CN108623691B - IgG样长效免疫融合蛋白及其应用 - Google Patents

IgG样长效免疫融合蛋白及其应用 Download PDF

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CN108623691B
CN108623691B CN201710159592.5A CN201710159592A CN108623691B CN 108623691 B CN108623691 B CN 108623691B CN 201710159592 A CN201710159592 A CN 201710159592A CN 108623691 B CN108623691 B CN 108623691B
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胡品良
何芸
邹敬
洪伟东
宋凌云
杨泽荣
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Abstract

本发明公开了一种IgG样长效免疫融合蛋白及其应用。本发明所提供的IgG样长效免疫融合蛋白由效应分子和IgG抗体恒定区组成，所述效应分子通过连接肽与所述IgG抗体恒定区相连；所述效应分子为能在体内发挥生理学功能的蛋白；所述IgG抗体恒定区为IgG抗体去除两个重链可变区和两个轻链可变区后的结构。本发明所提供的IgG样免疫融合蛋白，其在保证了与靶向分子具有较高亲和力以及具有较好体内活性的前提下，可以有效延长蛋白药物(效应分子)的生物半衰期，远远好于于同类的Fc免疫融合蛋白。本发明所提供的这种IgG样长效免疫融合蛋白可用于糖尿病、肿瘤、自身免疫性疾病和内分泌等多种疾病的治疗。

Description

IgG样长效免疫融合蛋白及其应用
技术领域
本发明属于生物技术领域，涉及一种IgG样长效免疫融合蛋白及其应用。
背景技术
重组蛋白药物是生物技术药物中很重要的一类，临床上一般通过静脉和皮下注射给药。经静脉和皮下注射后常伴有蛋白质降解，导致活性降低，生物利用度低；分子量较小者(小于60kD)甚至可以通过肾小球过滤到尿中，因而要达到需要的血药浓度和治疗效果需要反复给药，有的需要一天注射2-3次，不仅给患者带来不便，降低患者顺应性，从而影响疗效。因此临床上需要研制长效的重组蛋白药物。目前延长蛋白药物半衰期的方法主要有：
构建突变体通过构建突变体延长蛋白药物半衰期，常用方法有：1、增加蛋白药物的糖基化程度，使在蛋白药物表面形成保护，，阻碍了蛋白酶对蛋白药物的降解作用，增加蛋白质稳定性，同时使蛋白药物分子量增大，减少了肾小球滤过。已经研制成功并上市的药物如重组人EPO突变体(Amgen公司的Aranesp)和重组人胰岛素的突变体(Aventis公司的Lantus)。Aranesp采用定点突变技术，N连接的寡糖链从原来的3条增加到5条，从而使分子量从原来的30kDa增加到50kDa，半衰期由原来的4-13h延长到平均49h。2、通过形成缓释的微沉淀物，使释放游离型药物的时间延长。Lantus是从大肠杆菌K12株表达的重组人胰岛素的突变体，在人胰岛素A链第21个位点将Asp突变成Gly；在B链碳端最后第30个位点加两个Arg，使胰岛素等电点PI由原来的pH4.0变为pH6.7。这种突变使其在酸性环境下为完全澄清溶液，一旦注入皮下组织(pH值提高)则因为等电点特性，变成不溶的微沉淀物，可持续释放，与重组人胰岛素皮下注射后4-8h的持续时间相比，体内浓度相对恒定24小时以上。
PEG化修饰PEG化(PEGylation)，即聚乙二醇(polyethylene,PEG)共价修饰蛋白质。PEG化蛋白药物主要通过增加蛋白质的分子量，降低药物从肾小管过滤，并且PEG可以作为屏障挡住蛋白质分子表面的抗原决定簇，减少免疫原性，减少体内清除率，PEG的屏障作用还可以保护蛋白质不易被蛋白酶水解，PEG化的这些特点均有利于延长蛋白药物的半衰期。目前已上市的PEG化的蛋白药物如PEG化的天冬酰胺酶(Enzon公司的Oncaspar)、PEG化的IFNα2b(Schering公司的PEGIntron)、PEG化的IFNα2a(Roche公司的Pegasys)和PEG化的G-CSF(Amgen公司的Neulasta)。
血清白蛋白融合人血清，白蛋白(human serum albumin,HSA)是有585个氨基酸残基的单链无糖基化的球形蛋白质，分子量65kDa，大多数治疗用的蛋白和多肽半衰期一般较短，而HSA有将近长达19天的血清半衰期。临床试验结果表明，Albuferonα(HSA/IFNα融合蛋白)平均半衰期148小时，比Pegasys平均半衰期80小时(50-140小时)和PEGIntron平均半衰期40小时(22-60小时)更长。Albutropin是由HSA基因和重组人生长激素(rHGH)基因融合表达的蛋白。在猴子中，皮下注射albutropin(0.3mg/kg)和HGH(0.3mg/kg)相比，半衰期延长6倍，清除率慢8倍。
人Fc制备的融合蛋白。白蛋白融合蛋白和Fc制备的融合蛋白的主要通过二种机制提高药物在体内的生物半衰期，一个是增加其分子量的大小，使其不易被肾小管过滤出去；另一个是通过与新生儿Fc受体(neonatal Fc receptor,FcRn)的结合保护目标蛋白不轻易在血中清除(Rath T,Baker K,Dumont JA,et al.Crit Rev Biotechnol.2015Jun；35(2):235-54)。FcRn受体由fcgrt基因编码，也被称为Brambell受体，是通过非共价键与β2m微球蛋白形成异源二聚体的MHC I类样的受体分子。FcRn受体与白蛋白和免疫球蛋白的结合有严格的pH依赖性，FcRn在生理条件下(pH7.4)不与白蛋白和免疫球蛋白结合。但当白蛋白和免疫球蛋白通过胞饮途径内吞到细胞内，在酸性的溶酶体内(pH6.0-6.5)，FcRn与白蛋白和免疫球蛋白的亲和力提高，形成复合物，FcRn保护白蛋白和免疫球蛋白不被降解，并通过膜质循环，将白蛋白和免疫球蛋白再次释放到胞外。白蛋白和免疫球蛋白与FcRn结合可保护它们被代谢掉，延长二者的生物半衰期。目前，上市的白蛋白和免疫球蛋白产品有abatacept、aflibercept、belatacept、conbercept(四川康弘)、dulaglutide、etanercept和albutrepenonacog alfa等，其中etanercept和aflibercept为重磅炸弹级药物，2016年etanercept全球销售额达88.74亿美元，aflibercept达50亿美元，conbercept(四川康弘)在中国销售额有望突破5亿人民币。然而，这些Fc免疫融合蛋白具有一个普遍的缺点，那就是生物半衰期较短。
发明内容
为了有效解决现有延长蛋白药物生物半衰期方法的不足，本发明设计了IgG样免疫融合蛋白，其可以有效延长蛋白药物(效应分子)的生物半衰期，远远好于于同类的Fc免疫融合蛋白。
本发明所提供的IgG样免疫融合蛋白，具体是由效应分子和IgG抗体恒定区组成，所述效应分子通过连接肽与所述IgG抗体恒定区相连；所述效应分子为能在体内发挥生理学功能的蛋白；所述IgG抗体恒定区为IgG抗体去除两个重链可变区和两个轻链可变区后的结构。
即，所述IgG样免疫融合蛋白，与免疫球蛋白一样，是一类由两条轻链和两条重链组成的四肽链结构，重链之间和重、轻链之间由二硫键连接，其构象与英文大写字母Y形状类似。
轻链型别可为κ和λ型，包含轻链的恒定区(CL)。
重链类别可为IgG1，IgG2和IgG4，包含重链的恒定区(CH1、CH2和CH3)。
其中，所述连接肽的氨基酸序列可选自如下中任一：AKTTPKLEEGEFSEAR(序列1)；AKTTPKLEEGEFSEARV(序列2)；AKTTPKLGG(序列3)；SAKTTPKLGG(序列4)；SAKTTP(序列5)；RADAAP(序列6)；RADAAPTVS(序列7)；RADAAAAGGPGS(序列8)；RADAAAA(序列9)；SAKTTPKLEEGEFSEARV(序列10)；ADAAP(序列11)；DAAPTVSIFPP(序列12)；TVAAP(序列13)；TVAAPSVFIFPP(序列14)；QPKAAP(序列15)；QPKAAPSVTLFPP(序列16)；AKTTPP(序列17)；AKTTPPSVTPLAP(序列18)；AKTTAP(序列19)；AKTTAPSVYPLAP(序列20)；ASTKGP(序列21)；ASTKGPSVFPLAP(序列22)；GGGGSGGGGSGGGGS(序列23)；GENKVEYAPALMALS(序列24)；GPAKELTPLKEAKVS(序列25)；GHEAAAVMQVQYPAS(序列26)。
所述效应分子通过所述连接肽以下述8种方式中的任一种与所述IgG抗体恒定区相连：
方式1：所述效应分子通过所述连接肽分别连接于所述IgG抗体恒定区的两条重链和两条轻链的N端；分子式命名为：(E-L)2(E-H)2，E为效应分子。E-代表N端连接，L为轻链，H为重链。
方式2：所述效应分子通过所述连接肽连接于所述IgG抗体恒定区的两条重链的C端，不连接轻链；分子式命名为：L2(H-E)2，E为效应分子，-E代表C端连接，L为轻链，H为重链。
方式3：所述效应分子通过所述连接肽连接于所述IgG抗体恒定区的两条轻链的N端，不连接重链；分子式命名为：(E-L)2H2，E为效应分子，E-代表N端连接，L为轻链，H为重链。
方式4：所述效应分子通过所述连接肽连接于所述IgG抗体恒定区的两条轻链的C端，不连接重链；分子式命名为：(L-E)2H2，E为效应分子，-E代表C端连接，L为轻链，H为重链。
方式5：所述效应分子通过所述连接肽连接于所述IgG抗体恒定区的两条重链的N端，不连接轻链；分子式命名为：L2(E-H)2，E为效应分子，E-代表N端连接，L为轻链，H为重链。
方式6：所述效应分子通过所述连接肽分别连接于所述IgG抗体恒定区的两条重链的N端和两条轻链的C端。分子式命名为：(L-E)2(E-H)2，E为效应分子，-E代表C端连接，E-代表N端连接，L为轻链，H为重链。
方式7：所述效应分子通过所述连接肽分别连接于所述IgG抗体恒定区的两条重链和两条轻链的C端；分子式命名为：(L-E)2(H-E)2，E为效应分子，-E代表C端连接，L为轻链，H为重链。
方式8：所述效应分子通过所述连接肽分别连接于所述IgG抗体恒定区的两条轻链的N端和两条重链C端；分子式命名为：(E-L)2(H-E)2，E为效应分子，-E代表C端连接，E-代表N端连接，L为轻链，H为重链。
以上8种连接方式所形成的IgG样免疫融合蛋白的结构示意图具体参见图1。
所述效应分子作用的靶向分子可为糖尿病治疗的靶向分子、自身免疫性疾病治疗的靶向分子、肿瘤治疗的靶向分子或内分泌治疗的靶向分子。
其中，所述糖尿病治疗的靶向分子具体可为胰高血糖素样肽1受体(glucagon-likepeptide 1receptor,GLP-1R)或成纤维细胞生长因子受体(Fibroblast growthfactor receptor,FGFR)等分子；
所述自身免疫性疾病治疗的靶向分子具体可选自如下任一种：肿瘤坏死因子α(Tumor necrosis factor，TNF-α)、IL-1、IL-4、IL-5、IL-8、IL-13、IL-17、IL-23、CD80、CD86、CD2、CD110、CD257和B淋巴细胞刺激因子(B lymphocyte stimulator，BLyS)等分子；主要通过抑制炎性介质或通过阻碍淋巴细胞表面受体分子抑制淋巴细胞的激活。
所述肿瘤治疗的靶向分子具体可选自如下任一种：血管内皮生长因子A(vascularendothelial growth factor A，VEGF-A)、Fas配体(fas ligand,FasL)、生长分化因子2(Growth differentiation factor 2,GDF-2)、Wnt、骨形态发生蛋白11(Bonemorphogenetic protein 11,BMP-11)和激活素A(Activin A)等分子；主要通过抑制肿瘤血管生成、促进肿瘤细胞凋亡、抑制肿瘤细胞生长和转移等达到治疗肿瘤之目的。
所述内分泌治疗的靶向分子具体可为生长激素受体(growth hormone receptor，GHR)或甲状旁腺激素受体(Parathyroid Hormone receptor)等分子。通过生长激素和甲状旁腺激素及其类似物发挥生理作用，用于治疗矮小症、烧伤、和骨质疏松症等疾病。
相应的，作用于糖尿病治疗靶向分子胰高血糖素样蛋白1受体(GLP-1R)的所述效应分子为GLP-1或其类似物，其氨基酸序列为序列表中序列27或序列28或者是表1中的通式；作用于糖尿病治疗靶向分子成纤维细胞生长因子受体(FGFR)的所述效应分子为成纤维细胞生长因子21(FGF21)，其氨基酸序列具体为序列表中序列29。具体如表1所示。
表1 作用于糖尿病治疗靶向分子的效应分子的氨基酸残基序列
Figure BDA0001248108520000041
Figure BDA0001248108520000051
注：GLP-1类似物的通式中，Xaa8是Gly、Ser、Val或Ala；Xaa9是Glu、Asp或Gln；Xaa18是Ser或Lys；Xaa19是Tyr或Asn；Xaa22是Glu、Ala、Lys、Gly、Arg或Asp；Xaa23是Gln或Lys；Xaa26是Lys或Arg；Xaa29是Ile或Val；Xaa34是Lys或Arg；Xaa36是Arg或Gly。
作用于自身免疫性疾病治疗的靶向分子TNF-α的所述效应分子为肿瘤坏死因子受体1(TNFR1)或肿瘤坏死因子受体2(TNFR2)；所述TNFR1的氨基酸序列具体为序列表中序列30或序列31；所述TNFR2的氨基酸序列具体为序列表中序列32或序列33；作用于自身免疫性疾病治疗的靶向分子CD80和CD86的所述效应分子为细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)；所述CTLA-4的氨基酸序列具体为序列表中序列34或序列35；作用于自身免疫性疾病治疗的靶向分子CD2的所述效应分子为CD58(LFA-3)；所述CD58(LFA-3)的氨基酸序列具体为序列表中序列36；作用于自身免疫性疾病治疗的靶向分子B淋巴细胞刺激因子(BLyS)的所述效应分子为穿膜蛋白活化物(Transmembrane Activator And Caml Interactor，TACI)；所述TACI的氨基酸序列具体为序列表中序列37；作用于自身免疫性疾病治疗的靶向分子CD257的所述效应分子为CD268；所述CD268的氨基酸序列具体为序列表中序列38；作用于自身免疫性疾病治疗的靶向分子CD110的所述效应分子为免疫球蛋白重链恒定区γ1(immunoglobulin heavy constantgamma 1，IGHG1)；所述IGHG1的氨基酸序列具体为序列表中序列39。具体如表2所示。
表2 作用于自身免疫性疾病治疗的靶向分子的效应分子的氨基酸残基序列
Figure BDA0001248108520000052
Figure BDA0001248108520000061
作用于肿瘤治疗的靶向分子VEGF-A的所述效应分子为VEGFR-1或VEGFR1-D2/VEGFR2-D3(即表示VEGFR-1的功能区Ⅱ(D2)和VEGFR2的功能区Ⅲ(D3))或VEGFR1-D2/VEGFR2-D3-D4(即表示VEGFR-1的功能区Ⅱ(D2)和VEGFR2的功能区Ⅲ(D3)和VEGFR2的功能区Ⅳ(D4))；所述VEGFR-1的氨基酸序列具体为序列表中序列40；所述VEGFR1-D2/VEGFR2-D3的氨基酸序列具体为序列表中序列序列41；所述VEGFR1-D2/VEGFR2-D3-D4的氨基酸序列具体为序列表中序列序列42；作用于肿瘤治疗的靶向分子FasL的所述效应分子为CD95；所述CD95的氨基酸序列具体为序列表中序列43；作用于肿瘤治疗的靶向分子生长分化因子2(Growth differentiation factor 2,GDF-2)的所述效应分子为激活素A受体样激酶1(Activin Receptor-Like Kinase 1，ALK-1)；所述ALK-1的氨基酸序列具体为序列表中序列44；作用于肿瘤治疗的靶向分子Wnt的所述效应分子为Frizzled-8；所述Frizzled-8的氨基酸序列具体为序列表中序列45；作用于肿瘤治疗的靶向分子骨形态发生蛋白11(bonemorphogenetic protein，BMP-11)的所述效应分子为激活素A受体IIB型(activin Areceptor type IIB，ACVR2B)；所述ACVR2B的氨基酸序列具体为序列表中序列46或序列47；作用于肿瘤治疗的靶向分子激活素A的所述效应分子为激活素A受体IIA型(activin Areceptor type 2A,ACVR2A)；所述ACVR2A的氨基酸序列具体为序列表中序列48。具体如表3所示。
表3 作用于肿瘤治疗的靶向分子的效应分子的氨基酸残基序列
Figure BDA0001248108520000071
Figure BDA0001248108520000081
作用于内分泌治疗的靶向分子生长激素受体(growth hormone receptor，GHR)的所述效应分子为生长激素；所述生长激素的氨基酸序列具体为序列表中序列49；作用于内分泌治疗的靶向分子甲状旁腺激素受体(Parathyroid Hormone receptor)的所述效应分子为甲状旁腺激素或其类似物；所述甲状旁腺激素或其类似物的氨基酸序列具体为序列表中序列50或序列51。具体如表4所示。
表4 作用于内分泌治疗的靶向分子的效应分子的氨基酸残基序列
Figure BDA0001248108520000082
在本发明的一个实施例中，所述IgG样免疫融合蛋白(BY23.2)的轻链的氨基酸序列如序列表中序列58所示，重链的氨基酸序列如序列表中序列60所示。
更加具体的，所述IgG样免疫融合蛋白(BY23.2)是按照包括如下步骤的方法制备获得的：将序列表中序列58所示蛋白(BY23.2L)的编码基因插入到具有双表达盒的谷氨酰胺合成酶高效表达载体的酶切位点XhoI和EcoRI之间，得到中间载体；然后再将序列表中序列60所示蛋白(BY23.2H)的编码基因插入到所述中间载体的酶切位点XbaI和SaII之间，得到重组表达载体；所述重组表达载体表达出的蛋白即为所述IgG样免疫融合蛋白(BY23.2)。其中，所述具有双表达盒的谷氨酰胺合成酶高效表达载体具体参见申请号为201410441296.0的中国专利。
更进一步，所述序列表中序列58所示蛋白的编码基因具体为序列表中序列59所示DNA分子；所述序列表中序列60所示蛋白的编码基因具体为序列表中序列61所示DNA分子。
所述IgG样免疫融合蛋白在疾病治疗中的应用也属于本发明的保护范围。
所述IgG样免疫融合蛋白在制备用于疾病治疗的药物中的应用也属于本发明的保护范围。
其中，所述疾病可为糖尿病、自身免疫性疾病、肿瘤或内分泌相关疾病等多种疾病。
实验证明，本发明所提供的IgG样免疫融合蛋白，其在保证了与靶向分子具有较高亲和力以及具有较好体内活性的前提下，可以有效延长蛋白药物(效应分子)的生物半衰期，远远好于于同类的Fc免疫融合蛋白。本发明所提供的这种IgG样长效免疫融合蛋白可用于糖尿病、肿瘤、自身免疫性疾病和内分泌等多种疾病的治疗。
附图说明
图1为7种连接方式所形成的IgG样免疫融合蛋白的结构示意图。
图2为实施例4中实验组和对照组小鼠的血糖随时间变化图。纵坐标为血糖值，单位为mmol/L。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量实验均设置3次以上的重复，结果取均值。
实施例1、谷氨酰胺合成酶高效表达载体的构建
一、糖尿病相关IgG样免疫融合蛋白编码基因的克隆和表达载体的构建
1、Dulaglutide编码基因的合成及表达载体构建
Dulaglutide(Trulicity)是一种美国食品药品监督管理局(FDA)批准的胰高血糖素样肽-1(GLP-1)受体激动剂，也是一种用于帮助血糖水平正常化的免疫融合蛋白，其结构为GLP-1-Fc，属于现有典型Fc免疫融合蛋白。
根据International Nonproprietary Name(INN)数据库中和专利中有关Dulaglutide的氨基酸残基序列“HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(序列52)”委托上海捷瑞生物工程有限公司合成将Dulaglutide的编码基因“CACGGAGAGGGCACATTCACTTCCGACGTGTCTAGTTACTTGGAGGAACAGGCAGCTAAAGAGTTTATCGCATGGCTCGTCAAGGGTGGGGGCGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGCGCTGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGgccgccGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCTAA(序列53)”，经XhoI-EcoRI双酶切克隆至双表达盒的谷氨酰胺合成酶高效表达载体(具体参见专利申请号：201410441296.0)。并经测序验证重组载体构建正确。
2、GLP-1类似物IgG样融合蛋白编码基因的合成及表达载体构建
根据选择的GLP-1类似物的效应分子氨基酸残基序列，优化其编码序列，并分别与人IgG的轻链恒定区和重链恒定区的编码基因连接，分别构建(E-L)2(E-H)2、L2(H-E)2、(E-L)2H2、(L-E)2H2、L2(E-H)2、(L-E)2(E-H)2和(L-E)2(H-E)2七种分子结构IgG样融合蛋白。八种分子式表达载体的构建见如下表5。表6列举了几种GLP-1类似物IgG样融合蛋白的氨基酸残基和编码序列。
表5 八种分子式表达载体的构建
Figure BDA0001248108520000101
Figure BDA0001248108520000111
表6 几种GLP-1类似物IgG样融合蛋白的氨基酸残基和编码序列：
Figure BDA0001248108520000112
Figure BDA0001248108520000121
Figure BDA0001248108520000131
Figure BDA0001248108520000141
通过XhoI-EcoRI双酶切分别将BY23.1L、BY23.2L和BY23.4L编码基因连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(具体参见专利申请号：201410441296.0)；通过表5的组合通过XbaI-SaII分别将BY23.1H、BY23.2H和BY23.3H编码基因分别克隆至已连接对应编码轻链基因的双表达盒的谷氨酰胺合成酶高效表达载体(各重组表达载体经测序验证正确后备用)，所表达的IgG样融合蛋白分别命名为BY23.1、BY23.2、BY23.3、BY23.4、BY23.5、BY23.6、BY23.7和BY23.8。
二、自身免疫性疾病相关IgG样免疫融合蛋白编码基因的克隆和表达载体的构建
炎性介质TNF-α为三聚体，在通过TNFR1和TNFR2结合而发挥生理功能。TNFR1和TNFR2与TNF-α的亲和性较高，可达nM级别。TNFR1和TNFR2与TNF-α结合的功能区均位于两受体N端的4个富含半胱氨酸区。将其设计成为IgG样免疫融合蛋白可以在体内结合游离的TNF-α，达到治疗之目的。其效应分子氨基酸残基序列及编码基因见如下表7。
表7 自身免疫性疾病治疗相关IgG样融合蛋白的氨基酸残基和编码序列
Figure BDA0001248108520000142
Figure BDA0001248108520000151
通过XhoI-EcoRI双酶切将BY19.3L编码基因连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(具体参见专利申请号：201410441296.0)；通过XbaI-SaII分别将BY19.3H编码基因克隆至已连接BY19.3L编码轻链基因的双表达盒的谷氨酰胺合成酶高效表达载体(重组表达载体经测序验证正确后备用)，所表达的IgG样融合蛋白命名为BY19.3，分子式L2(H-E)2。
三、肿瘤治疗相关IgG样免疫融合蛋白编码基因的克隆和表达载体的构建
VEGF-A与其受体VEGFR1和VEGFR2均有较高亲和性，VEGFR均属于酪氨酸激酶受体超家族，在胞外段有7个免疫球蛋白样功能区(immunoglobulin-like domains)，其中第1-3个Ig样区和VEGF结合有关。将VEGFR1胞外第2功能结构域和VEGFR2胞外的第3功能结构域编码基因连接，构建成成为IgG样免疫融合蛋白可以在体内结合游离的VEGF-A和VEGF-B，抑制肿瘤血管的生成，从而达到治疗肿瘤之目的。其效应分子氨基酸残基序列及编码基因见如下表8。
表8 肿瘤治疗相关IgG样融合蛋白的氨基酸残基和编码序列
Figure BDA0001248108520000161
Figure BDA0001248108520000171
通过XhoI-EcoRI双酶切将BY25L编码基因连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(具体参见专利申请号：201410441296.0)；通过XbaI-SaII分别将BY25H编码基因克隆至已连接BY25L编码轻链基因的双表达盒的谷氨酰胺合成酶高效表达载体(重组载体测序验证正确后备用)，所表达的IgG样融合蛋白命名为BY25，分子式L2(E-H)2。
四、内分泌疾病相关IgG样免疫融合蛋白编码基因的克隆和表达载体的构建
生长激素通过与生长激素受体结合发挥生理作用，患矮小症缺乏生长激素，为治疗矮小症每天需注射重组生长激素一次，给矮小症患儿造成巨大的痛苦，在治疗期间会出现减少注射次数，甚至放弃治疗。长效生长激素的使用可以提高患儿治疗的顺应性和治疗效果。其效应分子氨基酸残基序列及编码基因见如下表9。
表9 内分泌疾病治疗相关IgG样融合蛋白的氨基酸残基和编码序列
Figure BDA0001248108520000172
Figure BDA0001248108520000181
Figure BDA0001248108520000191
Figure BDA0001248108520000201
通过XhoI-EcoRI双酶切将BY4.5L和BY3.4L编码基因连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(具体参见专利申请号：201410441296.0)；通过XbaI-SaII分别将BY4.5H和BY3.4H编码基因克隆至已连接BY4.5L和BY3.4L编码轻链基因的双表达盒的谷氨酰胺合成酶高效表达载体(重组载体测序验证正确后备用)，所表达的IgG样融合蛋白命名为BY4.5和BY3.4，分子式分别为(E-L)2H2和(E-L)2(E-H)2。
实施例2、IgG样免疫融合蛋白表达细胞的筛选、表达及纯化
一、IgG样免疫融合蛋白表达细胞的筛选
1、CHO-K1细胞(ATCC，货号：CCL-61)在10％D-FBS，4mM谷氨酰胺的CD OptiCHO(Invitrogen产品，货号：12681)中适应并贴壁生长。
2、24小时后，按Lipofectin 2000(Invitrogen货号：11668-019)操作说明，将实施例1构建的重组载体转染细胞，于10cm培养皿中进行：用无血清无抗生素的IMDM培养基洗2次，每次加入IMDM 100μl/孔。取实施例1构建好的表达质粒与IMDM培养基混均，取Lipofectin 2000与IMDM培养基混均。静置5分钟后，将两者混匀。室温静置15分钟后，将Lipofectin 2000CD和表达质粒混合物加入培养有CHO-K1细胞的10cm培养皿。
3、5小时后，换成完全培养基(10％透析血清，4mM谷氨酰胺的CD OptiCHO)。
4、转染24小时后胰酶消化转染细胞，按3-8×103/孔的细胞密度铺96孔培养板，并用含L-蛋氨酸砜亚胺(L-Methionine sulfoximine，MSX，Sigma产品，货号：M5379)25μM的CDOptiCHO培养基进行筛选。
5、大约2周后有细胞克隆长出，取50μl样品双抗体夹心法ELISA方法检测培养液中蛋白含量，筛选出目的蛋白表达量相对较高克隆。
二、IgG样免疫融合蛋白的表达和纯化
将步骤一获得高表达细胞置无血清的CD OptiCHO中培养，细胞活率在60％左右收集培养上清。用pH 7.4的PBS溶液平衡HiTrap MabSelect SuRe 1ml柱(GE HealthcareLife Sciences产品,Cat.No：11-0034-93)10个床体积，流速为0.5ml/min；培养上清液用0.45μm滤膜过滤上样，流速为0.5ml/min。用pH 7.4的PBS溶液再洗5-10个床体积，流速为0.5ml/min；用100mM柠檬酸缓冲液(pH 4.0)洗脱，流速为0.5ml/min，收集洗脱峰，目的蛋白存在于洗脱峰中。
实施例3、Biacore 3000检测IgG样免疫融合蛋白与其靶标的亲和力
一、实验方法
1、固定
分别将GLP-1R、VEGF-A、VEGF-B和TNF-α(均为义翘神州产品)稀释至浓度10μg/ml，采用氨基偶联的方法经由伯胺共价固定在羧甲基葡聚糖包被的CM5芯片(通用电气产品)，固定缓冲液10mM醋酸钠(pH5.0)，固定量为1000RU。
2、再生条件
再生液：pH为1.5-2.0的甘氨酸缓冲液；进样时间：30s；流速：30μl/min。
3、动力学分析
PBST(配方：PBS中含有0.005％体积百分含量的吐温20)为Running buffer；使用Kinetic Analysis Wizard模式；将Dulaglutide、BY23.2、BY25和BY19.3分别稀释为的600、300、150、75、37.5和18.75nM的浓度梯度；进样时间：2min，解离时间：5min，流速：30μl/min。
二、实验结果
拟和软件：BIAevaluation 4.1software。
Biacore方法检测Dulaglutide、BY23.2、BY25和BY19与各自的靶标的亲和力结果，具体如表10所示。
表10 Dulaglutide、BY23.2、BY25和BY19与各自的靶标的亲和力结果
Figure BDA0001248108520000211
从表10中Dulaglutide和BY23.2的结果可以看出，同样是针对GLP-1R靶标，两者所体现出来的亲和力(KD)是基本一致的。可见，与传统的Fc免疫融合蛋白相比，本发明的IgG样免疫融合蛋白仍然保留了较强的亲和力。
实施例4、IgG样免疫融合蛋白内活性检测
一、GPL-1的IgG样免疫融合蛋白体内活性检测
1、实验方法
(1)Ⅱ型糖尿病模型db/db小鼠(常州卡文斯实验动物有限公司)，禁食3h后测基础血糖，将血糖浓度大于10mmol/L入组；随几分2组，每组6只小鼠。
(2)按0.8mg/kg的量BY23.2(γ4，L2(H-E)2)皮下注射免疫融合蛋白，对照组为等体积缓冲液。
(3)分别在0小时、1小时、1天、2天、3天、4天、6天、8天、10天、12天和14天取小鼠尾血，测血糖(测血糖前小鼠禁食，不禁水3小时)。
2、实验结果
与对照组相比较，实验组BY23.2(γ4，L2(H-E)2)皮下注射1小时内血糖快速下降到10mmol/L以下，10天内基本维持血糖的稳定，到10天依然有显著的降糖效应。具体如图2所示。
二、生长激素IgG样免疫融合蛋白体内活性检测
1、实验方法
委托苏州西山中科药物研究开发有限公司完成。
SD大鼠，雌雄各半，3～4周龄，戊巴比妥钠(浓度15mg/mL)45mg/kg腹腔注射麻醉(给药容积3mL/kg)，麻醉后固定于立体定向垂体切除仪(Stoelting,USA)，门齿平面低于耳间线1mm，参考体重一深度和体重一角度相关曲线选择进针深度和角度，调整两侧耳杆角度为19°，取16G安全型带加药壶静脉留置针芯(1.7×50mm)经右侧中空耳杆针尖斜面向上进针，待针尖刚至外耳道处，用圆规测量垂体部位与针尖的直线距离,以此距离为尺度，开始进针至有落空感时，轻轻旋转针尖180°，用吸引器抽吸至粉红色垂体组织吸出并检查。手术后记录每只动物的体重和尾长(从尾末端到肛门的距离)。
手术完毕，术后3天内，每日单次皮下注射青霉素(40×104IU/ml)0.1ml/只，并让动物自由饮用5％葡萄糖水(装入给水瓶中)；自由饮水和进食。
术后3周，测量每只存活动物的体重，取术后2～3周体重变化小于手术前±10％去垂体大鼠为合格去垂体模型动物，并按体重随机均衡分为模型对照组(生理盐水)和BY4.5(γ4，(E-L)2H2)组(低剂量组0.45mg/kg，高剂量组1.8mg/kg)，人生长激素国际活性标准品(中国食品药品检定研究院，4.5IU(1.5mg)/支)组(低剂量组0.133mg/kg、高剂量组0.533mg/kg)，每组8只。标准品组每日一次，连续6天，BY4.5组6天注射2次，每3天1次。
第10天处死动物并刨取后两腿胫骨，剥离肌肉等组织(必要时实验结束后可进行尸检，切开蝶鞍区，肉眼检查有无垂体残留，剔除有垂体残存的动物)，从胫骨近心端顶部中间沿矢状面切开，置10％福尔马林中，水洗10min后，置丙酮中脱蛋白10min，水洗3min，2％硝酸银中染色2min，水洗后置水中强光照射至变棕黑色，10％硫代硫酸钠固定30s，置80％乙醇中保存供测量用，测量时沿刨面切1mm左右薄片置生物显微镜下(×100)测量胫骨骨骺板宽度(每鼠两腿胫骨测量10个数值取其平均值)。
2、实验结果
实验结果按“生物检定统计法”(《中国药典》附录ⅪⅤ)中的量反应平行线测定随机设计法列表的格式整理，按量反应平行线测定(2.2)法随机设计处理结果，并进行效价计算。
最终计算结果如下表11所示。IgG样免疫融合蛋白在体内仍然保持了很好的生物学活性。
表11 BY4.5的理论估计效价以及实际计算效价
供试品 理论估计效价 实际计算效价(IU/mg)
BY4.5 1.24IU/mg 1.08
实施例5、GLP-1类似物IgG样免疫融合蛋白食蟹猴体内生物半衰期测定
一、实验方法
(1)分组：实验分2组Dulaglutide和BY23.2(γ4，L2(H-E)2)，每组3只健康食蟹猴。
(2)动物给药：试验采用单一剂量、单次皮下注射给药。根据分子量大小，保证给药的量(摩尔数)一致，Dulaglutide给药量为0.52mg/0.2ml/kg，BY23.2(γ4，L2(H-E)2)，给药量为0.72mg/0.2ml/kg。
(3)样本采集：药前(0h)、给药后0.5、1、6h，1、2、3、5、7、10、14、17、21、24、28天。上或下肢静脉采集全血约0.5ml。
(4)样本处理：取血后放入预加5μl肝素钠(6250IU/ml)的1.5ml离心管中(取血前，含有肝素钠的离心管于2～8℃或冰浴保存)，手动颠倒至少3次混匀，置于碎冰中，2h内完成离心分离血浆，离心条件为：2400g，4℃，10min，血浆储存于-70℃以下，避免反复冻融。
(5)血药浓度测定：以Abcam公司的抗GLP-1抗体4F3为一抗(货号：ab23472)，以中杉金桥的辣根过氧化物酶标记的羊抗人抗体(货号：ZB-2304)为二抗，建立的ELISA方法测定血浆药物浓度，以供试品为标准品，制备标准曲线。
(6)数据采集及统计分析：用酶标仪自带软件计算各样本中含量。进而计算药物半衰期(T1/2)，即药物在血浆中最高浓度降低一半所需的时间。
二、实验结果
Dulaglutide(Fc免疫融合蛋白)的T1/2为62.1±5.8h，BY23.2(γ4，L2(H-E)2)的T1/2为179±9.46h，BY23.2在食蟹猴体内生物半衰期远远超过Dulaglutide，是Dulaglutide的3倍多。
综合以上各实施例的实验结果，可见：本发明所提供的IgG样免疫融合蛋白，其在保证了与靶向分子具有较高亲和力以及具有较好体内活性的前提下，可以有效延长蛋白药物(效应分子)的生物半衰期，远远好于于同类的Fc免疫融合蛋白。本发明所提供的这种IgG样长效免疫融合蛋白可用于糖尿病、肿瘤、自身免疫性疾病和内分泌等多种疾病的治疗。
<110> 北京比洋生物技术有限公司
<120> IgG样长效免疫融合蛋白及其应用
<130> GNCLN170597
<160> 81
<170> PatentIn version 3.5
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Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln
115 120 125
<210> 36
<211> 92
<212> PRT
<213> 人工序列
<220>
<223>
<400> 36
Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His
1 5 10 15
Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys
20 25 30
Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser
35 40 45
Phe Lys Asn Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile
50 55 60
Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro
65 70 75 80
Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val
85 90
<210> 37
<211> 81
<212> PRT
<213> 人工序列
<220>
<223>
<400> 37
Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly
1 5 10 15
Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr
20 25 30
Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
35 40 45
Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys
50 55 60
Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg
65 70 75 80
Ser
<210> 38
<211> 73
<212> PRT
<213> 人工序列
<220>
<223>
<400> 38
Asp Val Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala
1 5 10 15
Pro Thr Pro Cys Asn Pro Ala Glu Cys Phe Asp Pro Leu Val Arg His
20 25 30
Cys Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly
35 40 45
Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val
50 55 60
Gly Ala Gly Ala Gly Glu Ala Ala Val
65 70
<210> 39
<211> 14
<212> PRT
<213> 人工序列
<220>
<223>
<400> 39
Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
1 5 10
<210> 40
<211> 299
<212> PRT
<213> 人工序列
<220>
<223>
<400> 40
Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His
1 5 10 15
Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro
20 25 30
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro
35 40 45
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser
50 55 60
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val
65 70 75 80
Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
85 90 95
Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser
100 105 110
Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn
115 120 125
Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His
130 135 140
Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
145 150 155 160
Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp
165 170 175
Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys
180 185 190
Asn Ser Thr Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly
195 200 205
Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg
210 215 220
Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr
225 230 235 240
Lys Asn Gly Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His
245 250 255
Val Leu Thr Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr
260 265 270
Val Ile Leu Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val
275 280 285
Ser Leu Val Val Tyr Val Pro Pro Gly Pro Gly
290 295
<210> 41
<211> 205
<212> PRT
<213> 人工序列
<220>
<223>
<400> 41
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
35 40 45
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110
Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
115 120 125
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
130 135 140
His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
145 150 155 160
Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175
Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190
Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys
195 200 205
<210> 42
<211> 296
<212> PRT
<213> 人工序列
<220>
<223>
<400> 42
Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His
1 5 10 15
Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro
20 25 30
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro
35 40 45
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser
50 55 60
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val
65 70 75 80
Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
85 90 95
Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser
100 105 110
Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn
115 120 125
Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His
130 135 140
Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
145 150 155 160
Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp
165 170 175
Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys
180 185 190
Asn Ser Thr Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly
195 200 205
Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg
210 215 220
Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr
225 230 235 240
Lys Asn Gly Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His
245 250 255
Val Leu Thr Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr
260 265 270
Val Ile Leu Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val
275 280 285
Ser Leu Val Val Tyr Val Pro Pro
290 295
<210> 43
<211> 147
<212> PRT
<213> 人工序列
<220>
<223>
<400> 43
Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val
1 5 10 15
Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His Asp Gly Gln
20 25 30
Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cys
35 40 45
Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys
50 55 60
Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg Cys Arg
65 70 75 80
Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys Thr Arg
85 90 95
Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser
100 105 110
Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His Gly Ile
115 120 125
Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly
130 135 140
Ser Arg Ser
145
<210> 44
<211> 99
<212> PRT
<213> 人工序列
<220>
<223>
<400> 44
Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val Thr Cys Thr Cys Glu
1 5 10 15
Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly Ala Trp Cys Thr Val
20 25 30
Val Leu Val Arg Glu Glu Gly Arg His Pro Gln Glu His Arg Gly Cys
35 40 45
Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg Pro Thr Glu Phe Val
50 55 60
Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn His Asn Val Ser Leu
65 70 75 80
Val Leu Glu Ala Thr Gln Pro Pro Ser Glu Gln Pro Gly Thr Asp Gly
85 90 95
Gln Leu Ala
<210> 45
<211> 131
<212> PRT
<213> 人工序列
<220>
<223>
<400> 45
Ala Ser Ala Lys Glu Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys
1 5 10 15
Lys Gly Ile Gly Tyr Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His
20 25 30
Asp Thr Gln Asp Glu Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu
35 40 45
Val Glu Ile Gln Cys Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met
50 55 60
Tyr Thr Pro Ile Cys Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys
65 70 75 80
Arg Ser Val Cys Glu Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg
85 90 95
Gln Tyr Gly Phe Ala Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro
100 105 110
Glu Gln Gly Asn Pro Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp
115 120 125
Leu Thr Thr
130
<210> 46
<211> 107
<212> PRT
<213> 人工序列
<220>
<223>
<400> 46
Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg
1 5 10 15
Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg
20 25 30
Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu
35 40 45
Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln
50 55 60
Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys
65 70 75 80
Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly
85 90 95
Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
100 105
<210> 47
<211> 115
<212> PRT
<213> 人工序列
<220>
<223>
<400> 47
Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn
1 5 10 15
Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly
20 25 30
Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser
35 40 45
Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn
50 55 60
Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val
65 70 75 80
Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His
85 90 95
Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
100 105 110
Ala Pro Thr
115
<210> 48
<211> 116
<212> PRT
<213> 人工序列
<220>
<223>
<400> 48
Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn
1 5 10 15
Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly
20 25 30
Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser
35 40 45
Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn
50 55 60
Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val
65 70 75 80
Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr
85 90 95
Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro
100 105 110
Lys Pro Pro Thr
115
<210> 49
<211> 191
<212> PRT
<213> 人工序列
<220>
<223>
<400> 49
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
1 5 10 15
Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro
35 40 45
Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg
50 55 60
Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu
65 70 75 80
Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95
Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp
100 105 110
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140
Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr
145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175
Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190
<210> 50
<211> 34
<212> PRT
<213> 人工序列
<220>
<223>
<400> 50
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
20 25 30
Thr Ala
<210> 51
<211> 34
<212> PRT
<213> 人工序列
<220>
<223>
<400> 51
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210> 52
<211> 275
<212> PRT
<213> 人工序列
<220>
<223>
<400> 52
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
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
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 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 Gly Leu Pro Ser Ser
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 Gln 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 Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu 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 Leu Gly
275
<210> 53
<211> 828
<212> DNA
<213> 人工序列
<220>
<223>
<400> 53
cacggagagg gcacattcac ttccgacgtg tctagttact tggaggaaca ggcagctaaa 60
gagtttatcg catggctcgt caagggtggg ggcggcggcg gaggatctgg cggcggaggc 120
agtggaggcg gcggaagcgc tgagtccaag tacggccctc cttgccctcc ttgccctgcc 180
cctgaggccg ccggcggccc ttccgtgttc ctgttccctc ctaagcctaa ggacaccctg 240
atgatctccc gcacccctga ggtgacctgc gtggtggtgg acgtgtccca ggaggaccct 300
gaggtgcagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagcct 360
cgcgaggagc agttcaactc cacctaccgc gtggtgtccg tgctgaccgt cctgcaccag 420
gactggctga acggcaagga gtacaagtgc aaggtgtcca acaagggcct gccttcctcc 480
atcgagaaga ccatctccaa ggccaagggc cagcctcgcg agcctcaggt gtacaccctg 540
cctccttccc aggaggagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 600
ttctaccctt ccgacatcgc cgtggagtgg gagtccaacg gccagcctga gaacaactac 660
aagaccaccc ctcctgtgct ggactccgac ggctccttct tcctgtactc ccgcctgacc 720
gtggacaagt cccgctggca ggagggcaac gtgttctcct gctccgtgat gcacgaggcc 780
ctgcacaacc actacaccca gaagtccctg tccctgtccc tgggctaa 828
<210> 54
<211> 148
<212> PRT
<213> 人工序列
<220>
<223>
<400> 54
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Ser
35 40 45
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
50 55 60
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
65 70 75 80
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
85 90 95
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
100 105 110
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
115 120 125
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
130 135 140
Arg Gly Glu Cys
145
<210> 55
<211> 444
<212> DNA
<213> 人工序列
<220>
<223>
<400> 55
cacggagagg gcacattcac ttccgacgtg tctagttact tggaggaaca ggcagctaaa 60
gagtttatcg catggctcgt caagggtcgg ggcggcggag gatctggcgg cggaggcagt 120
ggaggcggcg gaagcgcccc cagcgtgttc atcttccctc ccagcgacga gcagctgaag 180
tctggcaccg ccagcgtggt gtgcctgctg aacaacttct acccccgcga ggccaaggtg 240
cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag 300
gactccaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac 360
gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gactgtctag ccccgtgacc 420
aagagcttca accggggcga gtgc 444
<210> 56
<211> 371
<212> PRT
<213> 人工序列
<220>
<223>
<400> 56
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr
35 40 45
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
50 55 60
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
65 70 75 80
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
85 90 95
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
100 105 110
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
115 120 125
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu
130 135 140
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Ala Ala
145 150 155 160
Ala Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
165 170 175
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala
180 185 190
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
195 200 205
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
210 215 220
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
225 230 235 240
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
245 250 255
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
260 265 270
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
275 280 285
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
290 295 300
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
305 310 315 320
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
325 330 335
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
340 345 350
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
355 360 365
Pro Gly Lys
370
<210> 57
<211> 1113
<212> DNA
<213> 人工序列
<220>
<223>
<400> 57
cacggagagg gcacattcac ttccgacgtg tctagttact tggaggaaca ggcagctaaa 60
gagtttatcg catggctcgt caagggtcgg ggcggcggag gatctggcgg cggaggcagt 120
ggaggcggcg gaagcgcctc caccaagggc ccttccgtgt tccctctggc cccttgctcc 180
cgctccacct ccgagtccac cgccgccctg ggctgcctgg tgaaggacta cttccctgag 240
cctgtgaccg tgtcctggaa ctccggcgcc ctgacctccg gcgtgcacac cttccctgcc 300
gtgctgcagt cctccggcct gtactccctg tcctccgtgg tgaccgtgcc ttcctccaac 360
ttcggcaccc agacatacac atgcaacgtg gaccacaagc cttctaacac aaaggtggac 420
aagaccgtgg agcggaagtg ctgcgtggag tgcccacctt gccccgctcc tcctgccgcc 480
gcatcctctg tgttcctgtt cccacctaag ccaaaggaca cactcatgat cagcagaacc 540
cctgaggtga cctgcgtggt ggtggacgtg agcgccgagg accccgaggt gcagttcaac 600
tggtatgtgg acggcgtgga ggtgcacaac gctaagacca agcctagaga agaacagttc 660
aacagcacat tcagagtggt gtccgtgctc accgtgctgc accaggactg gctgaacggc 720
aaagagtaca agtgcaaggt gtccaacaag ggcctgccat cttctatcga aaaaacaatc 780
agcaagacca agggccagcc tagagagcct caggtgtaca cactgcctcc atctcgggaa 840
gaaatgacaa agaaccaggt gtccctcaca tgcctcgtga agggcttcta cccatccgac 900
atcgctgtgg agtgggagtc taacggccag cccgagaaca actacaagac cacccctcct 960
atgctcgact ccgacggctc tttcttcctg tactctaagc tgaccgtgga caagtccaga 1020
tggcagcagg gcaacgtgtt ctcttgcagc gtgatgcacg aggctctcca caaccactac 1080
acccagaagt ccctgagcct gtctccaggc aag 1113
<210> 58
<211> 106
<212> PRT
<213> 人工序列
<220>
<223>
<400> 58
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 59
<211> 318
<212> DNA
<213> 人工序列
<220>
<223>
<400> 59
accgtggccg cccccagcgt gttcatcttc cctcccagcg acgagcagct gaagtctggc 60
accgccagcg tggtgtgcct gctgaacaac ttctaccccc gcgaggccaa ggtgcagtgg 120
aaggtggaca acgccctgca gagcggcaac agccaggaga gcgtgaccga gcaggactcc 180
aaggacagca cctacagcct gagcagcacc ctgaccctga gcaaggccga ctacgagaag 240
cacaaggtgt acgcctgcga ggtgacccac cagggactgt ctagccccgt gaccaagagc 300
ttcaaccggg gcgagtgc 318
<210> 60
<211> 370
<212> PRT
<213> 人工序列
<220>
<223>
<400> 60
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
325 330 335
Gly Gly Gly Ser His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
340 345 350
Tyr Leu Glu Glu Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
355 360 365
Gly Arg
370
<210> 61
<211> 1110
<212> DNA
<213> 人工序列
<220>
<223>
<400> 61
gcctccacca agggcccttc cgtgttccct ctggcccctt gctcccgctc cacctccgag 60
tccaccgccg ccctgggctg cctggtgaag gactacttcc ctgagcctgt gaccgtgtcc 120
tggaactccg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180
ggcctgtact ccctgtcctc cgtggtgacc gtgccttcct cctccctggg caccaagacc 240
tacacctgca acgtggacca caagccttcc aacaccaagg tggacaagcg cgtggagtcc 300
aagtacggcc ctccttgccc ttcctgccct gcccctgagt tcctgggcgg cccttccgtg 360
ttcctgttcc ctcctaagcc taaggacacc ctgatgatct cccgcacccc tgaggtgacc 420
tgcgtggtgg tggacgtgtc ccaggaggac cctgaggtgc agttcaactg gtacgtggac 480
ggcgtggagg tgcacaacgc caagaccaag cctcgcgagg agcagttcaa ctccacctac 540
cgcgtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 600
tgcaaggtgt ccaacaaggg cctgccttcc tccatcgaga agaccatctc caaggccaag 660
ggccagcctc gcgagcctca ggtgtacacc ctgcctcctt cccaggagga gatgaccaag 720
aaccaggtgt ccctgacctg cctggtgaag ggcttctacc cttccgacat cgccgtggag 780
tgggagtcca acggccagcc tgagaacaac tacaagacca cccctcctgt gctggactcc 840
gacggctcct tcttcctgta ctcccgcctg accgtggaca agtcccgctg gcaggagggc 900
aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 960
ctgtccctgt ccctgggagg cggaggatct ggcggcggag gcagtggagg cggcggaagc 1020
cacggagagg gcacattcac ttccgacgtg tctagttact tggaggaaca ggcagctaaa 1080
gagtttatcg catggctcgt caagggtcgg 1110
<210> 62
<211> 326
<212> PRT
<213> 人工序列
<220>
<223>
<400> 62
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Ala Ala Ala Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser Ala Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 63
<211> 978
<212> DNA
<213> 人工序列
<220>
<223>
<400> 63
gcctccacca agggcccttc cgtgttccct ctggcccctt gctcccgctc cacctccgag 60
tccaccgccg ccctgggctg cctggtgaag gactacttcc ctgagcctgt gaccgtgtcc 120
tggaactccg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180
ggcctgtact ccctgtcctc cgtggtgacc gtgccttcct ccaacttcgg cacccagaca 240
tacacatgca acgtggacca caagccttct aacacaaagg tggacaagac cgtggagcgg 300
aagtgctgcg tggagtgccc accttgcccc gctcctcctg ccgccgcatc ctctgtgttc 360
ctgttcccac ctaagccaaa ggacaccctc atgatcagca gaacccctga ggtgacctgc 420
gtggtggtgg acgtgagcgc cgaggacccc gaggtgcagt tcaactggta tgtggacggc 480
gtggaggtgc acaacgctaa gaccaagcct agagaagaac agttcaacag cacattcaga 540
gtggtgtccg tgctcaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 600
aaggtgtcca acaagggcct gccatcttct atcgaaaaaa caatcagcaa gaccaagggc 660
cagcctagag agcctcaggt gtacacactg cctccatctc gggaagaaat gacaaagaac 720
caggtgtccc tcacatgcct cgtgaagggc ttctacccat ccgacatcgc tgtggagtgg 780
gagtctaacg gccagcccga gaacaactac aagaccaccc ctcctatgct cgactccgac 840
ggctctttct tcctgtactc taagctgacc gtggacaagt ccagatggca gcagggcaac 900
gtgttctctt gcagcgtgat gcacgaggct ctccacaacc actacaccca gaagtccctg 960
agcctgtctc caggcaag 978
<210> 64
<211> 148
<212> PRT
<213> 人工序列
<220>
<223>
<400> 64
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
1 5 10 15
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
20 25 30
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
35 40 45
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
50 55 60
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
65 70 75 80
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
85 90 95
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser His Gly Glu Gly Thr Phe Thr Ser Asp Val
115 120 125
Ser Ser Tyr Leu Glu Glu Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
130 135 140
Val Lys Gly Arg
145
<210> 65
<211> 444
<212> DNA
<213> 人工序列
<220>
<223>
<400> 65
gcccccagcg tgttcatctt ccctcccagc gacgagcagc tgaagtctgg caccgccagc 60
gtggtgtgcc tgctgaacaa cttctacccc cgcgaggcca aggtgcagtg gaaggtggac 120
aacgccctgc agagcggcaa cagccaggag agcgtgaccg agcaggactc caaggacagc 180
acctacagcc tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg 240
tacgcctgcg aggtgaccca ccagggactg tctagccccg tgaccaagag cttcaaccgg 300
ggcgagtgcg gcggcggagg atctggcggc ggaggcagtg gaggcggcgg aagccacgga 360
gagggcacat tcacttccga cgtgtctagt tacttggagg aacaggcagc taaagagttt 420
atcgcatggc tcgtcaaggg tcgg 444
<210> 66
<211> 106
<212> PRT
<213> 人工序列
<220>
<223>
<400> 66
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 67
<211> 318
<212> DNA
<213> 人工序列
<220>
<223>
<400> 67
accgtggccg cccccagcgt gttcatcttc cctcccagcg acgagcagct gaagtctggc 60
accgccagcg tggtgtgcct gctgaacaac ttctaccccc gcgaggccaa ggtgcagtgg 120
aaggtggaca acgccctgca gagcggcaac agccaggaga gcgtgaccga gcaggactcc 180
aaggacagca cctacagcct gagcagcacc ctgaccctga gcaaggccga ctacgagaag 240
cacaaggtgt acgcctgcga ggtgacccac cagggactgt ctagccccgt gaccaagagc 300
ttcaaccggg gcgagtgc 318
<210> 68
<211> 505
<212> PRT
<213> 人工序列
<220>
<223>
<400> 68
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly
325 330 335
Gly Ser Gly Gly Gly Gly Ser Leu Pro Ala Gln Val Ala Phe Thr Pro
340 345 350
Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp
355 360 365
Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala
370 375 380
Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu
385 390 395 400
Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser
405 410 415
Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr
420 425 430
Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala
435 440 445
Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys
450 455 460
Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val
465 470 475 480
Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser
485 490 495
Thr Asp Ile Cys Arg Pro His Gln Ile
500 505
<210> 69
<211> 1515
<212> DNA
<213> 人工序列
<220>
<223>
<400> 69
gccagcacca agggccccag cgtcttccca ctggctcctt cctctaaaag cactagcgga 60
gggaccgcag ccctgggctg tctggtgaaa gactacttcc ccgagcccgt gaccgtctcc 120
tggaactctg gagccctgac ctccggggtg cacacctttc ccgccgtgct gcagtcttct 180
ggactgtact ccctgtcctc cgtcgtgact gtgcccagct cctccctggg aactcagaca 240
tacatctgca acgtgaacca caagccttcc aacacaaagg tggacaagaa agtcgagccc 300
aagagctgtg ataagaccca tacatgtccc ccatgccccg ctccagaact gctgggcgga 360
ccttccgtgt ttctgttccc acccaaacca aaggacacac tgatgatcag cagaacccct 420
gaggtgactt gcgtggtcgt ggacgtgagc catgaggacc ccgaggtgaa gttcaactgg 480
tatgtggatg gcgtggaagt gcataatgcc aagacaaaac ctagggaaga gcagtacaac 540
agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggattggct gaacggcaag 600
gaatacaagt gcaaggtgtc caataaggct ctgcctgcac ctatcgagaa gaccatcagc 660
aaagccaagg gccaacccag agagcctcaa gtctacaccc tgcccccaag cagggatgag 720
ctgaccaaaa atcaagtgag cctgacatgc ctggtcaaag gcttctaccc tagcgacatc 780
gccgtggagt gggagagcaa tggccagcct gagaacaact acaagaccac tccccccgtc 840
ctggatagcg acggcagctt cttcctgtac tccaaactga cagtcgataa aagcaggtgg 900
cagcaaggca atgtctttag ctgtagcgtg atgcacgagg ccctgcataa ccactacact 960
caaaagtccc tgtccctgag ccccggcggc ggaggatctg gcggcggagg cagtggaggc 1020
ggcggaagcc tgcccgcaca agtggccttc accccctacg ccccagagcc cggctctact 1080
tgtaggctga gggagtacta cgaccagacc gcccagatgt gctgctccaa gtgtagcccc 1140
ggacagcacg ccaaggtgtt ctgtacaaag acctccgaca ccgtgtgcga ctcctgcgag 1200
gactccacct acacccagct gtggaactgg gtgcccgagt gcctgtcctg cggctccagg 1260
tgctcctctg accaggtcga gacccaagcc tgtaccaggg agcagaacag gatctgcact 1320
tgcaggccag gctggtattg cgccctgtcc aagcaggaag gctgcaggct gtgcgcccca 1380
ctgaggaaat gtaggcctgg gttcggcgtg gctaggcccg gaaccgagac ttccgacgtg 1440
gtgtgcaagc cctgtgcccc tggcaccttt tccaacacca cctcctccac cgacatctgt 1500
aggccacacc agatt 1515
<210> 70
<211> 106
<212> PRT
<213> 人工序列
<220>
<223>
<400> 70
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 71
<211> 318
<212> DNA
<213> 人工序列
<220>
<223>
<400> 71
accgtggccg cccccagcgt gttcatcttc cctcccagcg acgagcagct gaagtctggc 60
accgccagcg tggtgtgcct gctgaacaac ttctaccccc gcgaggccaa ggtgcagtgg 120
aaggtggaca acgccctgca gagcggcaac agccaggaga gcgtgaccga gcaggactcc 180
aaggacagca cctacagcct gagcagcacc ctgaccctga gcaaggccga ctacgagaag 240
cacaaggtgt acgcctgcga ggtgacccac cagggactgt ctagccccgt gaccaagagc 300
ttcaaccggg gcgagtgc 318
<210> 72
<211> 537
<212> PRT
<213> 人工序列
<220>
<223>
<400> 72
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
35 40 45
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110
Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
115 120 125
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
130 135 140
His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
145 150 155 160
Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175
Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190
Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Ser Ser Ala
195 200 205
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
210 215 220
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
225 230 235 240
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
245 250 255
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
260 265 270
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
275 280 285
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
290 295 300
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
305 310 315 320
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
325 330 335
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
340 345 350
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
355 360 365
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
370 375 380
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
385 390 395 400
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
405 410 415
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
420 425 430
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
435 440 445
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
450 455 460
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
465 470 475 480
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
485 490 495
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
500 505 510
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
515 520 525
Lys Ser Leu Ser Leu Ser Pro Gly Lys
530 535
<210> 73
<211> 1611
<212> DNA
<213> 人工序列
<220>
<223>
<400> 73
tccgacaccg gccgcccttt cgtggagatg tactccgaga tccctgagat catccacatg 60
accgagggcc gcgagctggt gatcccttgc cgcgtgacct cccctaacat caccgtgacc 120
ctgaagaagt tccctctgga caccctgatc cctgacggca agcgcatcat ctgggactcc 180
cgcaagggct tcatcatctc caacgccacc tacaaggaga tcggcctgct gacctgcgag 240
gccaccgtga acggccacct gtacaagacc aactacctga cccaccgcca gaccaacacc 300
atcatcgacg tggtgctgtc cccttcccac ggcatcgagc tgtccgtggg cgagaagctg 360
gtgctgaact gcaccgcccg caccgagctg aacgtgggca tcgacttcaa ctgggagtac 420
ccttcctcca agcaccagca caagaagctg gtgaaccgcg acctgaagac ccagtccggc 480
tccgagatga agaagttcct gtccaccctg accatcgacg gcgtgacccg ctccgaccag 540
ggcctgtaca cctgcgccgc ctcctccggc ctgatgacca agaagaactc caccttcgtg 600
cgcgtgcacg agaagtcttc cgccagcacc aagggcccca gcgtcttccc actggctcct 660
tcctctaaaa gcactagcgg agggaccgca gccctgggct gtctggtgaa agactacttc 720
cccgagcccg tgaccgtctc ctggaactct ggagccctga cctccggggt gcacaccttt 780
cccgccgtgc tgcagtcttc tggactgtac tccctgtcct ccgtcgtgac tgtgcccagc 840
tcctccctgg gaactcagac atacatctgc aacgtgaacc acaagccttc caacacaaag 900
gtggacaaga aagtcgagcc caagagctgt gataagaccc atacatgtcc cccatgcccc 960
gctccagaac tgctgggcgg accttccgtg tttctgttcc cacccaagcc aaaggacacc 1020
ctgatgatca gcagaacccc tgaggtgact tgcgtggtcg tggacgtgag ccatgaggac 1080
cccgaggtga agttcaactg gtatgtggat ggcgtggaag tgcataatgc caagacaaaa 1140
cctagggaag agcagtacaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 1200
caggattggc tgaacggcaa ggaatacaag tgcaaggtgt ccaataaggc tctgcctgca 1260
cctatcgaga agaccatcag caaagccaag ggccaaccca gagagcctca agtctacacc 1320
ctgcccccaa gcagggatga gctgaccaaa aatcaagtga gcctgacatg cctggtcaaa 1380
ggcttctacc ctagcgacat cgccgtggag tgggagagca atggccagcc tgagaacaac 1440
tacaagacca ctccccccgt cctggatagc gacggcagct tcttcctgta ctccaaactg 1500
acagtcgata aaagcaggtg gcagcaaggc aatgtcttta gctgtagcgt gatgcacgag 1560
gccctgcata accactacac tcaaaagtcc ctgtccctga gccccggaaa a 1611
<210> 74
<211> 297
<212> PRT
<213> 人工序列
<220>
<223>
<400> 74
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
1 5 10 15
Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro
35 40 45
Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg
50 55 60
Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu
65 70 75 80
Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95
Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp
100 105 110
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140
Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr
145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175
Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Thr
180 185 190
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
195 200 205
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
210 215 220
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
225 230 235 240
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
245 250 255
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
260 265 270
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
275 280 285
Thr Lys Ser Phe Asn Arg Gly Glu Cys
290 295
<210> 75
<211> 891
<212> DNA
<213> 人工序列
<220>
<223>
<400> 75
ttccccacca ttcctctgtc caggctgttc gacaacgcca tgctgagggc ccacaggctg 60
catcaactgg ccttcgacac ctaccaggag ttcgaggaag cctacatccc taaggagcag 120
aaatactcct tcctgcagaa cccccagact tctctgtgct tctccgagtc catcccaacc 180
ccctccaaca gggaggaaac ccaacagaag tccaacctgg aactgctgag gatctctctg 240
ctgctgattc agtcctggct ggagcccgtg caattcctga ggtctgtgtt cgcaaactcc 300
ctggtgtacg gcgcctccga ctccaacgtg tacgatctgc tgaaggacct ggaggaggga 360
atccagacac tgatgggcag gctggaagac ggctccccaa ggaccggcca aatcttcaag 420
cagacctact ccaagtttga caccaactcc cacaacgatg acgccctgct gaaaaactac 480
ggactgctgt actgctttag gaaagatatg gacaaagtgg agacctttct gaggattgtg 540
cagtgcaggt ccgtggaggg ctcctgcggc ttcaccgtgg ccgcccccag cgtgttcatc 600
ttccctccca gcgacgagca gctgaagtct ggcaccgcca gcgtggtgtg cctgctgaac 660
aacttctacc cccgcgaggc caaggtgcag tggaaggtgg acaacgccct gcagagcggc 720
aacagccagg agagcgtgac cgagcaggac tccaaggaca gcacctacag cctgagcagc 780
accctgaccc tgagcaaggc cgactacgag aagcacaagg tgtacgcctg cgaggtgacc 840
caccagggac tgtctagccc cgtgaccaag agcttcaacc ggggcgagtg c 891
<210> 76
<211> 324
<212> PRT
<213> 人工序列
<220>
<223>
<400> 76
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
1 5 10 15
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
20 25 30
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
35 40 45
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
50 55 60
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
65 70 75 80
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
100 105 110
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 77
<211> 972
<212> DNA
<213> 人工序列
<220>
<223>
<400> 77
accaagggcc cttccgtgtt ccctctggcc ccttgctccc gctccacctc cgagtccacc 60
gccgccctgg gctgcctggt gaaggactac ttccctgagc ctgtgaccgt gtcctggaac 120
tccggcgccc tgacctccgg cgtgcacacc ttccctgccg tgctgcagtc ctccggcctg 180
tactccctgt cctccgtggt gaccgtgcct tcctcctccc tgggcaccaa gacctacacc 240
tgcaacgtgg accacaagcc ttccaacacc aaggtggaca agcgcgtgga gtccaagtac 300
ggccctcctt gcccttcctg ccctgcccct gagttcctgg gcggcccttc cgtgttcctg 360
ttccctccta agcctaagga caccctgatg atctcccgca cccctgaggt gacctgcgtg 420
gtggtggacg tgtcccagga ggaccctgag gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagcctcgc gaggagcagt tcaactccac ctaccgcgtg 540
gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 600
gtgtccaaca agggcctgcc ttcctccatc gagaagacca tctccaaggc caagggccag 660
cctcgcgagc ctcaggtgta caccctgcct ccttcccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaagggcttc tacccttccg acatcgccgt ggagtgggag 780
tccaacggcc agcctgagaa caactacaag accacccctc ctgtgctgga ctccgacggc 840
tccttcttcc tgtactcccg cctgaccgtg gacaagtccc gctggcagga gggcaacgtg 900
ttctcctgct ccgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgtccctgg ga 972
<210> 78
<211> 155
<212> PRT
<213> 人工序列
<220>
<223>
<400> 78
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
20 25 30
Thr Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
50 55 60
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
65 70 75 80
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
85 90 95
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
100 105 110
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
115 120 125
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
130 135 140
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
145 150 155
<210> 79
<211> 465
<212> DNA
<213> 人工序列
<220>
<223>
<400> 79
gccgtgtccg agcaccagct gctccacgac aagggcaagt ctatccagga cctcaggcgc 60
agattcttcc tgcaccacct cattgctgag attcacactg ctggcggggg agggagcggc 120
ggagggggat ccggaggagg cggatccacc gtggccgccc ccagcgtgtt catcttccct 180
cccagcgacg agcagctgaa gtctggcacc gccagcgtgg tgtgcctgct gaacaacttc 240
tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc 300
caggagagcg tgaccgagca ggactccaag gacagcacct acagcctgag cagcaccctg 360
accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 420
ggactgtcta gccccgtgac caagagcttc aaccggggcg agtgc 465
<210> 80
<211> 380
<212> PRT
<213> 人工序列
<220>
<223>
<400> 80
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
20 25 30
Thr Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
35 40 45
Ser Val Thr Val Ser Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
50 55 60
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
65 70 75 80
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
85 90 95
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
100 105 110
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
115 120 125
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
130 135 140
Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
145 150 155 160
Pro Pro Cys Pro Ala Pro Pro Ala Ala Ala Ser Ser Val Phe Leu Phe
165 170 175
Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val
180 185 190
Thr Cys Val Val Val Asp Val Ser Ala Glu Asp Pro Glu Val Gln Phe
195 200 205
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
210 215 220
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
225 230 235 240
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
245 250 255
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr
260 265 270
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
275 280 285
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
290 295 300
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
305 310 315 320
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
325 330 335
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
340 345 350
Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His
355 360 365
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 81
<211> 1140
<212> DNA
<213> 人工序列
<220>
<223>
<400> 81
gccgtgtccg agcaccagct gctccacgac aagggcaagt ctatccagga cctcaggcgc 60
agattcttcc tgcaccacct cattgctgag attcacactg ctggcggggg agggagcggc 120
ggagggggat ccggaggagg cggatccgtg accgtgtcca ccgcctccac caagggccct 180
tccgtgttcc ctctggcccc ttgctcccgc tccacctccg agtccaccgc cgccctgggc 240
tgcctggtga aggactactt ccctgagcct gtgaccgtgt cctggaactc cggcgccctg 300
acctccggcg tgcacacctt ccctgccgtg ctgcagtcct ccggcctgta ctccctgtcc 360
tccgtggtga ccgtgccttc ctccaacttc ggcacccaga catacacatg caacgtggac 420
cacaagcctt ctaacacaaa ggtggacaag accgtggagc ggaagtgctg cgtggagtgc 480
ccaccttgcc ccgctcctcc tgccgccgca tcctctgtgt tcctgttccc acctaagcca 540
aaggaccagc tcatgatcag cagaacccct gaggtgacct gcgtggtggt ggacgtgagc 600
gccgaggacc ccgaggtgca gttcaactgg tatgtggacg gcgtggaggt gcacaacgct 660
aagaccaagc ctagagaaga acagttcaac agcacattca gagtggtgtc cgtgctcacc 720
gtgctgcacc aggactggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 780
ctgccatctt ctatcgaaaa aacaatcagc aagaccaagg gccagcctag agagcctcag 840
gtgtacacac tgcctccatc tcgggaagaa atgacaaaga accaggtgtc cctcacatgc 900
ctcgtgaagg gcttctaccc atccgacatc gctgtggagt gggagtctaa cggccagccc 960
gagaacaact acaagaccac ccctcctatg ctcgactccg acggctcttt cttcctgtac 1020
tctaagctga ccgtggacaa gtccagatgg cagcagggca acgtgttctc ttgcagcgtg 1080
ctgcacgagg ctctccacaa ccactacacc cagaagtccc tgagcctgtc tccaggcaag 1140

Claims (8)

1.IgG样免疫融合蛋白，由效应分子和IgG抗体恒定区组成，所述效应分子通过连接肽与所述IgG抗体恒定区相连；
所述IgG样免疫融合蛋白的轻链的氨基酸序列如序列表中序列74所示，重链的氨基酸序列如序列表中序列76所示。
2.根据权利要求1所述的IgG样免疫融合蛋白，其特征在于：所述IgG样免疫融合蛋白是按照包括如下步骤的方法制备获得的：将序列表中序列74所示蛋白的编码基因插入到具有双表达盒的谷氨酰胺合成酶高效表达载体的酶切位点XhoI和EcoRI之间，得到中间载体；然后再将序列表中序列76所示蛋白的编码基因插入到所述中间载体的酶切位点XbaI和SaII之间，得到重组表达载体；所述重组表达载体表达出的蛋白即为所述IgG样免疫融合蛋白。
3.根据权利要求2所述的IgG样免疫融合蛋白，其特征在于：所述序列表中序列74所示蛋白的编码基因为序列表中序列75所示DNA分子；所述序列表中序列76所示蛋白的编码基因为序列表中序列77所示DNA分子。
4.IgG样免疫融合蛋白，由效应分子和IgG抗体恒定区组成，所述效应分子通过连接肽与所述IgG抗体恒定区相连；
所述IgG样免疫融合蛋白的轻链的氨基酸序列如序列表中序列58所示，重链的氨基酸序列如序列表中序列60所示。
5.根据权利要求4所述的IgG样免疫融合蛋白，其特征在于：所述IgG样免疫融合蛋白是按照包括如下步骤的方法制备获得的：将序列表中序列58所示蛋白的编码基因插入到具有双表达盒的谷氨酰胺合成酶高效表达载体的酶切位点XhoI和EcoRI之间，得到中间载体；然后再将序列表中序列60所示蛋白的编码基因插入到所述中间载体的酶切位点XbaI和SaII之间，得到重组表达载体；所述重组表达载体表达出的蛋白即为所述IgG样免疫融合蛋白。
6.根据权利要求5所述的IgG样免疫融合蛋白，其特征在于：所述序列表中序列58所示蛋白的编码基因为序列表中序列59所示DNA分子；所述序列表中序列60所示蛋白的编码基因为序列表中序列61所示DNA分子。
7.权利要求1-3中任一所述的IgG样免疫融合蛋白在制备用于治疗与生长激素相关疾病的药物中的应用。
8.权利要求4-6中任一所述的IgG样免疫融合蛋白在制备用于治疗糖尿病的药物中的应用。
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