一种牛呼吸道胞合体病毒抗原蛋白的制作方法

文档序号:12092184阅读:334来源:国知局
一种牛呼吸道胞合体病毒抗原蛋白的制作方法与工艺

本发明属于生物产品制备技术领域,涉及一种牛呼吸道胞合体病毒抗原蛋白及其制备方法。



背景技术:

牛呼吸道胞合体病毒(Bovine Respiration Syncytial Virus – BRSV)感染可引起牛呼吸道疾病,在欧共体国家被列为仅次于牛粘膜病(BVD)及牛传染性鼻气管炎(IBR)的重要牛病之一。本病呈世界范围性流行,其危害性在于BRSV感染发病率高达到60%-80%,死亡率在一些爆发中达到20%以上,给养牛业造成很大的经济损失。虽然这种BRSV从上个世纪70年代就开始列入研发范畴,由于种种原因并没有开发出理想的药物和疫苗。尽管已有关于BRSV的多种灭活,弱毒,重组以及DNA疫苗的报道,但距离临床使用还有一定距离,目前国际上尚没有理想的病毒疫苗。

传统疫苗的研制和生产主要是通过改变培养条件,或在不同寄主动物上传代使致病微生物毒性减弱,或通过物理、化学方法将其灭活来完成的。随着人类知识的不断进步,传统疫苗的局限性也日益显露出来:(1)动物和人类的病毒需要在动物细胞中培养,这使得疫苗生产的成本很高;(2)疫苗中的致病物质在疫苗生产过程中有可能没有完全杀死或充分减毒,这会导致疫苗中含有强毒性致病物质,进而使得疾病在更大的范围内传播;(3)减毒菌株有可能会发生突变,引起病害。

牛呼吸道胞合体病毒是副黏液病毒科家族的成员,其上的F-蛋白是高度保守的并形成三聚体刺突,其在激活下发生构象变化。F-蛋白介导病毒和细胞膜的融合,让病毒核壳体进入细胞质,抑制F-蛋白介导的步骤阻抑感染周期的起始阶段并中和病毒感染性可以有效的防止病毒侵害,即针对F-蛋白的抗体,抑制它的活性,可以中和病毒感染性并可以保护免受BRSV 感染。施一公开创了中国结构生物学基础理论研究的先河,本发明将结构生物学应用到实际中,合成牛呼吸道胞合体病毒抗原蛋白。



技术实现要素:

本发明的目的在于克服现有技术存在的缺点,提供一种牛呼吸道胞合体病毒抗原蛋白及其制备方法,该抗原蛋白应用蛋白质晶体学的技术和理论,确定蛋白在病毒侵染和致病过程中的结构变化,再根据结构变化的结果对这个关键的病毒蛋白进行基因工程改造,使其成为只侵染不致病、并引起动物体抗体高效反应的蛋白抗原。

为了实现上述目的,本发明解决其技术问题所采取的技术方案是:

一种牛呼吸道胞合体病毒抗原蛋白,编码牛呼吸道胞合体病毒关键蛋白F-蛋白野生种的氨基酸序列简称SEQ 048055.1,具体序列为MATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAVASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK,其特征在于:所述牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQ 048055.1至少有两个位点被半胱氨酸替代,且替代后的半胱氨酸之间由二硫键连接。

作为优化的,所述位点为氨基酸序列第148位缬氨酸和288位异亮氨酸。

作为优化的,所述位点为氨基酸序列第158位亮氨酸和290位丝氨酸。

作为优化的,所述牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQ 048055.1,在第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充。

作为优化的,所述针对牛呼吸道胞合体病毒抗原蛋白的氨基酸序列剔除SEQ 048055.1序列中的103位点至138位点的氨基酸序列,采用sgsgs连接102位点和139位点。

作为优化的,所述针对牛呼吸道胞合体病毒抗原蛋白的氨基酸序列剔除SEQ 048055.1序列中的103位点至142位点的氨基酸序列,采用sgsgs连接102位点和143位点。

作为优化的,所述编码牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQ 048055.1序列,第99位点天冬氨酸由半胱氨酸替代,第362位点丝氨酸由半胱氨酸替代,并在替代后的半胱氨酸之间连接内二硫键。

一种牛呼吸道胞合体病毒抗原蛋白的制备方法,其特征在于:具体工艺如下:

(1)根据结构生物学的原理,进行蛋白结晶,结晶后用X-射线观测,确定侵染病毒关键病原F-蛋白侵染前后的结构变化;

(2)根据步骤(1)结构变化设计出抗原蛋白的序列和结构;

(3)经多种抗原测试和稳定性特性检测确定出需要抗原蛋白的合格性;

(4)将经过验证的抗原蛋白序列进行相应的蛋白合成、表达、提纯和检测,得到用来作为动物试验和临床试验的针对牛呼吸道胞合体病毒抗原蛋白。

牛胞合体病毒关键F-蛋白介导病毒和细胞膜的融合,让病毒核壳体进入细胞。针对牛呼吸道胞合体病毒设计的抗原蛋白,便是根据F-蛋白侵染前后的结构变化设定的,在基因工程的基础上使F-蛋白结构始终保持在融合前的状态,使其具有只侵染不致病、并引起动物体抗体高效反应的蛋白抗原等特点。

本发明能够使F-蛋白在侵染前后都保持三聚体刺突状态(闭合状态),得到只侵染不致病、并引起动物抗体高效反应的蛋白抗原;为了保证来自于病原病毒上的F-蛋白处在稳定的侵染前的状态,给F-蛋白上增添了系列的连接链,包括内二硫键、二硫键、空穴填充和单链连接。

本发明与现有技术相比,其有益效果是:采用蛋白质晶体学方法制备的牛呼吸道胞合体病毒抗原蛋白能够有效的预防和治疗牛呼吸道胞合体病毒感染,以此为基础可以开发出理想的病毒疫苗;有效控制和防范病毒感染带来的损失,具有重大的经济意义和社会意义。

附图说明

图1 本发明涉及利用Phenix软件进行牛合胞体病毒F-蛋白的三维立体结构模拟图,左图为浸染前三聚体三维立体结构模拟图,右图为左图右下角方框位置的局部放大图。

图2 本发明涉及的牛合胞体病毒呼吸道病毒侵染前后过程的关键的F-蛋白结构变化示意图。在图中显示侵染前(左),F-蛋白三聚体呈现三聚体刺突状态,侵染后(右)F-蛋白被激活,发生构象变化,病毒和细胞膜的融合,病毒核壳体进入细胞质,进而侵染牛体并使其致病。

图3 本发明涉及的经过改造成功的牛呼吸道胞合体蛋白抗原侵染牛体后的电镜照片状态。左图显示RD-BRSV-DB1-CF1改造的牛呼吸道胞合体蛋白抗原侵染牛体后的状态,可见呈现稳定的融合前三聚体刺突状态,右图为未经过改造的牛呼吸道胞合体病毒侵染牛体后的电镜照片。

图4 本发明涉及的F-蛋白上添加连接链示意图,包括内二硫键、二硫键、空穴填充和单链连接,这些链接保证F-蛋白保持三聚体刺突状态。

具体实施方式

下面通过实施例并结合附图对本发明进一步说明。

实施例1:将SEQ 048055.1进行如下改造得到本发明的一种牛呼吸道胞合体病毒抗原蛋白:

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第148位缬氨酸由半胱氨酸替代,第288位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1,其氨基酸序列简称为SEQ DB1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第158位亮氨酸由半胱氨酸替代,第290位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2,其氨基酸序列简称为SEQ DB2。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第148位缬氨酸由半胱氨酸替代,第288位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1,其氨基酸序列简称为SEQ DB1-CF1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第158位亮氨酸由半胱氨酸替代,第290位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1,其氨基酸序列简称为SEQ DB2-CF1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第148位缬氨酸由半胱氨酸替代,第288位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接;同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点138位点的氨基酸序列,采用sgsgs连接102及139位点,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC1,其氨基酸序列简称为SEQ DB1-CF1-SC1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第158位亮氨酸由半胱氨酸替代,第290位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点138位点的氨基酸序列,采用sgsgs连接102及139位点,将其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC1,其氨基酸序列简称为SEQ DB2-CF1-SC1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第148位缬氨酸由半胱氨酸替代,第288位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接;同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点138位点的氨基酸序列,采用sgsgs连接102及139位点,第99位点天冬氨酸由半胱氨酸替代,第362位点丝氨酸由半胱氨酸替代,并在替代后的半胱氨酸之间连接内二硫键,进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC1-ID1,其氨基酸序列简称为SEQ DB1-CF1-SC1-ID1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第148位缬氨酸由半胱氨酸替代,第288位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接;同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点142位点的氨基酸序列,采用sgsgs连接102及143位点,第99位点天冬氨酸由半胱氨酸替代,第362位点丝氨酸由半胱氨酸替代,并在替代后的半胱氨酸之间连接内二硫键,进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC2-ID1,其氨基酸序列简称为SEQ DB1-CF1-SC2-ID1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第158位亮氨酸由半胱氨酸替代,第290位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点138位点的氨基酸序列,采用sgsgs连接102及139位点;第99位点天冬氨酸由半胱氨酸替代,第362位点丝氨酸由半胱氨酸替代,并在替代后的半胱氨酸之间连接内二硫键。其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC1-ID1,其氨基酸序列简称为SEQ DB2-CF1-SC1-ID1。

牛呼吸道胞合体病毒F-蛋白氨基酸序列SEQ 048055.1第158位亮氨酸由半胱氨酸替代,第290位异亮氨酸由半胱氨酸替代,并由二硫键将两位点连接,同时将第260位点的亮氨酸由半胱氨酸替代,并在该位点实现空穴填充;剔除SEQ 048055.1序列中103至位点142位点的氨基酸序列,采用sgsgs连接102及143位点;第99位点天冬氨酸由半胱氨酸替代,第362位点丝氨酸由半胱氨酸替代,并在替代后的半胱氨酸之间连接内二硫键。其进行相应的蛋白合成、表达、提纯,得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC2-ID1,其氨基酸序列简称为SEQ DB2-CF1-SC2-ID1。

免疫原性又称抗原性,指抗原刺激机体产生免疫应答能力的特性。免疫反应性指抗原分子能与相应免疫应答的产物(抗体或致敏淋巴细胞),在体内或体外发生特异性结合的性能,采用ELISA法对抗原与抗体的亲和力-免疫进行检验。ELISA法具有灵敏、特异、简单、快速、稳定及易于自动化操作等特点,作为免疫诊断中的一项新技术,已成功地应用于大分子抗原和小分子抗原的定量测定。高亲合力的抗体与抗原的结合力强,即抗原浓度很低时也有较多的抗体结合抗原形成免疫复合物。亲和性用平衡常数K来表示,K值越大,亲和性越高,与抗原结合也越牢固。表1给出了本发明制备的抗原蛋白分别在SiteØ、SiteQP、SiteⅤ、SiteⅡ位点与抗体结合的亲和性数据。

表1 实施例牛呼吸道胞合体病毒抗原蛋白的特性检测

本发明经过设计改造的F-蛋白具有较高的抗体库亲和性。经过改造后的抗原蛋白都保持融合前三聚体刺突状态(Trimer),且都能得到目的抗原蛋白。

本发明获得纯化的抗原蛋白与D25抗体进行特异性结合,经蛋白免疫电泳和ELISA方法检测,结果表明不仅具有较好的免疫原性,而且能够诱导小鼠产生较高的抗体水平,表2给出了D25抗体与本发明抗原蛋白特异性结合的免疫原性加权平均值,对照组为非变异野生种。

表2 实施例牛呼吸道胞合体病毒抗原蛋白与D25特异性结合的特性检测

对本发明中的抗原蛋白进行物理化学稳定性进行测定,包括温度、pH、渗透压等方面,详细数据见表3,表中数据为在相应条件下抗原蛋白活性与在最佳状态时的抗原蛋白活性的比值,结果表明经过设计改造的抗原蛋白对温度、PH值、渗透压等呈现较好的稳定性,具有较高的反应活性。

表3实施例牛呼吸道胞合体病毒抗原蛋白的反应活性稳定性

实施例2:制备实施例1中RD-BRSV-DB1-CF1-SC1-ID1的具体工艺。看到蛋白机构和形状可以通过以下三种途径,分别为x-射线(x-ray Crystallography)、核磁共振(NMR)、电镜,本发明采用X-射线法对蛋白结构进行观察。采用Solution maker 600 plux系列将目标蛋白进行结晶,将结晶后的目标蛋白采用X-射线系统(SER-CAT22)收集结晶体数据,并使其成像,获得蛋白结构的电子云图,得到关键蛋白F-蛋白侵染前后的数据变化,侵染前后结构变化如图 2所示。

对上述电子云图进行云数据处理,利用相应的软件(Phenix)和野生蛋白序列进行数据模拟,形成蛋白结构的三维立体结构模拟图形。根据蛋白序列模拟出的三维蛋白结构图形得到F-蛋白的三聚体结构,详见图1。

按照晶体化学的原理,利用模拟软件进行抗原蛋白结构设计,其设计包括如图4所示的四种方式:内二硫键(intra disulphide bond)、二硫键(disulphide bond)、空穴填充(cavity filling)和单链连接(single chain),对野生种048055.1进行设计。

将设计好的蛋白序列转译成DNA序列,采用invitrogen公司提供的Expi293F作为细胞载体在293Fectin培养基中进行培养5天,收集细胞培养液,将目标蛋白进行提纯精制得到设计好的纯化的BRSV F-蛋白,即RD-BRSV-DB1-CF1-SC1-ID1,改造后的牛呼吸道胞合体蛋白抗原侵染牛体后呈现稳定的融合前三聚体刺突状态见图3(左)。

上述具体实施方式仅是本发明的具体个案,本发明的专利保护范围包括但不限于上述具体实施方式的产品形态和式样,任何符合本发明权利要求书的一种牛呼吸道胞合体病毒抗原蛋白且任何所属技术领域的普通技术人员对其所做的适当变化或修饰,皆应落入本发明的专利保护范围。

SEQUENCE LISTING

<110> 烟台偌帝生物工程有限公司

<120> 一种牛呼吸道胞合体病毒抗原蛋白

<130>

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 574

<212> PRT

<213> 牛呼吸道胞合体病毒F-蛋白野生种

<400> 1

Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser

1 5 10 15

Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile

50 55 60

Gln Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu

85 90 95

Met Gln Asn Glu Pro Ala Ser Phe Ser Arg Ala Lys Arg Gly Ile Pro

100 105 110

Glu Leu Ile His Tyr Thr Arg Asn Ser Thr Lys Lys Phe Tyr Gly Leu

115 120 125

Met Gly Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Ile

130 135 140

Gly Ser Ala Val Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro Gln Val Asn

195 200 205

Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Val Val Lys Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro

290 295 300

Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Thr Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly

450 455 460

Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn

485 490 495

Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu

500 505 510

Leu His Ser Val Asp Val Gly Lys Ser Thr Thr Asn Val Val Ile Thr

515 520 525

Thr Ile Ile Ile Val Ile Val Val Val Ile Leu Met Leu Ile Ala Val

530 535 540

Gly Leu Leu Phe Tyr Cys Lys Thr Lys Ser Thr Pro Ile Met Leu Gly

545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Leu Ser Phe Ser Lys

565 570

<210> 2

<211> 489

<212> PRT

<213> 人工序列

<400> 2

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Thr Arg Asn Ser

85 90 95

Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 3

<211> 489

<212> PRT

<213> 人工序列

<400> 3

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Thr Arg Asn Ser

85 90 95

Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Val Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 4

<211> 489

<212> PRT

<213> 人工序列

<400> 4

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Thr Arg Asn Ser

85 90 95

Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 5

<211> 489

<212> PRT

<213> 人工序列

<400> 5

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Thr Arg Asn Ser

85 90 95

Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Val Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 6

<211> 494

<212> PRT

<213> 人工序列

<400> 6

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Ser Phe Ser Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His

85 90 95

Tyr Thr Arg Asn Ser Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys

100 105 110

Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys

115 120 125

Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val

130 135 140

Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser

145 150 155 160

Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys

165 170 175

Asn Tyr Ile Asp Lys Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys

180 185 190

Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn

195 200 205

Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr

210 215 220

Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe

225 230 235 240

Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser

245 250 255

Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val

260 265 270

Lys Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val

275 280 285

Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr

290 295 300

Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly

305 310 315 320

Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu

325 330 335

Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser

340 345 350

Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn

355 360 365

Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser

370 375 380

Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr

385 390 395 400

Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser

405 410 415

Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val

420 425 430

Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr

435 440 445

Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro

450 455 460

Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn

465 470 475 480

Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His

485 490

<210> 7

<211> 454

<212> PRT

<213> 人工序列

<400> 7

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Val Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Val

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln Ser Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

<210> 8

<211> 458

<212> PRT

<213> 人工序列

<400> 8

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

130 135 140

Lys Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile

210 215 220

Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val

225 230 235 240

Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro

245 250 255

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu

260 265 270

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

275 280 285

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val

290 295 300

Gln Cys Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro

305 310 315 320

Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp

325 330 335

Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr

340 345 350

Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

355 360 365

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

370 375 380

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

385 390 395 400

Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu

405 410 415

Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

420 425 430

Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala

435 440 445

Phe Ile Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 9

<211> 454

<212> PRT

<213> 人工序列

<400> 9

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Val Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Val

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln Cys Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

<210> 10

<211> 458

<212> PRT

<213> 人工序列

<400> 10

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

130 135 140

Lys Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile

210 215 220

Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val

225 230 235 240

Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro

245 250 255

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu

260 265 270

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

275 280 285

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val

290 295 300

Gln Cys Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro

305 310 315 320

Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp

325 330 335

Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr

340 345 350

Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

355 360 365

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

370 375 380

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

385 390 395 400

Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu

405 410 415

Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

420 425 430

Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala

435 440 445

Phe Ile Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 11

<211> 454

<212> PRT

<213> 人工序列

<400> 11

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Val Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Val Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Val

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln Cys Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

当前第1页1 2 3 
网友询问留言 已有0条留言
  • 还没有人留言评论。精彩留言会获得点赞!
1