505207A2 (en) | 2012-10-03 |
| HK1095045A1 (en) | 2007-04-20 |
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