针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合及其设计方法

摘要

本发明公开了一种针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合及其设计方法，设计方法包括如下步骤：收集靶向药物耐药突变数据；截取耐药突变多肽序列截取及预测MHC分子亲和力及免疫原性；靶向药物与耐药位点相互关系及药物聚类；人工设计相对应的疫苗多肽序列；通过这样的设计方法得到的靶向药物联合多肽疫苗组合能够覆盖常见的靶向治疗药物，可以有效降低肿瘤耐药发生概率，延长靶向药有效作用时间，增加患者的疾病响应率，具有广谱性，缩短个体化多肽疫苗从分析到治疗的时间，降低医疗成本。

说明书

本案是申请号为：201910086371.9，发明名称为：针对肿瘤靶向药物耐药位点的多肽疫苗及其设计方法的专利的分案。

技术领域

本发明涉及肿瘤疫苗领域，特别是一种针对肿瘤靶向药物耐药位点的多肽疫苗及其设计方法。

背景技术

近年来,肿瘤靶向治疗的进展随着分子生物学技术的发展和对发病机制从细胞、分子水平的进一步认识已经进入了一个全新的时代。这些领域的进展很快，迄今为止，很多靶向药物已经在临床起了极其重要甚至是奇迹般的作用。分子靶向治疗已成为外科、放化疗等肿瘤治疗的又一重要手段，在肿瘤治疗中发挥着越来越重要的作用。由于靶向治疗的高效率和低毒性，与传统的治疗和化疗相比，其优点是很明显的。靶向治疗发展迅速，有些已经按照循证医学的原则进入了国际肿瘤学界公认的标准治疗方案和规范。更多、更有希望的药物也在快马加鞭地研制和早期临床试验中。靶向药已成肿瘤治疗的一大利器。但是，随着治疗时间越来越长，多数患者出现耐药。至于耐药的具体机制，目前主要有3种。首先，通过基因突变产生耐药。基因检测阳性的患者约40％的基因会由原来基因产生新的基因，这会导致对原来的药物不敏感，从而产生耐药反应。其次，狡猾的癌细胞常常会“明修栈道暗度陈仓”，绕弯走另外一条路。这种情况在耐药患者中约占20％左右。除了以上两种耐药途径，剩下30％左右的患者的耐药机制，尚不明确。靶向后的“变身”(耐药)是不可避免的，几乎所有的靶向药物治疗都会出现耐药现象，其根本原因是由于癌细胞的异质性以及动态变化。目前一种靶向药物是针对癌细胞的某一个蛋白、某一个分子起作用，所以只能抑制肿瘤生长的一条通路而已。当一条通路受到抑制时，肿瘤细胞会自寻新的“生路”，选择其他通路合成自身生长所需要的物质，久而久之，分子靶向药物就失去了作用，从而产生耐药性，比如EGFR突变患者服用靶向药后出现耐药，50％～60％是因为再次发生基因突变，大多数突变为T790M突变，此时患者可以使用第三代靶向药物如奥斯替尼。大多数患者在接受EGFR-TKI治疗后的8-14个月会出现继发耐药，如何解决靶向药物治疗的耐药问题成为研究的热点。

近年来，由于肿瘤相关抗原和肿瘤特异性抗原的发现，以及人们对肿瘤诱导免疫应答和肿瘤逃避免疫监视机制的深入研究和理解，肿瘤多肽疫苗的研究已经取得了可喜的成就。新生抗原是基因突变导致的肿瘤细胞表面表达的异常蛋白，与常规抗原靶点不同的是新生抗原只在肿瘤细胞表面表达不在正常细胞表面表达且能被免疫系统识别并激活免疫系统。2017年7月13日，Nature杂志同一天发布了两项基于neoantigen的个性化肿瘤疫苗治疗恶性黑色素瘤的成功案例。德国的Carmen Loquai教授和

发明内容

为解决现有技术的不足，本发明的目的在于提供一种针对肿瘤靶向药物耐药位点的多肽疫苗及其设计方法，提供的靶向药物联合多肽疫苗组合方案覆盖常见的靶向治疗药物，可以有效降低肿瘤耐药发生概率，延长靶向药有效作用时间，增加患者的疾病响应率，具有广谱性，缩短个体化多肽疫苗从分析到治疗的时间，降低医疗成本。

为了实现上述目标，本发明采用如下的技术方案：

针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合，达拉菲尼的多肽疫苗组合针对如下突变：MAP2K1-P124S，MAP2K2-Q60P，NRAS-G12D，NRAS-Q61R；MAP2K1-P124S的序列为：LQVLHECNSSYIVGFYGAFYKK；MAP2K2-Q60P的序列为：KKRLEAFLTPKAKVGELK；NRAS-G12D的序列为：YKLVVVGADGVGKSAL；NRAS-Q61R的序列为：CLLDILDTAGREEYSAMRDQYKKK；派姆单抗的多肽疫苗针对如下突变：NRAS-Q61R；NRAS-Q61R的序列为：CLLDILDTAGREEYSAMRDQYKKK；威罗菲尼的多肽疫苗组合针对如下突变：MAP2K1-G128V，MAP2K1-P124L，MAP2K1-Q56P，NRAS-Q61R；MAP2K1-G128V的序列为：LHECNSPYIVVFYGAFYSDGEKK；MAP2K1-P124L的序列为：ELQVLHECNSLYIVGFYGAFYKKK；MAP2K1-Q56P的序列为：RKRLEAFLTPKQKVGELK；NRAS-Q61R的序列为：CLLDILDTAGREEYSAMRDQYKKK；司美替尼的多肽疫苗针对如下突变：MAP2K1-P124L；MAP2K1-P124L的序列为：ELQVLHECNSLYIVGFYGAFYKKK。

针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合的设计方法，包括如下步骤：寻找靶向药物耐药突变数据的数据；截取耐药突变多肽序列及预测MHC分子亲和力及免疫原性：对于点突变截取覆盖突变位点上下游16个氨基酸的多肽序列，对于移码突变截取向前延伸16个长度的氨基酸、向后延伸直到终止密码子的多肽序列作为耐药突变位点的突变型多肽，同时截取相应位置对应的野生型多肽序列，用至少一个数据库作为来源统计高频HLA分型及频率，将统计出的HLA合并去重之后作为用于预测的候选HLA分型，使用多款软件分析这些突变位点对应的多肽与HLA分子结合亲和力，综合多款软件将亲和力分为三类：强亲和力-SB、弱亲和力-WB和无亲和力，并与相对野生型多肽比较确定其亲和力变化，A类变化为由无亲和力变为有强亲和力，B类变化为由无亲和力变为弱亲和力，C类变化为由弱亲和力变为强亲和力，D类变化为无变化，内部排序认为A类优于B类优于C类优于D类，使用免疫原性预测工具预测其免疫原性，保留突变型多肽亲和力强、亲和力变化大并且免疫原性强的表位；靶向药物与耐药位点相互关系及药物聚类：对耐药位点亲和力打分：综合考虑位点有亲和力的表位个数及各表位亲和力变化大小，对A、B、C、D四类不同亲和力变化给予相对应的权重，根据每个表位对应的HLA频率大小给予权重,将位点的各表位累加求和；

第一类针对SMO突变产生耐药的Hedgehog信号通路拮抗剂维莫德吉，第二类针对BTK突变产生耐药的依鲁替尼，第三类针对AR耐药突变的一些抗雄激素类药物如阿比特龙等，第四类针对BRAF耐药突变的一些菲尼类药物，第五类针对ALK、MET等融合基因的一些络氨酸激酶抑制剂，第六类针对EGFR通路的络氨酸激酶抑制剂，第七类针对PI3K/AKT/mTOR通路的依维莫司等激酶抑制剂；

设计相对应的疫苗多肽序列。

前述的针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合的设计方法，截取耐药突变多肽序列及预测MHC分子亲和力及免疫原性：对于点突变截取覆盖突变位点上下游16个氨基酸的多肽序列，对于移码突变截取向前延伸16个长度的氨基酸、向后延伸直到终止密码子的多肽序列作为耐药突变位点的突变型多肽，同时截取相应位置对应的野生型多肽序列，用至少一个数据库作为来源统计高频HLA分型及频率，将统计出的HLA合并去重之后作为用于预测的候选HLA分型，所述数据库包括：公共数据库，临床患者数据库，使用多款软件分析这些突变位点对应的多肽与HLA分子结合亲和力，综合多款软件将亲和力分为三类：强亲和力-SB、弱亲和力-WB和无亲和力，并与相对野生型多肽比较确定其亲和力变化，所述多款软件包括：netMHCpan、netMHC及Pickpocket这三款软件，A类变化为由无亲和力变为有强亲和力，B类变化为由无亲和力变为弱亲和力，C类变化为由弱亲和力变为强亲和力，D类变化为无变化，内部排序认为A类优于B类优于C类优于D类，使用免疫原性预测工具预测其免疫原性，保留突变型多肽亲和力强、亲和力变化大并且免疫原性强的表位。

前述的针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合的设计方法，使用cytoscape对所有的靶向药物及耐药位点作网络图，耐药位点的大小代表其亲和力得分数，综合分析靶向药物和耐药位点间相互关系结合靶向药物作用机制对靶向药物聚类从而将其分为7类：第一类针对SMO突变产生耐药的Hedgehog信号通路拮抗剂维莫德吉，第二类针对BTK突变产生耐药的依鲁替尼，第三类针对AR耐药突变的一些抗雄激素类药物如阿比特龙等，第四类针对BRAF耐药突变的一些菲尼类药物，第五类针对ALK、MET等融合基因的一些络氨酸激酶抑制剂，第六类针对EGFR通路的络氨酸激酶抑制剂，第七类针对PI3K/AKT/mTOR通路的依维莫司等激酶抑制剂。

本发明的有益之处在于：

本发明提供的靶向药物联合多肽疫苗组合方案覆盖常见的靶向治疗药物；对比单独的靶向治疗，可以有效降低肿瘤耐药发生概率，延长靶向药有效作用时间；对比单独的多肽疫苗，我们提供的方案增加患者的疾病响应率；

无需经过患者遗传信息测序、专门测定患者HLA分型，具有一定的广谱性，大大缩短个体化多肽疫苗从分析到治疗的时间，降低医疗成本；另外多肽疫苗也产生毒副作用；

多肽疫苗本身可以保持长期的肿瘤杀伤效果，疫苗肽在体内被酶切成短多肽并MHC分子递呈出去被TCR识别，可以刺激机体产生特异性杀伤性T细胞和记忆T细胞，识别并清除携带突变的细胞，并且这种效果在体内可以长期保持；

提供了31个靶向药物的多肽疫苗，基本覆盖常见肿瘤靶向药物；

将靶向药物根据其机制和耐药突变聚类分析归为7大类，多靶点联合可以有效增加多肽疫苗对肿瘤细胞的杀伤效果，显著延长靶向药物有效作用时间。

附图说明

图1是本发明的靶向药物和耐药位点相互作用图；原点代表靶向药物，三角形代表耐药位点，三角形大小表示该位点对应多肽的亲和力得分，大圆圈将网络图分为7个部分，代表7个产品；

图2是本发明实验1的A-D的阴性对照和137条肽段的ELISPOT斑点数图；

图3是本发明实验2-1的各组的效应T细胞杀伤效率示意图；

图4是本发明实验2-2的各组的效应T细胞杀伤效率示意图；

图5是本发明实验2-3的各组的效应T细胞杀伤效率示意图；

图6是本发明实验2-4的各组的效应T细胞杀伤效率示意图；

图7是本发明实验2-5的各组的效应T细胞杀伤效率示意图；

图8是本发明实验2-6的各组的效应T细胞杀伤效率示意图；

图9是本发明实验2-7的各组的效应T细胞杀伤效率示意图。

具体实施方式

以下结合附图和具体实施例对本发明作具体的介绍。

针对肿瘤靶向药物耐药位点的多肽疫苗的设计方法，包括如下步骤：

1、靶向药物耐药突变数据收集：人类癌症相关体细胞突变目录Cosmic(数据库官网https://cancer.sanger.ac.uk/cosmic)对临床研究中的获得性耐药和原发性耐药的报道进行了整理和注释，包括基因、突变、药物，癌种及参考文献等信息。我们下载了FDA或者CFDA已批准的或处于临床试验III/IV期的所有靶向治疗药物耐药突变位点，对所有候选靶向药物和耐药突变尤其是支持的样本量较少的突变，进一步查找文献确定其可靠性，确定真正全面可靠的靶向药物耐药位点用于下游分析，例如cosmic收录有一条确定EGFR-T790M突变是奥斯替尼耐药位点的报道，但经过查找文献发现T790M不是奥斯替尼的耐药位点。

2、耐药突变多肽序列截取及MHC分子亲和力及免疫原性预测：

2.1、对于点突变截取覆盖突变位点上下游16个氨基酸的多肽序列，对于移码突变截取向前延伸16个长度的氨基酸、向后延伸直到终止密码子的多肽序列作为耐药突变位点的突变型多肽，同时截取相应位置对应的野生型多肽序列。

2.2、统计高频HLA分型及频率：高频HLA的来源主要有两部分，一是来自公共数据库的统计(IMGT/HLA及中华骨髓库)，结合近年文献报道的信息，汇总了中国人群比较常见的HLA等位基因分型；二是从临床患者的数据库中统计了出现频率比较高的一组HLA分型，具体的数据库如表2所示。将两组HLA合并去重之后作为用于预测的候选HLA分型。需要说明的是，数据库的选择不受限制，这只是一种优选实施例，也可以是其他几种数据库的组合统计。

2.3、我们使用netMHCpan、netMHC及Pickpocket三款软件分析了这些突变位点对应的多肽与2.2步中得到的HLA分子结合亲和力综合三款软件将亲和力分为三类(强亲和力-SB、弱亲和力-WB和无亲和力)，并与相对野生型多肽比较确定其亲和力变化(由无亲和力变为有强亲和力定义为A类变化、由无亲和力变为弱亲和力定义为B类变化、由弱亲和力变为强亲和力定义为C类变化、其它无变化的定义为D类变化，内部排序认为A类优于B类优于C类优于D类)，另外还使用IEDB提供的免疫原性预测工具预测其免疫原性，保留突变型多肽亲和力强、亲和力变化大并且免疫原性强的表位，共筛选得到31种靶向药物对应的137个耐药突变。

3、靶向药物与耐药位点相互关系及药物聚类：3.1对耐药位点亲和力打分：综合考虑位点有亲和力的表位个数及各表位亲和力变化大小(a.对A、B、C、D四类不同亲和力变化给予不同的权重，b.根据每个表位对应的HLA频率大小给予权重,c.将位点的各表位累加求和)。

3.2使用cytoscape对所有的靶向药物及耐药位点作网络图(如图1)，耐药位点的大小代表其亲和力得分数，综合分析靶向药物和耐药位点间相互关系结合靶向药物作用机制对靶向药物聚类从而将其分为7类如图1所示。第一类针对SMO突变产生耐药的Hedgehog信号通路拮抗剂维莫德吉，第二类针对BTK突变产生耐药的依鲁替尼，第三类针对AR耐药突变的一些抗雄激素类药物如阿比特龙等，第四类针对BRAF耐药突变的一些菲尼类药物，第五类针对ALK、MET等融合基因的一些络氨酸激酶抑制剂，第六类针对EGFR通路的络氨酸激酶抑制剂，第七类针对PI3K/AKT/mTOR通路的依维莫司等激酶抑制剂。由于以上靶向药物聚类综合考虑了药物作用机制和与耐药位点之间的相互关系，将有相似作用机制和交叉耐药位点的靶向药物聚为一类，从而最大化的覆盖靶向药物的有免疫原性的耐药位点，并且可以显著降低由于患者自身HLA分子差异导致的“脱靶”现象，从而使得多肽疫苗可以最大范围内的对有类似耐药突变的肿瘤细胞起到杀伤作用，相对单药疫苗显著提高了其有效性。

4、多肽疫苗序列设计：作为一种实施例，可以通过人工设计相对应的疫苗多肽序列，也可以输入我们自主研发的疫苗设计程序iNeo-VaDes(V1.2)，设计多肽疫苗序列。作为一种针对靶向药物耐药的多肽疫苗疗法，在疫苗多肽设计过程中，本发明考虑了抗原表位分布，尽可能多的覆盖含有突变位点的表位，另外还考虑多肽的长度、疏水率等影响氨基酸合成效率的因素，以及多肽的毒性、同源性等影响多肽安全性的因素。本发明的疫苗多肽组合由包含以下137个突变的多肽序列组成，具体如下表1所示。5、疫苗制备：多肽疫苗的制备既可以直接使用化学合成的方式制备，也可以使用核酸分子(例如DNA和RNA)通过转录和翻译的方式获得，或者也可以通过细菌或病毒为载体表达得到。在本例中我们使用化学合成方式获得多肽疫苗。

表1：靶向药物耐药突变及多肽疫苗序列

表2：临床患者的数据库：

△本发明提供了31个靶向药物的多肽疫苗，基本覆盖常见肿瘤靶向药物，用以下实验对各个多肽的免疫原性进行实验验证：

实验1，多肽的免疫原性测定；实验目的：通过ELISpot实验，验证本发明中7个产品对应的多肽在人源化小鼠体内均能引起免疫反应。实验方法：实验多肽：7个产品多肽由南京金斯瑞生物科技有限公司代合成，多肽纯度均大于90％，内毒素含量低于0.5EU/mg。为了检测多肽的免疫反应，实施IFN-γ酶联免疫吸附(ELISPOT)测定法。详细的实验过程如下：选用8周龄人源化小鼠B-NSG(CD34+)24只，随机分为8组，每组3只。适应一周后，分为多肽组1(对应产品1)、多肽组2(对应产品2)、多肽组3(对应产品3)……多肽组7(对应产品7)，共计7组及阴性对照组编号8。采用CpG为佐剂(0.2μg/只)，多肽50μg每只,再与弗氏不完全佐剂Freund’s adjuvant(FIA，Sigma-Aldrich)1：1混匀，乳化30分钟，PBS与弗氏不完全佐剂1：1混合乳化30分钟作为阴性对照，四次于颈背部右胸皮下免疫，总剂量0.5mL/只，1周一次，共三周，第三次免疫后10天，取的小鼠脾脏，制备小鼠淋巴细胞悬液，用于ELISPOT检测。ELISPOT检测结果中，IFN-γ呈阳性结果的多肽，即判定为阳性候选多肽。实验按组别分别进行单肽有ELISPOT试验，即将小鼠淋巴细胞稀释成浓度为1-2*10

实验结果：为了进一步明确了各个单独多肽的免疫反应，分别进行了各个多肽组中其相应的单肽的ELISPOT实验，各多肽产生的斑点数见图2A-D，各个均能引起免疫反应，但各个多肽产生的斑点差异比较大，从10个斑点到200多个斑点不等，而对照组基本无斑点产生。结果分析：本发明中7个产品对应的多肽在人源化小鼠体内均能引起免疫反应。

实验二，验证本发明中7类靶向药物的耐药多肽疫苗的治疗及预防效果；

为了验证本发明中胃癌耐药多肽疫苗的治疗及预防效果，因此需要构建一套含本发明中特定突变位点的稳转细胞系，针对7类靶向药物分别进行如下7个实施例的实验准备；实施例1，第一类针对SMO突变产生耐药的Hedgehog信号通路拮抗剂维莫德吉(Vismodegib)的特定突变位点的稳转细胞系的构建：Vismodegib购于LC Laboratories，胃癌细胞株系AGS购于ATCC，FITC-CD44购于BD公司(BD555478)。细胞的制备：胃癌细胞株系AGS于含有10％FBS,100U/mL penicillin和100μg/mL streptomycin，2mmol/L l-glutamine的DMEM培养48h，换液并补加20ng/mL of EGF,bFGF,N-2(1×),和B27，继续培养72h,促进其形成椭球体(“spheroid formation media”)。椭球体经Accutase(InnovativeCell Technologies)酶解形成单细胞，用FITC-CD44流式抗体进行分选出阳性细胞作为后续的转染细胞，记为CD44

实验目的：为了验证本发明中胃癌耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽。a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部15条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associated membraneglycoprotein 1，LAMP1)，15条突变多肽和MHC class I trafficking domain(MITD)，15条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为smo15-pcDNA3.1(+)。同时合成相应野生型多肽作为对照(由于其中相邻的多肽可归到一条野生型长肽，故总共有7条野生型多肽)，命名为smow7-pcDNA3.1(+)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建，smo15的氨基酸序列为:SEQ ID：NO 6所示；Smow7的氨基酸序列为：SEQ ID：NO 7所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列；

b.构建能够稳定表达突变多肽的细胞系：胃癌细胞株系CD44

实施例2，第二类针对BTK突变产生耐药的依鲁替尼Ibrutinib的特定突变位点的稳转细胞系的构建：Ibrutinib(依鲁替尼)购于Selleck Chemicals，人多发性骨髓瘤细胞系RPMI8226(

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽

a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部5条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associatedmembrane glycoprotein 1，LAMP1)，5条突变多肽和MHC class I trafficking domain(MITD)，5条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为BTK5-pcDNA3.1(+)。同时合成相应野生型多肽作为对照，命名为BTKw2-pcDNA3.1(+)(由于其中4条多肽为SNP位点突变，故总共有2条野生型多肽)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建，

BTK5的氨基酸序列为：SEQ ID：NO 8所示；BTKW2的氨基酸序列为：SEQ ID：NO 9所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

b.构建能够稳定表达突变多肽的细胞系：人多发性骨髓瘤细胞系RPMI8226以2*10

实施例3，第三类针对AR耐药突变的一些抗雄激素类药物如阿比特龙等的特定突变位点的稳转细胞系的构建；前列腺癌细胞系PC-3(

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽。

a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部4条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associatedmembrane glycoprotein 1，LAMP1)，4条突变多肽和MHC class I trafficking domain(MITD)，4条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为AR4-pcDNA3.1(+)。同时合成相应野生型多肽作为对照，命名为ARw2-pcDNA3.1(+)(由于其中4条多肽为SNP位点突变，故总共有2条野生型多肽)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建。AR4的氨基酸序列为：SEQ ID：NO 10所示；ARW2的氨基酸序列为：SEQ ID：NO 11所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

b.构建能够稳定表达突变多肽的细胞系：前列腺癌细胞系PC-3以2*10

实施例4，第四类针对BRAF耐药突变的一些菲尼类药物的特定突变位点的稳转细胞系的构建；

人黑素瘤细胞系A378购于ATCC,培养于DMEM培养基加10％的胎牛血清。

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽

a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部7条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associatedmembrane glycoprotein 1，LAMP1)，7条突变多肽和MHC class I trafficking domain(MITD)，7条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为ME7-pcDNA3.1(+)。同时合成相应野生型多肽作为对照，命名为MEw5-pcDNA3.1(+)(其中MAP2K1 124氨基酸位点突变体来源于同一条野生型多肽，可以合并为一条野生型长肽)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建。

ME7的氨基酸序列为：SEQ ID：NO 12所示；MEw5的氨基酸序列为：SEQ ID：NO 13所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

b.构建能够稳定表达突变多肽的细胞系：人黑素瘤细胞系A378以2*10

实施例5，第五类针对ALK、MET等融合基因的一些络氨酸激酶抑制剂的特定突变位点的稳转细胞系的构建；

人非小细胞肺癌H2228(EML4-ALK variant 3a/b E6；A20)购于ATCC,培养于RPMI1640培养基加10％的胎牛血清。

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽

a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部10条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associatedmembrane glycoprotein 1，LAMP1)，10条突变多肽和MHC class I trafficking domain(MITD)，10条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为ALK10-pcDNA3.1(+)。同时合成相应野生型多肽作为对照，命名为ALKw5-pcDNA3.1(+)(其中ALK 1171氨基酸位点和1174氨基酸位点附近突变体来源于同一条野生型多肽，ALK 1196氨基酸位点和1202及1203氨基酸位点附近突变体来源于同一条野生型多肽，MET 1246氨基酸位点来源于同一条野生型多肽可以合并为一条野生型长肽，共计5条野生型长肽)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建。

ALK10的氨基酸序列为：SEQ ID：NO 14所示；ALKw5的氨基酸序列为：SEQ ID：NO 15所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

a.构建能够稳定表达突变多肽的细胞系

人非小细胞肺癌H2228以2*10

实施例6，第六类针对EGFR通路的络氨酸激酶抑制剂的特定突变位点的稳转细胞系的构建；

人慢性粒细胞白血病细胞系THP-1(ATCC TIB-202)购于ATCC,培养于RPMI-1640培养基加10％的胎牛血清。

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽。

a.构建突变位点真核表达质粒：为了实现突变多肽的表达，本发明拟通过串连各个多肽基因(即mini-gene)在人慢性粒细胞白血病细胞系THP-1中过表达。但由于这些突变热点比较从集中(基因片段同源性高)，将这92条多肽对应的4个基因，分成2组，每组含有46个多肽的基因，分别克隆于真核表达载体pcDNA3.1-hygro(+)和pcDNA3.1-zeo(+)。每个载体能够表达本发明中突变的全部46条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associated membrane glycoprotein 1，LAMP1)，13条突变多肽和MHC classI trafficking domain(MITD)，46条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为mut46-hygro(+)和mut46-zeo(+)。同时合成相应野生型多肽作为对照，命名为wid8-hygro(+)(其中ABL1蛋白直接选取第234-505氨基酸位点，FLT3蛋白选取第835氨基酸位点附近一条，EGFR蛋白选取第760和790氨基酸位点附近2条，KIT蛋白3条和PDGFRA蛋白1条野生型多肽)，其克隆到真核表达载体pcDNA3.1-hygro(+)中，wid8-hygro(+)所有基因片段由南京金斯瑞生物科技有限公司代合成和构建。

Mut46-hy的氨基酸序列为：SEQ ID：NO 16所示；Mut46-zeo的氨基酸序列为：SEQID：NO 17所示；Wid8-hy的氨基酸序列为：SEQ ID：NO 18所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

b.构建能够稳定表达突变多肽的细胞系：人慢性粒细胞白血病细胞系THP-1以2*10

将mut46(mut46-hygro(+))重新活化，并按上述转染流程，将mut46-zeo(+)导入上述细胞系，用400μg/mL zeo进行筛选，命名为mut92(mut46-hygro(+)，mut46-zeo(+))。

实施例7，第七类针对PI3K/AKT/mTOR通路的依维莫司等激酶抑制剂的特定突变位点的稳转细胞系的构建；

人胚肾成纤维细胞株HEK 293(

实验目的：为了验证本发明中耐药多肽诱导的T细胞具有杀伤活性，因此需要构建一套含本发明中特定突变位点的稳转细胞系，能够表达递呈这些多肽

a.构建突变位点真核表达质粒：采用人工合成的方法获得能够表达本发明中突变的全部3条疫苗多肽的mini-gene，由以下几部分组成：信号肽部分(lysosome-associatedmembrane glycoprotein 1，LAMP1)，3条突变多肽和MHC class I trafficking domain(MITD)，3条突变多肽之间用柔性连接肽GGSGGGGSGG连接，基因进行密码子优化后，上游引入GATATC(EcoR V)，下游引入CTCGAG(Xho I)，将其克隆到真核表达载体pcDNA3.1-hygro(+)中，命名为MEM3-pcDNA3.1(+)。同时合成相应野生型多肽作为对照，命名为MEMw3-pcDNA3.1(+)。所有基因片段由南京金斯瑞生物科技有限公司代合成和构建。

MEM3的氨基酸序列为：SEQ ID：NO 19所示；MEMw3的氨基酸序列为：SEQ ID：NO 20所示；其中第1-28个氨基酸为信号肽区域，用加粗斜体表示；下划线标注部分为MITD序列。

b.构建能够稳定表达突变多肽的细胞系：人胚肾成纤维细胞株HEK 293以2*10

根据以上实施例1-7得到的多肽组1小鼠的淋巴细胞悬液做体外细胞杀伤实验，实验内容如下：

实验2-1：第一类靶向药物的多肽疫苗组的体外细胞杀伤实验；

实验目的：验证15条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记smo15-ASG(含有smo15-pcDNA3.1(+))和smow7-AGS(含有smow7-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例1中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验结果：如图3所示，实验组(图3)诱导的效应T细胞其杀伤效率在40％-80％不等，杀伤效率明显高于对照组，说明突变多肽组对靶细胞起到了杀伤作用。突变多肽组中，随着效靶比的升高，T细胞的杀伤作用越来越强，当效靶比为20:1时，其对靶细胞杀伤效率达75％以上。

实验2-2：第二类靶向药物的多肽疫苗组的体外细胞杀伤实验

实验目的：验证5条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记BTK5(含有BTK5-pcDNA3.1(+))和BTKw2(含有BTKw2-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例2中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验结果：如图4所示，实验组(图4)诱导的效应T细胞其杀伤效率在40％-80％不等，杀伤效率明显高于对照组，说明突变多肽组对靶细胞起到了杀伤作用。突变多肽组中，随着效靶比的升高，T细胞的杀伤作用越来越强，当效靶比为20:1时，其对靶细胞杀伤效率达75％以上。

实验2-3：第三类靶向药物的多肽疫苗组的体外细胞杀伤实验

实验目的：验证4条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记AR4(含有AR4-pcDNA3.1(+))和ARw2(含有ARw2-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例3中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验结果：如图5所示，实验组(图5)诱导的效应T细胞其杀伤效率在40％-90％不等，杀伤效率明显高于对照组，说明突变多肽组对靶细胞起到了杀伤作用。突变多肽组中，随着效靶比的升高，T细胞的杀伤作用越来越强，当效靶比为20:1时，其对靶细胞杀伤效率达80％以

实验2-4：第四类靶向药物的多肽疫苗组体外细胞杀伤实验

实验目的：验证7条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记ME7(ME7-pcDNA3.1(+))和MEw5(含有MEw5-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例4中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验结果：如图6所示，实验组(图6)诱导的效应T细胞其杀伤效率在45％-90％不等，杀伤效率明显高于对照组，说明突变多肽组对靶细胞起到了杀伤作用。突变多肽组中，随着效靶比的升高，T细胞的杀伤作用越来越强，当效靶比为20:1时，其对靶细胞杀伤效率达85％以上。

实验2-5：第五类靶向药物的多肽疫苗组体外细胞杀伤实验；

实验目的：验证10条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记ALK10(ALK10-pcDNA3.1(+))和ALKw5(含有ALKw5-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例5中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验2-6：第六类靶向药物的多肽疫苗组体外细胞杀伤实验；

实验目的：验证92条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。

实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记mut92(mut46-hygro(+)，mut46-zeo(+))和wid8(含有wid8-hygro(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例6中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

实验结果：如图8所示，实验组(图8)诱导的效应T细胞其杀伤效率在40％-90％不等，杀伤效率明显高于对照组，说明突变多肽组对靶细胞起到了杀伤作用。突变多肽组中，随着效靶比的升高，T细胞的杀伤作用越来越强，当效靶比为20:1时，其对靶细胞杀伤效率达84％以上。

实验2-7：第七类靶向药物的多肽疫苗组体外细胞杀伤实验；实验目的：验证3条多肽的在体外细胞水平能够引起肿瘤细胞的杀伤效果。实验方法：(1)5-(6)-Carboxy-fluorescein succinimidyl ester(CFSE)染料购自Invitrogen公司。操作步骤按照试剂盒说明书进行。无菌条件下用CFSE标记MEM3(含有MEM3-pcDNA3.1(+))和MEMw3(含有MEMw3-pcDNA3.1(+))靶细胞，分别作为实验组和对照组用的靶细胞。(2)杀伤实验a.准备效应细胞：将实施例7中留取的多肽组1小鼠的淋巴细胞悬液，用RPMI 1640培养基重悬，台盼蓝染色计数。b.效应细胞CTL的诱导培养：将组2淋巴细胞稀释浓度为(1-2)*10

根据以上7个实验，可以得知通过本发明的设计方法将靶向药物根据其机制和耐药突变聚类分析归位7大类，多靶点联合可以有效增加多肽疫苗对肿瘤细胞的杀伤效果，显著延长靶向药物有效作用时间。本发明提供一种针对肿瘤靶向药物耐药位点的多肽疫苗及其设计方法，提供的靶向药物联合多肽疫苗组合方案覆盖常见的靶向治疗药物，可以有效降低肿瘤耐药发生概率，延长靶向药有效作用时间，增加患者的疾病响应率，具有广谱性，缩短个体化多肽疫苗从分析到治疗的时间，降低医疗成本。

以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。

序列表

<110> 杭州纽安津生物科技有限公司

<120> 针对靶向药物达拉菲尼、派姆单抗、威罗菲尼、司美替尼的多肽疫苗组合及其设计方法

<141> 2020-11-24

<160> 25

<170> SIPOSequenceListing 1.0

<210> 16

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 16

Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr Ile Val Gly Phe Tyr

1 5 10 15

Gly Ala Phe Tyr Lys Lys

20

<210> 17

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 17

Lys Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Ala Lys Val Gly Glu

1 5 10 15

Leu Lys

<210> 18

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 18

Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu

1 5 10 15

<210> 19

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 19

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 20

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 20

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 6

<211> 630

<212> PRT

<213> Artificial Sequence

<400> 6

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Gly Gly

20 25 30

Ser Gly Gly Gly Gly Ser Gly Gly Met Leu Arg Leu Gly Ile Phe Gly

35 40 45

Phe Leu Val Phe Gly Phe Val Leu Ile Thr Phe Ser Cys Lys Lys Lys

50 55 60

Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Phe Gly

65 70 75 80

Phe Val Leu Ile Thr Phe Ser Tyr His Phe Tyr Asp Phe Phe Asn Gln

85 90 95

Ala Glu Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

100 105 110

Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr Gly Phe Phe Asn Gln

115 120 125

Ala Glu Trp Glu Arg Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Leu Gly Phe Val Leu

145 150 155 160

Ile Thr Phe Ser Cys His Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Gly Tyr Val Leu Cys Gln Ala Asn Val Thr Ile

180 185 190

Trp Leu Pro Thr Lys Gln Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly

195 200 205

Gly Gly Gly Ser Gly Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr

210 215 220

His Phe Phe Asn Gln Ala Glu Trp Glu Arg Ser Lys Lys Lys Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Phe Thr Glu Ala Glu His Gln Asp

245 250 255

Met Arg Ser Tyr Ile Ala Ala Phe Gly Ala Val Thr Lys Lys Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Met Phe Gly Thr Gly Ile Ala Met

275 280 285

Ser Thr Leu Val Trp Thr Lys Ala Thr Leu Leu Ile Trp Lys Lys Lys

290 295 300

Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val

305 310 315 320

Leu Ile Thr Phe Ser Cys His Phe Tyr Tyr Phe Phe Asn Gln Ala Glu

325 330 335

Trp Glu Arg Ser Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

340 345 350

Gly Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Glu Ala Glu Trp Glu

355 360 365

Arg Ser Phe Arg Asp Tyr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys

385 390 395 400

Thr Leu Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly

405 410 415

Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile

420 425 430

Phe Val Ile Ala Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys

435 440 445

Lys Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

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

465 470 475 480

Phe Met Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

485 490 495

Gly Met Phe Gly Thr Gly Ile Ala Met Ser Thr Arg Val Trp Thr Lys

500 505 510

Ala Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly

515 520 525

Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val Ile

530 535 540

Met Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys Lys

545 550 555 560

Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

565 570 575

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

580 585 590

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

595 600 605

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

610 615 620

Asp Val Ser Leu Thr Ala

625 630

<210> 7

<211> 348

<212> PRT

<213> Artificial Sequence

<400> 7

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Met Leu

20 25 30

Arg Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe Gly Phe Val Leu

35 40 45

Ile Thr Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Gln Ala Glu Trp

50 55 60

Glu Arg Ser Phe Arg Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

65 70 75 80

Tyr Val Leu Cys Gln Ala Asn Val Thr Ile Gly Leu Pro Thr Lys Gln

85 90 95

Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

100 105 110

Phe Thr Glu Ala Glu His Gln Asp Met His Ser Tyr Ile Ala Ala Phe

115 120 125

Gly Ala Val Thr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys Thr Leu

145 150 155 160

Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser

165 170 175

Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val

180 185 190

Ile Val Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys

195 200 205

Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

210 215 220

Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Trp Leu Ala Gln Phe Met

225 230 235 240

Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met

245 250 255

Phe Gly Thr Gly Ile Ala Met Ser Thr Leu Val Trp Thr Lys Ala Thr

260 265 270

Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly

275 280 285

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

290 295 300

Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg

305 310 315 320

Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser

325 330 335

Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

340 345

<210> 8

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 8

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile

20 25 30

Ile Thr Glu Tyr Met Ala Asn Gly Phe Leu Leu Asn Tyr Leu Arg Glu

35 40 45

Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

50 55 60

Ile Ile Thr Glu Tyr Met Ala Asn Gly Arg Leu Leu Asn Tyr Leu Arg

65 70 75 80

Glu Met Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val

85 90 95

Arg Asp Ser Ser Lys Ala Gly Lys Tyr Ala Val Ser Val Phe Ala Lys

100 105 110

Ser Thr Gly Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Ile

115 120 125

Thr Glu Tyr Met Ala Asn Gly Ser Leu Leu Asn Tyr Leu Arg Glu Met

130 135 140

Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Phe Ile Ile

145 150 155 160

Thr Glu Tyr Met Ala Asn Gly Tyr Leu Leu Asn Tyr Leu Arg Glu Met

165 170 175

Arg His Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

180 185 190

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

195 200 205

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

210 215 220

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

225 230 235 240

Asp Val Ser Leu Thr Ala

245

<210> 9

<211> 150

<212> PRT

<213> Artificial Sequence

<400> 9

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile

20 25 30

Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu Arg Glu

35 40 45

Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

50 55 60

Val Arg Asp Ser Ser Lys Thr Gly Lys Tyr Ala Val Ser Val Phe Ala

65 70 75 80

Lys Ser Thr Gly Asp Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

85 90 95

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

100 105 110

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

115 120 125

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

130 135 140

Asp Val Ser Leu Thr Ala

145 150

<210> 10

<211> 208

<212> PRT

<213> Artificial Sequence

<400> 10

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Pro Ile

20 25 30

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

35 40 45

Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Pro

50 55 60

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

65 70 75 80

His Met Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu

85 90 95

Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro

100 105 110

Gly Phe Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Ile Ala Arg

115 120 125

Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys Ser His Met Gly

130 135 140

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

145 150 155 160

Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val

165 170 175

Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln

180 185 190

Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

195 200 205

<210> 11

<211> 147

<212> PRT

<213> Artificial Sequence

<400> 11

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Val Gln

20 25 30

Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys

35 40 45

Ser His Met Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu

50 55 60

Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro

65 70 75 80

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

85 90 95

Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val

100 105 110

Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser

115 120 125

Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser

130 135 140

Leu Thr Ala

145

<210> 12

<211> 300

<212> PRT

<213> Artificial Sequence

<400> 12

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu His

20 25 30

Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe Tyr Ser

35 40 45

Asp Gly Glu Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr

50 55 60

Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu Gly

65 70 75 80

Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp

85 90 95

Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Lys Lys

100 105 110

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn

145 150 155 160

Ser Leu Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Lys Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu Ala Phe

180 185 190

Leu Thr Pro Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser Gly Gly

195 200 205

Gly Gly Ser Gly Gly Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr

210 215 220

Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Gly Gly Ser Leu Gly

225 230 235 240

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

245 250 255

Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg

260 265 270

Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser

275 280 285

Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

290 295 300

<210> 13

<211> 238

<212> PRT

<213> Artificial Sequence

<400> 13

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Tyr Lys

20 25 30

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

35 40 45

Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp Thr

50 55 60

Ala Gly Gln Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Lys Lys Lys

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu Ala

85 90 95

Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn Ser

115 120 125

Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Lys

130 135 140

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu

145 150 155 160

Ala Phe Leu Thr Gln Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser

165 170 175

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

180 185 190

Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys

195 200 205

Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala

210 215 220

Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

225 230 235

<210> 14

<211> 390

<212> PRT

<213> Artificial Sequence

<400> 14

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val

20 25 30

Ala Asp Phe Gly Leu Ala Arg His Met Tyr Asp Lys Glu Tyr Tyr Ser

35 40 45

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

50 55 60

Leu Ile Arg Gly Leu Ser His Gly Ala Phe Gly Glu Val Tyr Glu Gly

65 70 75 80

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp Phe Leu Met Glu

85 90 95

Ala Leu Ile Thr Ser Lys Phe Asn His Gln Asn Ile Val Arg Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Arg Phe Ile Leu Leu Glu Leu Met

115 120 125

Ala Gly Arg Asp Leu Lys Ser Phe Leu Arg Glu Thr Arg Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Cys Pro Gly Pro Gly Arg Val Ala Lys

145 150 155 160

Ile Ala Asp Phe Gly Met Ala Arg Asp Ile Tyr Arg Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Gly Phe Leu Met Glu Ala Leu Ile Ile Ser Lys

180 185 190

Leu Asn His Gln Asn Ile Val Arg Cys Ile Gly Lys Gly Gly Ser Gly

195 200 205

Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ala Arg Asn

210 215 220

Met Tyr Asp Lys Glu Tyr Tyr Ser Val His Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Ile Leu Leu Glu Leu Met Ala Gly Gly Asn Leu Lys

245 250 255

Ser Phe Leu Arg Glu Thr Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala

275 280 285

Gly Gly Asp Leu Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Phe

290 295 300

Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile Val

305 310 315 320

Arg Cys Ile Gly Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

325 330 335

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

340 345 350

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

355 360 365

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

370 375 380

Asp Val Ser Leu Thr Ala

385 390

<210> 15

<211> 247

<212> PRT

<213> Artificial Sequence

<400> 15

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val

20 25 30

Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser

35 40 45

Val His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp

50 55 60

Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Phe Asn His Gln Asn Ile

65 70 75 80

Val Arg Cys Ile Gly Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

85 90 95

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

100 105 110

Tyr Glu Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Pro Gly Pro

115 120 125

Gly Arg Val Ala Lys Ile Gly Asp Phe Gly Met Ala Arg Asp Ile Tyr

130 135 140

Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Leu Gln Ser Leu

145 150 155 160

Pro Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Gly Asp Leu Lys Ser

165 170 175

Phe Leu Arg Glu Thr Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

180 185 190

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

195 200 205

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

210 215 220

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

225 230 235 240

Ser Asp Val Ser Leu Thr Ala

245

<210> 16

<211> 1559

<212> PRT

<213> Artificial Sequence

<400> 16

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Ser

20 25 30

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

35 40 45

Val Asp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Cys Leu

50 55 60

Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe Gly Cys Leu

65 70 75 80

Leu Asp Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

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

100 105 110

Val Trp Ala Phe Gly Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu

130 135 140

Lys Glu Ala Ala Val Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly

165 170 175

Val Cys Thr Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys

180 185 190

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

195 200 205

Arg Leu Met Thr Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys

210 215 220

Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr Gly

225 230 235 240

Asn Leu Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gln Leu Ile Thr Gln Leu Met Pro Phe Asp Cys Leu Leu Asp Tyr Val

260 265 270

Arg Glu His Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Glu Asn His Leu Val Lys Val Ala Asp Leu Gly Leu Ser Arg Leu Met

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val

305 310 315 320

Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met Glu Arg Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly

340 345 350

Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr

370 375 380

Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly

385 390 395 400

Ser Gly Gly Leu Thr Ser Thr Val Gln Leu Ile Thr Gln His Met Pro

405 410 415

Phe Gly Cys Leu Leu Asp Tyr Val Lys Lys Lys Lys Lys Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile

435 440 445

Ile Asn Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Lys Glu Ala Ala Val Met Lys Glu

465 470 475 480

Ile Arg Gly Gly His Pro Asn Leu Val Gln Leu Leu Gly Val Gly Gly

485 490 495

Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly Gln

500 505 510

Tyr Gly Lys Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly Ser

515 520 525

Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg

530 535 540

Met Met Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Gly Gly Ser

545 550 555 560

Gly Gly Gly Gly Ser Gly Gly Lys Trp Glu Met Glu Arg Thr Asp Ile

565 570 575

Thr Val Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Gly Ser Gly Gly

580 585 590

Gly Gly Ser Gly Gly Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Tyr

595 600 605

His Leu Val Lys Val Ala Asp Phe Lys Lys Lys Gly Gly Ser Gly Gly

610 615 620

Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu

625 630 635 640

Tyr Ile Ile Thr Glu Phe Met Thr Tyr Gly Lys Lys Lys Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly

660 665 670

Gly Met Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly

675 680 685

Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu

690 695 700

Leu Lys Asp Tyr Arg Met Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly

705 710 715 720

Ser Gly Gly Leu Met Thr Gly Asp Thr Tyr Thr Ala His Pro Gly Ala

725 730 735

Lys Phe Pro Ile Lys Trp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

740 745 750

Gly Gly Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Met Glu Cys Asn

755 760 765

Arg Gln Glu Val Asn Ala Val Lys Lys Gly Gly Ser Gly Gly Gly Gly

770 775 780

Ser Gly Gly Asn Cys Leu Val Gly Glu Asn His Leu Val Arg Val Ala

785 790 795 800

Asp Phe Gly Leu Ser Arg Leu Met Gly Gly Ser Gly Gly Gly Gly Ser

805 810 815

Gly Gly Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr

820 825 830

Gly Asn Leu Leu Asp Tyr Leu Lys Lys Gly Gly Ser Gly Gly Gly Gly

835 840 845

Ser Gly Gly Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly

850 855 860

Ala Lys Phe Pro Ile Lys Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser

865 870 875 880

Gly Gly Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Ala Ile His Arg

885 890 895

Asp Leu Ala Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile

900 905 910

Thr Met Lys His Lys Leu Gly Gly Gly Arg Tyr Gly Glu Val Tyr Glu

915 920 925

Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu

930 935 940

Lys Glu Asp Thr Met Glu Val Glu Gly Phe Leu Lys Glu Ala Ala Val

945 950 955 960

Met Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Glu

965 970 975

Glu Phe Leu Lys Glu Ala Ala Phe Met Lys Glu Ile Lys His Pro Asn

980 985 990

Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met Ala Thr

995 1000 1005

Gln Ile Ser Ser Ala Met Gly Tyr Leu Glu Lys Lys Asn Phe Ile His

1010 1015 1020

Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp

1025 1030 1035 1040

Leu Ser Gln Val Tyr Ala Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg

1045 1050 1055

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp

1060 1065 1070

Phe Gly Leu Ala Arg Phe Ile Met Ser Asp Ser Asn Tyr Val Val Arg

1075 1080 1085

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys Asp Phe Gly

1090 1095 1100

Leu Ala Arg Ala Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Gly

1105 1110 1115 1120

Ser Gly Gly Gly Gly Ser Gly Gly Asp Phe Gly Leu Ala Arg Asp Ile

1125 1130 1135

Lys Asn Val Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Gly Gly Ser

1140 1145 1150

Gly Gly Gly Gly Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val

1155 1160 1165

Ile Ile Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly

1170 1175 1180

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Tyr Leu Gly Asn His Met

1185 1190 1195 1200

Asn Ile Val Thr Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Lys Gly

1205 1210 1215

Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp Leu Ser

1220 1225 1230

Gln Val Tyr Lys Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Gly Gly

1235 1240 1245

Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe Gly Leu

1250 1255 1260

Ala Arg Tyr Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser

1265 1270 1275 1280

Gly Gly Gly Gly Ser Gly Gly Thr Gln Ile Ser Ser Ala Met Glu Tyr

1285 1290 1295

Leu Ala Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly

1300 1305 1310

Gly Ser Gly Gly Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Ser Ala

1315 1320 1325

Glu Ile His Gln Ala Phe Glu Thr Met Lys Gly Gly Ser Gly Gly Gly

1330 1335 1340

Gly Ser Gly Gly Trp Ala Phe Gly Val Leu Leu Trp Glu Ile Thr Thr

1345 1350 1355 1360

Tyr Gly Met Ser Pro Tyr Pro Gly Ile Lys Lys Lys Gly Gly Ser Gly

1365 1370 1375

Gly Gly Gly Ser Gly Gly Val Ala Val Lys Thr Leu Lys Glu Asp Ala

1380 1385 1390

Met Glu Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Lys Gly Gly Ser

1395 1400 1405

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

1410 1415 1420

Arg Val Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Gly Ser Gly

1425 1430 1435 1440

Gly Gly Gly Ser Gly Gly Asp Ser Asn Tyr Val Val Lys Gly Asn Pro

1445 1450 1455

Arg Leu Pro Val Lys Trp Met Ala Gly Gly Ser Gly Gly Gly Gly Ser

1460 1465 1470

Gly Gly Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Lys Asn Asp

1475 1480 1485

Ser Asn Tyr Val Val Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

1490 1495 1500

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

1505 1510 1515 1520

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

1525 1530 1535

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

1540 1545 1550

Ser Asp Val Ser Leu Thr Ala

1555

<210> 17

<211> 1511

<212> PRT

<213> Artificial Sequence

<400> 17

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu Ile

20 25 30

Thr Gln Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val Arg Glu His

35 40 45

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

50 55 60

Leu Lys Glu Asp Thr Met Tyr Val Glu Glu Phe Leu Lys Glu Ala Ala

65 70 75 80

Val Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg

85 90 95

Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr Gly Asn Leu

100 105 110

Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Leu

115 120 125

Ser Gln Val Tyr Glu Leu Leu Gly Lys Asp Tyr Arg Met Glu Arg Lys

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Thr Tyr Thr Ala His

145 150 155 160

Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Lys Lys Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Phe Leu Lys Glu Ala Ala

180 185 190

Val Met Lys Val Ile Lys His Pro Asn Leu Val Gln Leu Leu Lys Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Leu Ile Thr Gln Leu

210 215 220

Met Pro Phe Arg Cys Leu Leu Asp Tyr Val Arg Glu His Lys Lys Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Met Arg Ala Cys Trp

245 250 255

Gln Trp Asn Leu Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Glu Ser Leu Ala Tyr Asn Lys Phe

275 280 285

Ser Ile Glu Ser Asp Val Trp Ala Phe Gly Val Leu Leu Lys Lys Gly

290 295 300

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

305 310 315 320

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

325 330 335

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr

340 345 350

Glu Leu Leu Ala Lys Asp Tyr Arg Met Glu Arg Lys Gly Gly Ser Gly

355 360 365

Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly

370 375 380

Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Val Glu

405 410 415

Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Ala Val Lys Thr Leu

420 425 430

Lys Glu Asp Thr Met Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly

450 455 460

Gln Tyr Gly Val Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Gly Gly Lys Thr Leu Lys Glu Asp Thr Met

485 490 495

Ala Val Glu Glu Phe Leu Lys Glu Ala Ala Val Lys Lys Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg

515 520 525

Leu Leu Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Lys Gly Gly Ser

530 535 540

Gly Gly Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro

545 550 555 560

Ser Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr Lys Lys Lys Lys Gly

565 570 575

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu

580 585 590

Gly Gly Gly Lys Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly

595 600 605

Ser Gly Gly Gly Gly Ser Gly Gly Met Thr Gly Asp Thr Tyr Thr Ala

610 615 620

His Ala Arg Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Lys Lys Lys

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Glu Ser Leu Ala Tyr

645 650 655

Asn Lys Phe Ser Val Lys Ser Asp Val Trp Ala Phe Gly Val Gly Gly

660 665 670

Ser Gly Gly Gly Gly Ser Gly Gly Pro Phe Tyr Ile Ile Thr Glu Phe

675 680 685

Met Thr Cys Gly Asn Leu Leu Asp Tyr Leu Arg Lys Lys Lys Gly Gly

690 695 700

Ser Gly Gly Gly Gly Ser Gly Gly Gln Ile Ser Ser Ala Met Glu Tyr

705 710 715 720

Leu Gly Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly

725 730 735

Gly Ser Gly Gly Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Phe Val

740 745 750

Leu Leu Tyr Met Ala Thr Gln Ile Lys Gly Gly Ser Gly Gly Gly Gly

755 760 765

Ser Gly Gly Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly

770 775 780

Ala Lys Phe Pro Ile Lys Trp Thr Lys Gly Gly Ser Gly Gly Gly Gly

785 790 795 800

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

805 810 815

Arg Asp Leu Ala Ala Arg Asn Gly Gly Ser Gly Gly Gly Gly Ser Gly

820 825 830

Gly Thr Leu Lys Glu Asp Thr Met Glu Val Glu Lys Phe Leu Lys Glu

835 840 845

Ala Ala Val Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

850 855 860

Val Glu Glu Phe Leu Lys Glu Ala Ala Ala Met Lys Glu Ile Lys His

865 870 875 880

Pro Asn Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met

885 890 895

Ala Thr Gln Ile Ser Ser Ala Met Asp Tyr Leu Glu Lys Lys Asn Phe

900 905 910

Ile His Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly

915 920 925

Ile Asp Leu Ser Gln Val Tyr Gly Leu Leu Glu Lys Asp Tyr Arg Met

930 935 940

Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys

945 950 955 960

Asp Phe Gly Leu Ala Arg His Ile Met Ser Asp Ser Asn Tyr Val Val

965 970 975

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

980 985 990

Gly Leu Ala Arg Glu Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly

995 1000 1005

Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Ala Arg Asp Ile Lys Asn

1010 1015 1020

Gly Ser Asn Tyr Val Val Lys Gly Asn Gly Gly Ser Gly Gly Gly Gly

1025 1030 1035 1040

Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Glu Glu Tyr

1045 1050 1055

Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly Gly Ser Gly Gly

1060 1065 1070

Gly Gly Ser Gly Gly Leu Ser Tyr Leu Gly Asn His Met Asn Ile Ala

1075 1080 1085

Asn Leu Leu Gly Ala Cys Thr Ile Gly Lys Lys Gly Gly Ser Gly Gly

1090 1095 1100

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

1105 1110 1115 1120

Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly Gly Gly

1125 1130 1135

Gly Ser Gly Gly Ser Phe Ala Glu Ile His Gln Ala Phe Glu Arg Met

1140 1145 1150

Phe Gln Glu Ser Ser Ile Ser Asp Glu Lys Lys Lys Gly Gly Ser Gly

1155 1160 1165

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

1170 1175 1180

Lys Asp Thr Met Glu Val Glu Glu Phe Leu Lys Gly Gly Ser Gly Gly

1185 1190 1195 1200

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

1205 1210 1215

Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Lys Gly Gly Ser Gly Gly

1220 1225 1230

Gly Gly Ser Gly Gly Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe

1235 1240 1245

Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Gly Gly Ser Gly Gly Gly

1250 1255 1260

Gly Ser Gly Gly Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met

1265 1270 1275 1280

Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Lys Gly Gly Ser Gly

1285 1290 1295

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

1300 1305 1310

Leu Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Gly Gly Ser Gly Gly

1315 1320 1325

Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser Glu Leu Glu Val

1330 1335 1340

Leu Ser Tyr Leu Gly Asn His Met Asn Lys Lys Gly Gly Ser Gly Gly

1345 1350 1355 1360

Gly Gly Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Phe

1365 1370 1375

Lys Glu Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly

1380 1385 1390

Ser Gly Gly Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Tyr Ser Asn

1395 1400 1405

Tyr Val Val Lys Gly Asn Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser

1410 1415 1420

Gly Gly Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Phe Asn Tyr Val

1425 1430 1435 1440

Val Lys Gly Asn Ala Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

1445 1450 1455

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

1460 1465 1470

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

1475 1480 1485

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

1490 1495 1500

Ser Asp Val Ser Leu Thr Ala

1505 1510

<210> 18

<211> 625

<212> PRT

<213> Artificial Sequence

<400> 18

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Trp

20 25 30

Glu Met Glu Arg Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly

35 40 45

Gln Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr

50 55 60

Val Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe

65 70 75 80

Leu Lys Glu Ala Ala Val Met Lys Glu Ile Lys His Pro Asn Leu Val

85 90 95

Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr

100 105 110

Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn

115 120 125

Arg Gln Glu Val Asn Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile

130 135 140

Ser Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp

145 150 155 160

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

165 170 175

Ala Asp Phe Gly Leu Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala

180 185 190

His Ala Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu

195 200 205

Ala Tyr Asn Lys Phe Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val

210 215 220

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

225 230 235 240

Asp Leu Ser Gln Val Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu

245 250 255

Arg Pro Glu Gly Cys Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys

260 265 270

Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gln

275 280 285

Ala Phe Glu Thr Met Phe Gln Glu Ser Ser Ile Ser Asp Gly Gly Ser

290 295 300

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

305 310 315 320

Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly

325 330 335

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

340 345 350

Asp Glu Ala Tyr Val Met Ala Ser Val Asp Lys Gly Gly Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr

370 375 380

Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys

385 390 395 400

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe

405 410 415

Gly Leu Ala Arg Asp Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly

420 425 430

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser

435 440 445

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

450 455 460

Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu

465 470 475 480

Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Gly Gly Ser Gly Gly Gly Gly

485 490 495

Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Ser Lys Glu

500 505 510

Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly Ser Gly

515 520 525

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

530 535 540

Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala

545 550 555 560

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

565 570 575

Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr

580 585 590

Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser

595 600 605

Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr

610 615 620

Ala

625

<210> 19

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 19

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln

20 25 30

Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys Gln Leu

35 40 45

Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu Cys Asn

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu Tyr Tyr Ser

100 105 110

Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly

115 120 125

Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val

130 135 140

Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly

145 150 155 160

Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val

165 170 175

Ser Leu Thr Ala

180

<210> 20

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 20

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln

20 25 30

Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu

35 40 45

Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu Cys Asn

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser

100 105 110

Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly

115 120 125

Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val

130 135 140

Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly

145 150 155 160

Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val

165 170 175

Ser Leu Thr Ala

180

<210> 21

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 21

Leu His Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe

1 5 10 15

Tyr Ser Asp Gly Glu Lys Lys

20

<210> 22

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 22

Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe

1 5 10 15

Tyr Gly Ala Phe Tyr Lys Lys Lys

20

<210> 23

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 23

Arg Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Gln Lys Val Gly Glu

1 5 10 15

Leu Lys

<210> 24

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 24

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 25

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 25

Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe

1 5 10 15

Tyr Gly Ala Phe Tyr Lys Lys Lys

20