CN101195822A - 高亲和性靶向融合蛋白 - Google Patents
高亲和性靶向融合蛋白 Download PDFInfo
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- CN101195822A CN101195822A CNA200610162005XA CN200610162005A CN101195822A CN 101195822 A CN101195822 A CN 101195822A CN A200610162005X A CNA200610162005X A CN A200610162005XA CN 200610162005 A CN200610162005 A CN 200610162005A CN 101195822 A CN101195822 A CN 101195822A
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Abstract
本发明主要涉及由人促黄体生成激素释放因子衍生物(GnRH)和假单胞菌外毒素A的缺失体PE40或PE38及其突变体串联组成的融合蛋白，本发明重点对LHRH的N末端5，7，8位氨基酸进行了突变，使得突变后的融合蛋白与已知的该类靶向融合蛋白相比具有更强的肿瘤细胞亲和性和特异杀伤性。
Description
一.技术领域
本发明属于生物基因工程中的导向融合蛋白领域，目前癌症仍然是死亡率位居第二的疾病，目前人们还没有特别有效的手段来进行治疗，化疗仍然是目前最常用的治疗方法，虽然有一定的疗效，但是其副作用大，治疗效果只有部分理想，治疗时容易产生耐药株癌细胞等缺点影响了这种方法的疗效和有效率。
导向治疗是一种有望代替化疗的新的治疗手段，有多篇文献和专利(中国专利：应用嵌合毒素诊断癌症的方法，99806580.3；中国专利：一种能特异杀死肿瘤细胞的基因工程重组蛋白，03137587.1；中国专利：高效低毒的系列功能蛋白，200410033621.6)报道了关于使用人促黄体激素释放因子及其衍生物作为靶向融合蛋白的导向部分，但是其存在着与GnRH受体亲和力低，靶向性差，从而存在非特异性毒性高的特点。本发明通过选用人促黄体激素释放因子衍生物作为导向，选用PE40和PE38及其的KDEL衍生物作为毒素部分，得到了与现有已知的人促黄体生成激素释放因子-假单胞杆菌毒素A(即GnRH-PE)类靶向融合分子相比，受体亲和性特异性和活性大大提高的基因工程融合蛋白，在肿瘤治疗方面有很高的应用价值。
二.背景技术
目前癌症的治疗仍然是一个难题，人们应用的主要的治疗手段仍然是手术，放疗和化疗，传统的治疗法在带来疗效的同时，副作用也往往很大，并且对于复发的肿瘤治疗效果更加有限。传统的疗法的缺点主要在于其在杀死肿瘤细胞的同时也大量地杀死了正常细胞，导向治疗的出现使我们有可能对肿瘤细胞实现导向治疗，即药品分子可以导向地到达肿瘤细胞并发挥作用，达到导向治疗的效果。目前免疫毒素或导向毒素是达到导向治疗的一个重要的发展方向，通过特异性抗体或细胞因子、激素等与毒素连接，前者作为导向部分，即配体，可以引导分子到达肿瘤部位并与肿瘤细胞表面的过量表达的该种受体结合，后者作为毒素部分，即杀灭肿瘤细胞的部分。目前以GnRH或其衍生物为导向部分，PE40及其衍生物为毒素部分的靶向治疗剂已经有报道(中国专利：应用嵌合毒素诊断癌症的方法，99806580.3；中国专利：一种能特异杀死肿瘤细胞的基因工程重组蛋白，03137587.1；中国专利：高效低毒的系列功能蛋白，200410033621.6)，但其存在与GnRH受体的亲和性不强，稳定性差的缺点，导致了其作为肿瘤的治疗剂具有特异性差，非特异性毒性强的特点，所以目前该类分子还无法作为有效的肿瘤治疗药物进行应用。
三.发明内容：
本发明涉及一种GnRH-PE本发明人通过对已知GnRH-PE融合蛋白进行蛋白质三级结构分析，采用基因工程方法对所述蛋白质的N-末端作为导向的GnRH加以修饰，通过大量的筛选，出人意料的获得了在靶向性方面明显优于现有GnRH-PE衍生物的新的GnRH-PE衍生物。
本发明中的GnRH-PE衍生物包括将天然人促黄体生成激素释放因子的第5位氨基酸突变为His，第6位突变为Trp，第7位突变为Trp，第8位突变为Leu所得到的(His5，Trp6，Trp7，Leu8)GnRH-PE衍生物和将天然人促黄体生成激素释放因子的第五位氨基酸突变为His，第7位突变为Trp，第8位突变为Leu所得到的(His5，Trp7，Leu8)GnRH-PE衍生物，这两种衍生物具有高亲和性，高特异性的特点。
通过上述发明得到的靶向融合蛋白具有高活性，受体高亲和性和高特异性的特点，对肿瘤细胞具有特异性杀伤作用，是潜在的肿瘤治疗药物。
本发明的一个方面，涉及一种GnRH-PE衍生物，其分别为SEQ ID NO：1所示的(His5，Trp6，Trp7，Leu8)GnRH-PE40，SEQ ID NO：2所示的(His5，Trp6，Trp7，Leu8)GnRH-PE38，NO：3所示的(His5，Trp6，Trp7，Leu8)GnRH-PE40KDEL，NO：4所示的(His5，Trp6，Trp7，Leu8)GnRH-PE38KDEL，以及SEQ ID NO：5所示的(His5，Trp7，Leu8)GnRH-PE40，SEQ ID NO：6所示的(His5，Trp7，Leu8)GnRH-PE38，NO：7所示的(His5，Trp7，Leu8)GnRH-PE40KDEL，和NO：8所示的(His5，Trp7，Leu8)GnRH-PE38KDEL。
本领域普通技术人员知晓，所述上述融合蛋白可以通过基因重组表达的方法或者化学合成的方法制备。在本发明中，所述GnRH-PE衍生物是通过基因工程重组表达的方法获得的。
在本发明的一个实施方案中，通过如下方法获得GnRH-PE衍生物，包括如下步骤：
1)表达载体的构建
依据已知氨基酸序列(SEQ ID NO：1)，选用大肠杆菌偏好密码子全基因合成基因序列，同时在相应于所编码的氨基酸序列之N末端和C末端加上限制性酶切位点。
应用PCR的方法对SEQ ID NO：1的基因序列进行N-末端突变和C末端突变，得到编码SEQ ID NO：2-8的融合蛋白的核酸序列。
再分别将如上述制备的GnRH-PE衍生物序列以及筛选质粒pBSK用限制性内切酶处理好，经计算各样品浓度后按一定摩尔比加入T4连接体系。经转化、筛选成功的重组子。
用限制性酶切下融合蛋白基因，再将该基因与用限制性酶处理好的NOVAGEN公司质粒pET21b连接，经筛选得到正确地重组子。
2)重组靶向融合蛋白的表达及分离纯化
将如上述所得重组子转化到表达宿主菌，经表达筛选出高表达基因工程菌，然后经发酵，得到目的蛋白高表达的工程菌。使用渗透压方法破碎细菌，然后经过离心、澄清的步骤，通过一系列常用的层析方法，如离子交换，凝胶过滤和疏水层析等，纯化最终得到纯度大于95％的目的蛋白。
3)目的蛋白生物活性的测定：用MTT法，首先对Hela细胞进行消化和收集，将细胞用RPMI1640培养液稀释成6-8×104个/毫升的细胞悬液，接种于96孔板，100ul/孔，37度培养4小时。对照加入100ul培养液。样品先用RPMI 1640培养液稀释成100ul/mL，作为起始孔，然后按每孔与上孔2.5倍的关系稀释样品，各加入稀释的样品每孔100ul，37度，培养24小时，取出，用MTT法染色，用酶标仪570nm进行测定并计算IC50值。
4)目的蛋白与GnRH受体的亲和性测定：
制备胎盘质膜，取胎盘绒毛，于冰冷的25mM PB，pH7.4，1mM MgCl2中匀浆，过滤，并且10000g，离心20min，去上清，沉淀用上述缓冲液洗一次，再10000g，离心15min，沉淀按湿重60mg/mL的比例悬浮于上述缓冲液中备用，用Lowry法定蛋白浓度。靶向融合蛋白的碘标记：在1.5mL的doff管中加入3.8×107Bq Na125I，20uL0.5M PB，pH7.5，500ug靶向融合蛋白，2.3mU乳过氧化氢酶β-D(+)-葡萄糖开始反应，20℃反应10min，然后加入50uL0.1M硼酸缓冲液pH9.2终止反应。然后使用1mL的Q Sepharose High Performance进行层析纯化。结合试验：聚丙烯管内加入100uL已标记的GnRH-PE40和100uL已标记的靶向融合蛋白，100uL 25mM PB，pH7.4，1mM MgCl2，0.2％BSA，100uL胎盘绒毛质膜液，20℃水浴震荡1小时，加入2mL冰冷的10mM PB，pH7.5缓冲液终止反应。然后用GF/C滤片过滤，并用上述冰冷磷酸缓冲液2mL洗反应管和滤片2次，最后滤片在γ-计数器上测定并作出竞争曲线，求出靶向融合蛋白与GnRH-PE40竞争50％绒毛质膜GnRH受体时的浓度，即IC50。
本发明的另一方面，涉及一种药物组合物，其含有本发明所述的GnRH-PE衍生物，以及任选的药物上可接受的载体。
在本发明知，术语“药物上可接受的”意味着制药领域公认的可用于动物，更特别的是可用于人的。术语“载体”指稀释剂、佐剂(例如(完全或不完全)弗氏佐剂)、赋形剂、或用于容纳或施用治疗剂的介质。
这些药用载体可以是无菌液体，诸如水和油，包括源自石油、动物、植物、或合成的油，诸如花生油、大豆油、矿物油、芝麻油、诸如此类。静脉内施用药用组合物时，水是优选的载体。盐水溶液以及水性右旋糖和甘油溶液也可用作液态载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油、滑石、氯化钠、奶粉、甘油、丙烯、乙二醇、水、乙醇、诸如此类。如果需要，组合物还可以包含较少数量的润湿或乳化剂如透明质酸钠，或是pH缓冲剂。这些组合物可以采取溶液、悬浮液、乳状液、片剂、丸剂、胶囊粉剂、缓释配方、诸如此类的形式。
在本发明的一个实施方案中，所述药物组合物为冻干注射剂形式，其中含有0.01％-0.2％的GnRH-PE衍生物，以及5％的甘露醇，以及药物上可接受的载体。
将本发明的药用组合物配制成与其意向施用路径相容。施用路径的实例包括但不限于肠胃外，例如静脉内、皮内、皮下、口腔、鼻内(例如吸入)、经皮(例如局部)、经粘膜、和直肠施用。在一个具体实施方案中，依照常规流程将组合物配制成适合静脉内、皮下、肌肉内、口腔、鼻内、或局部施用于人类的药用组合物。通常，用于静脉内施用的组合物是无菌等渗水性缓冲液中的溶液。如果需要，组合物还可以包含增溶剂和局部麻醉剂诸如麦角胺以减轻注射部位的疼痛。
若要局部施用本发明的组合物，则可以将组合物配制成软膏、乳膏、皮肤贴、洗剂、凝胶、洗发剂、喷雾剂、气雾剂、溶液、乳液的形式或是本领域技术人员众所周知的其它形式。参阅例如《雷明顿氏药物科学和药物剂型导论》(Remington′s Pharmaceutical Sciencesand Introduction to Pharmaceutical Dosage Forms)，第19版，Mack出版公司，伊斯顿，宾夕法尼亚，1995年。
对于非可喷雾局部剂量形式，通常采用包含与局部应用相容的载体或一种或多种赋形剂且具有优选大于水的动态粘滞度的粘性至半固体或固体形式。合适的配方包括但不限于溶液、悬浮液、乳状液、乳膏、软膏、粉剂、搽剂、药膏、诸如此类，如果需要，是无菌的或与辅助试剂(例如防腐剂、稳定剂、润湿剂缓冲剂、或盐)混和以改变各种特性，诸如例如渗透压。其它合适的局部剂量形式包括可喷雾气雾剂制剂，其中优选联合固相或液相惰性载体的活性成分包装在含有增压挥发物(例如压缩气体，诸如氟利昂)的混合物或是挤瓶中。如果需要，还可以向药物组合物和剂量形式中添加增湿剂或湿润剂。这些额外成分的实例在本领域是众所周知的。
在本发明的又一实施方案中，所述药物组合物为软膏形式，其中含有0.01％-0.2％的GnRH-PE衍生物，和药物上可接受的赋形剂如凡士林。
若本发明的方法包括组合物的鼻内施用，则组合物可以配制成气雾剂、喷雾剂、轻雾剂、或滴剂的形式。具体而言，依照本发明使用的预防性或治疗性试剂可以使用合适的推进剂(例如二氯二氟甲烷、三氯单氟甲烷、二氯四氟乙烷、二氧化碳、或其它合适其它)以气雾剂喷雾呈现的形式由增压包装或喷雾器进行方便的投递。在增压气雾剂的情况中，可以通过提供阀门来确定剂量单位，以投递计量数量。吸入器或吹入器中所使用的胶囊和药筒(由例如明胶构成)可以配制成含有化合物及合适粉末基质诸如乳糖或淀粉的粉末混合物。
在本发明的又一实施方案中，所述药物组合物为喷剂，其中含有5-500μg/ml的GnRH-PE衍生物，以及0.003％的尼泊金乙酯，和药物上可接受的载体。
若本发明的方法包括口服施用，则组合物可以配制成片剂、胶囊、药包、软胶囊、溶液、悬浮液、诸如此类的形式。可以通过常规手段用药用可接受赋形剂诸如粘合剂(例如预胶凝玉米淀粉、聚乙烯吡咯烷酮、或羟丙基甲基纤维素)；填料(例如乳糖、微晶纤维素、或磷酸氢钙)；润滑剂(例如硬脂酸镁、滑石、或硅石)；分解质(例如马铃薯淀粉或淀粉乙醇酸钠)；或润湿剂(例如月桂醇硫酸钠)片剂或胶囊。可以通过本领域众所周知的方法包被片剂。用于口腔施用的液体制剂可以采取但不限于溶液、糖浆、或悬浮液的形式，或者它们可以以干燥产品的形式存在，使用前用水或其它合适介质溶解。可以通过常规手段用药用可接受添加剂，诸如悬浮剂(例如山梨醇糖浆、纤维素衍生物、或氢化食用脂肪)；乳化剂(例如卵磷脂或阿拉伯树胶)；非水性介质(例如杏仁油、油性酯、乙醇、或分馏植物油)；以及防腐剂(例如甲基或丙基-对-羟基苯甲酸酯或山梨酸)制备这些液体制剂。制剂还可以适当包含缓冲盐、芳香剂、着色剂、和甜味剂。可以适当配制用于口腔施用的制剂以缓慢释放、受控释放、或持续释放预防性或治疗性试剂。
本发明的方法还包括配制用于通过注射(例如通过推注或连续输液)肠胃外施用的组合物的施用。用于注射的配方可以以含有额外防腐剂的单位剂量形式(例如在安瓿中或在多剂量容器中)存在。组合物可以采取诸如油性或水性介质中的悬浮液、溶液、或乳状液等形式，而且可以含有配制剂，诸如悬浮剂、稳定剂、和/或分散剂。或者，活性成分可以是粉末形式，在使用前用合适介质(例如无菌、无热原水)溶解。
可用于提供本发明肠胃外剂量形式的合适介质对于本领域技术人员而言是众所周知的。在某些实施方案中，适于肠胃外剂量形式的介质包括但不限于注射用水USP；水性介质包括但不限于氯化钠注射液、Ringer氏注射液、葡萄糖注射液、葡萄糖和氯化钠注射液、以及乳酸化Ringer氏注射液；水易混介质包括但不限于乙醇、聚乙二醇、和聚丙二醇；以及非水性介质包括但不限于玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯、和苯甲酸苯甲酯。
本发明的再一方面，涉及选自GnRH-PE衍生物用于制备治疗肿瘤细胞表面表达GnRH受体的癌症患者的药物的用途。
本发明人出人意料地发现，在通过模式动物实验研究对肿瘤的体内靶向性杀伤的活性方面，本发明的GnRH-PE衍生物均具有明显高于目前已知GnRH-PE衍生物的活性。
本发明人首先研究了GnRH-PE衍生物对体内肿瘤杀伤的作用。研究人员将120只裸鼠接种上A549肺癌细胞，从而得到肺癌裸小鼠模型。隔天注射(His5，Trp7，Leu8)GnRH-PE40KDEL共10次的裸鼠肿瘤块减小最明显，抑瘤率最高，治疗效果明显优于(Ala6)GnRH-PE40KDEL以及目前已知的GnRH-PE衍生物。
上述结果表明，本发明的GnRH-PE衍生物在治疗肿瘤方面活性明显高于目前已知的GnRH-PE衍生物。
本领域普通技术人员知晓，施用的方式、频率和剂量将根据所治疗的病症、病况和个体的不同而异。一般来说，可以通过注射(例如皮内、肌内、静脉内或皮下)、局部施用(例如表皮施用)或滴加施用(例如滴眼剂)等方式施用。也可以根据患者个体的不同选择合理的施用途径和施用方案。合适的剂量为当施用上述的药物组合物后能够有效治疗肿瘤细胞表面表达GnRH受体的癌症患者的量。
一般来说，对于含有本发明所述的含有GnRH-PE衍生物的药物组合物，存在于每一个剂量中的量大约为100μg-5mg。合适的剂量的多少将因患者病症以及给药方式而异，但一般大约范围为0.5mg-2mg，优选1mg。
四.具体实施方式：
下面的实例目的在于进一步阐述本发明，而不会构成对本发明专利待批权利要求的限制。
实例一.
根据大肠杆菌偏爱密码子的原则，通过全基因合成的方法结合PCR的方法合成出SEQ IDNO：1基因序列，然后通过PCR突变缺失的方法得到SEQ ID NO：2-8以及目前已知的GnRH-PE衍生物，三种对照试验融合蛋白GnRH-PE40(SEQ ID NO：9)，(Ala6)GnRH-PE40(SEQ IDNO：10)和(Ala6)GnRH-PE40KDEL(SEQ ID NO：11)的基因序列。经过测序验证后，将目的基因连接入表达载体，将验证后的重组质粒转导入到宿主菌中，经过表达、发酵得到目的菌体，然后通过渗透压法破菌后，离心取上清，通过疏水层析和离子交换层析的方法纯化得到纯度大于95％的8种本发明中的靶向融合蛋白以及三种对照试验融合蛋白GnRH-PE40，(Ala6)GnRH-PE40和(Ala6)GnRH-PE40KDEL纯品，然后分别同时进行细胞活性试验，亲和性试验和药效学试验。
实例二.
取八种本发明中的靶向融合蛋白以及三种对照试验融合蛋白进行细胞活性试验：用MTT法，首先对Hela细胞进行消化和收集，将细胞用RPMI 1640培养液稀释成6-8×104个/毫升的细胞悬液，接种于96孔板，100ul/孔，37度培养4小时。对照加入100ul培养液。11种样品先用RPMI 1640培养液稀释成100ul/mL，作为起始孔，然后按每孔与上孔2.5倍的关系稀释样品，各加入稀释的样品每孔100ul，37度，培养24小时，取出，用MTT法染色，用酶标仪570nm进行测定并计算IC50值。活性测定的结果见下表，
实例三
取八种本发明中的靶向融合蛋白以及三种对照试验融合蛋白进行亲和性试验(目的蛋白与GnRH受体的亲和性力测定)：制备胎盘质膜，取胎盘绒毛，于冰冷的25mM PB，pH7.4，1mMMgCl2中匀浆，过滤，并且10000g，离心20min，去上清，沉淀用上述缓冲液洗一次，再10000g，离心15min，沉淀按湿重60mg/mL的比例悬浮于上述缓冲液中备用，用Lowry法定蛋白浓度。靶向融合蛋白的碘标记：在1.5mL的doff管中加入3.8×107Bq Na125I，20uL0.5M PB，pH7.5，500ug靶向融合蛋白，2.3mU乳过氧化氢酶β-D(+)-葡萄糖开始反应，20℃反应10min，然后加入50uL 0.1M硼酸缓冲液pH9.2终止反应。然后使用1mL的Q Sepharose High Performance进行层析纯化。结合试验：聚丙烯管内加入100uL已标记的GnRH-PE40和100uL已标记的靶向融合蛋白，100uL 25mM PB，pH7.4，1mM MgCl2，0.2％BSA，100uL胎盘绒毛质膜液，20℃水浴震荡1小时，加入2mL冰冷的10mM PB，pH7.5缓冲液终止反应。然后用GF/C滤片过滤，并用上述冰冷磷酸缓冲液2mL洗反应管和滤片2次，最后滤片在γ-计数器上测定并作出竞争曲线，求出靶向融合蛋白与GnRH-PE40竞争50％绒毛质膜GnRH受体时的浓度，即IC50。亲和性试验结果见下表：
| 融合蛋白 | IC50(nmol/L) |
| GnRH-PE40 | 220.1 |
| (Ala6)GnRH-PE40 | 150.2 |
| (Ala6)GnRH-PE40KDEL | 162.6 |
| (His5，Trp6，Trp7，Leu8)GnRH-PE40 | 54.3 |
| (His5，Trp6，Trp7，Leu8)GnRH-PE38 | 43.9 |
| (His5，Trp6，Trp7，Leu8)GnRH-PE40KDEL | 32.8 |
| (His5，Trp6，Trp7，Leu8)GnRH-PE38KDEL | 48.9 |
| (His5，Trp7，Leu8)GnRH-PE40 | 49.3 |
| (His5，Trp7，Leu8)GnRH-PE38 | 38.7 |
| (His5，Trp7，Leu8)GnRH-PE40KDEL | 32.6 |
| (His5，Trp7，Leu8)GnRH-PE38KDEL | 50.5 |
实例四：
体内的药效试验：120只雄性BALB/C裸鼠(SPF级)接种上A549肺癌细胞，从而得到肺癌裸小鼠模型。随机分为12组，每组10只裸鼠，分别为生理盐水组，GnRH-PE衍生物SEQID NO：1-8组和GnRH-PE衍生物SEQ ID NO：9-11组，接种后7天起，隔一天静脉注射75ug/kg，共给药10次，每4天测瘤块的长径(a)、短径(b)，根据公式V＝ab2/2计算肿瘤体积(mm3)，接种后27天处死动物，解剖取瘤块，称瘤重，计算抑瘤率。
试验结果：
| 融合蛋白 | 抑瘤率 |
| GnRH-PE40 | 32.3％ |
| (Ala6)GnRH-PE40 | 37.2％ |
| (Ala6)GnRH-PE40KDEL | 42.9％ |
| (His5，Trp6，Trp7，Leu8)GnRH-PE40 | 53.8％ |
| (His5，Trp6，Trp7，Leu8)GnRH-PE38 | 57.5％ |
| (His5，Trp6，Trp7，Leu8)GnRH-PE40KDEL | 72.5％ |
| (His5，Trp6，Trp7，Leu8)GnRH-PE38KDEL | 66.4％ |
| (His5，Trp7，Leu8)GnRH-PE40 | 45.8％ |
| (His5，Trp7，Leu8)GnRH-PE38 | 56.1％ |
| (His5，Trp7，Leu8)GnRH-PE40KDEL | 75.5％ |
| (His5，Trp7，Leu8)GnRH-PE38KDEL | 61.8％ |
结论：从以上结果可以看出，本发明中的8种GnRH-PE衍生物(SEQ ID NO：1-8)的体外细胞活性、与GnRH受体的亲和性以及体内药效学的抑瘤活性都显著高于目前已知的GnRH-PE衍生物(SEQ ID NO：9-11)，说明本发明通过对靶向融合蛋白GnRH部分的突变增强了靶向融合蛋白分子对肿瘤细胞的GnRH受体亲和力和识别力，从而提高了抑瘤率。
序列表
SEQ ID NO：1
Met-Glu-His-Trp-Ser-His-Trp-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：2
Met-Glu-His-Trp-Ser-His-Trp-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：3
Met-Glu-His-Trp-Ser-His-Trp-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
SEQ ID NO：4
Met-Glu-His-Trp-Ser-His-Trp-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Lys-Asp-Glu-Leu
SEQ ID NO：5
Met-Glu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：6
Met-Glu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：7
Met-Glu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
SEQ ID NO：8
Met-Glu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Lys-Asp-Glu-Leu
SEQ ID NO：9
Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Ieu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：10
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
SEQ ID NO：11
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu -Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
Claims (7)
1.一种基因工程重组融合蛋白，其特征是由人促黄体生成激素释放因子衍生物(GnRH)和假单胞菌外毒素A的缺失体PE40或PE38及其突变体串联组成的融合蛋白，上述发明的融合蛋白与已知的该类靶向融合蛋白相比具有更强的肿瘤细胞亲和性和特异杀伤性。
2.根据权利要求1，所说的基因工程重组融合蛋白中的人促黄体生成激素释放因子衍生物的特征是：将天然人促黄体生成激素释放因子的第5位氨基酸突变为His，第6位突变为Trp，第7位突变为Trp，第8位突变为Leu所得到的衍生物。
3.根据权利要求1，所说的基因工程重组融合蛋白中的人促黄体生成激素释放因子衍生物的特征是：将天然人促黄体生成激素释放因子的第五位氨基酸突变为His，第7位突变为Trp，第8位突变为Leu所得到的衍生物。
4.根据权利要求1，所述的基因工程重组融合蛋白PE40部分的突变体是指将PE40 C末端5个氨基酸(REDLK)突变为KDEL。
5.根据权利要求1，所述的基因工程重组融合蛋白PE38部分的突变体是指将PE38 C末端5个氨基酸(REDLK)突变为KDEL。
6.权利要求1中的融合蛋白是使用大肠杆菌、酵母或真核细胞为宿主菌，应用基因工程的方法进行表达、纯化得到的。
7.权利要求1中所述的基因工程重组融合蛋白为组合物成分，用于治疗细胞表面过量表达GnRH受体的癌症。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010022639A1 (zh) * | 2008-08-25 | 2010-03-04 | 北京博翱泰生物技术有限公司 | 靶特异性双突变体融合蛋白质 |
| CN102321178A (zh) * | 2011-05-10 | 2012-01-18 | 北京易德新奥生物科技有限公司 | 一种新的促性腺激素释放激素导向融合蛋白突变体 |
| CN102516395A (zh) * | 2011-12-22 | 2012-06-27 | 中国医学科学院生物医学工程研究所 | 提高药物/基因的靶向性和转染效率的多肽载体及用途 |
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| US6140066A (en) * | 1998-03-24 | 2000-10-31 | Lorberboum-Galski; Haya | Methods of cancer diagnosis using a chimeric toxin |
| CN1294149C (zh) * | 2003-06-18 | 2007-01-10 | 北京诺思兰德生物技术有限责任公司 | 一种能特异杀死肿瘤细胞的基因工程重组蛋白 |
| CN1768862A (zh) * | 2004-10-26 | 2006-05-10 | 北京诺思兰德生物技术有限责任公司 | 一类能与表达GnRH受体的癌细胞特异结合且用锝－99m标记的蛋白 |
| CN1846785A (zh) * | 2005-04-05 | 2006-10-18 | 北京诺思兰德生物技术有限责任公司 | 含有基因工程融合蛋白的冻干组合物 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010022639A1 (zh) * | 2008-08-25 | 2010-03-04 | 北京博翱泰生物技术有限公司 | 靶特异性双突变体融合蛋白质 |
| CN102321178A (zh) * | 2011-05-10 | 2012-01-18 | 北京易德新奥生物科技有限公司 | 一种新的促性腺激素释放激素导向融合蛋白突变体 |
| CN102321178B (zh) * | 2011-05-10 | 2013-07-31 | 北京易德新奥生物科技有限公司 | 一种新的促性腺激素释放激素导向融合蛋白突变体 |
| CN102516395A (zh) * | 2011-12-22 | 2012-06-27 | 中国医学科学院生物医学工程研究所 | 提高药物/基因的靶向性和转染效率的多肽载体及用途 |
| CN102516395B (zh) * | 2011-12-22 | 2013-10-23 | 中国医学科学院生物医学工程研究所 | 提高药物/基因的靶向性和转染效率的多肽载体及用途 |
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