CN109836503A - 一种治疗代谢疾病的多重活性蛋白 - Google Patents

一种治疗代谢疾病的多重活性蛋白 Download PDF

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CN109836503A
CN109836503A CN201711195354.6A CN201711195354A CN109836503A CN 109836503 A CN109836503 A CN 109836503A CN 201711195354 A CN201711195354 A CN 201711195354A CN 109836503 A CN109836503 A CN 109836503A
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黄岩山
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Abstract

本发明属于生物药物领域，具体涉及一种治疗代谢疾病的多重活性蛋白。本发明的多重活性蛋白的结构式为：A‑L‑F或A‑L1‑F‑L2‑B。与现有技术相比，本发明的多重活性蛋白具有如下有益效果：所述多重活性蛋白半衰期长、支持一周一次的给药频率；所述多重活性蛋白的GLP‑1R激动活性最高提高到了200倍以上，GCG与GLP‑1比例仅达到了1：1左右；所述多重活性蛋白稳定性好，免疫原性低。

Description

一种治疗代谢疾病的多重活性蛋白
技术领域
本发明属于生物药物领域，具体涉及一种治疗代谢疾病的多重活性蛋白。
背景技术
糖尿病按病理特征可分为一型糖尿病和二型糖尿病两种。一型糖尿病主要表现为胰岛素分泌不足，需要每天注射胰岛素；而二型糖尿病则是由于人体无法有效利用胰岛素造成。其中二型糖尿病患者占绝大多数。据估计大约80-90％二型糖尿病患者明显肥胖(Center for disease control and prevention(CDC)National Diabetes Fact Sheet,2014)。
用于治疗二型糖尿病的常规化药如磺酰脲类，噻唑烷二酮类等降糖效果明显，但主要缺点是会导致体重增加(Kahn SE,Haffner SM,Heise MA,Herman WH,Holman RR,Jones NP,et al.Glycemic durability of rosiglitazone,metformin,or glyburidemonotherapy.N Engl J Med 2006；355(23):2427-43.)。而用于二型糖尿病的蛋白类药物主要是GLP-1R(GLP-1受体)激动剂，如杜拉鲁肽(Dulaglutide，商品名：)、阿必鲁肽(Albiglutide，商品名)、利拉鲁肽(Liraglutide，商品名及分别用于治疗肥胖和糖尿病)、艾塞那肽(Exenatide，商品名)、利西拉肽(Lixisenatide，商品名)及可能即将上市的索马鲁肽(Semaglutide)等。GLP-1R激动剂具有显著的降血糖效应，而且与胰岛素不同的是，GLP-1R激动剂的降糖作用是严格地血糖依赖性的，不容易造成低血糖，还有降体重的效果。例如，杜拉鲁肽的减重大约为2.9公斤，而被批准用于减肥的Liraglutide(一天一次，剂量3mg)减重约8公斤。这些药物的减重主要是通过食欲控制，而且大部分都不超过平均体重的10％。减肥手术(Bariatricsurgery)虽然可以显著改善肥胖症和治疗糖尿病，然而其应用并不广泛，因为大部分的患者出于手术风险及长期后遗症的考虑，并不愿意接受这种手术(Obesity and Diabetes，New Surgical and Nonsurgical Approaches，Springer出版社，2015)。
据报道，经外科减肥手术的患者肠降血糖素(Incretin)分泌会激增(Obesity andDiabetes，New Surgical and Nonsurgical Approaches，Springer出版社，2015)。因此，目前新一代的糖尿病药物主要集中于双效或多效肠降血糖素(Incretin)受体激动剂的研究，如GLP-1R/GIPR和GLP-1R/GCGR双效激动剂，甚至GLP-1R/GIPR/GCGR三效激动剂。
其中，胰高血糖素(Glucagon)和GLP-1(Glucagon-like peptide-1)的受体在结构上相关，但是这两种激素在控制葡萄糖中表现出截然相反的作用。临床上，GLP-1及其类似物主要用于糖尿病人的血糖控制，而胰高血糖素(Glucagon)则用于急性低血糖症。近年来，越来越多的研究证明，胰高血糖素(Glucagon)尽管存在升血糖的风险，但是却能有效减少体重；更重要的是，GLP-1与Glucagon似乎有正向的附加或协同生理作用，例如Glucagon受体(GCGR)与GLP-1受体(GLP-1R)双激动剂比GLP-1R单激动剂能更有效地减重。虽然GCGR激动可能会导致血糖水平增加，但是这个风险可以通过GLP-1R激动而适当地抵消。
目前GLP-1R和GCGR的双效激动剂普遍都是基于胃泌酸调节素(Oxyntomodulin)或Glucagon，并且为了改善其短效及酶解的缺陷而进行改造(Oxyntomodulin类似物或Glucagon类似物)。这些类似物大都将第二位的丝氨酸(Ser)突变成非天然氨基酸Aib来抵抗DPP-IV的酶解。这是因为天然的Glucagon与胃泌酸调节素都与天然GLP-1类似，极易遭受血清中DPP-IV蛋白酶的水解而导致失活(Victor A.Gault等，A novel GLP-1/glucagonhybrid peptide with triple-acting agonist activity at GIP,GLP-1and glucagonreceptors and therapeutic potential in high-fat fed mice，J Biol Chem.,288(49):35581-91.2013；Bhat VK等，A DPP-IV-resistant triple-acting agonist of GIP,GLP-1and glucagon receptors with potent glucose-lowering and insulinotropicactions in high-fat-fed mice，Diabetologia，56(6):1417-24.2013；JohnA.Pospisilik等；Metabolism of glucagon by dipeptidyl peptidase IV(CD26)，Regulatory Peptides96:133–141,2001；Hinke SA等，Dipeptidyl peptidase IV(DPIV/CD26)degradation of glucagon.Characterization of glucagon degradationproducts and DPIV-resistant analogs，J Biol Chem 275:3827–3834,2000；AlessiaSantoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。突变产生的免疫原性风险极高。也有少数报道第二位保持天然Ser的交联脂肪酸的Glucagon类似物(Henderson SJ等，Robust anti-obesity andmetaboliceffects of a dual GLP-1/Glucagon receptor peptide agonist in rodentsand non-human primates,Diabetes Obes Metab，2016)。该类似物(MEDI0382)包括30个氨基酸，与天然Glucagon相比，突变了9个氨基酸。同时，虽然MEDI0382的第二位也保留了天然的Ser氨基酸，但是仅能支持一天一次的给药频率。
胃泌酸调节素类似物虽然表现出了初步的降糖及减脂效果，但是其作用机制仍不确切：胃泌酸调节素受体一直没有发现，目前仅仅是通过GCGR或GLP-1R敲除的小鼠或者细胞试验来验证胃泌酸调节素能与这2种受体结合而起作用。另外，胃泌酸调节素虽然能激动GLP-1R及GCGR，但是活性相当的低(大约分别是天然GLP-1和Glucagon的十分之一和百分之一左右)，而一般研究所用的胃泌酸调节素类似物在GLP-1R及GCGR的激动活性一般是设计成1:1左右，多数研究认为活性1:1左右时的降糖效果及减脂效果最好(Peptide-basedGLP-1/Glucagon co-agonists:a double-edged sword to combat diabesity，HiteshSoni，95:5–9，2016)。另外也有在基于Glucagon序列基础上进行改造的方法，同样的也大多是采用引入非天然氨基酸的形式。由于降糖药物的安全性要求极高，这类受体激动剂开发的难点之一为需要综合考虑到免疫原性的影响，一般情况下与人源序列具有越高的同源性，那么在人体中免疫原性风险就相对的越低。罗氏与益普生合作研发的GLP-1R激动剂降糖药Taspoglutide(引入了非天然氨基酸Aib)，抗体生成率达到了49％，最终停止了所有Ⅲ期临床研究(Julio Rosenstock等，The Fate of Taspoglutide,a Weekly GLP-1ReceptorAgonist,Versus wice-Daily，Exenatide for Type 2,Diabetes Care,36:498-504,2013)。
另一个问题则涉及GLP-1R/GCGR双效激动剂的半衰期问题。目前大多研究都采用脂肪酸或PEG交联的方式。脂肪酸交联可以最大程度地减少活性损失，然而半衰期大多只能维持12小时左右，因此只能采取每天给药的方式；PEG交联相对于脂肪酸虽然更有效地延长半衰期，但是导致的活性损伤问题非常严重。更重要的是，目前还未有报道能有效抵御DPP-IV降解的融合Glucagon类似物或者GLP-1R/GCGR双特异性激动剂。根据现有技术，将Glucagon类似物与FC或人血清白蛋白等长效单元融合表达并在第二位引入非天然氨基酸Aib是极难实现的。而如果模仿天然GLP-1，将天然Glucagon的第二位Ser突变为其他天然氨基酸，则会导致GCGR激动活性明显下降(Alessia Santoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。常用的方法是先化学合成带非天然氨基酸的小肽，再用FC、PEG或脂肪酸等进行交联。如HM12525A就是在GLP-1R/GCGR双特异性小肽的基础上交联了人源FC制备而成。(Jahoon Kang等，The ultra-long acting LAPSGLP/GCG dual agonist,HM12525A,demonstrated safety andprolonged pharmacokinetics in healthy volunteers:a phase 1first-in-humanstudy，51st European Association for the Study of Diabetes(EASD),Stockholm,Sweden；September 14-18,2015)。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种治疗代谢及相关疾病的多结构域活性蛋白及其制备与应用。本发明的多重活性蛋白具有显著的减重效果，临床可以用于治疗糖尿病、减肥、非酒精性脂肪肝、高血脂等相关疾病。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面提供一种多重活性蛋白，所述多重活蛋白的结构中包括如式I所
示结构，如如式I所示结构为：A-L-F式I
式I中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，L为连接所述A与F的连接链。
进一步地，所述GCGR/GLP-1R双效激动剂选自天然Glucagon(SEQ ID NO.44)的类似物或者其他具有GCGR/GLP-1R双效激动活性的多肽或蛋白。在本发明的实施例中，所述A的结构中包括如式II所示结构，如式II所示结构为：
HSQGTFTSDYSKYLDX16X17X18 AQDFVQWLMNX29 Xz(SEQ ID NO.141)式II，其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18除选自P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS(SEQ IDNO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。
所述A能抵御体内蛋白酶水解。
所述A的氨基酸序列可以如SEQ ID NO.46、SEQ ID NO.55、SEQ ID NO.59、SEQ IDNO.68、SEQ ID NO.74之任一所示。
目前，无论是基于Glucagon还是胃泌酸调节素的杂合肽，大多在第二位上进行了改造，将Ser突变为非天然的氨基酸Aib或者D型氨基酸(D-Ser)(详见综述文献Peptide-based GLP-1/Glucagon co-agonists:a double-edged sword to combat diabesity，Hitesh Soni，95:5–9，2016)以抵御血清DPP-IV酶的水解。与天然GLP-1(SEQ ID NO.1,HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG)类似，天然的Glucagon(SEQ ID NO.44)极易遭受血清中DPP-IV的水解而导致失活(Victor A.Gault等，A novel GLP-1/glucagon hybridpeptide with triple-acting agonist activity at GIP,GLP-1 and glucagonreceptors and therapeutic potential in high-fat fed mice，J Biol Chem.,288(49):35581-91.2013；Bhat VK等，A DPP-IV-resistant triple-acting agonist of GIP,GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropicactions in high-fat-fed mice，Diabetologia，56(6):1417-24.2013；JohnA.Pospisilik等；Metabolism of glucagon by dipeptidyl peptidase IV(CD26)，Regulatory Peptides 96:133–141,2001；Hinke SA等，Dipeptidyl peptidase IV(DPIV/CD26)degradation of glucagon.Characterization of glucagon degradationproducts and DPIV-resistant analogs，J Biol Chem 275:3827–3834,2000)。然而本发明人发现，本发明筛选获得的GCG类似物即使保留了天然第二位的Ser，在与FC融合后，稳定性也足以支持一周一次的给药频率，降低了免疫原性的潜在风险。而在16、17和18位上的改造，除了减弱内肽酶对GCG类似物的降解外，也能较好地保持GCGR激动活性。在本发明的其中一个实施例中，多重活性蛋白A-L-F结构中的A为GCG类似物，当与不含L-F部分的对应小肽相比，A-L-F结构的多重活性蛋白具有极高的抵御DPP-IV水解的能力。相反地，如报道所述，不含L-F部分的小肽第二位Ser极易受到DPP-IV攻击而失活。而且，A-L-F结构的多重活性蛋白在血清中的稳定性与第二位引入非天然氨基酸Aib或D-Ser的类似物相当。目前并无现有技术揭示可通过融合FC来增加GCG类似物的DPP-IV酶抵抗能力。众所周知，GLP-1的第二位为A，即便与FC融合后，GLP-1仍极易被DPP-IV降解水解前两个氨基酸HA。因此，目前上市及临床中一周一次给药频率的长效GLP-1类似物必须将第二位氨基酸突变成甘氨酸Gly(如杜拉鲁肽和阿必鲁肽)或Aib(如索马鲁肽)才能保持N端的稳定性。同样地，对于Glucagon类似物多肽，如上所述有众多的报道表明，第二位氨基酸为天然Ser的GCG类似物极易受DPP-IV攻击而失活，同样必须通过将第二位突变为非天然氨基酸才能避免降解，同时保留GCGR激动活性。根据现有技术，将GCG类似物与FC融合并进行第二位氨基酸的修饰，是无法实现的。而如果模仿天然GLP-1，将天然Glucagon的第二位氨基酸Ser突变为其他天然氨基酸，很可能会导致GCGR激动活性明显下降(Alessia Santoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。唯一的既能实现一周一次给药周期(长半衰期)又能保留高活性的方法，只有将第二位的Ser替换为非天然氨基酸，同时用PEG或者FC等大分子进行交联反应(Jahoon Kang等，Theultra-long acting LAPSGLP/GCG dual agonist,HM12525A,demonstrated safety andprolonged pharmacokinetics in healthy volunteers:a phase 1first-in-humanstudy，51st European Association for the Study of Diabetes(EASD),Stockholm,Sweden；September 14-18,2015)。而本发明人惊奇地发现，本发明提供的优选GCG类似物与FC融合后，获得了极高的DPP-IV抵抗能力，而且药效学实验表明稳定性能支持一周一次的给药频率。通过融合的方式，达到这种效果，是目前未有所见的。保留第二位的Ser，一方面降低了免疫原性风险，另一方面则是最大程度地保留了GCGR的激动活性，能有效地达到减重效果。
Ritzel U等报道了一种第二位从Ala突变为Ser的GLP-1类似物多肽(Ritzel U等，Asynthetic glucagon-like peptide-1analog with improved plasma stability，JEndocrinol.，159(1):93-102，1998)具有抵抗DPP-IV效果。基于此，Picha KM等(Picha KM等，Protein engineering strategies for sustained glucagon-like peptide-1receptor-dependent control of glucose homeostasis，Diabetes，57(7):1926-34，2008)亦报道了一种N端两位氨基酸为HS的GLP-1与抗体FC融合的活性蛋白(CNTO736)。然而，如上所述，众多报道表明Glucagon类似物的N端保留天然HS序列时，是非常不稳定的，即便是交联了脂肪酸，半衰期都很难持续到12小时以上；而且发明人也发现，将具有HS序列的天然Glucagon直接与FC融合(SEQ ID NO.75)，或将Joseph R.Chabenne等报道的Glucagon-cex序列(Joseph R.Chabenne等，Optimization of the Native Glucagon Sequence forMedicinal Purposes，J Diabetes Sci Technol.4(6):1322–1331，2010)与FC融合(SEQ IDNO.76)，仍不具备显著的抵抗DPP-IV酶的性质。这说明虽然GLP-1与Glucagon同属于肠降血糖素(Incretin)家族成员，然而不同的序列本身具有较大的构象差异，因而对于蛋白酶的抵抗能力也各有不同。
另外，有报道表明Glucagon的16-18位也是易遭受降解的位点，因此大多数的Glucagon类似物都在此位点进行改造或修饰(PEG或脂肪酸等)，而为了平衡GCGR和GLP-1R激动活性，还需要在另外的位点引入更多突变，从而获得对两个受体都有高活性的多肽。比如目前处于临床研究中的MEDI0382(Henderson SJ等，Robust anti-obesity andmetabolic effects of a dual GLP-1/Glucagon receptor peptide agonist inrodents and non-human primates,Diabetes Obes Metab，2016)，包括30个氨基酸。与天然Glucagon相比，引入了9个突变。同时，虽然MEDI0382的第二位也保留了天然的Ser氨基酸，但是仅能支持一天一次的给药频率。在本发明中的GCG类似物一方面保留了N端第二位的天然S氨基酸，仅进行了不超过3个氨基酸的突变，再与长效单元(如FC)融合后即足以支持一周一次的给药频率。在本发明的特定实施例中，为了降低GCGR的激动活性以平衡GCGR与GLP-1R的活性比例，还进一步把C末端的T删除。C末端的T删除对于Glucagon类似物的稳定性并无影响。因此，本发明的最大优势是采用了较少的位点突变达到了最理想的受体激动活性及稳定性的目的，并且突变时不引入非天然氨基酸，在降低了潜在的免疫原性的同时便于利用重组技术直接制备产物。
研究发现，将Joseph R.Chabenne等报道的Glucagon-cex序列与FC融合的制备获得的活性蛋白(C002G12S3A1F4，SEQ ID NO.76)并无显著的DPP-IV抵御能力(实施例4)。在ICR鼠的IPGTT试验及DIO鼠减肥试验(实施例8和9)中显示，C002G12S3A1F4也无明显的降血糖及持续的减重效果。同时，实施例4中还可见，虽然同样是16-18位突变的FC融合的GCG类似物，不同二聚体双效活性蛋白的稳定性差异却极大。蛋白的空间构象极其复杂，因此符合式II中的突变不但提高了肽链内部的稳定性，更有可能是改变了GCG类似物与FC链间的相互作用构象，进一步地提高了融合蛋白N端的稳定性。更重要的是，本发明提供的A-L-F结构蛋白与现有的GCG及突变体相比，具有显著提升的GLP-1R激动活性。虽然JosephR.Chabenne等和Richard D.DiMarchi等曾报道，在Glucagon的C末端增加一段Exendin-4的C末端小肽cex(SEQ ID NO.4,GPSSGAPPPS)后，使得其GLP-1R激动活性从0.7％增至1.6％，提高了约2倍左右(Optimization of the Native Glucagon Sequence for MedicinalPurposes，J Diabetes Sci Technol.4(6):1322–1331，2010及专利US9018164B2)，但是GCGR激动活性与GLP-1R激动活性的比例仅达到35：1左右。另外，Evers A等报道(Evers A等，Design of Novel Exendin-Based Dual Glucagon-like Peptide 1(GLP-1)/GlucagonReceptor Agonists，J Med Chem.；60(10):4293-4303.2017)在GCG类似物的C末端加上cex序列后，GLP-1R激动活性反而下降了原来的3倍左右，GCG的活性下降了14倍左右(文章中表2，肽7和8)。
就是说，仅简单在天然Glucagon的C末端增加Exendin-4的C末端肽cex序列(如GPSSGAPPPS)并不会显著提高甚至会进一步减弱其GLP-1R的激动活性。另一方面，Glucagon、GLP-1这类小肽与Fc，白蛋白这类载体融合蛋白进行融合表达的时候，由于空间位阻效应，活性往往是显著下降的(YAN-SHAN HUANG等,Preparation andcharacterization of a novel exendin-4 human serum albumin fusion proteinexpressed in Pichia pastoris，J.Pept.Sci.2008；14:588–595)，并且这种活性变化是不可预测的。本发明人意外地发现，将GCG类似物与Fc融合以后，对GLP-1R与GCGR的活性影响完全是不一样的。GCG类似物的C末端额外加入cex或类似序列(SEQ ID NO.3-10)再进一步融合至FC链时，与GCG类似物直接与FC链融合相比，含有cex序列的结构其GLP-1R激动活性保留率明显提高，最高提高了200倍以上，但是GCG活性保留率却是基本不变，甚至略有下降的。
另外，对于本领域技术人员，尤其是重组蛋白药的科研技术人员，一个公知的概念是：在蛋白序列上任意一个位点引入突变，都是无法准确预料其结果的，尤其是对于GLP-1、Exendin-4或Glucagon这类氨基酸数量只有30-40个的小肽而言，单个位点突变或者若干个位点同时突变的效果更是难以预测。比如Joseph Chabenne等报道(Joseph Chabenne等，AGlucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia，Molecular Metabolism,3：293-300，2014)对Glucagon(SEQID NO.44)进行了丙氨酸扫描(Ala scan)，Glucagon的各个位点独立地被丙氨酸取代后，相对残留活性保留跨度从0.2％-100％，并认为Glucagon的第1、2、3、4、6-12、14、15、22、23、25-27、29位突变都会使GCGR激动活性大幅减弱(文章中的表4)。然而我们也可以在其他的报道中看到在上述这些位点中取若干个位点同时进行突变，用其他的氨基酸取代时，活性的变化并不总是与丙氨酸扫描的结果一致。如Jonathan W Day等(Jonathan W Day等，Anew Glucagon and GLP-1 co-agonist eliminates obesity in rodents，NatureChemical Biology，5：749-757，2009)报道的杂合肽(文中的Chimera 2)，将23位的Val突变为Ile，会使GCGR的激动活性略微提升。但是丙氨酸扫描结果显示，在23位用丙氨酸取代，将导致GCGR激动活性基本完全丧失(降低至仅剩1.1％)。
最重要的是，本发明提供一种具有GCGR/GLP-1R双效激动活性的多重活性蛋白。目前GLP-1R和GCGR的双效激动剂普遍都是基于天然Oxyntomodulin(SEQ ID NO.2,HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN IA)或Glucagon(SEQ ID NO.44)序列而改造的。而一般研究所用的类似物在GLP-1R及GCGR的激动活性一般是设计成1:1左右，多数研究认为活性1:1的降糖效果及减脂效果最好(Peptide-based GLP-1/Glucagon co-agonists:adouble-edged sword to combat diabesity，Hitesh Soni，95:5–9，2016)。
Oxyntomodulin(SEQ ID NO.2)对于两个受体的激动活性本身就比较低，而Glucagon(SEQ ID NO.44)经序列突变后，虽然对GLP-1R的激动活性有所提高，但是在加上PEG或者脂肪酸后，活性仍会不可避免地下降，尤其是PEG修饰的情况。然而本发明的双效活性蛋白保持了大部分完整的GLP-1R和GCGR激动活性。活性的提高预示着剂量的减少，更低剂量即可达到更平稳的血糖控制，从而很好的提高了用药便利性，并减少了潜在的免疫原性的风险。众所周知，GLP-1类似物及其融合蛋白的副作用包括头晕、恶心等，是剂量依赖的，降低剂量可以减少胃肠道副作用。同时Glucagon可以提高代谢速率，增加脂肪消耗，起到更显著的减重效果，同时也能减少低血糖的风险，适合于与其他降糖药，如胰岛素等联合用药。
GLP-1R和GCGR激动和下游信号传递与其生理效应是极其复杂的，至今尚未完全清楚。目前一致认为大致是：葡萄糖通过GLUT2进入胰腺β细胞后发生糖酵解并产生丙酮酸。丙酮酸进入线粒体进行氧化代谢及产生ATP。细胞内的ATP增加将会关闭KATP(对ATP敏感的钾离子，ATP-sensitive potassium)通道，使得胞膜去极化，打开钙离子通道，增加胞外钙离子的流入，这一系列的变化导致了胰岛素的外分泌。GLP-1通过一系列的机制增加胰岛素的外分泌：GLP-1R与Gαs结合，激活腺苷酸环化酶，腺苷酸环化酶将ATP转化为cAMP，并调动下游的PKA和Epac信号因子。这会导致一系列的细胞反应，包括关闭KATP通道，促进胰岛素分泌颗粒与胞膜的融合(Chris de Graaf等，Glucagon-Like Peptide-1 and Its Class B GProtein–Coupled Receptors:A Long March to Therapeutic Successes，Pharmacological Reviews,68(4)954-1013，2016)。GCGR与GLP-1R类似，在与Glucagon结合后，胞内出现cAMP上调。GLP-1R及GCGR都属于GPCR家族，具有7个跨膜区域。在与各自的配体结合后，受体的C末端磷酸化，β-抑制蛋白(β-arrestin)富集并与受体结合，最终导致受体的内吞(Jorgensen,R等，Oxyntomodulin differentially affects Glucagon-likepeptide-1receptor-arrestin recruitment and signaling through Gα，J.Pharmacol.Exp.Ther.322,148–154，2007)。
有研究表示，GCGR跟GLP-1R的内吞效率不一样，而受体的内吞又影响着下游信号转导(Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talkand Trafficking，JBiol Chem，Sarah Noerklit Roed等290(2):1233–1243，2015)，最终可能影响组织细胞的生理功能。如Kuna,R.S.等表示，GLP-1R内吞受阻会减少胰腺细胞的胰岛素释放(Glucagon-like peptide-1 receptor-mediated endosomal cAMP generationpromotes glucose-stimulated insulin secretion in pancreatic-cells，Kuna,R.S.等，Am.J.Physiol.Endocrinol Metab，305,E161–E170，2013)。另外，两型受体的内吞效率也存在差异，如GCGR的内吞效率比GLP-1R低。为了避免GLP-1R及GCGR双效激动剂同时结合于GLP-1R及GCGR，现有的GLP-1R及GCGR的双效激动剂，绝大多数是基于一条肽链结构的。但是，如果在FC二价蛋白基础上构建GLP-1R和GCGR双效激动剂，有可能会同时分别结合GLP-1R及GCGR两个受体，造成两个受体异二聚化交联而影响了各自受体的内吞，胞内信号传递从而影响其正常的生理功能。目前已经不少提及GCGR与GLP-1R在同一组织细胞表面共表达的报道(Dominik Schelshorn等，Lateral Allosterism in the Glucagon ReceptorFamily:Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor HeteromerFormation，Molecular Pharmacology，81(3)309-318，2012)。在本发明研究过程中也发现，不同结构的GCGR/GLP-1R双效激动活性蛋白在大鼠BRIN-BD11细胞诱生胰岛素的能力是完全不一样的。多种因素会影响其异二聚化产生的效应。因此，如何获得正常生理效应的GLP-1R及GCGR双效激动剂是一件极其困难的工作。如GLP-1R/GCGR受体激动剂与FC之间的连接肽长度和结构不仅会影响蛋白的活性，也与GLP-1R及GCGR交联有关。在一个优选的实施方案中，所述连接链为一段合适长度的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，并优选获得合适的序列和长度减少潜在的受体异二聚化，使得二价的双效激动剂仍具有最好的GLP-1R和GCGR激动活性。
本发明优选的连接链包括含富含G、S和/或A的单元，示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA、等，n为1-10的整数，在一个优选的实施方案中，所述的连接链的氨基酸长度为5-26。示例性的连接链各自独立的选自表2。
进一步地，所述式I中的连接链L的氨基酸序列可如SEQ ID NO.21～43之任一所示。
进一步地，所述F为源自哺乳动物免疫球蛋白的FC部分。所述免疫球蛋白是含有二硫键的多肽链分子，一般具有两条轻链和两条重链。此处用到的免疫球蛋白的FC部分具有免疫学领域术语的常用含义。具体的，该术语是指通过从抗体中去除两个抗原结合区(Fab片段)而得到的抗体片段。FC部分可以包括铰链区并且延伸通过CH2和CH3结构域到达抗体C末端。FC部分可以进一步包括一个或多个糖基化位点。人体有5种具有不同效应特征和药代动力学特性的人免疫球蛋白：IgG、IgA、IgM、IgD和IgE。IgG是血清中含量最高的免疫球蛋白。IgG在所有的免疫球蛋白中也是血清半衰期最长的(约23天)。
进一步地，F可选自免疫球蛋白的完整FC部分、免疫球蛋白的FC部分的片段或免疫球蛋白的FC部分的突变体。
用于本发明的免疫球蛋白FC部分来源于哺乳动物IgG1、IgG2或IgG4的FC区或其突变体；优选的，可以是来源自人的IgG1、IgG2或IgG4的FC区或其突变体；更优选的，可以来源自人IgG1或IgG4的FC区或其突变体。在一个优选的实施方案中，FC结构域的第297位被甘氨酸或丙氨酸替换。上述内容是依照kabat的EU索引编号(kabat,E.A.等，sequences ofproteins of immunological interest，第五版，public health service,NationalInstitutes of Health,Bethesda,MD(1991))。
在一个优选的实施方案中，FC结构域来自人IgG1，并如SEQ ID NO.12所示。在一个优选的实施方案中，所述FC结构域来自人IgG4，如SEQ ID NO.16所示。所述FC链末端的K可去除便于提高表达产物的均一性。
所述F的氨基酸序列可以如SEQ ID NO.11～20之任一所示。
本发明提供的多重活性蛋白是一种FC融合蛋白，保留了FC的常规特性，如与FcRn结合而延长体内半衰期。另外，这些多重活性蛋白除了能有效地抵抗血清内的蛋白酶对蛋白内部的降解外，还能有效地防止N端的降解。对于Incretin类多肽如Glucagon或GLP-1而言，N端的完整性是决定其生物学活性的关键。天然Glucagon和GLP-1在体内的半衰期短，除了分子量小这个原因外，更重要的是由于受体内的DPP-IV酶水解。在本发明的一个实施例中，天然Glucagon与FC融合后，依然会被DPP-IV快速地降解而失活；而对应的Glucagon类似物却能明显地抵抗DPP-IV的攻击。利拉鲁肽与上述的MEDI0382虽然都保持了第二位的天然氨基酸，但是给药周期只能支持一天一次。而本发明的多重活性蛋白即使保留了第二位的天然氨基酸，稳定性的显著提高却足以支持一周一次的给药频率。
进一步地，本发明第二方面提供一种多重活性蛋白，具有三效激动活性，其结构式中包括如式III所示结构，如式III所示结构为：A-L1-F-L2-B，其中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，B为天然FGF21(SEQ ID NO.143)或FGF21类似物，L1为连接链，序列选自SEQ ID NO.21-43之任一；L2不存在或选自SEQ ID NO.21-43之任一。
进一步地，A的结构中包括如式II所示结构，如式II所示结构为：
HSQGTFTSDYSKYLDX16X17X18 AQDFVQWLMNX29 Xz(SEQ ID NO.141)式II，其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18选自除P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS(SEQ IDNO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。
所述A的氨基酸序列可以如SEQ ID NO.46、SEQ ID NO.54、SEQ ID NO.55、SEQ IDNO.68之任一所示。
式III所述的FGF21类似物可选自如US20140213512、US8188040、US9493530、WO2016114633、US 20150291677、US 9422353、US 8541369、US7622445、US7576190、US20070142278、US 9006400或US 20130252884等专利或专利申请中所描述的FGF21类似物或突变体。进一步地，所述FGF21类似物的氨基酸序列如SEQ ID NO.144、SEQ ID NO.145或SEQ ID NO.146所示。在本发明的另一个动物体内药效实施例中，三活性蛋白组与相同剂量的双效活性蛋白+FGF21类似物联合给药组相比具有更显著的减重效果。但是对于食欲的影响却并无太大影响。证明三活性蛋白的副作用可能更低，安全性提高(实施例12)。
式III所述的B还可以是天然瘦素(SEQ ID NO.155)及其类似物，可选自US7307142、US 7423113或US 20140018290等专利或专利申请中所描述的变体，衍生物或类似物；
式III所述的B还可以是Amylin及其类似物。
本发明的第二方面，提供一种分离的多核苷酸，所述分离的多核苷酸编码前述多重活性蛋白。
本发明的第三方面，提供一种重组表达载体，包含前述分离的多核苷酸。
本发明的第四方面，提供一种宿主细胞，所述细胞含有前述重组表达载体或基因组中整合有外源的前述分离的多核苷酸。
本发明的第五方面，提供前述多重活性蛋白的制备方法，包括在合适的条件下培养前述宿主细胞，使之表达所述多重活性蛋白，而后分离及纯化获得所述多重活性蛋白。
本发明的第六方面，提供前述多重活性蛋白在制备治疗糖尿病代谢相关疾病的药物中的用途。
本发明提供的多重活性蛋白可以用于治疗用于治疗代谢综合征。代谢综合征的特征通常在于簇集至少三种以上的以下风险因素：(1)腹部肥胖(腹部内或周围脂肪组织过多)，(2)致动脉粥样硬化血脂异常，血脂失调，包括高甘油三酯、低HDL胆固醇及高LDL胆固醇，其增强动脉壁中斑块的积累，(3)血压升高，(4)胰岛素抗性或葡萄糖耐受不良，(5)血栓样状态，例如血液中高纤维蛋白或纤溶酶原活化因子抑制剂-1，以及(6)促发炎状态，例如血液中C反应性蛋白升高。其他风险因素可包括老化、激素失衡及遗传因素。
此外，本发明的多重活性蛋白还可用于治疗肥胖症。在一些方面中，本发明的多重活性蛋白通过降低食欲、减少食物摄取、降低患者体内脂肪水平、提高能量消耗等机制来治疗肥胖症。
本发明的第七方面，提供一种治疗代谢相关疾病的方法，包括向对象施用前述多重活性蛋白。
本发明进一步提供一种促进体重减少或者防止体重增加的方法，包括在对象中施用所述的多重活性蛋白。
本发明的第八方面，提供一种组合物，含有前述多重活性蛋白或前述宿主细胞的培养物，以及药学上可接受的载体。
本发明的第九方面，提供前述多重活性蛋白在制备融合蛋白中的用途。
本发明的第十方面，提供一种多结构域蛋白，所述多结构域蛋白的结构中含有前述多重活性蛋白。
综上所述，与现有技术相比，本发明具有如下有益效果：
(1)本发明的多重活性蛋白半衰期长、支持一周一次的给药频率；
(2)本发明的多重活性蛋白的GLP-1R激动活性最高提高到了200倍以上；
(3)本发明的多重活性蛋白体内外稳定性好，且免疫原性低。
(4)由于不需要引入非天然氨基酸，无需涉及化学合成及交联步骤，因此可通过重组方法制备，极大地简化了制备工序。
附图说明
图1为纯化获得的部分重组蛋白电泳图(10％SDS-PAGE)，泳道1-6分别为C240G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4及C462G12S3A1F4非还原处理的样品；7-12分别为C240G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4及C462G12S3A1F4还原处理的样品；M为蛋白标准品：97.2、66.4、44.3、29、20.1、14.3KD。
图2A为GCGR测活结果图。
图2B为GLP-1R测活结果图。
图3A：血清稳定性随时间变化的结果图。
图3B：血清稳定性随时间变化的结果图。
图3C：血清稳定性随时间变化的结果图。
图3D：血清稳定性随时间变化的结果图。
图4：实施例6和7中由GCG类似物与不同长度连接链及F(SEQ ID NO.12)融合而获得的二聚体重组蛋白对胰岛细胞的刺激作用图。
图5：实施例8中的二聚体重组蛋白在正常ICR小鼠体内的降糖效果图。
图6：实施例9中的二聚体重组蛋白对DIO小鼠体重的影响作用。
图7：实施例11纯化获得的融合蛋白的电泳图(10％SDS-PAGE)，泳道1-6分别为C382F4FGF1、C382F4FGF2、C382F4FGF3、C495F4FGF1、C495F4FGF2及C495F4FGF3非还原处理的样品；7-12分别为C382F4FGF1、C382F4FGF2、C382F4FGF3、C495F4FGF1、C495F4FGF2及C495F4FGF3还原处理的样品；M为自制蛋白标准品：140、97.2、66.4、44.3、29、20.1、14.3KD。
图8：实施例12中的三活性蛋白的对DIO小鼠体重的影响作用。
图9：实施例12中的三活性蛋白对DIO小鼠摄食量的影响作用。以PBS组DIO鼠的摄食量为100％，纵坐标为其他组的摄食量与之相比的百分比。
图10：实施例14中的三活性蛋白对DIO小鼠体重的影响作用。
图11：实施例14中的三活性蛋白对DIO小鼠摄食量的影响作用。以PBS组DIO鼠的摄食量为100％，纵坐标为其他组的摄食量与之相比的百分比。
具体实施方式
术语解释：
术语“糖尿病”包括一型糖尿病、二型糖尿病、妊娠糖尿病以及引起高血糖症的其他症状。该术语用于代谢紊乱，其中胰腺产生不了足够的胰岛素，或身体的细胞未能适当响应胰岛素，因此组织细胞吸收葡萄糖效率下降导致葡萄糖在血液中积累。
一型糖尿病也称为胰岛素依赖性糖尿病和幼年发病型糖尿病，通过β细胞破坏引起，通常导致绝对胰岛素缺乏。
二型糖尿病也称为非胰岛素依赖性糖尿病和成年发病型糖尿病，普遍与胰岛素抗性相关。
术语“肥胖”意指脂肪组织的过量，当能量摄取超过能量消耗时，过量卡里路贮存于脂肪中，则导致肥胖。在本文中，肥胖最佳地被视为任何程度的危害健康的过量脂肪组织的形成。在本文中体重指数(BMI＝体重(千克)除以身高(米)的平方)超过25的个体视为肥胖。
肠降血糖素(Incretin)：肠降血糖素是通过增强葡萄糖刺激的胰岛素分泌(亦称为葡萄糖依赖的胰岛素分泌，GSIS)来调控血糖的胃肠激素(Drucker.D J,Nauck,MA,Lancet368:1696-705,2006)。肠降血糖素还可通过延缓胃排空的方式减慢营养吸收的速度并直接地减少食物吸收。同时，肠降血糖素还抑制肠道α细胞分泌胰高血糖素(Glucagon)。迄今为止有两种已知的肠降血糖素：胰高血糖素样肽-1(GLP-1)和葡萄糖依赖性促胰岛素多肽(GIP)。
前胰高血糖素原(preproGlucagon)：是158个氨基酸组成的前体多肽，其在组织中被差异性加工而形成多种结构上相关的胰高血糖素原衍生肽，包括胰高血糖素(Glucagon)、胰高血糖素样肽-1(GLP-1)、胰高血糖素样肽-2(GLP-2)和胃泌酸调节素(Oxyntomodulin，OXM)。
GIP：是由133个氨基酸的前体(pre-pro-GIP)通过蛋白水解加工得到的42个氨基酸的肽，这些分子参与多种生物功能，包括葡萄糖体内平衡、胰岛素分泌、胃排空和肠生长以及食物摄取调节。
胰高血糖素样肽(GLP-1)：是从肠L-细胞分泌的30或31个氨基酸的多肽肠促胰岛素激素，有GLP-1(7-36)和GLP-1(7-37)两种活性形式。GLP-1在进餐后释放到循环中，并通过激活GLP-1受体发挥生物活性。GLP-1具有许多生物学作用，包括葡萄糖依赖性的促胰岛素分泌，抑制胰高血糖素生成，延缓胃排空和抑制食欲(Tharakan G,Tan T,BloomS.Emerging therapies in the treatment of‘diabesity’:beyond GLP-1.TrendsPharmacol Sci2011；32(1):8-15.)等。天然GLP-1由于能够被二肽基肽酶-4(DPP-4)，中性肽链内切酶(NEP)，血浆激肽释放酶或纤溶酶等快速降解因而限制了其治疗潜力。由于天然GLP-1在体内仅有大约2分钟的超短半衰期，因此，出现了通过利用化学修饰和/或制剂形式来改善功效以治疗糖尿病和肥胖症的方法(Lorenz M,Evers A,Wagner M.Recentprogress and future options in the development of GLP-1receptor agonists forthe treatment of diabesity.Bioorg Med Chem Lett 2013；23(14):4011-8.TomlinsonB,Hu M,Zhang Y,Chan P,Liu ZM.An overview of new GLP-1receptor agonists fortype 2diabetes.Expert Opin Investig Drugs 2016；25(2):145-58)。
胃泌酸调节素(Oxyntomodulin)是37个氨基酸的小肽，序列如SEQ ID NO：2所示；其包含胰高血糖素Glucagon(SEQ ID NO.44)完整的29个氨基酸序列。胃泌酸调节素是GLP-1R和GCGR的双重激动剂，在进餐后通过肠L-细胞与GLP-1一起分泌。与胰高血糖素Glucagon类似，胃泌酸调节素在人和啮齿动物中产生显着的体重减轻。胃泌酸调节素的减肥活性已在肥胖小鼠中与等摩尔剂量的选择性GLP-1激动剂进行比较。已经发现，与选择性的GLP-1R激动剂相比，胃泌酸调节素具有抗高血糖作用，能够显著的减轻体重和具有降脂活性(TheGlucagon receptor is involved in mediating the body weight-lowering effectsof oxyntomodulin，Kosinski JR等，Obesity(Silver Spring)，20):1566-71，2012)。在超重和肥胖患者中，皮下施用天然胃泌酸调节素在四周内减少体重1.7公斤。胃泌酸调节素也被证明可以减少人类的食物摄取和增加能量消耗(Subcutaneous oxyntomodulin reducesbody weight in overweight and obese subjects:a double-blind,randomized,controlled trial，Wynne K等，Diabetes，54:2390-5，2005；Oxyntomodulin increasesenergy expenditure in addition to decreasing energy intake in overweight andobese humans:a andomized controlled trial；Wynne K等，Int J Obes(Lond)，30:1729-36，2006)。但是同样由于分子量偏小及DPP-IV的降解，胃泌酸调节素具有较短的半衰期。目前GLP-1受体(GLP-1R)和胰高血糖素受体(GCGR)的双效激动剂普遍都是基于胃泌酸调节素的，并且为了改善胃泌酸调节素的短效及酶解的缺陷而做了突变(胃泌酸调节素类似物)，且大都采用第二位丝氨酸Ser突变为α-氨基异丁酸(Aib)的方法，通过引入非天然氨基酸来抵抗DPP-IV的酶解。胃泌酸调节素类似物虽然表现出了初步的降糖及减脂效果，但是其作用机制仍不确切，胃泌酸调节素受体一直没有发现，目前仅仅是通过GCGR或GLP-1R敲除的小鼠或者细胞试验验证胃泌酸调节素能与这2种受体结合而起作用。
胰高血糖素(Glucagon)是29个氨基酸的肽，其对应于前胰高血糖素原的53-81位氨基酸，序列如SEQ ID NO.44所示(C.G.Fanelli等，Nutrition,Metabolism&Cardiovascular Diseases(2006)16,S28-S34)。胰高血糖素受体活化已显示在啮齿类动物和人两者中增加能量消耗且减少食物摄入(Habegger K.M.等人，the metabolic actionsof Glucagon revisited,Nat.Rev.Endocrinol.2010,6,689-697)并且这些效应在啮齿类动物中是稳定和持续的。胰高血糖素具有许多生理效应，例如通过刺激糖原分解和糖异生，增加低血糖状况下的血糖水平，调节肝酮生成，调节胆汁酸代谢和通过迷走神经的饱腹效应。在治疗上，胰高血糖素已经用于急性低血糖症，胰高血糖素受体激活减少食物摄取并促进动物和人的脂肪分解和体重减轻。
术语“受体激动剂”可以定义为与受体结合且引发天然配体的通常应答的多肽、蛋白或其他小分子。
“GLP-1受体(GLP-1R)激动剂”可以定义为与GLP-1R结合且能够引发与天然GLP-1相同或类似的特征性反应的多肽、蛋白或其他小分子。GLP-1R激动剂通过完全或部分激活GLP-1R，继而引起一系列细胞内的下游信号通路反应，产生相应的细胞活性：如β细胞分泌胰岛素；典型的GLP-1R激动剂包括天然GLP-1及其突变体、类似物，如埃塞那肽、利拉鲁肽(Liraglutide)等。
GLP-1R类似物：在本文中，“GLP-1类似物”或“GLP-1突变体”皆意为GLP-1R激动剂，可互相通用。
胰高血糖素受体(GCGR)激动剂:即Glucagon受体激动剂，可以定义为与GCGR结合且能够引发与天然胰高血糖素(Glucagon)相同或类似特征性反应的多肽、蛋白或其他小分子。GCGR激动剂通过完全或部分激活GCGR，继而引起一系列细胞内的下游信号通路反应，产生相应的细胞活性：如肝细胞糖原分解、糖质新生、脂肪酸氧化及生酮作用等。
Glucagon类似物：在本文中，“Glucagon类似物”、“GCG类似物”、“Glucagon突变体”和“GCG突变体”皆意为Glucagon受体激动剂，可互相通用。
GCGR/GLP-1R双效激动剂：本发明的GCGR/GLP-1R双效激动剂包括能同时激动GLP-1R和GCGR的蛋白或多肽。如Alessandro Pocai等报道的基于Oxyntomodulin的双效激动剂(Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity inMice，Diabetes；58(10):2258-2266，2009)，或者Richard D.DiMarchi等报道的基于Glucagon的双效激动剂(US9018164B2)。在本文中，“双效激动剂“或“双特异性活性蛋白”或“双效活性蛋白”皆同义。
FGF21：成纤维细胞生长因子(fibroblast growth factor，FGF)又叫肝素结合生长因子(heparin binding growth factor)，是主要由垂体和下丘脑分泌的一类多肽物质。FGF具有多种功效，如促进成纤维细胞有丝分裂、中胚层细胞的生长，刺激血管形成等。而FGF21是FGF家族中的重要一员，目前该激素被当作药物开发成减肥药以及治疗糖尿病药物，并且该药物已经进入了临床试验阶段。FGF21通过FGF21受体及其共受体β-klotho发挥生理作用。
瘦素(leptin)：主要由白色脂肪组织产生。其前体由167个氨基酸残基组成，包含N端21个氨基酸的信号肽，前体的信号肽在血液中被切掉而成为146氨基酸的瘦素成熟肽(leptin)。瘦素具有广泛的生物学效应，如作用于下丘脑的代谢调节中枢、发挥抑制食欲、减少能量摄取、增加能量消耗及抑制脂肪合成等的作用。
二聚体：本发明所指的二聚体是通过免疫球蛋白的恒定区(FC)天然的非共价及共价作用而形成。如果没有其他特别指出，FC形成的二聚体皆为同二聚体，如本发明提供的二聚体所述。
二聚体双效活性蛋白：在本文中，“双效活性蛋白”、“二聚体双效活性”、“双效激动活性蛋白”皆同义，可互换使用。意指同时具有GCGR和GLP-1R激动活性的融合蛋白，该种融合蛋白由于具有FC部分，因此具有两条肽链，通过两条肽链之间的非共价和共价作用而形成了二聚体的结构。
二聚体三效活性蛋白：在本文中，“三效活性蛋白”、“三效激动活性蛋白”、“三活性激动活性蛋白”、“二聚体三效活性蛋白”皆同义，可互换使用。意指同时具有GCGR、GLP-1R激动活性及FGF21活性(或瘦素活性)的融合蛋白，该种融合蛋白由于具有FC部分，因此具有两条肽链，通过两条肽链之间的非共价和共价作用而形成了二聚体的结构。
IC50(half maximal inhibitory concentration)是指被测量的拮抗剂的半抑制浓度。它能指示某一药物或者物质(抑制剂)在抑制其相应的50％生物学反应(或者是包含在此反应中的某些物质，比如酶，细胞受体或是微生物)时所需的浓度。IC50值越低，表明该药物或物质的抑制能力越强，例如，更直观地表现为与受体的结合亲和力越佳。是物质在抑制特定生物或生化功能中的有效性的量度标准。
EC50(concentration for 50％of maximal effect)是指某一药物或者物质在刺激其相应的生物学反应的50％时所需的浓度。EC50值越低，表明该药物或物质的刺激或激动能力越强，例如，更直观地可表现为引起的细胞内信号越强，从而诱导某激素产生的能力越佳。
低密度脂蛋白(LDL)：属于血浆脂蛋白的一种，是血液中胆固醇的主要载体,其倾向于将胆固醇沉积在动脉壁上。白细胞试图消化低密度脂蛋白，但在这个过程却将它们变成毒素。越来越多的白细胞被吸引到发生变化的区域，致使动脉壁可能发炎。随着时间的推移，随着该过程继续，这些斑块沉积物可以积聚在动脉壁上，使得通道变得非常窄和缺乏韧性。如果太多的斑块累积，则动脉可以完全阻塞。当LDL与胆固醇形成的复合物(LDL-C)在动脉壁上产生太多斑块时，血液将不能自由流过动脉。斑块随时可能会在动脉中突然塌陷，导致血管堵塞，最终引发心脏病。
高密度蛋白(HDL)：有助于清除动脉上的LDL，起到清道夫的作用，将LDL从动脉上清走并回到肝脏。
甘油三酯(TG)：是另一种类型的脂肪，用于储存饮食过多的能量。血液中高水平的甘油三酯与动脉粥样硬化有关。高甘油三酯可以由超重和肥胖，身体缺乏运动，吸烟，过量的酒精消耗和高碳水化合物(超过总卡路里的60％)摄入引起。有时基础疾病或遗传疾病是高甘油三酯的原因。具有高甘油三酯的人通常具有高的总胆固醇水平，包括高LDL胆固醇和低HDL胆固醇，许多患有心脏病或糖尿病的人也具有高甘油三酯水平。
细胞生物学活性
本发明的GLP-1R和GCGR激动活性体外细胞活性测定都采用荧光素酶报告基因检测法。此法基于GLP-1R和GCGR激动后能激活下游cAMP途径的原理。FGF21及其类似物的活性测定则是通过将FGF21R与β-klotho共转染同一个CHO细胞，检测信号引起的荧光变化而获得。
Joseph R.Chabenne等和Richard D.DiMarchi等曾报道，在Glucagon的C末端增加一段Exendin-4的C末端小肽cex(GPSSGAPPPS)能使GLP-1R的激动活性提高2倍左右(Optimization of the Native Glucagon Sequence for Medicinal Purposes，JDiabetes Sci Technol.4(6):1322–1331，2010及专利US9018164B2)。另外，Evers A等报道(Evers A等，Design of Novel Exendin-Based Dual Glucagon-like Peptide 1(GLP-1)/Glucagon Receptor Agonists，J Med Chem.；60(10):4293-4303.2017)在GCG类似物的C末端加上cex序列后，GLP-1R激动活性反而下降了3倍左右，GCG的活性下降了14倍左右(文章中表2，肽7和8)。
在本发明的一个实施例中，含Exendin-4的C末端延伸肽的GCG类似物进一步融合至FC链时，GLP-1R激动活性提高到了惊人的200倍以上(EC50约1.1nM)。而根据US9018164B2及Joseph R.Chabenne等在相应专利及文献中披露的数据计算获得的GCG类似物，其GLP-1R激动活性比例在增加Exendin-4的C末端延伸肽cex序列前后变化仅在2倍左右(如文章中天然Glucagon的GLP-1R激动活性百分比为0.7％，增加了GPSSGAPPPS序列后提高至1.6％)。就是说，在Glucagon多肽的C末端增加GPSSGAPPPS序列并不会显著提高其GLP-1R的激动活性。
多重活性蛋白的稳定性
天然Glucagon有多处敏感的降解位点，包括第二位的DPP-IV降解位点，以及16-18位的SRR位点。尽管有报道认为FC能提高活性蛋白的化学稳定性及血清稳定性，然而对于N端必须暴露的GLP-1或Glucagon类似物，FC的作用似乎是无法一概而论的。天然的GLP-1或Glucagon与FC融合后，仍能观察到其在37度血清条件下的明显降解。本发明在天然Glucagon的基础上引入抵抗16-18位蛋白酶水解的突变，以增加其稳定性。这些突变体与FC融合后，稳定性进一步获得提高。在本发明的实施例中，在72小时仍然能检测到50％左右的GCGR激动活性。而此时含cex序列的天然Glucagon与FC融合形成的二聚体(SEQID NO.76)已几乎检测不到任何的活性。
目前几乎所有基于Oxyntomodulin和Glucagon设计开发的GCGR/GLP-1R双效激动剂都在第二位上引入抵抗DPP-IV的突变，如L型突变为D型氨基酸(L-Ser突变为D-Ser)，或引入非天然氨基酸Aib等(Matthias H.等，Unimolecular Polypharmacy forTreatment of Diabetes and Obesity，24：51–62,2016)。然而，在本发明的实施例中，第二位保存天然L-Ser的二聚体活性蛋白呈现出非常高的血清稳定性，在24小时仍并没有显著的DPP-IV降解的迹象，而没有融合FC的对应多肽则快速地被DPP-IV水解(表5)。本发明人制备了天然Glucagon与FC融合的活性蛋白C001G12S3A1F4(SEQ ID NO.75)以及JosephR.Chabenne等报道的Glucagon-cex与FC融合的活性蛋白C002G12S3A1F4(SEQ ID NO.76)作为对照验证是否FC融合提高了稳定性。然而，C001G12S3 A1F4(SEQ ID NO.75)和C002G12S3A1F4(SEQ ID NO.76)均无呈现出明显的抵抗DPP-IV的迹象。虽然有报道指出结合在血清白蛋白(如HSA)上可能会有助于提高蛋白的稳定性(如利拉鲁肽)，然而如果第二位不进行突变，根本无法使半衰期持续12小时以上，即不可能支持一周一次的给药频率。而本发明提供的GCG类似物药代及药效试验显示足以支持一周一次的给药频率，而非普遍报道的一天一次(如结合白蛋白的利拉鲁肽)。天然氨基酸的保留进一步减低免疫原性的风险，避免了化学交联还使制备过程更简易方便。
葡萄糖刺激的胰岛素分泌实验(GSIS)
众所周知，GLP-1或其类似物通过激动GLP-1R，作用于胰岛β细胞，促进胰岛素基因的转录、胰岛素的合成和分泌。临床上，GLP-1类似物常与胰岛素联合使用。虽然GLP-1R激动的详细机制仍未完全揭开，然而受体激动后的cAMP信号产生及受体迅速内吞却是比较明确的事实。有多个研究表明，cAMP信号与GLP-1R受体内吞属于不同的信号通路，但是都会影响着胰岛素的分泌(Agonist-induced internalization of the Glucagon-like peptide-1receptor is mediated by the Gaq pathway，Aiysha Thompson,等，BiochemicalPharmacology，93:72–84，2015；Molecular Characterisation of Small MoleculeAgonists Effect on the Human Glucagon Like Peptide-1Receptor Internalisation，Aiysha Thompson等，PLOS ONE)。因此，cAMP信号与受体内吞的减弱，理论上都会破坏胰岛素的分泌从而影响GLP-1类似物的生理效能。GLP-1R与GCGR交联形成异二聚体后，内吞明显减弱，而影响胰岛素的分泌。
在本发明的一个实施例中，部分GCGR/GLP-1R激动剂诱导的胰岛素分泌明显减少。已有研究显示多种人体细胞表面存在同时表达多种肠道激素受体的现象(DominikSchelshorn等，Lateral Allosterism in the Glucagon Receptor Family:Glucagon-Like Peptide 1Induces G-Protein-Coupled Receptor Heteromer Formation，Molecular Pharmacology，81(3)309-318，2012)。因此，若GCGR/GLP-1R双效激动剂同时结合于GLP-1R和GCGR受体，不仅可能降低了其原有的生理效应，一些潜在未知作用更是很难预测的。除了胰岛素分泌的减少，还有可能会产生难以预料的毒副作用。糖尿病用药的安全性要求极高，因此，本发明人认为不会使受体产生交联的二聚体应该是更为优越的。
腹腔糖耐量测试(IPGTT)
在其中一个实施例中，进行了IPGTT实验。施用了双特异性活性蛋白的小鼠在注射葡萄糖后，呈现出极平稳的血糖波动。
DIO小鼠的减重实验
GCGR激动剂具有潜在的减重效果已有多篇报道。然而天然Glucagon由于易被降解且分子量极小，因此成药的潜力极小。目前Glucagon类似物主要用于急性的低血糖症状。长效GCG类似物用于糖尿病患者减重的临床报道也不断涌现。众所周知，肥胖是导致糖尿病患者胰岛素抵抗的原因之一，而体重减轻量是评价一个降糖药物的重要指标。此外，本发明的多重活性蛋白在DIO小鼠施用后，诱导了体重的显著降低。大鼠药代动力学研究本发明的双特异性活性蛋白具有改善的药物代谢动力学特性，即，它们在体内具有延长的半衰期。在一个实施例中，在大鼠皮下施用各种双特异性活性蛋白，并在不同时间点取血清测定血药浓度，以评价其药物代谢动力学特性。
临床应用展望
在临床上，本发明的多重活性蛋白具有潜在的适合每周或以上施用一次的药物代谢动力学特性。剂量取决于施用频率和模式，受治疗的受试者的年龄、性别、重量和一般状况，治疗的状况和严重性，待治疗的任何伴随性疾病以及对于本领域技术人员显而易见的其他因素。同时，根据受治疗者的情况和其他病理状况，本发明的多重活性蛋白可以与一种或多种其他的治疗活性化合物或物质组合施用或应用，例如可以选择的其他的治疗活性化合物包括但不限于抗糖尿病药、抗高血脂药、抗肥胖药，抗高血压药和用于治疗起因于糖尿病或与糖尿病有关的并发症的试剂。
代谢综合征与冠心病和血管斑块积累相关的其他病症风险增加相关，例如中风和外周血管疾病，成为粥样动脉硬化心血管病(ASCVD)。患有代谢综合征的患者可自处于早期的胰岛素抗性状态发展成完全成熟的二型糖尿病，并且ASCVD的风险进一步增加。不旨在受限于任何特定理论，胰岛素抗性、代谢综合征及血管疾病之间的关系可以涉及一种或多种共同发病机制，包括胰岛素刺激的血管舒张障碍、由氧化应力增强所致的胰岛素抗性相关性可用性降低，以及脂肪细胞衍生激素(诸如脂联素)异常(Lteif,Mather,Can.J.Cardiol.20(增刊B)：66B-76B，2004)
本发明的活性蛋白还可用于治疗肥胖症。在一些方面中，本发明的活性蛋白通过降低食欲、减少食物摄取、降低患者体内脂肪水平、提高能量消耗等机制来治疗肥胖症。
在一些潜在的实施方案中，本发明的活性蛋白可用于治疗非酒精性脂肪肝病(NAFLD)。NAFLD是指广谱肝脏疾病，范围自单纯脂肪肝(脂肪变性)至非酒精性脂肪变性肝炎(NASH)至肝硬化(肝脏的不可逆晚期瘢痕形成)。NAFLD的所有病期均具有在肝脏细胞中的脂肪累积。单纯脂肪肝为某类型的脂肪、甘油三酯在肝脏细胞中异常积累，但无发炎或瘢痕形成。在NASH中，脂肪累积与不同程度的肝脏发炎(肝炎)和瘢痕形成(纤维化)相关。发炎性细胞可破坏肝脏细胞(肝细胞坏死)。在术语“脂肪变性肝炎”和“脂肪变性坏死”中，脂肪变性是指脂肪浸润，肝炎是指肝脏中的炎症，并且坏死是指经破坏的肝脏细胞。NASH可最终导致肝脏瘢痕形成(纤维化)且接着导致不可逆晚期瘢痕形成(肝硬化)，由NASH导致的肝硬化为NAFLD谱内的最后且最严重的病期。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1GCG类似物的筛选(胰高血糖类似物的筛选)
本发明的GCG类似物记为A，A的结构式如式II(SEQ ID NO.141)所示：
HSQGTFTSDYSKYLDX16X17X18 AQDFVQWLMNX29 Xz(SEQ ID NO.141)。其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18选自除P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS(SEQ IDNO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。
表1中列举了部分本发明GCG类似物的氨基酸序列：
表1
进一步地，本发明的重组蛋白的结构式如式I所示：A-L-F 式I，
式I中，A的结构式如式II所示：HSQGTFTSDYSKYLDX16X17X18 AQDFVQWLMNX29 Xz(SEQ IDNO.141)，其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18选自除P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS(SEQ ID NO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。
式I中，F为长效蛋白单元，F可选自免疫球蛋白的完整FC部分、免疫球蛋白的FC部分的片段或免疫球蛋白的FC部分的突变体，如SEQ ID NO.11-20所示。
例如，F可以是天然IgG1的完整FC部分，氨基酸序列如SEQ ID NO.11所示，具体为：
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
F可以是天然IgG1的FC部分的突变体，氨基酸序列如SEQ ID NO.12所示，具体为：
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
F可以是天然IgG2的完整FC部分，氨基酸序列如SEQ ID NO.13所示，具体为：
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
F可以是天然IgG4的完整FC部分，氨基酸序列如SEQ ID NO.14所示，具体为：
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。
F可以是天然IgG4的FC部分的突变体，氨基酸序列如SEQ ID NO.15所示，具体为：
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。
F可以是天然IgG4的FC部分的突变体，氨基酸序列如SEQ ID NO.16所示，具体为：
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
F可以是天然IgG4的FC部分的突变体，氨基酸序列可以如SEQ ID NO.17所示，具体为：
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
F可以是天然IgG4的FC部分的突变体，氨基酸序列可以如SEQ ID NO.18所示，具体为：
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
F可以是天然IgG1的FC部分的突变体，氨基酸序列可以如SEQ ID NO.19所示，具体为：
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
F可以是天然IgG1的FC部分的突变体，氨基酸序列可以如SEQ ID NO.20所示，具体为：
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
式I中，L为连接链，所述连接链为一段合适长度的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动。当有必要保证所述两个相邻结构域空间上彼此互不干扰时，可以使用较长的连接链。所述连接链示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA等，n为1-10的整数，在一个优选的实施方案中，所述的连接链的氨基酸长度为5-26。示例性的连接链各自独立的选自表2。
表2
SEQ ID NO. 代号 连接链
21 G<sub>4</sub>S<sub>1</sub> GGGGS
22 G<sub>4</sub>A<sub>1</sub> GGGGA
23 G<sub>6</sub>S<sub>2</sub> GGGSGGGS
24 G<sub>4</sub>S<sub>4</sub> GSGSGSGS
25 G<sub>8</sub>S<sub>2</sub> (GGGGS)<sub>2</sub>
26 G<sub>8</sub>A<sub>2</sub> (GGGGA)<sub>2</sub>
27 G<sub>9</sub>S<sub>3</sub> GGSGGGSGGGGS
28 G<sub>9</sub>S<sub>2</sub>A<sub>1</sub> GGSGGGAGGGGS
29 G<sub>12</sub>S<sub>3</sub> (GGGGS)<sub>3</sub>
30 G<sub>12</sub>A<sub>3</sub> (GGGGA)<sub>3</sub>
31 G<sub>12</sub>A<sub>1</sub>S<sub>3</sub> (GGSGG)<sub>3</sub>A
32 G<sub>12</sub>A<sub>4</sub> (GGGGA)<sub>3</sub>A
33 G<sub>12</sub>S<sub>3</sub>A<sub>1</sub> (GGGGS)<sub>3</sub>A
34 G<sub>12</sub>S<sub>1</sub>A<sub>3</sub> (GGGGA)<sub>3</sub>S
35 G<sub>13</sub>S<sub>4</sub> GS(GGGGS)<sub>3</sub>
36 G<sub>16</sub>A<sub>4</sub> (GGGGA)<sub>4</sub>
37 G<sub>16</sub>S<sub>4</sub> (GGGGS)<sub>4</sub>
38 G<sub>17</sub>S<sub>5</sub> GS(GGGGS)<sub>4</sub>
39 G<sub>20</sub>S<sub>5</sub> (GGGGS)<sub>5</sub>
40 G<sub>20</sub>S<sub>5</sub>A<sub>1</sub> (GGGGS)<sub>5</sub>A
41 G<sub>24</sub>S<sub>6</sub> (GGGGS)<sub>6</sub>
42 G<sub>28</sub>S<sub>7</sub> (GGGGS)<sub>7</sub>
43 G<sub>32</sub>S<sub>8</sub> (GGGGS)<sub>8</sub>
实施例2二聚体双效活性蛋白的制备
实施例1中，由GCG类似物与连接链及F融合而获得的二聚体双效活性蛋白(结构式如式I所示)的氨基酸序列如下：
C001G12S3A1F4的氨基酸序列如SEQ ID NO.75所示，具体为：
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C002G12S3A1F4的氨基酸序列如SEQ ID NO.76所示，具体为：
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C240G12S3A1F4的氨酸酸序列如SEQ ID NO.77所示，具体为：
HSQGTFTSDYSKYLDERAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C320G12S3A1F4的氨基酸序列如SEQ ID NO.78所示，具体为：
HSQGTFTSDYSKYLDYQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C276G12S3A1F4的氨基酸序列如SEQ ID NO.79所示，具体为：
HSQGTFTSDYSKYLDSRAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C368G12S3A1F4的氨基酸序列如SEQ ID NO.80所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C225G12S3A1F4的氨基酸序列如SEQ ID NO.81所示，具体为：
HSQGTFTSDYSKYLDERAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C302G12S3A1F4的氨基酸序列如SEQ ID NO.82所示，具体为：
HSQGTFTSDYSKYLDEPAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C163G12S3A1F4的氨基酸序列如SEQ ID NO.83所示，具体为：
HSQGTFTSDYSKYLDSRAAQDFVQWLMNTGPSSGAPPPS GGGGS GGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C350G12S3A1F4的氨基酸序列如SEQ ID NO.84所示，具体为：
HSQGTFTSDYSKYLDNQEAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C271G12S3A1F4的氨基酸序列如SEQ ID NO.85所示，具体为：
HSQGTFTSDYSKYLDGRAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C232G12S3A1F4的氨基酸序列如SEQ ID NO.86所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C495G12S3A1F4的氨基酸序列如SEQ ID NO.87所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C307G12S3A1F4的氨基酸序列如SEQ ID NO.88所示，具体为：
HSQGTFTSDYSKYLDSLAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C382G12S3A1F4的氨基酸序列如SEQ ID NO.89所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C227G12S3A1F4的氨基酸序列如SEQ ID NO.90所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C266G12S3A1F4的氨基酸序列如SEQ ID NO.91所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C137G12S3A1F4的氨基酸序列如SEQ ID NO.92所示，具体为：
HSQGTFTSDYSKYLDSERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C399G12S3A1F4的氨基酸序列如SEQ ID NO.93所示，具体为：
HSQGTFTSDYSKYLDGERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C395G12S3A1F4的氨基酸序列如SEQ ID NO.94所示，具体为：
HSQGTFTSDYSKYLDEQSAQDFVQWLMNTPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C394G12S3A1F4的氨基酸序列如SEQ ID NO.95所示，具体为：
HSQGTFTSDYSKYLDSQPAQDFVQWLMNTSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C392G12S3A1F4的氨基酸序列如SEQ ID NO.96所示，具体为：
HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C353G12S3A1F4的氨基酸序列如SEQ ID NO.97所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C352G12S3A1F4的氨基酸序列如SEQ ID NO.98所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C228G12S3A1F4的氨基酸序列如SEQ ID NO.99所示，具体为：
HSQGTFTSDYSKYLDEERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C462G12S3A1F4的氨基酸序列如SEQ ID NO.100所示，具体为：
HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C187G12S3A1F4的氨基酸序列如SEQ ID NO.101所示，具体为：
HSQGTFTSDYSKYLDSQRAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C334G12S3A1F4的氨基酸序列如SEQ ID NO.102所示，具体为：
HSQGTFTSDYSKYLDSTAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C364G12S3A1F4的氨基酸序列如SEQ ID NO.103所示，具体为：
HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C209G12S3A1F4的氨基酸序列如SEQ ID NO.104所示，具体为：
HSQGTFTSDYSKYLDSEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C289G12S3A1F4的氨基酸序列如SEQ ID NO.105所示，具体为：
HSQGTFTSDYSKYLDGEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C0382G12S3A1F4的氨基酸序列如SEQ ID NO.142所示，具体为：
HSQGTFTSDYSEYLDSERARDFVAWLEAGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
在知晓了二聚体双效活性蛋白的氨基酸序列的基础上，本领域技术人员能够采用现有技术对其进行制备：由于带有FC序列，因此可通过高亲和力和高特异性的Protein A树脂层析进行蛋白纯化，在这里仅示例性地给出一种可行的制备方法。
制备过程如下：
(1)根据蛋白序列及氨基酸密码子表，设计其DNA序列。分别制备重组蛋白中A、L、F对应的多核苷酸DNA片段，各DNA片段均可由常规的固相合成技术合成拼接；
(2)设计引物进行巢式PCR扩增，拼接A、L、F分别对应的DNA片段，获得目的基因，PCR拼接技术(包括引物设计、PCR引入突变及酶切等)为本领域技术人员所熟知的公知技术。本领域技术人员应当知晓，本实施例的PCR拼接过程并非是唯一的方法，例如，通过基因合成也可以获得目的基因。成功获得目的基因后，将目的基因克隆至哺乳动物细胞表达载体pTT5(Yves Durocher)，转化大肠杆菌Top10F’；阳性克隆鉴定后，接种于500ml LB培养基中，过夜培养，离心收集菌体，使用Omega E.Z.N.A.Endo-Free PlasmidMaxi Kit提取质粒。
(3)转染Hek293F细胞及细胞表达：取1.0mg质粒，使用Freestyle 293表达培养基(Thermofisher)稀释至25ml；取3.0mg PEI(线性，25KD)，使用Freestyle 293表达培养基稀释至25ml，加入到质粒溶液中，混匀，室温孵育30分钟；与此同时，取对数期生长的Hek293F细胞(活率>95％)，计数；1100RPM，离心10分钟，弃上清；使用450ml Freestyle293表达培养基重悬细胞；PEI质粒混合液孵育结束后，加入到细胞悬液中，37℃，5％CO2，140RPM震荡培养；7小时后，使用1000ml 293SFM II培养基(Thermofisher)置换Freestyle293表达培养基，继续培养7天。
(4)重组蛋白的纯化：将细胞培养液以8000rpm，高速离心10min，得到上清上样到预先用平衡液(20mM PB，0.5M NaCl，pH7)平衡好的Protein A柱(博格隆(上海)生物技术有限公司)，再次平衡后，100％洗脱(洗脱液为0.1M Gly-HCl，pH3.0)；收集管中预先加入中和液为(1M Tris-HCl，pH8.0)，收集洗脱样品；最后加中和液到洗脱样品体积的1/10，并采用常规Bradford法测定蛋白浓度。
(5)重组蛋白的理化性质鉴定：对纯化获得的重组蛋白进行SDS-PAGE电泳，图1为示例性的纯化样品的SDS-PAGE电泳图。
实施例3体外细胞活性检测
将实施例2中获得的双效活性蛋白进行体外活性测定，包括GLP-1R激动活性检测和GCGR激动活性检测。
GLP-1R激动活性检测：
GLP-1R激动活性检测采用荧光素酶报告基因检测法。将人源GLP-1R基因克隆至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GLP-1R，同时荧光素酶(luciferase)全长基因克隆至pCRE-EGFP质粒上，替换EGFP基因，得到pCRE-Luc重组质粒。pCDNA3.1-GLP-1R和pCRE-Luc质粒按摩尔比1:10的比例转染CHO细胞，筛选稳转表达株，获得重组CHO/GLP-1R稳转细胞株。
在9-cm细胞培养皿中用含10％FBS和300μg/ml G418的DMEM/F12培养基培养细胞，等汇合度至90％左右时，弃去培养上清，加入2ml胰酶消化3min后，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基中和，转移至15ml离心管中，1000rpm离心5min后，弃去上清，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基重悬，计数。用含10％FBS的DMEM/F12培养基稀释细胞至3×105，96孔板中每孔铺100μl，即5×104/孔，贴壁后换成含0.2％FBS的DMEM/F12培养基培养。铺在96孔板的细胞弃去上清后，将纯化的重组蛋白(表3)或天然Glucagon(杭州中肽生化有限公司，GLUC-004)与天然GLP-1(杭州中肽生化有限公司,GLUC-016B)作为对照，用含0.1％FBS的DMEM/F12培养基稀释至一系列指定浓度，加入到细胞培养孔中，100μl/孔，刺激6h后检测。根据萤光素酶报告检测试剂盒(Lucifersae reporterkit，Ray Biotech,Cat:68-LuciR-S200)说明书进行检测。
GCGR激动活性检测方法：
GCGR激动活性检测同样也采用荧光素酶报告基因检测法。将GCGR基因克隆至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GCGR，转染HEK 293T细胞及稳转细胞株HEK 293T/GCGR的构建同上。图2为部分二聚体双效活性蛋白的测活EC50结果图。
结果如表3所示：
表3
此外，还对其他不带有Exendin-4C末端序列的二聚体活性蛋白：CG283G12S3A1F4(SEQ ID NO.106)、CG214G12S3A1F4(SEQ ID NO.107)、CG267G12S3A1F4(SEQ ID NO.108)、C308G12S3A1F4(SEQ ID NO.109)、C224G12S3A1F4(SEQ ID NO.110)、CG308G12S3A1F4(SEQID NO.111)、C319G12S3A1F4(SEQ ID NO.112)、C214G12S3A1F4(SEQ ID NO.113)、C303G12S3A1F4(SEQ ID NO.114)、CG303G12S3A1F4(SEQ ID NO.115)进行体外活性测定，包括GLP-1R激动活性检测和GCGR激动活性检测。
上述CG283G12S3A1F4的氨基酸序列如SEQ ID NO.106所示，具体为：
HSQGTFTSDYSKYLDERAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
CG214G12S3A1F4的氨基酸序列如SEQ ID NO.107所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
CG267G12S3A1F4的氨基酸序列如SEQ ID NO.108所示，具体为：
HSQGTFTSDYSKYLDSRAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C308G12S3A1F4的氨基酸序列如SEQ ID NO.109所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C224G12S3A1F4的氨基酸序列如SEQ ID NO.110所示，具体为：
HSQGTFTSDYSKYLDERAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
CG308G12S3A1F4的氨基酸序列如SEQ ID NO.111所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C319G12S3A1F4的氨基酸序列如SEQ ID NO.112所示，具体为：
HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C214G12S3A1F4的氨基酸序列如SEQ ID NO.113所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
C303G12S3A1F4的氨基酸序列如SEQ ID NO.114所示，具体为：
HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
CG303G12S3A1F4的氨基酸序列如SEQ ID NO.115所示，具体为：
HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
测定结果与本发明的重组蛋白进行比较，结果如表4所示：
表4
说明：a.为加上Exendin-4的C末端延伸肽Cex(在本发明中为SEQ ID NO.3-10之任一)前后的GLP-1R激动活性比例。
b.为根据US9018164B2表2中披露的天然Glucagon与Glucagon Cex的GLP-1R激动活性数据计算获得的比例。
c.为根据Joseph R.Chabenne等(Joseph R.Chabenne等，Optimization of theNative Glucagon Sequence for Medicinal Purposes，J Diabetes Sci Technol.4(6):1322–1331，2010)文章中表1披露的天然Glucagon与Glucagon Cex的GLP-1R激动活性数据计算获得的比例。
如表3和表4所示，当把含延伸肽的序列通过(GGGGS)3A(SEQ ID NO.33)与F(SEQID NO.16)融合制备成二聚体后，对GLP-1R的激动活性提高了200倍以上，而对GCGR的激动活性则无明显差异。
实施例4DPP-IV耐酶稳定性
将纯化的二聚体双效活性蛋白5uM溶解于10mM的HEPES缓冲液(含0.05mg/ml BSA中，加入终浓度为10nM的重组DPP-IV酶，于37℃温浴24小时后，检测体外GCGR细胞活性检测。活性保留率＝(DPP-IV酶处理后活性/处理前活性)×100％。
本实施例中采用了第二位引入非天然氨基酸Aib或D-Ser的GCG类似物作为对照：
GDSerGS：H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.116)；
GAibGS：H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.117)；
C364(SEQ ID NO.72)、C382(SEQ ID NO.59)、C495(SEQ ID NO.55)、C462(SEQ IDNO.68)、C225(SEQ ID NO.49)及C209(SEQ ID NO.73)作为本实施例中稳定性实验的对照。
结果如表5所示：
表5
实施例5血清稳定性试验
体外细胞检测法：
(1)取二聚体双效活性蛋白，先超滤浓缩，再用20mM PB pH7.4稀释到1.6mg/ml，除菌过滤后，血清(FBS，GEMINI 900-108，A97E00G)稀释10倍，混匀，分装到无菌离心管中；
(2)另外取Glucagon(SEQ ID NO.44，杭州中肽生化有限公司，GLUC-004)，稀释到0.2mg/ml，除菌过滤后，血清稀释10倍，混匀，分装到无菌离心管中；
(3)上述样品1-2管于-20度冻存作为对照，另几管置37℃培养箱，于不同时间点取样检测GCGR激动活性；
(4)HEK 293T/GCGR细胞传代两次后铺96孔板，检测样品活性。相对活性随着时间的变化如图3A-D所示。从图3结果可见C240G12S3A1F4、C276G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C163G12S3A1F4、C232G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4、C271G12S3A1F4、C227G12S3A1F4、C266G12S3A1F4、C399G12S3A1F4、C392G12S3A1F4、C353G12S3A1F4、C137G12S3A1F4、C289G12S3A1F4、C209G12S3A1F4、C352G12S3A1F4、C228G12S3A1F4、C462G12S3A1F4、C187G12S3A1F4、C364G12S3A1F4在血清中的稳定性相对其他二聚体明显较高。
残留活性：以0小时的活性值为100％，后续时间点测得的值与之相比而获得。
实施例6不同连接链长度的二聚体双效活性蛋白的活性测定
将实施例1中的GCG类似物通过不同长度的连接链融合于人IgG1 FC(SEQ IDNO.12)的N端。亦即，由GCG类似物与不同长度连接链及F(SEQ ID NO.12)融合而获得的二聚体双效活性蛋白的氨基酸序列如下：
C382G4A1F1的氨基酸序列如SEQ ID NO.118所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G4S1F1的氨基酸序列如SEQ ID NO.119所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G6S2F1的氨基酸序列如SEQ ID NO.120所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G4S4F1的氨基酸序列如SEQ ID NO.121所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGSGSGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G8S2F1的氨基酸序列如SEQ ID NO.122所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G8A2F1的氨基酸序列如SEQ ID NO.123所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G9S3F1的氨基酸序列如SEQ ID NO.124所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G9S2A1F1的氨基酸序列如SEQ ID NO.125所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGAGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12S3F1的氨基酸序列如SEQ ID NO.126所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12A3F1的氨基酸序列如SEQ ID NO.127所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12A1S3F1的氨基酸序列如SEQ ID NO.128所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGGSGGGGSGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12S3A1F1的氨基酸序列如SEQ ID NO.129所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12A4F1的氨基酸序列如SEQ ID NO.130所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G12S1A3F1的氨基酸序列如SEQ ID NO.131所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G13S4F1的氨基酸序列如SEQ ID NO.132所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G16A4F1的氨基酸序列如SEQ ID NO.133所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G16S4F1的氨基酸序列如SEQ ID NO.134所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G17S5F1的氨基酸序列如SEQ ID NO.135所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G20S5F1的氨基酸序列如SEQ ID NO.136所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G20S5A1F1的氨基酸序列如SEQ ID NO.137所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G24S6F1的氨基酸序列如SEQ ID NO.138所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G28S7F1的氨基酸序列如SEQ ID NO.139所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
C382G32S8F1的氨基酸序列如SEQ ID NO.140所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
在知晓了二聚体双效活性蛋白的氨基酸序列的基础上，本领域技术人员能够采用现有技术对其进行制备。
本实施例中所使用的制备方法同实施例2。
将本实施例中获得的二聚体双效活性蛋白进行体外活性测定，包括GLP-1R激动活性检测和GCGR激动活性检测。检测方法同实施例3。
检测结果如表6所示。
表6
实施例7葡萄糖刺激的胰岛素分泌实验(GSIS)
本实施例参照Aisling M.Lynch等人的方法(A novel DPP IV-resistant C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1receptor activation，Aisling M.Lynch等，Diabetologia，57:1927–1936，2014)采用大鼠BRIN-BD11细胞用于测定活性蛋白刺激引起的胰岛素释放，但稍作修改，即在24孔板(Orange Scientific,Brainel’Alleud,Belgium)中每孔加入1.0×106个细胞，37℃培养过夜后离心去上清，再每孔加入1.0ml KRB(115mM NaCl、4.7mM KCl、1.28mM CaCl2、1.2mMMgSO4、1.2mM KH2PO4、25mM HEPES、10mM NaHCO3，NaOH调节pH至7.4)、0.1％(wt/vol.)BSA和1.1mM葡萄糖。细胞置于37℃培养40分钟后，离心去上清并替换成1.0ml新鲜的KRB溶液与梯度浓度的表6中的活性蛋白。37℃培养20分钟后，离心去除缓冲液并于-20℃储存过夜，再作免疫放射检测胰岛素含量。结果如图4所示。
实施例8正常ICR小鼠的葡萄糖耐量试验(IPGTT)
正常ICR小鼠分组，每组8只。禁食过夜，尾部采血(记为t＝0分钟血糖样)，皮下注射二聚体双效活性蛋白(40nmol/kg，醋酸盐缓冲液)或生理盐水PBS，其中C002G12S3A1F4给药剂量为250nmol/kg(醋酸盐缓冲液)。15分钟后腹腔注射葡萄糖(2克/千克体重)，并在t＝30分钟、t＝60分钟、t＝120分钟和t＝240分钟测量血液葡萄糖水平。在实验期间动物仍禁食以防止食物摄取的干扰。结果如图5所示。
实施例9连续给药在诱导肥胖(DIO)小鼠中的药效研究
本实施例的目的是研究不同的二聚体双效活性蛋白对DIO小鼠体重的影响作用。7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下给药处理。按照50nmol/千克体重的剂量给药，4天一次，其他日期予以PBS假注射，连续给药28天；阴性对照组按5ul/克体重注射生理盐水(PBS)；阳性对照组注射利拉鲁肽(40nmol/千克体重)，每天一次给药，每天测量小鼠体重。最后一次给药后的第5天处死。眼眶取血。将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化。结果如图6所示。
实施例10大鼠的药物代谢动力学参数(PK)测定
将6周左右的雄性SD大鼠分组，每组8只。根据表7的方案给药或生理盐水。给药结束后所有大鼠开始自由饮水进食；给药结束的时间点设置为0，并作为后续实验的时间参照。二聚体双效活性蛋白的药代检测采用夹心ELISA法。即鼠抗人IgG4FC单抗(9002-01，I2013-NG 56，0.25mg/ml)；包板(96孔板，corning，42592)，200ng/孔，4℃过夜；PBST洗板4次后用5％奶粉溶液封闭，室温1h；再PBST洗板4次；用PBST稀释大鼠血清后，37℃孵育2h；PBST洗板6次；再用1％BSA溶液稀释兔抗Glucagon抗体(自制：用天然Glucagon多肽(SEQ IDNO.44)免疫家兔，通过Protein G纯化得到抗Glucagon抗体(杭州华安生物技术有限公司)，生物素标记，200×，0.2mg/ml)，1:2000，37℃孵育2h；PBST洗板6次；1％BSA溶液稀释strep-HRP，1:5万，37℃孵育1.5h；PBST洗板6次；最后TMB显色，OD450读数。
表7
结果如表8所示：
表8
二聚体双效活性蛋白 T<sub>1/2</sub>(小时) T<sub>max</sub>(小时)
C240G12S3A1F4(SEQ ID NO.77) 41.2 24
C382G12S3A1F4(SEQ ID NO.89) 42.5 24
C495G12S3A1F4(SEQ ID NO.87) 43.7 24
C364G12S3A1F4(SEQ ID NO.103) 42.6 24
C462G12S3A1F4(SEQ ID NO.100) 39.5 24
C227G12S3A1F4(SEQ ID NO.90) 42.2 24
C368G12S3A1F4(SEQ ID NO.80) 37.8 24
C266G12S3A1F4(SEQ ID NO.91) 38.4 24
C002G12S3A1F4(SEQ ID NO.76) 8.5 6
实施例11融合FGF21类似物的二聚体三效活性蛋白的构建及活性测定
在前述二聚体双效活性蛋白的C末端，进一步融合FGF21类似物，构建获得表9中的融合蛋白(融合FGF21类似物的三效活性蛋白)。表9中的各融合蛋白的氨基酸序列如下：
天然FGF21的氨基酸序列如SEQ ID NO.143所示，具体为：
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。
FGF21类似物1的氨基酸序列如SEQ ID NO.144所示，具体为：
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
FGF21类似物2的氨基酸序列如SEQ ID NO.145所示，具体为：
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
FGF21类似物3的氨基酸序列如SEQ ID NO.146所示，具体为：
DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。
C382F4FGF的氨基酸序列如SEQ ID NO.147所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。
C382F4FGF1的氨基酸序列如SEQ ID NO.148所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
C382F4FGF2的氨基酸序列如SEQ ID NO.149所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
C382F4FGF3的氨基酸序列如SEQ ID NO.150所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。
C495F4FGF的氨基酸序列如SEQ ID NO.151所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。
C495F4FGF1的氨基酸序列如SEQ ID NO.152所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
C495F4FGF2的氨基酸序列如SEQ ID NO.153所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。
C495F4FGF3的氨基酸序列如SEQ ID NO.154所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。
所述融合蛋白的制备方法属于现有技术：由于带有FC序列，因此可通过高亲和力和高特异性的Protein A树脂层析进行蛋白纯化，可参照实施例2中的方法。纯化获得的融合蛋白的电泳图如图7所示。天然FGF21、FGF21类似物的制备参考Xu J等的报道(Xu J等，Polyethylene glycol modified FGF21engineered to maximize potency and minimizevacuoleformation，Bioconjug Chem.；24(6):915-25，2013)并作如下修改：表达载体为PET30，宿主菌为BL21(DE3)(默克中国)。包涵体用洗涤液(50mM Tris,150mM NaCl,2M尿素，pH 8.0)洗涤四次，称重；按照(1:10的质量体积比)每0.1g包涵体加入1ml变性液(50mMTris,150mMNaCl,8M urea,10mM DTT，pH 8.5)，室温摇床轻轻混合溶解5h以上；按照1:100-200的比例稀释复性。将变性液缓慢滴加到复性液中，过程中不断搅拌；滴加完成之后，将含有蛋白的复性液放于4℃，24h；结束后用0.45um滤膜抽滤用于层析纯化。
测活细胞的构建：
PCR扩增嘌呤霉素抗性基因pac，克隆到pcDNA3.1(+)，替换原有的G418抗性基因。PCR扩增GAL4DBD-ELK1、IRES、KLB(β-klotho)基因，依次克隆到pcDNA-Puro质粒上，构建质粒pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro用于细胞转染筛选。质粒采用OmegaEndo-Free Plasmid Midi Kit提取备用。细胞转染过程如下：Hek293T细胞铺6孔板，每孔3x105个细胞，培养过夜。
Opti-MEM培养基洗涤细胞两次后，加入2ml Opti-MEM培养基。按照如下比例配制细胞转染试剂：Lipofectamine 2000(6μl)：pFR-Luc(4.6μg)：pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro(1μg)。静置20min后，缓慢加入到6孔板中，边加边混匀。培养6h后换DMEM+10％FBS培养基，37℃，5％CO2继续培养。筛选获得具有FGF21活性响应的稳转细胞株。
细胞测活：
细胞长满培养皿后，胰酶消化，制备细胞悬液(1x 105细胞/ml,DMEM+5％FBS+1μg/ml puromycin)，铺96孔板，每孔100μl，过夜培养。添加梯度浓度的待测样品，作用6h，使用Luciferase Reporter Assay Kit(68-LucifR-S200)进行荧光检测。结果如表9所示：
表9
实施例12融合FGF21及其类似物的三活性蛋白在DIO小鼠中的药效研究
7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下给药处理(表10)。双效活性蛋白按照30nmol/千克体重或90nmol/千克体重的剂量给药，4天一次；FGF21类似物按30nmol/千克体重，每天两次给药；阴性对照组按5ul/克体重注射生理盐水(PBS)；阳性对照组注射利拉鲁肽(30nmol/千克体重)，每天一次给药，连续给药28天，每天测量小鼠体重及进食量。最后一次给药后的第5天处死。眼眶取血。将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化。体重变化结果如图8所示；总摄食量的变化如图9所示。
表10
实施例13融合瘦素的三活性蛋白的构建及活性测定
在前述二聚体双效活性蛋白的C末端，可进一步融合天然瘦素(SEQ ID NO.155)及其类似物，构建获得表11中的融合蛋白(融合瘦素的三活性蛋白)。表11中的各融合蛋白的氨基酸序列如下：
表11
天然瘦素的氨基酸序列如SEQ ID NO.155所示，具体为：
VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
C382F4Lep的氨基酸序列如SEQ ID NO.156所示，具体为：
HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
C495F4Lep的氨基酸序列如SEQ ID NO.157所示，具体为：
HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
C462F4Lep的氨基酸序列如SEQ ID NO.158所示，具体为：
HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPSGGGGS GGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
C364F4Lep的氨基酸序列如SEQ ID NO.159所示，具体为：
HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGPSSGAPPPSGGGGS GGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
C289F4Lep的氨基酸序列如SEQ ID NO.160所示，具体为：
HSQGTFTSDYSKYLDGEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
实施例14融合瘦素的三活性蛋白在DIO小鼠中的药效研究
7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下给药处理(表12)。按照30nmol/千克体重的剂量给药，阴性对照组按5ul/克体重注射生理盐水(PBS)；双效活性蛋白4天一次，其他日期予以PBS假注射，连续给药28天，瘦素按30nmol/千克体重每天两次给药，每天测量小鼠体重及进食量。最后一次给药后的第5天处死。眼眶取血。将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化。体重变化结果如图10所示；总摄食量的变化如图11所示。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
序列表
<110> 浙江道尔生物科技有限公司
<120> 一种治疗代谢疾病的多重活性蛋白
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Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Pro Gly
225
<210> 14
<211> 229
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 15
<211> 229
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 16
<211> 228
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly
225
<210> 17
<211> 228
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly
225
<210> 18
<211> 228
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly
225
<210> 19
<211> 227
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 20
<211> 226
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly
225
<210> 21
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Gly Gly Gly Gly Ser
1 5
<210> 22
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Gly Gly Gly Gly Ala
1 5
<210> 23
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Gly Gly Gly Ser Gly Gly Gly Ser
1 5
<210> 24
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gly Ser Gly Ser Gly Ser Gly Ser
1 5
<210> 25
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 26
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala
1 5 10
<210> 27
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 28
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Gly Gly Ser Gly Gly Gly Ala Gly Gly Gly Gly Ser
1 5 10
<210> 29
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 30
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala
1 5 10 15
<210> 31
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala
1 5 10 15
<210> 32
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Ala
1 5 10 15
<210> 33
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
1 5 10 15
<210> 34
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Ser
1 5 10 15
<210> 35
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser
<210> 36
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Gly
1 5 10 15
Gly Gly Gly Ala
20
<210> 37
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 38
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Gly Ser 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> 39
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 40
<211> 26
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
20 25
<210> 41
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
<210> 42
<211> 35
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser
35
<210> 43
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser
35 40
<210> 44
<211> 29
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr
20 25
<210> 45
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 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(Artificial Sequence)
<400> 46
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 47
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Tyr
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 48
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 49
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 50
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Pro Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 51
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 52
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Asn
1 5 10 15
Gln Glu Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 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(Artificial Sequence)
<400> 53
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 54
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 55
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 55
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 56
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 56
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Ser
35
<210> 57
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 57
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser
35
<210> 58
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Leu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 59
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met 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(Artificial Sequence)
<400> 60
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 61
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 61
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 62
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 62
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser
35
<210> 63
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 63
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 64
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 64
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 65
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 65
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ser Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Pro Ser Ser
20 25 30
Gly Ala Pro Pro Ser
35
<210> 66
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 66
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Pro Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Ser Ser Gly
20 25 30
Ala Pro Pro Pro Ser
35
<210> 67
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 67
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 68
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 68
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 69
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 69
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 70
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 70
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 71
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 71
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Thr Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 72
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 72
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 73
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 73
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn 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(Artificial Sequence)
<400> 74
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 75
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 75
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 76
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 76
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 77
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 77
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 78
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 78
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Tyr
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 79
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 79
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 80
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 80
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 81
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 81
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 82
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 82
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Pro Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 83
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 83
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 84
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 84
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Asn
1 5 10 15
Gln Glu Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 85
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 85
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 86
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 86
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 87
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 87
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 88
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 88
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Leu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 89
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 89
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 90
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 90
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
50 55 60
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
65 70 75 80
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
85 90 95
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
100 105 110
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
115 120 125
Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
130 135 140
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
145 150 155 160
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
165 170 175
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
180 185 190
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
195 200 205
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
210 215 220
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
225 230 235 240
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
245 250 255
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
260 265 270
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 91
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 91
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
50 55 60
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
65 70 75 80
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
85 90 95
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
100 105 110
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
115 120 125
Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
130 135 140
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
145 150 155 160
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
165 170 175
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
180 185 190
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
195 200 205
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
210 215 220
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
225 230 235 240
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
245 250 255
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
260 265 270
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 92
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 92
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 93
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 93
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 94
<211> 281
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 94
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ser Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Pro Ser Ser
20 25 30
Gly Ala Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
35 40 45
Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
50 55 60
Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
65 70 75 80
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
85 90 95
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
100 105 110
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
115 120 125
Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
130 135 140
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
145 150 155 160
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
165 170 175
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
180 185 190
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
195 200 205
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
210 215 220
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
225 230 235 240
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
245 250 255
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
260 265 270
Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 95
<211> 281
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 95
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Pro Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Ser Ser Gly
20 25 30
Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
35 40 45
Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
50 55 60
Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
65 70 75 80
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
85 90 95
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
100 105 110
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
115 120 125
Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
130 135 140
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
145 150 155 160
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
165 170 175
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
180 185 190
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
195 200 205
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
210 215 220
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
225 230 235 240
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
245 250 255
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
260 265 270
Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 96
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 96
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 97
<211> 281
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 97
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
35 40 45
Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
50 55 60
Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
65 70 75 80
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
85 90 95
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
100 105 110
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
115 120 125
Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
130 135 140
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
145 150 155 160
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
165 170 175
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
180 185 190
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
195 200 205
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
210 215 220
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
225 230 235 240
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
245 250 255
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
260 265 270
Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 98
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 98
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
35 40 45
Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
50 55 60
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
65 70 75 80
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
85 90 95
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
100 105 110
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
115 120 125
Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
130 135 140
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
145 150 155 160
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
165 170 175
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
180 185 190
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
195 200 205
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
210 215 220
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
225 230 235 240
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
245 250 255
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
260 265 270
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 99
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 99
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 100
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 100
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 101
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 101
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 102
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 102
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Thr Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 103
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 103
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 104
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 104
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 105
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 105
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
275 280
<210> 106
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 106
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 107
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 107
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 108
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 108
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 109
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 109
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 110
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 110
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 111
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 111
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 112
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 112
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 113
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 113
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 114
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 114
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 115
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 115
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
35 40 45
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
50 55 60
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
65 70 75 80
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
85 90 95
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
100 105 110
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg
115 120 125
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
130 135 140
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
145 150 155 160
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
165 170 175
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
195 200 205
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 116
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 116
His Asp Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp
1 5 10 15
Ser Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 117
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 117
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 118
<211> 275
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 118
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Glu Pro Lys Ser
35 40 45
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Pro Gly
275
<210> 119
<211> 275
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 119
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser
35 40 45
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Pro Gly
275
<210> 120
<211> 278
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 120
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
35 40 45
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
50 55 60
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
115 120 125
Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
145 150 155 160
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Pro Gly
275
<210> 121
<211> 278
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 121
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Ser Gly Ser Gly Ser Glu
35 40 45
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
50 55 60
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
115 120 125
Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
145 150 155 160
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Pro Gly
275
<210> 122
<211> 280
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 122
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
50 55 60
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
65 70 75 80
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
85 90 95
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
100 105 110
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
115 120 125
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
130 135 140
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
145 150 155 160
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
165 170 175
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
180 185 190
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
195 200 205
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
210 215 220
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
225 230 235 240
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
245 250 255
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
260 265 270
Lys Ser Leu Ser Leu Ser Pro Gly
275 280
<210> 123
<211> 280
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 123
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly
35 40 45
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
50 55 60
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
65 70 75 80
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
85 90 95
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
100 105 110
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
115 120 125
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
130 135 140
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
145 150 155 160
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
165 170 175
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
180 185 190
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
195 200 205
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
210 215 220
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
225 230 235 240
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
245 250 255
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
260 265 270
Lys Ser Leu Ser Leu Ser Pro Gly
275 280
<210> 124
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 124
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Ser Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
50 55 60
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
65 70 75 80
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
85 90 95
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
100 105 110
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
115 120 125
Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
130 135 140
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
145 150 155 160
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
165 170 175
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
180 185 190
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
195 200 205
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
210 215 220
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
225 230 235 240
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
245 250 255
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
260 265 270
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280
<210> 125
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 125
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Ser Gly Gly Gly Ala Gly Gly
35 40 45
Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
50 55 60
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
65 70 75 80
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
85 90 95
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
100 105 110
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
115 120 125
Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
130 135 140
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
145 150 155 160
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
165 170 175
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
180 185 190
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
195 200 205
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
210 215 220
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
225 230 235 240
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
245 250 255
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
260 265 270
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280
<210> 126
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 126
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
50 55 60
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
65 70 75 80
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
85 90 95
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
100 105 110
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
115 120 125
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
130 135 140
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
145 150 155 160
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
165 170 175
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
180 185 190
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
195 200 205
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
210 215 220
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
225 230 235 240
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
245 250 255
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
260 265 270
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 127
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 127
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly
35 40 45
Ala Gly Gly Gly Gly Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr
50 55 60
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
65 70 75 80
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
85 90 95
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
100 105 110
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
115 120 125
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
130 135 140
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
145 150 155 160
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
165 170 175
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
180 185 190
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
195 200 205
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
210 215 220
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
225 230 235 240
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
245 250 255
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
260 265 270
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 128
<211> 286
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 128
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly
35 40 45
Gly Gly Gly Ser Gly Gly Ala Glu Pro Lys Ser Cys Asp Lys Thr His
50 55 60
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
65 70 75 80
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
85 90 95
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
100 105 110
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
115 120 125
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser
130 135 140
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
145 150 155 160
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
180 185 190
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
195 200 205
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
210 215 220
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
225 230 235 240
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
245 250 255
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
260 265 270
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 129
<211> 286
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 129
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Pro Lys Ser Cys Asp Lys Thr His
50 55 60
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
65 70 75 80
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
85 90 95
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
100 105 110
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
115 120 125
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser
130 135 140
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
145 150 155 160
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
180 185 190
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
195 200 205
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
210 215 220
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
225 230 235 240
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
245 250 255
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
260 265 270
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 130
<211> 286
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 130
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly
35 40 45
Ala Gly Gly Gly Gly Ala Ala Glu Pro Lys Ser Cys Asp Lys Thr His
50 55 60
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
65 70 75 80
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
85 90 95
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
100 105 110
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
115 120 125
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser
130 135 140
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
145 150 155 160
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
180 185 190
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
195 200 205
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
210 215 220
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
225 230 235 240
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
245 250 255
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
260 265 270
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 131
<211> 286
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 131
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly
35 40 45
Ala Gly Gly Gly Gly Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His
50 55 60
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
65 70 75 80
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
85 90 95
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
100 105 110
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
115 120 125
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser
130 135 140
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
145 150 155 160
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
180 185 190
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
195 200 205
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
210 215 220
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
225 230 235 240
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
245 250 255
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
260 265 270
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 132
<211> 287
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 132
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr
50 55 60
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
65 70 75 80
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
85 90 95
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
100 105 110
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
115 120 125
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
130 135 140
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
145 150 155 160
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
165 170 175
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
180 185 190
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
195 200 205
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
210 215 220
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
225 230 235 240
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
245 250 255
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
260 265 270
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
275 280 285
<210> 133
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 133
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly
35 40 45
Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Glu Pro Lys Ser Cys
50 55 60
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
65 70 75 80
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
85 90 95
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
100 105 110
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
115 120 125
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
130 135 140
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
145 150 155 160
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
165 170 175
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
180 185 190
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
195 200 205
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
210 215 220
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
225 230 235 240
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
245 250 255
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
260 265 270
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
275 280 285
Pro Gly
290
<210> 134
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 134
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys
50 55 60
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
65 70 75 80
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
85 90 95
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
100 105 110
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
115 120 125
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
130 135 140
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
145 150 155 160
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
165 170 175
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
180 185 190
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
195 200 205
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
210 215 220
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
225 230 235 240
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
245 250 255
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
260 265 270
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
275 280 285
Pro Gly
290
<210> 135
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 135
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys
50 55 60
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
65 70 75 80
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
85 90 95
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
100 105 110
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
115 120 125
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser
130 135 140
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
145 150 155 160
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
165 170 175
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
180 185 190
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
195 200 205
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
210 215 220
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
225 230 235 240
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
245 250 255
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
260 265 270
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
275 280 285
Leu Ser Pro Gly
290
<210> 136
<211> 295
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 136
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
65 70 75 80
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
85 90 95
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
100 105 110
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
115 120 125
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
130 135 140
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
145 150 155 160
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
165 170 175
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
180 185 190
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
195 200 205
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
210 215 220
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
225 230 235 240
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
245 250 255
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
260 265 270
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
275 280 285
Ser Leu Ser Leu Ser Pro Gly
290 295
<210> 137
<211> 296
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 137
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
65 70 75 80
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
85 90 95
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
100 105 110
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
115 120 125
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
130 135 140
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
145 150 155 160
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
165 170 175
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
180 185 190
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
195 200 205
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
210 215 220
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
225 230 235 240
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
245 250 255
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
260 265 270
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
275 280 285
Lys Ser Leu Ser Leu Ser Pro Gly
290 295
<210> 138
<211> 300
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 138
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
65 70 75 80
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
85 90 95
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
100 105 110
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
115 120 125
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
130 135 140
Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
145 150 155 160
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
165 170 175
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
180 185 190
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
195 200 205
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
210 215 220
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
225 230 235 240
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
245 250 255
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
260 265 270
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
275 280 285
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
290 295 300
<210> 139
<211> 305
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 139
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp
65 70 75 80
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
85 90 95
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
100 105 110
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
115 120 125
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
130 135 140
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
145 150 155 160
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
165 170 175
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
180 185 190
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
195 200 205
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
210 215 220
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
225 230 235 240
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
245 250 255
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
260 265 270
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
275 280 285
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
290 295 300
Gly
305
<210> 140
<211> 310
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 140
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
50 55 60
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
65 70 75 80
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
85 90 95
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
100 105 110
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
115 120 125
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
130 135 140
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
145 150 155 160
Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
165 170 175
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
180 185 190
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
195 200 205
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
210 215 220
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
225 230 235 240
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
245 250 255
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
260 265 270
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
275 280 285
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
290 295 300
Leu Ser Leu Ser Pro Gly
305 310
<210> 141
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 141
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Xaa Xaa Ala Gln Asp Phe Val Gln Trp Leu Met Asn Xaa Xaa
20 25 30
<210> 142
<211> 274
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 142
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Glu Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser
35 40 45
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
50 55 60
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
65 70 75 80
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
85 90 95
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
100 105 110
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr
115 120 125
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
130 135 140
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
145 150 155 160
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
165 170 175
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
180 185 190
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
195 200 205
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
210 215 220
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
225 230 235 240
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
245 250 255
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
260 265 270
Leu Gly
<210> 143
<211> 181
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 143
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser
180
<210> 144
<211> 181
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 144
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Gly Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Glu Ser
180
<210> 145
<211> 181
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 145
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Cys Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Cys Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Gly Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Glu Ser
180
<210> 146
<211> 177
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 146
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
1 5 10 15
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
20 25 30
Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
35 40 45
Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
50 55 60
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly
65 70 75 80
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu
85 90 95
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu
100 105 110
His Cys Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
115 120 125
Pro Cys Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu
130 135 140
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
145 150 155 160
Pro Leu Ala Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
165 170 175
Ser
<210> 147
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 147
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
465 470 475
<210> 148
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 148
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Arg Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Gly Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser
465 470 475
<210> 149
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 149
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Arg Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Cys
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Cys
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Gly Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser
465 470 475
<210> 150
<211> 475
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 150
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Asp Ser Ser Pro Leu Leu
290 295 300
Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala
305 310 315 320
Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly
325 330 335
Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu
340 345 350
Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu
355 360 365
Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
370 375 380
Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val
385 390 395 400
Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Cys Pro Gly Asn Lys
405 410 415
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Cys Arg Phe Leu Pro
420 425 430
Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala
435 440 445
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ala Met Val Gly
450 455 460
Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
465 470 475
<210> 151
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 151
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
465 470 475
<210> 152
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 152
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Arg Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Gly Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser
465 470 475
<210> 153
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 153
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala His Pro Ile Pro Asp Ser
290 295 300
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
305 310 315 320
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
325 330 335
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
340 345 350
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
355 360 365
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
370 375 380
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Arg Leu Leu Glu Asp
385 390 395 400
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Cys
405 410 415
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Cys
420 425 430
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
435 440 445
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
450 455 460
Ser Met Val Gly Gly Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser
465 470 475
<210> 154
<211> 475
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 154
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ala
275 280 285
Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Asp Ser Ser Pro Leu Leu
290 295 300
Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala
305 310 315 320
Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly
325 330 335
Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu
340 345 350
Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu
355 360 365
Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
370 375 380
Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val
385 390 395 400
Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Cys Pro Gly Asn Lys
405 410 415
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Cys Arg Phe Leu Pro
420 425 430
Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala
435 440 445
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ala Met Val Gly
450 455 460
Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
465 470 475
<210> 155
<211> 146
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 155
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
1 5 10 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
20 25 30
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
65 70 75 80
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
<210> 156
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 156
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Pro Ile Gln Lys
290 295 300
Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile
305 310 315 320
Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr
325 330 335
Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys
340 345 350
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro
355 360 365
Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp
370 375 380
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala
385 390 395 400
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
405 410 415
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu
420 425 430
Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
435 440 445
<210> 157
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 157
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Pro Ile Gln Lys
290 295 300
Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile
305 310 315 320
Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr
325 330 335
Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys
340 345 350
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro
355 360 365
Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp
370 375 380
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala
385 390 395 400
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
405 410 415
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu
420 425 430
Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
435 440 445
<210> 158
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 158
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Pro Ile Gln Lys
290 295 300
Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile
305 310 315 320
Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr
325 330 335
Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys
340 345 350
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro
355 360 365
Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp
370 375 380
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala
385 390 395 400
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
405 410 415
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu
420 425 430
Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
435 440 445
<210> 159
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 159
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Pro Ile Gln Lys
290 295 300
Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile
305 310 315 320
Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr
325 330 335
Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys
340 345 350
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro
355 360 365
Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp
370 375 380
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala
385 390 395 400
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
405 410 415
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu
420 425 430
Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
435 440 445
<210> 160
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 160
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
50 55 60
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
65 70 75 80
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
85 90 95
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
100 105 110
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
115 120 125
Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
130 135 140
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
145 150 155 160
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
165 170 175
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
180 185 190
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
195 200 205
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
210 215 220
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
225 230 235 240
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
245 250 255
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
260 265 270
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Pro Ile Gln Lys
290 295 300
Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile
305 310 315 320
Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr
325 330 335
Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys
340 345 350
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro
355 360 365
Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp
370 375 380
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala
385 390 395 400
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
405 410 415
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu
420 425 430
Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
435 440 445

Claims (19)

1.一种多重活性蛋白，所述多重活蛋白的结构中包括如式I所示结构，如式I所示结构为：A-L-F，式I中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，L为连接所述A与F的连接链。
2.根据权利要求1所述的多重活性蛋白，其特征在于，所述A的结构中包括如式II所示结构，如式II所示结构为：HSQGTFTSDYSKYLDX16X17X1 8AQDFVQWLMNX2 9Xz式II，其中，其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18除选自P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS、GPSSGAPPPS、PSSGAPPPS、SSGAPPPS、GGPSSGAPPS、GPSSGAPPS、PSSGAPPS或SSGAPPS之任一。
3.根据权利要求1所述的多重活性蛋白，其特征在于，还包括以下特征的任一项或多项：(1)所述F为源自哺乳动物免疫球蛋白的FC部分；(2)所述L为富含G、S和/或A的连接链。
4.根据权利要求1所述的多重活性蛋白，其特征在于，还包括以下特征的任一项或多项：(1)所述A的氨基酸酸序列如SEQ ID NO.46、SEQ ID NO.55、SEQ ID NO.59、SEQ IDNO.68、SEQ ID NO.74之任一所示；(2)所述F的氨基酸序列如SEQ ID NO.11～20之任一所示；(3)所述L的氨基酸序列如SEQ ID NO.21～43之任一所示。
5.根据权利要求1所述的多重活性蛋白，其特征在于，所述多重活性蛋白的氨基酸序列如SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.100之任一所示。
6.一种多重活性蛋白，所述多重活蛋白的结构中包括如式III所示结构，如式III所示结构为：A-L1-F-L2-B，其中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，B为天然FGF21或FGF21类似物，L1为连接A与F的连接链，L2不存在或为连接B与F的连接链。
7.根据权利要求6所述的多重活性蛋白，其特征在于，所述A的结构中包括如式II所示结构，如式II所示结构为：HSQGTFTSDYSKYLDX16X17X1 8AQDFVQWLMNX2 9Xz式II，其中，X16选自除Y、N、W、和H外的氨基酸之任一；X17选自除P、L、T、F和H外的氨基酸之任一；X18选自除P、F、H和W外的氨基酸之任一；另外X17与X18不能同时为R，X29为T或缺失，Xz选自GGPSSGAPPPS、GPSSGAPPPS、PSSGAPPPS、SSGAPPPS、GGPSSGAPPS、GPSSGAPPS、PSSGAPPS或SSGAPPS之任一。
8.根据权利要求6所述的还包括以下特征的任一项或多项：(1)所述F为源自哺乳动物免疫球蛋白的FC部分；(2)所述L1为富含G、S和/或A的连接链；(3)所述L2为富含G、S和/或A的连接链。
9.根据权利要求6所述的还包括以下特征的任一项或多项：
(1)所述A的氨基酸酸序列如SEQ ID NO.46、SEQ ID NO.54、SEQ ID NO.55、SEQ IDNO.68之任一所示；(2)所述F的氨基酸序列如SEQ ID NO.11～20之任一所示；(3)所述L1的氨基酸序列如SEQ ID NO.21～43之任一所示；(4)所述L2的氨基酸序列如SEQ ID NO.21～43之任一所示；(5)所述B氨基酸序列如SEQ ID NO.143-146、SEQ ID NO.155之任一所示。
10.根据权利要求6所述的还包括以下特征的任一项或多项：所述多重活性蛋白的氨基酸序列如SEQ ID NO.147-154或SEQ ID NO.156-160之任一所示。
11.一种分离的多核苷酸，所述分离的多核苷酸编码如权利要求1-10之任一项所述多重活性蛋白。
12.一种重组表达载体，包含如权利要求11所述分离的多核苷酸。
13.一种宿主细胞，所述细胞含有如权利要求12所述重组表达载体或基因组中整合有外源的如权利要求11所述分离的多核苷酸。
14.如权利要求1-10之任一项所述多重活性蛋白的制备方法，其特征在于，
在合适的条件下培养如权利要求13所述宿主细胞，使之表达所述多重活性蛋白，而后分离及纯化获得所述多重活性蛋白。
15.如权利要求1-10之任一项所述多重活性蛋白在制备治疗代谢相关疾病的药物中的用途。
16.一种促进体重减少或者防止体重增加的方法，包括在对象中施用如权利要求1-10之任一项所述多重活性蛋白。
17.一种组合物，含有如权利要求1-10之任一项所述多重活性蛋白或如权利要求13所述宿主细胞的培养物，以及药学上可接受的载体。
18.如权利要求1-10之任一项所述多重活性蛋白在制备融合蛋白中的用途。
19.一种融合蛋白，其结构中含有如权利要求1-10之任一项所述多重活性蛋白。
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021083306A1 (zh) * 2019-10-31 2021-05-06 东莞市东阳光生物药研发有限公司 Glp-1/gcg双受体激动剂多肽
WO2021139763A1 (zh) * 2020-01-08 2021-07-15 上海翰森生物医药科技有限公司 Fgf21突变体蛋白及其融合蛋白
WO2021203864A1 (zh) * 2020-04-08 2021-10-14 浙江道尔生物科技有限公司 一种肠促胰岛素类似物及其制备方法和用途
CN115536739A (zh) * 2022-07-04 2022-12-30 北京惠之衡生物科技有限公司 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法
WO2023280144A1 (zh) * 2021-07-05 2023-01-12 上海翰森生物医药科技有限公司 一种融合蛋白及其医药用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113332416B (zh) * 2021-05-17 2022-02-22 宁波大学 丙谷二肽在制备治疗非酒精性脂肪肝药物中的应用
CN113209270B (zh) * 2021-05-17 2022-02-22 宁波大学 丙谷二肽在制备防治急性肝衰竭药物中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (zh) * 2000-12-07 2004-03-17 Glp-1融合蛋白
TW200920395A (en) * 2007-10-30 2009-05-16 Univ Indiana Res & Tech Corp Compounds exhibiting glucagon antagonist and GLP-1 agonist activity
CN101730523A (zh) * 2007-07-10 2010-06-09 伊莱利利公司 GLP-1-Fc融合蛋白质制剂
US20110034373A1 (en) * 2007-08-03 2011-02-10 Eli Lilly And Company Use of an fgf-21 compound and a glp-1 compound for the treatment of obesity
CN106046145A (zh) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Glp-1r/gcgr双靶点激动剂多肽治疗脂肪肝病、高脂血症和动脉硬化
CN106604739A (zh) * 2014-05-30 2017-04-26 韩美药品株式会社 包括胰岛素和glp‑1/胰高血糖素双重激动剂的用于治疗糖尿病的组合物

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2525809B1 (en) * 2010-01-20 2016-08-03 Zealand Pharma A/S Glucagon-glp1 dual agonists for use in the treatment of cardiac conditions
KR102670157B1 (ko) * 2015-10-28 2024-05-29 주식회사유한양행 이중 작용 단백질 및 이를 포함하는 약학적 조성물

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (zh) * 2000-12-07 2004-03-17 Glp-1融合蛋白
CN101730523A (zh) * 2007-07-10 2010-06-09 伊莱利利公司 GLP-1-Fc融合蛋白质制剂
US20110034373A1 (en) * 2007-08-03 2011-02-10 Eli Lilly And Company Use of an fgf-21 compound and a glp-1 compound for the treatment of obesity
TW200920395A (en) * 2007-10-30 2009-05-16 Univ Indiana Res & Tech Corp Compounds exhibiting glucagon antagonist and GLP-1 agonist activity
CN106604739A (zh) * 2014-05-30 2017-04-26 韩美药品株式会社 包括胰岛素和glp‑1/胰高血糖素双重激动剂的用于治疗糖尿病的组合物
CN106046145A (zh) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Glp-1r/gcgr双靶点激动剂多肽治疗脂肪肝病、高脂血症和动脉硬化

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HYE-SHIN CHUNG等: "The N-terminal alanine-extended GLP-1/IgG-Fc fusion protein confers resistance to DPP-IV and reduces serum glucose level in db/db mice", 《REGULATORY PEPTIDES》 *
JAHOON KANG等: "The ultra-long acting LAPSGLP/GCG dual agonist, HM12525A, demonstrated safety and prolonged pharmacokinetics in healthy volunteers: a phase 1 first-in-human study", 《51ST EUROPEAN ASSOCIATION FOR THE STUDY OF DIABETES (EASD)》 *
M KUMAR等: "Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose level in db/db mice", 《GENE THERAPY》 *
WOLFGANG GLAESNER等: "Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein", 《DIABETES/METABOLISM RESEARCH AND REVIEWS》 *
杨懿等: "长效GLP-1-IgG2σ Fc融合蛋白的真核表达与生物活性鉴定", 《生物技术通讯》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021083306A1 (zh) * 2019-10-31 2021-05-06 东莞市东阳光生物药研发有限公司 Glp-1/gcg双受体激动剂多肽
WO2021139763A1 (zh) * 2020-01-08 2021-07-15 上海翰森生物医药科技有限公司 Fgf21突变体蛋白及其融合蛋白
CN113366024A (zh) * 2020-01-08 2021-09-07 上海翰森生物医药科技有限公司 Fgf21突变体蛋白及其融合蛋白
WO2021203864A1 (zh) * 2020-04-08 2021-10-14 浙江道尔生物科技有限公司 一种肠促胰岛素类似物及其制备方法和用途
WO2023280144A1 (zh) * 2021-07-05 2023-01-12 上海翰森生物医药科技有限公司 一种融合蛋白及其医药用途
CN115536739A (zh) * 2022-07-04 2022-12-30 北京惠之衡生物科技有限公司 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法

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