CN114716560A - 一种人乳头瘤病毒18型嵌合蛋白及其用途 - Google Patents

一种人乳头瘤病毒18型嵌合蛋白及其用途 Download PDF

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CN114716560A
CN114716560A CN202110002251.3A CN202110002251A CN114716560A CN 114716560 A CN114716560 A CN 114716560A CN 202110002251 A CN202110002251 A CN 202110002251A CN 114716560 A CN114716560 A CN 114716560A
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许雪梅
张婷
王志荣
夏百成
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Abstract

本发明涉及一种人乳头瘤病毒18型嵌合蛋白及其用途。具体地，本发明涉及一种乳头瘤病毒嵌合蛋白，其包含HPV18型L1蛋白或HPV18型L1蛋白的突变体以及插入所述HPV18型L1蛋白或HPV18型L1蛋白的突变体的表面区的来自HPV59型L2蛋白的多肽、或由其组成，其中所述HPV18型L1蛋白的氨基酸序列如SEQ ID NO.1所示，所述HPV59型L2蛋白的氨基酸序列如SEQ ID NO.2所示。

Description

一种人乳头瘤病毒18型嵌合蛋白及其用途
技术领域
本发明涉及生物技术领域，具体涉及一种人乳头瘤病毒嵌合蛋白，及由其形成的五聚体或病毒样颗粒，以及人乳头瘤病毒嵌合蛋白、人乳头瘤病毒嵌合五聚体或人乳头瘤病毒嵌合病毒样颗粒在制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
背景技术
人乳头瘤病毒(human papillomavirus,HPV)是一类感染上皮组织的无包膜小DNA病毒。根据人乳头瘤病毒的主要外壳蛋白L1氨基酸的同源性，已鉴定了200 多型的该病毒，分为α、β、γ、μ、η属。根据感染部位不同又分为黏膜型及皮肤型。黏膜型HPV主要感染泌尿生殖器、肛周及口咽部的粘膜皮肤，均为α属，并进一步分为有转化活性的致癌型(oncogenic HPV)及诱发良性增生的低危型 (low-risk HPV，LR-HPV)。致癌型HPV包括12种常见的高危型(包括HPV16、 -18、-31、-33、-35、-39、-45、-51、-52、-56、-58、-59型等)，1种可能的高危型(HPV68)，及10余种十分少见的可疑高危型(HPV26、-30、-34、-53、-66、 -67、-69、-70、-73、-82、-85型等)。研究发现，所有致癌型HPV阳性癌组织均呈现特异性E6*I mRNA表达、抑癌基因Rb/P53及细胞周期蛋白CD1的表达降低及p16INK 4a表达增高，表明感染任何一种致癌型HPV罹患癌症的风险是一样的。低危型HPV有约12种(HPV6、-7、-11、-13、-32、-40、-42、-43、-44、 -54、-74、-91型等)，其中HPV6、-11型合计诱发90％的肛周生殖器尖锐湿疣及绝大多数呼吸道复发性乳头瘤。皮肤型HPV主要感染上述部位之外的皮肤组织，其中一些型别(HPV2、-27、-57)诱发皮肤疣状增生，另一些型别(HPV5、-8、 -38等)与皮肤鳞状细胞癌及基底细胞癌的发生相关。
致癌型HPV感染相关的恶性肿瘤目前已确定的有：宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌及口腔癌，其中以宫颈癌的危害最大。宫颈癌是世界范围第三高发的妇女恶性肿瘤，年发病率约52.7万，其中亚洲地区28.5万；中国的年发病数7.5万。12种常见高危型HPV累计诱发 95.2％-96.5％的宫颈癌，其余10多种少见的可能及可疑的高危型累计诱发约 3.29％的宫颈癌。HPV16型是全球范围内的优势流行高危型，在HPV相关的肿瘤如宫颈癌、肛周癌、阴茎癌、外阴癌等及癌前病变中的检出率最高。HPV16及 HPV18在世界范围内宫颈癌中的检出率分别达50-60％及～20％。12种常见高危型HPV累计诱发95.2％-96.5％的宫颈癌，其余10多种少见的可能及可疑的高危型累计诱发约3.29％的宫颈癌。
HPV L1病毒样颗粒(L1 virus-like particle,L1 VLP)主要诱发型别特异性中和抗体和保护反应，只能通过增加L1 VLP的型别来扩大疫苗的保护范围。上市的3 种HPV疫苗均为L1 VLP疫苗，分别是GSK的二价苗(Cervarix，HPV16/-18)， Merck的四价苗(Gardasil，HPV6/-11/-16/-18)和九价苗(Gardasil-9， HPV6/-11/-16/-18/-31/-33/-45/-52/-58)，其中保护范围最宽的九价苗才只涵盖有限的7种高危型、2种低危型(HPV6/-11)，而且不能预防皮肤型。另外L1 VLP疫苗不能通过无限制的增加L1 VLP的型别来扩大保护范围，因此L1 VLP疫苗难以满足HPV感染相关疾病的预防要求。
HPV的次要衣壳蛋白L2在天然状态下没有免疫活性，但L2的N端多肽可诱发交叉中和抗体及交叉保护反应，只是免疫原性弱，诱发抗体的滴度低，而且单型L2抗血清的交叉中和型别有限。目前仅在16L2N中发现了多种可诱发中和抗体的保守表位肽，其中aa.17-38为其主要中和表位区，识别该区域的单抗RG-1交叉中和的型别最多，因此该区域又称RG-1表位肽，其中aa.21-31为其中和表位的核心序列，RG-1表位肽的相关研究，无论序列的长短，均保留aa.21-31的同源区。
已经报道的用于疫苗研究的RG-1型别有HPV4型RG-1、HPV6型RG-1、 HPV16型RG-1、HPV17型RG-1、HPV31型RG-1、HPV33型RG-1、HPV45型 RG-1、HPV51型RG-1、HPV58型RG-1等[C.Schellenbacher et al.,The Journal of investigative dermatology 2013,133(12):2706-13；H.Seitz et al.,Vaccine 2014, 32(22):2610-2617；B.Huber et al.,PLoS One 2015,10(3):e0120152；B.Huber et al., PLoS One 2017,12(1):e0169533；X.Chen et al.,Oncotarget 2017,8(38):63333-63344； X.Chen et al.,HumanVaccines&Immunotherapeutics 2018,14(8):2025-2033； PCT/CN2017/075402]，采用的方式包括VLP表面展示、细菌蛋白表面展示(细菌硫氧还蛋白Trx、鞭毛蛋白、霍乱毒素突变体CRM197)、靶向IgγR改造抗体及含RG-1表位的多型L2多肽串联融合。但研究表明，多种RG-1表位肽相关疫苗的活性结果较差，如表面展示HPV4型RG-1、HPV6型RG-1、HPV17型RG-1的 3种16型cVLP诱发产生的HPV16的中和抗体滴度很低，交叉中和滴度未能检测到[B.Huber etal.,PLoS One 2017,12(1):e0169533；X.Chen et al.,Oncotarget 2017, 8(38):63333-63344]；展示HPV45型RG-1的18cVLP诱发产生的HPV18的中和抗体滴度很低(仅为18型L1VLP的1/100)，而且仅交叉中和致癌型HPV45、70 及39，滴度很低，最高的仅为100[B.Huber etal.,PLoS One 2015, 10(3):e0120152]；表面展示51型RG1的Trx融合蛋白抗血清的交叉范围窄，交叉中和抗体滴度最高的仅为500[H.Seitz et al.,Vaccine 2014,32(22):2610-2617]。相反，Schellenbacher等人报道的16型RG1-cVLP及本发明人之前报道的31型RG1-cVLP、33型RG1-cVLP及58型RG1-cVLP的免疫活性较好，骨架型别VLP 诱发的HPV16中和抗体滴度高达105(与16型L1 VLP诱发的相当)，相应RG-1 表位诱发的L2依赖的交叉中和抗体中和范围广、滴度相对较高(最高的可达 6400)[C.Schellenbacher et al.,The Journal ofinvestigative dermatology 2013, 133(12):2706-13；X.Chen et al.,Oncotarget2017,8(38):63333-63344；X.Chen et al.,Human Vaccines&Immunotherapeutics 2018,14(8):2025-2033； PCT/CN2017/075402]。
上述数据提示，不同型别HPV来源的RG-1表位肽的免疫原性存在非常大的差异。本发明人在之前的文献中比较了58型RG-1及6型RG-1的免疫原性，发现58型RG-1表位肽抗血清交叉中和的型别多(13个型别)、滴度也较高(最高的达3200)，而6型RG-1表位肽抗血清的中和型别较少(9个型别)、滴度很低 (最高的仅为100)[X.Chen et al.,Oncotarget 2017,8(38):63333-63344]。表明尽管 RG-1表位肽区在不同型别间具有较强的保守性，但不同型别的RG-1的免疫原性存在差异，因此任选1种L2 aa.17-36同源多肽，构建嵌合蛋白疫苗，其免疫活性是无法预测的。
另一方面，Schellenbacher和Wang的报道的HPV16 cVLP疫苗研究显示，同是将16型RG-1表位肽插入16型L1 VLP载体的表面区，由于16型RG-1核心表位肽序列的旁侧序列及插入位点和插入方式的差异，获得的多种不同的16型 RG1-cVLP的免疫活性具有显著差异，其中最好的是在16型L1的DE环区插入 16型RG-1的cVLP，最差的是在16型L1的h4区插入16型RG-1核心序列的 cVLP。另外，Chen和Boxus均报道了33型RG-1的cVLP，但采用的载体不同，分别是HPV16 L1 VLP及18L1 VLP，虽然两篇报道均选择了DE环作为插入位点，但是插入区相差1个氨基酸，表位肽长度相差2个氨基酸，获得的两种33型 RG1-cVLP诱发产生的33型RG-1依赖的交叉中和抗体的活性差异十分显著，33 型RG1-cVLP抗血清可交叉中和至少12种型别(其中2种型别的滴度>1000)，而33型RG1-18cVLP抗血清仅交叉中和7种型别，其中6种型别的中和滴度(其中4种型别的滴度均＜100)均远较33RG1-16cVLP抗血清的低。
因而，目前需要开发一种能够针对更多HPV型别的病毒产生高滴度中和抗体的基于HPV L1与HPV L2嵌合蛋白的疫苗。
发明内容
为解决上述技术问题，本发明人选用了多种不同长度的59型RG-1表位肽，用于HPVcVLP的研究，结果显示，本发明获得的HPV18型cVLP的免疫原性很强，其诱发的血清中和抗体可高滴度中和α7亚属的多个型别的HPV。
本发明人在实施例1中对8个不同型别RG-1表位免疫血清的中和活性比较分析，发明人意外的发现59型RG-1表位肽的免疫血清可交叉中和至少17种型别，特别是中和HPV45、59及16型的滴度均在103以上，是目前报道的中和α7属高危型HPV型别活性最好的，且同时对HPV16的交叉中和活性又可与目前报道的免疫原性较强的16RG-1相当。
有鉴于此，本发明的目的在于提供一种人乳头瘤病毒嵌合蛋白，用于制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗。
本发明基于本发明人以下的意想不到的发现：在全长或截短型HPV18型L1 蛋白的表面区插入HPV59型L2蛋白多肽，可提高HPV59型L2蛋白多肽的免疫原性，获得的嵌合蛋白在大肠杆菌或昆虫细胞表达系统中可高水平表达，该嵌合蛋白可组装成VLP，并可诱发针对来自不同属/亚属的多种型别HPV的广谱保护性免疫反应。在本文实施例中提供了相关的实验结果。
基于上述目的，本发明一方面提供了一种人乳头瘤病毒嵌合蛋白，其骨架是HPV18 L1蛋白或HPV18 L1蛋白的突变体，所述的骨架上嵌合至少一个来自 HPV59型L2蛋白的多肽。
即，在本发明的第一方面中，本发明提供了一种人乳头瘤病毒嵌合蛋白，其包含HPV18型L1蛋白或HPV18型L1蛋白的突变体以及插入所述HPV18型L1 蛋白或HPV18型L1蛋白的突变体的表面区的来自HPV59型L2蛋白的多肽、或由其组成，其中所述HPV18型L1蛋白的氨基酸序列如SEQ ID NO.1所示，所述 HPV59型L2蛋白的氨基酸序列如SEQ ID NO.2所示。
在根据本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述的HPV18 型L1蛋白的突变体是在所述的HPV18型L1蛋白的N端截短0-8个氨基酸和/或C 端截短0-32个氨基酸的所获得的蛋白。
在根据本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述HPV18型 L1蛋白的突变体选自：
将SEQ ID No.1所示的氨基酸序列的C端截短32个氨基酸的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、504、 506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体 (mut1)；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、499、504、 506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体 (mut2)；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、502、 506置换为甘氨酸(G)、将氨基酸485、500置换为丝氨酸(S)且将氨基酸504 置换为天冬氨酸(D)的突变体(mut3)；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、502、506置换为甘氨酸(G)、将氨基酸486、500置换为丝氨酸(S)且将氨基酸499、504 置换为天冬氨酸(D)的突变体(mut4)；
将SEQ ID No.1所示氨基酸序列的氨基酸477、484、496、499、504、506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体(mut5)；和
将SEQ ID No.1所示氨基酸序列的氨基酸477、485、496、499、504、506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体(mut6)。
在根据本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述来自 HPV59型L2蛋白的多肽选自SEQ ID No.2所示氨基酸的1-50区域内的任意连续 8-33个氨基酸的片段；优选地，所述来自HPV59型L2蛋白的多肽为HPV型59 型L2蛋白RG-1表位肽或其突变体表位肽；进一步优选地，所述来自HPV59型 L2蛋白的多肽由选自以下的氨基酸序列组成：SEQID No.2所示的氨基酸17至32、 SEQ ID No.2所示的氨基酸16至35、SEQ ID No.2所示的氨基酸17至37、SEQ ID No.2所示的氨基酸16至37、以及在上述氨基酸序列的N端和/或C端延长或截短 1至7个氨基酸的序列。
最优选地，所述来自HPV59型L2蛋白的多肽的氨基酸序列如SEQ ID No.3、 SEQ IDNo.4、SEQ ID No.5或SEQ ID No.6所示。
可选地，所述来自HPV59型L2蛋白的多肽是在SEQ ID No.3所示的氨基酸序列的N端延长或截短1-7个氨基酸和/或C端延长或截短1-7个氨基酸所获得的多肽。
可选地，所述来自HPV59型L2蛋白的多肽还可以是与SEQ ID No.3、SEQ ID No.4、SEQ ID No.5或SEQ ID No.6所示的氨基酸序列具有大于60％、优选大于 70％、优选大于80％、大于90％、甚至更优选大于95％序列同一性的多肽。
在根据本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述HPV18型 L1蛋白可来自，例如但不限于NCBI数据库中的ATL15214.1、ATL14646.1、 ARS43458.1、ARS43428.1、ARS43449.1、AGU90430.1等来自HPV18变异株的 L1蛋白。优选地，所述的HPV18型L1蛋白的氨基酸序列如SEQ ID No.1所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述来自HPV59型 L2蛋白的多肽插入HPV18型L1蛋白或HPV18型L1蛋白的突变体的表面区；优选地，插入所述的HPV18型L1蛋白或HPV18型L1蛋白的突变体的DE环或h4 区；更优选地，所述来自HPV59型L2蛋白的多肽通过直接插入的方式插入所述的HPV18型L1蛋白或所述HPV18型L1蛋白的突变体的氨基酸134和氨基酸135 之间、或氨基酸137和138之间、或氨基酸432和433之间、或氨基酸434和435 之间，或者通过非等长置换的方式插入所述的HPV18型L1蛋白或所述HPV18型 L1蛋白的突变体的氨基酸121至124区域、或氨基酸131至138区域、或氨基酸 431-433区域、或氨基酸432-435区域。
如本文所用，术语“直接插入”是指在相邻两个氨基酸之间插入所选择的肽片段。例如，在SEQ ID NO.1的氨基酸134和氨基酸135之间的直接插入指的是将所选择的肽片段直接插入到SEQ ID NO.1的氨基酸134和氨基酸135之间。
如本文所用，术语“非等长置换”指的是在删除指定氨基酸区间的序列后，将所选的肽片段插入到指定的氨基酸区间。例如，在SEQ ID NO.1的氨基酸121 至124区域的非等长置换指的是，删除SEQ ID NO.1的氨基酸122-123之后，将所选择的肽片段插入到SEQ IDNO.1的氨基酸氨基酸121至124之间。可选地，在所述直接插入或非等长置换的方式中，所述来自HPV59型L2蛋白的多肽在其 N端和/或C端包含1至3个氨基酸残基长的连接子。
可选地，所述的连接子由选自甘氨酸(G)、丝氨酸(S)、丙氨酸(A)及脯氨酸(P)的氨基酸任意组合构成。优选地，N端选用G(甘氨酸)P(脯氨酸) 连接子，C端选用P(脯氨酸)连接子。
在本发明的乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是SEQ ID No.4或SEQ ID No.5，插入位点为所述的HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18 型L1蛋白的突变体的氨基酸137和氨基酸138之间，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.7、SEQ IDNo.8、SEQ ID No.9或SEQ ID No.10所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是SEQ ID No.3，插入位点为所述的HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸432和433之间或氨基酸434和435之间，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.11、SEQ IDNo.12、SEQ ID No.13或SEQ ID No.14所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是N端含有GP连接子且C端含有P连接子的SEQ ID No.4或SEQ ID No.6所示序列，插入位点为所述的HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸134和氨基酸135之间，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.15、SEQ ID No.16、SEQ ID No.17或SEQ ID No.18所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述非等长置换的方式中，删除所述HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型 L1蛋白的突变体的氨基酸132-137区域后，在HPV18型L1蛋白或C端截短32 个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸131及138之间插入来自 HPV59型L2蛋白的多肽，所述来自HPV59型L2蛋白的多肽其N端增加了甘氨酸-脯氨酸连接子，所述来自HPV59型L2蛋白的多肽的氨基酸序列如SEQ ID No.3所示，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.19、或SEQ IDNo.20所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述非等长置换的方式中，删除所述HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型 L1蛋白的突变体的氨基酸122-123区域后，在HPV18型L1蛋白或C端截短32 个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸121及124之间插入来自 HPV59型L2蛋白的多肽，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是SEQ ID No.3所示，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.21或SEQ ID No.22所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述非等长置换的方式中，删除所述HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型 L1蛋白的突变体的氨基酸432后，在HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸431及433之间插入来自HPV59型 L2蛋白的多肽，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是SEQ ID No.3所示，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.23或SEQ ID No.24所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，在所述非等长置换的方式中，删除所述HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型 L1蛋白的突变体的氨基酸433-434后，在HPV18型L1蛋白或C端截短32个氨基酸的所述HPV18型L1蛋白的突变体的氨基酸432及435之间插入来自HPV59 型L2蛋白的多肽，在所述直接插入的方式中，所述来自HPV59型L2蛋白的多肽的氨基酸序列是SEQ ID No.3所示，获得的乳头瘤病毒嵌合蛋白氨基酸序列如 SEQ ID No.25或SEQ ID No.26所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述SEQ ID No.4 所示的多肽通过直接插入的方式嵌合于所述HPV18型L1蛋白突变体的氨基酸137 和138之间，所述HPV18型L1蛋白突变体选自：
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、504、 506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.27所示(mut1)；或者，
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、499、504、 506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.28所示(mut2)；或者，
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、502、 506置换为甘氨酸(G)、将氨基酸485、500置换为丝氨酸(S)且将氨基酸504 置换为天冬氨酸(D)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.29所示(mut3)；或者，
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、502、506置换为甘氨酸(G)、将氨基酸486、500置换为丝氨酸(S)且将氨基酸499、504 置换为天冬氨酸(D)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.30所示(mut4)；或者，
将SEQ ID No.1所示氨基酸序列的氨基酸477、484、496、499、504、506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.31所示(mut5)；或者，
将SEQ ID No.1所示氨基酸序列的氨基酸477、485、496、499、504、506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.32所示(mut6)。
本发明的另一方面涉及编码上述的乳头瘤病毒嵌合蛋白的多核苷酸。
本发明还提供了包含上述的多核苷酸的载体，以及包含所述的载体的细胞。
本发明涉及的编码上述的乳头瘤病毒嵌合蛋白的多核苷酸序列适用于不同的表达系统。可选地，这些核苷酸序列采用大肠杆菌密码子进行全基因优化，可在大肠杆菌表达系统中高水平表达；或采用昆虫细胞密码子进行全基因优化，可在昆虫细胞表达系统中高水平表达。
本发明还提供了一种多聚物，优选地，该多聚物为乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒，其含有上述的乳头瘤病毒嵌合蛋白，或者由上述的乳头瘤病毒嵌合蛋白所形成。
本发明还提供了上述的乳头瘤病毒嵌合蛋白、乳头瘤病毒嵌合五聚体或上述的乳头瘤病毒嵌合病毒样颗粒在制备预防乳头瘤病毒感染和/或所述乳头瘤病毒感染诱发的疾病的疫苗中的用途，优选地，所述乳头瘤病毒感染诱发的疾病包括但不限于宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌及口腔癌；
优选地，所述乳头瘤病毒感染为一种或多种选自以下乳头瘤病毒型别的感染：HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、 HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV70、HPV73； HPV6、HPV11、HPV2、HPV5、HPV27和HPV57。
本发明还提供了一种用于预防乳头瘤病毒感染及感染诱发的疾病的疫苗，其包含上述的乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒、佐剂、以及疫苗用赋形剂或载体，优选地，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。其中，这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
可选地，所述佐剂为人用佐剂。
发明中相关术语的说明及解释
根据本发明，术语“昆虫细胞表达系统”包括昆虫细胞、重组杆状病毒、重组Bacmid及表达载体。其中昆虫细胞来源于市场上可得到的细胞，在此举例但不限于：Sf9，Sf21，High Five。
根据本发明，术语“原核表达系统”包括但不限于大肠杆菌表达系统。其中表达宿主菌来源于市场上可得到的菌株，在此举例但不限于：BL21(DE3)， BL21(DE3)plysS，C43(DE3)，Rosetta-gami B(DE3)。
根据本发明，术语“全长HPV18型L1蛋白”的例子包括但不限于NCBI数据库中编号为ATL15070.1的蛋白等长的全长L1蛋白。
“截短型HPV18型L1蛋白”的基因片段指的是其与野生型HPV 18型L1蛋白基因相比，在其5’端和/或3’端缺失编码1个或多个氨基酸的核苷酸，其中“野生型 HPV18型L1蛋白”的全长序列例如但不限于NCBI数据库中的如下序列： ATL15214.1、ATL14646.1、ARS43458.1、ARS43428.1、ARS43449.1、 AGU90430.1等。
根据本发明，术语“疫苗用赋形剂或载体”是指选自一种或多种，包括但不限于：pH调节剂，表面活性剂，离子强度增强剂。例如，pH调节剂举例但不限于磷酸盐缓冲液，表面活性剂包括阳离子、阴离子或非离子型表面活性剂，举例但不限于聚山梨酯80(Tween-80)，离子强度增强剂举例但不限于氯化钠。
根据本发明，术语“人用佐剂”是指在临床上可应用于人体的佐剂，包括当前已获得批准的和将来可能获得批准的各种佐剂，例如但不限于铝佐剂、MF59及各种形式的佐剂组合物。
根据本发明，本发明的疫苗可采用患者可接受的形式，包括但不限于口服或者注射，优选注射。
根据本发明，本发明疫苗优选单位剂型使用，其中单位剂型中蛋白病毒样颗粒的剂量为5μg-100μg，例如5、10、15、20、25、30、35、40、45、50、55、60、 65、70、75、80、85、90、95、100μg、以及上述任意两个数值之间的范围；优选 30μg-60μg。
附图说明
图1A-图1B：本发明实施例6中嵌合蛋白在大肠杆菌及昆虫细胞中的表达鉴定。结果显示，26种嵌合蛋白均可在大肠杆菌或昆虫细胞中表达，其中有6种嵌合蛋白可在两种表达系统中表达。
图1A：嵌合蛋白在大肠杆菌中的表达鉴定：1为18L1DE137-138/59dES；2为18L1DE137-138/59dE；3为18L1h4432-433/59dE；4为18L1h4434-435/59dE；5为 18L1DE134-135/59dES；6为18L1DE134-135/59dE；7为18L1DE131-138/59dE；8为18L1DE121-124/59dE，9为18L1h4431-433/59dE；10为18L1h4432-435/59dE；11为 18L1DE137-138/59dES-mut1；12为18L1DE137-138/59dES-mut2；13为 18L1DE137-138/59dES-mut3；14为18L1DE137-138/59dES-mut4；15为 18L1DE137-138/59dES-mut5；16为18L1DE137-138/59dES-mut6；
图1B：嵌合蛋白在昆虫细胞中的表达鉴定：1为18L1ΔCDE137-138/59dES；2 为18L1ΔCDE137-138/59dE；3为18L1ΔCh4432-433/59dE；4为18L1ΔCh4434-435/59dE； 5为18L1ΔCDE134-135/59dES；6为18L1ΔCDE134-135/59dE；7为 18L1ΔCDE131-138/59dE；8为18L1ΔCDE121-124/59dE，9为18L1ΔCh4431-433/59dE；10 为18L1ΔCh4432-435/59dE；11为18L1DE137-138/59dES-mut1；12为 18L1DE137-138/59dES-mut2；13为18L1DE137-138/59dES-mut3；14为18L1DE137-138/59dES-mut4；15为18L1DE137-138/59dES-mut5；16为 18L1DE137-138/59dES-mut6。
图2A-图2D：本发明实施例6中纯化后获得的cVLP的动态光散射分析结果。结果显示18L1ΔCDE134-135/59dE、18L1ΔCDE134-135/59dES、18L1ΔCDE137-138/59dE 及18L1ΔCDE137-138/59dES重组蛋白形成的病毒样颗粒水化动力学直径分别为 106.8nm、113.3nm、114.7nm和122.9nm，颗粒组装的百分比均为100％。
图2A：18L1ΔCDE134-135/59dE；图2B：18L1ΔCDE134-135/59dES；图2C： 18L1ΔCDE137-138/59dE；图2D：18L1ΔCDE137-138/59dES。
图3A-图3D：本发明实施例7中纯化后获得的cVLP的透射电镜观察结果。视野中可见大量的病毒样颗粒，颗粒均一度好。cVLP直径为50nm左右，与L1 蛋白的VLP的大小相似。Bar＝50nm。
图3A：18L1ΔCDE134-135/59dE；图3B：18L1ΔCDE134-135/59dES；图3C： 18L1ΔCDE137-138/59dE；图3D.18L1ΔCDE137-138/59dES。
图4：本发明实施例10中的嵌合VLP小鼠免疫血清对α7亚属HPV假病毒的中和活性检测。*：P<0.05。
具体实施方式
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围，因为实施方案必然是多样的。本说明书中使用的用语仅是为了阐述特定的实施方案，而非作为限制，本发明的范围已界定在所附的权利要求中。
除非特别说明，本说明书中所使用的所有技术和科学用语均和本案所属技术领域的技术人员所普遍明了的意义相同。下面就本发明的优选方法和材料加以叙述，但是与本说明书中所述方法和材料类似或等效的任何方法和材料均可用以实施或测试本发明。下述实验方法如无特别说明，均为常规方法或产品说明书所描述的方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本说明书中所提到的所有公开文献均被并入于此作为参考，以揭示并说明所述公开文献中的方法和/或材料。
实施例1：不同型别RG-1表位肽的免疫活性检测
采用化学合成法合成HPV35、-39、-51、-53、-56、-59、-68、-82的RG-1表位肽，表位肽序列如表1所示，多肽由上海吉尔生化有限公司合成，为了提高合成肽的免疫原性，各合成肽通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐 (EDC，CAS No.25952-53-8)活化羧基后与钥孔血蓝蛋白(KLH)偶联。
取2.0-2.5kg体重的新西兰大白兔，随机分组，每组2-4只，于免疫前4天背部多点皮下注射15mg灭活的与等体积弗氏完全佐剂充分混匀的DH5a(含0.5％v/v 甲醛的PBS，37℃处理24-48h)进行免疫刺激，首次免疫采用背部、大腿内侧多点皮下注射1mg与等体积弗氏完全佐剂充分混匀的KLH-多肽。加强免疫4次，每次间隔2周，加强免疫的抗原为0.5mg与等体积弗氏不完全佐剂(充分混匀的 KLH-多肽。最后一次免疫后2周采血，分离血清。
使用17种HPV假病毒对免疫血清的中和抗体滴度进行检测，结果如表2所示。59RG-1表位肽的免疫活性最好，其抗血清可中和所有17种检测型别，其中 HPV45,-59,-16的中和抗体的滴度均在103以上，HPV5,-31,-18,-39,-68,-57的中和抗体滴度在500-1000之间。值得注意的是，59RG-1表位肽抗血清对检测的5种α7-HPV的中和抗体水平均很高。
多肽合成、假病毒制备及假病毒中和实验的方法均是公开的，例如专利CN104418942A及108676057A。
表1.合成的不同型别RG-1表位肽的序列
型别 合成肽序列 SEQ ID NO.
HPV35 TQLYRTCKAAGTCPPDVIPKVEG 44
HPV39 STLYRTCKQSGTCPPDVVDKVEG 45
HPV51 TQLYSTCKAAGTCPPDVVNKVEG 46
HPV53 TQLYQTCKQSGTCPEDVINKIEH 47
HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 48
HPV59 LYKTCKQ AGTCP SDVIN KVEGTT 49
HPV68 STLYKTCKQSGTCPPDVINKVEG 50
HPV82 TQLYSTCKAAGTCPPDVIPKVKG 51
表2.不同的RG1-KLH偶联肽在兔子中诱发的血清中和抗体滴度
Figure BDA0002881944270000121
实施例2：嵌合L1蛋白的基因的合成及表达载体构建
26种嵌合L1蛋白，分别为：
1)嵌合L1蛋白18L1DE137-138/59dES：骨架为全长HPV18型L1蛋白(序列如SEQ IDNo.1所示)，在其DE环aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32多肽(如SEQ IDNo.4所示)，18L1DE137-138/59dES嵌合蛋白的氨基酸序列如SEQ ID No.7所示。编码18L1DE137-138/59dES的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
2)嵌合L1蛋白18L1DE137-138/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，在其DE环aa.137/138位点，直接插入HPV59型L2蛋白的 aa.16-35多肽(如SEQID No.5所示)，18L1DE137-138/59dE嵌合蛋白的氨基酸序列如SEQ ID No.9所示。编码18L1DE137-138/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
3)嵌合L1蛋白18L1h4432-433/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，在其h4区aa.432/433位点，直接插入HPV59型L2蛋白的 aa.17-37多肽(如SEQID No.3所示)，18L1h4432-433/59dE嵌合蛋白的氨基酸序列如SEQ ID No.11所示。编码18L1h4432-433/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
4)嵌合L1蛋白18L1h4434-435/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，在其h4区aa.434/435位点，直接插入HPV59型L2蛋白的 aa.17-37多肽(如SEQID No.3所示)，18L1h4434-435/59dE嵌合蛋白的氨基酸序列如SEQ ID No.13所示。编码18L1h4434-435/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
5)嵌合L1蛋白18L1DE134-135/59dES：骨架为全长HPV18型L1蛋白(序列如SEQ IDNo.1所示)，在其DE环aa.134/135位点，直接插入N端含GP连接子且C端含P连接子的HPV59型L2蛋白的aa.17-32多肽(即在SEQ ID No.4所示序列的N端添加甘氨酸-脯氨酸，且在C端添加脯氨酸)，18L1DE134-135/59dES嵌合蛋白的氨基酸序列如SEQ ID No.15所示。编码18L1DE134-135/59dES的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
6)嵌合L1蛋白18L1DE134-135/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，在其DE环aa.134/135位点，直接插入N端含GP连接子且 C端含P连接子的HPV59型L2蛋白的aa.16-37多肽(即在SEQ ID No.6所示序列的N端添加甘氨酸-脯氨酸，且在C端添加脯氨酸)，18L1DE134-135/59dE嵌合蛋白的氨基酸序列如SEQ ID No.17所示。编码18L1DE134-135/59dES的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
7)嵌合L1蛋白18L1DE131-138/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，删除其aa.132-137区域，并在aa.131/138之间融合N端包含 GP连接子的HPV59型L2蛋白的aa.17-37多肽(在HPV 18型L1蛋白的aa.132-137 区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.3所示序列的N端添加甘氨酸-脯氨酸，18L1DE131-138/59dE嵌合蛋白的氨基酸序列如SEQ ID No.19 所示。编码18L1DE131-138/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
8)嵌合L1蛋白18L1DE121-124/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，删除其aa.122-123区域，并在aa.121/124之间融合HPV59 型L2蛋白的aa.17-37多肽(在HPV 18型L1蛋白的aa.122-133区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.3所示，18L1DE121-124/59dE嵌合蛋白的氨基酸序列如SEQ ID No.21所示。编码18L1DE121-124/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
9)嵌合L1蛋白18L1h4431-433/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，删除其aa.432，并在aa.431/433之间融合HPV59型L2蛋白的aa.17-37多肽(在HPV 18型L1蛋白的aa.431-433区域非等长置换插入)，插入片段的氨基酸序列如SEQ IDNo.3所示，18L1h4431-433/59dE嵌合蛋白的氨基酸序列如SEQ ID No.23所示。编码18L1h4431-433/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
10)嵌合L1蛋白18L1h4432-435/59dE：骨架为全长HPV18型L1蛋白(序列如 SEQ IDNo.1所示)，删除其aa.433-434区域，并在aa.432/435之间融合HPV59 型L2蛋白的aa.17-37多肽(在HPV 18型L1蛋白的aa.432-435区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.3所示，18L1h4432-435/59dE嵌合蛋白的氨基酸序列如SEQ ID No.25所示。编码18L1h4433-434/59dE的多核苷酸序列经大肠杆菌密码子优化设计，采用全基因合成的方式构建；
11)嵌合L1蛋白18L1ΔCDE137-138/59dES：骨架为C端截短32个氨基酸的HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其DE环 aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32多肽(如SEQ ID No.4所示)，18L1ΔCDE137-138/59dES嵌合蛋白的氨基酸序列如SEQ ID No.8所示。编码 18L1ΔCDE137-138/59dES的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.33所示；
12)嵌合L1蛋白18L1ΔCDE137-138/59dE：骨架为C端截短32个氨基酸的 HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其DE环 aa.137/138位点，直接插入HPV59型L2蛋白的aa.16-35多肽(如SEQ ID No.5所示)，18L1ΔCDE137-138/59dE嵌合蛋白的氨基酸序列如SEQ ID No.10所示。编码 18L1ΔCDE137-138/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.34所示；
13)嵌合L1蛋白18L1ΔCh4432-433/59dE：骨架为C端截短32个氨基酸的HPV18 型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其h4区aa.432/433 位点，直接插入HPV59型L2蛋白的aa.17-37多肽(如SEQ ID No.3所示)， 18L1ΔCh4432-433/59dE嵌合蛋白的氨基酸序列如SEQ ID No.12所示。编码 18L1ΔCh4432-433/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
14)嵌合L1蛋白18L1ΔCh4434-435/59dE：骨架为C端截短32个氨基酸的HPV18 型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其h4区aa.434/435 位点，直接插入HPV59型L2蛋白的aa.17-37多肽(如SEQ ID No.3所示)， 18L1ΔCh4434-435/59dE嵌合蛋白的氨基酸序列如SEQ ID No.14所示。编码 18L1ΔCh4434-435/59dE的多核苷酸序列经昆虫细胞码子优化设计，采用全基因合成的方式构建；
15)嵌合L1蛋白18L1ΔCDE134-135/59dES：骨架为C端截短32个氨基酸的 HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其DE环 aa.134/135位点，直接插入N端含GP连接子且C端含P连接子的HPV59型L2 蛋白的aa.17-32多肽(即在SEQ ID No.4所示序列的N端添加甘氨酸-脯氨酸，且在C端添加脯氨酸)，18L1ΔCDE134-135/59dES嵌合蛋白的氨基酸序列如SEQ ID No.16所示。编码18L1ΔCDE134-135/59dES的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.35所示；
16)嵌合L1蛋白18L1ΔCDE134-135/59dE：骨架为C端截短32个氨基酸的 HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，在其DE环 aa.134/135位点，直接插入N端含GP连接子且C端含P连接子的HPV59型L2 蛋白的aa.16-37多肽(即在SEQ ID No.6所示序列的N端添加甘氨酸-脯氨酸，且在C端添加脯氨酸)，18L1ΔCDE134-135/59dE嵌合蛋白的氨基酸序列如SEQ ID No.18所示。编码18L1ΔCDE134-135/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.36所示；
17)嵌合L1蛋白18L1ΔCDE131-138/59dE：骨架为C端截短32个氨基酸的 HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，删除其aa.132-137 区域，并在aa.131/138之间融合N端包含GP连接子的HPV59型L2蛋白的aa.17-37 多肽(在HPV 18型L1蛋白的aa.132-137区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.3所示序列的N端添加甘氨酸-脯氨酸，18L1DE131-138/59dE 嵌合蛋白的氨基酸序列如SEQ ID No.20所示。编码18L1DE131-138/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.37所示；
18)嵌合L1蛋白18L1ΔCDE121-124/59dE：骨架为C端截短32个氨基酸的 HPV18型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，删除其aa.122-123 区域，并在aa.121/124之间融合HPV59型L2蛋白的aa.17-37多肽(在HPV 18型 L1蛋白的aa.122-133区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.3所示，18L1ΔCDE121-124/59dE嵌合蛋白的氨基酸序列如SEQ ID No.22所示。编码18L1ΔCDE121-124/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
19)嵌合L1蛋白18L1ΔCh4431-433/59dE：骨架为C端截短32个氨基酸的HPV18 型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，删除其aa.432，并在 aa.431/433之间融合HPV59型L2蛋白的aa.17-37多肽(在HPV 18型L1蛋白的 aa.431-433区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.3所示， 18L1ΔCh4431-433/59dE嵌合蛋白的氨基酸序列如SEQ ID No.24所示。编码 18L1ΔCh4431-433/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
20)嵌合L1蛋白18L1ΔCh4432-435/59dE：骨架为C端截短32个氨基酸的HPV18 型L1蛋白(序列SEQ ID No.1的C端截短32个氨基酸)，删除其aa.433-434区域，并在aa.432/435之间融合HPV59型L2蛋白的aa.17-37多肽(在HPV 18型 L1蛋白的aa.432-435区域非等长置换插入)，插入片段的氨基酸序列如SEQ ID No.3所示，18L1ΔCh4432-435/59dE嵌合蛋白的氨基酸序列如SEQ ID No.26所示。编码18L1ΔCh4432-435/59dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
21)嵌合L1蛋白18L1DE137-138/59dES-mut1：骨架为全长HPV18型L1蛋白的突变体mut1(即将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、504、506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体)，在其DE环aa.137/138位点，直接插入HPV59型L2蛋白的 aa.17-32多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut1嵌合蛋白的氨基酸序列如SEQ ID No.27所示。编码18L1DE137-138/59dES-mut1的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut1的多核苷酸序如 SEQ IDNo.38所示；
22)嵌合L1蛋白18L1DE137-138/59dES-mut2：骨架为全长HPV18型L1蛋白的突变体mut2(即将SEQ ID No.1所示序列的氨基酸477、478、485、496、499、 504、506置换为甘氨酸且将氨基酸486、500、502置换为丝氨酸)，在其DE环 aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut2嵌合蛋白的氨基酸序列如SEQ ID No.28所示。编码18L1DE137-138/59dES-mut2的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut2的多核苷酸序如SEQ ID No.39所示；
23)嵌合L1蛋白18L1DE137-138/59dES-mut3：骨架为全长HPV18型L1蛋白的突变体mut3(即将SEQ ID No.1所示序列的氨基酸477、478、484、496、499、 502、506置换为甘氨酸、将氨基酸485、500置换为丝氨酸且将氨基酸504置换为天冬氨酸)，在其DE环aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32 多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut3嵌合蛋白的氨基酸序列如SEQ ID No.29所示。编码18L1DE137-138/59dES-mut3的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut3的多核苷酸序如SEQ IDNo.40所示；
24)嵌合L1蛋白18L1DE137-138/59dES-mut4：骨架为全长HPV18型L1蛋白的突变体mut4(即将SEQ ID No.1所示序列的氨基酸477、478、485、496、502、 506置换为甘氨酸、将氨基酸486、500置换为丝氨酸且将氨基酸499、504置换为天冬氨酸)，在其DE环aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32 多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut4嵌合蛋白的氨基酸序列如SEQ ID No.30所示。编码18L1DE137-138/59dES-mut4的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut4的多核苷酸序如SEQ IDNo.41所示；
25)嵌合L1蛋白18L1DE137-138/59dES-mut5：骨架为全长HPV18型L1蛋白的突变体mut5(即将SEQ ID No.1所示序列的氨基酸477、484、496、499、504、 506置换为甘氨酸且将氨基酸485、500、502置换为丝氨酸)，在其DE环 aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut5嵌合蛋白的氨基酸序列如SEQ ID No.31所示。编码18L1DE137-138/59dES-mut5的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut5的多核苷酸序如SEQ ID No.42所示；
26)嵌合L1蛋白18L1DE137-138/59dES-mut6：骨架为全长HPV18型L1蛋白的突变体mut6(即将SEQ ID No.1所示序列的氨基酸477、485、496、499、504、 506置换为甘氨酸且将氨基酸486、500、502置换为丝氨酸)，在其DE环 aa.137/138位点，直接插入HPV59型L2蛋白的aa.17-32多肽(如SEQ ID No.4所示)，18L1DE137-138/59dES-mut6嵌合蛋白的氨基酸序列如SEQ ID No.32所示。编码18L1DE137-138/59dES-mut6的多核苷酸序列经大肠杆菌密码子优化设计或昆虫细胞密码子优化设计，采用全基因合成的方式构建，其中采用昆虫细胞密码子优化的18L1DE137-138/59dES-mut5的多核苷酸序如SEQ ID No.43所示。
大肠杆菌密码子优化的嵌合蛋白基因经NdeI/XhoI酶切后，分别插入商业化的表达载体pET22b(Novagen公司生产)。昆虫细胞密码子优化的嵌合蛋白基因经BamHI/EcoRI酶切后，分别插入商业化表达载体pFastBac1(Invitrogen公司生产)中。得到包含嵌合蛋白基因的表达载体，用于大肠杆菌的表达载体共16个，分别为：pET22b-18L1DE137-138/59dES，pET22b-18L1DE137-138/59dE， pET22b-18L1h4432-433/59dE，pET22b-18L1h4434-435/59dE，pET22b-18L1DE134-135/59dES，pET22b-18L1DE134-135/59dE， pET22b-18L1DE131-138/59dE，pET22b-18L1DE121-124/59dE， pET22b-18L1h4431-433/59dE，pET22b-18L1h4432-435/59dE，pET22b-18L1DE137-138/59dES-mut1，pET22b-18L1DE137-138/59dES-mut2， pET22b-18L1DE137-138/59dES-mut3，pET22b-18L1DE137-138/59dES-mut4， pET22b-18L1DE137-138/59dES-mut5，pET22b-18L1DE137-138/59dES-mut6；用于昆虫细的表达载体共16个，分别为：pFastBac1-18L1ΔCDE137-138/59dES， pFastBac1-18L1ΔCDE137-138/59dE，pFastBac1-18L1ΔCh4432-433/59dE， pFastBac1-18L1ΔCh4434-435/59dE，pFastBac1-18L1ΔCDE134-135/59dES，pFastBac1-18L1ΔCDE134-135/59dE，pFastBac1-18L1ΔCDE131-138/59dE， pFastBac1-18L1ΔCDE121-124/59dE，pFastBac1-18L1ΔCh4431-433/59dE， pFastBac1-18L1ΔCh4432-435/59dE，pFastBac1-18L1DE137-138/59dES-mut1， pFastBac1-18L1DE137-138/59dES-mut2，pFastBac1-18L1DE137-138/59dES-mut3， pFastBac1-18L1DE137-138/59dES-mut4，pFastBac1-18L1DE137-138/59dES-mut5， pFastBac1-18L1DE137-138/59dES-mut6。上述酶切、连接及克隆构建的方法都是公知的，例如专利CN101293918B。
本发明中使用的多肽的氨基酸序列如下所示：
HPV18型L1全长氨基酸
Figure BDA0002881944270000181
HPV59型L2全长氨基酸序列
Figure BDA0002881944270000182
Figure BDA0002881944270000191
HPV 59型L2 aa.17-37
LYKTCKQAGTCPSDVINKVEG SEQ ID NO.3
HPV 59型L2 aa.17-32
LYKTCKQAGTCPSDVI SEQ ID NO.4
HPV 59型L2 aa.16-35
DLYKTCKQAGTCPSDVINKV SEQ ID NO.5
HPV 59型L2 aa.16-37
DLYKTCKQAGTCPSDVINKVEG SEQ ID NO.6
18L1DE137-138/59dES
Figure BDA0002881944270000192
18L1ΔCDE137-138/59dES
Figure BDA0002881944270000193
18L1DE137-138/59dE
Figure BDA0002881944270000194
Figure BDA0002881944270000201
18L1ΔCDE137-138/59dE
Figure BDA0002881944270000202
18L1h4432-433/59dE
Figure BDA0002881944270000203
18L1ΔCh4432-433/59dE
Figure BDA0002881944270000204
18L1h4434-435/59dE
Figure BDA0002881944270000205
Figure BDA0002881944270000211
18L1ΔCh4434-435/59dE
Figure BDA0002881944270000212
18L1DE134-135/59dES
Figure BDA0002881944270000213
18L1ΔCDE134-135/59dES
Figure BDA0002881944270000214
18L1DE134-135/59dE
Figure BDA0002881944270000215
Figure BDA0002881944270000221
18L1ΔCDE134-135/59dE
Figure BDA0002881944270000222
18L1DE131-138/59dE
Figure BDA0002881944270000223
18L1ΔCDE131-138/59dE
Figure BDA0002881944270000224
18L1DE121-124/59dE
Figure BDA0002881944270000225
Figure BDA0002881944270000231
18L1ΔCDE121-124/59dE
Figure BDA0002881944270000232
18L1h4431-433/59dE
Figure BDA0002881944270000233
18L1ΔCh4431-433/59dE
Figure BDA0002881944270000234
Figure BDA0002881944270000241
18L1h4432-435/59dE
Figure BDA0002881944270000242
18L1ΔCh4432-435/59dE
Figure BDA0002881944270000243
18L1DE137-138/59dES-mut1
Figure BDA0002881944270000244
18L1DE137-138/59dES-mut2
Figure BDA0002881944270000245
Figure BDA0002881944270000251
18L1DE137-138/59dES-mut3
Figure BDA0002881944270000252
18L1DE137-138/59dES-mut4
Figure BDA0002881944270000253
18L1DE137-138/59dES-mut5
Figure BDA0002881944270000254
18L1DE137-138/59dES-mut6
Figure BDA0002881944270000261
编码本发明嵌合蛋白的核苷酸序列如下所示：
18L1ΔCDE137-138/59dES nt
Figure BDA0002881944270000262
Figure BDA0002881944270000271
18L1ΔCDE137-138/59dE nt
Figure BDA0002881944270000272
18L1ΔCDE134-135/59dES nt
Figure BDA0002881944270000273
Figure BDA0002881944270000281
18L1ΔCDE134-135/59dE nt
Figure BDA0002881944270000282
Figure BDA0002881944270000291
18L1ΔCDE131-138/59dE nt
Figure BDA0002881944270000292
Figure BDA0002881944270000301
18L1DE137-138/59dES-mut1 nt
Figure BDA0002881944270000302
18L1DE137-138/59dES-mut2 nt
Figure BDA0002881944270000303
Figure BDA0002881944270000311
18L1DE137-138/59dES-mut3 nt
Figure BDA0002881944270000312
18L1DE137-138/59dES-mut4 nt
Figure BDA0002881944270000321
18L1DE137-138/59dES-mut5 nt
Figure BDA0002881944270000322
Figure BDA0002881944270000331
18L1DE137-138/59dES-mut6 nt
Figure BDA0002881944270000332
实施例3：嵌合L1蛋白的基因的重组Bacmid及重组杆状病毒的构建
分别使用包含嵌合L1基因的重组表达载体pFastBac1-18L1ΔCDE137-138/59dES，pFastBac1-18L1ΔCDE137-138/59dE， pFastBac1-18L1ΔCh4432-433/59dE，pFastBac1-18L1ΔCh4434-435/59dE， pFastBac1-18L1ΔCDE134-135/59dES，pFastBac1-18L1ΔCDE134-135/59dE，pFastBac1-18L1ΔCDE131-138/59dE，pFastBac1-18L1ΔCDE121-124/59dE， pFastBac1-18L1ΔCh4431-433/59dE，pFastBac1-18L1ΔCh4432-435/59dE， pFastBac1-18L1DE137-138/59dES-mut1，pFastBac1-18L1DE137-138/59dES-mut2， pFastBac1-18L1DE137-138/59dES-mut3，pFastBac1-18L1DE137-138/59dES-mut4， pFastBac1-18L1DE137-138/59dES-mut5，pFastBac1-18L1DE137-138/59dES-mut6转化大肠杆菌DH10Bac感受态，筛选获得重组Bacmid，然后用重组Bacmid转染昆虫细胞Sf9，在Sf9内扩增重组杆状病毒。重组Bacmid的筛选及重组杆状病毒的扩增方法都是公知的，例如专利CN101148661B。
实施例4：嵌合L1蛋白的基因在Sf9细胞中的表达
Sf9细胞分别接种16种嵌合L1基因的重组杆状病毒，进行嵌合L1蛋白的表达，27℃培养约88h后收发酵液，3000rpm离心15min，弃上清，用PBS洗涤细胞后，用于表达鉴定及纯化。感染表达的方法是公开的，例如专利CN 101148661 B。
实施例5：嵌合L1蛋白的基因在大肠杆菌中的表达
分别使用包含嵌合L1基因的重组表达载体pET22b-18L1DE137-138/59dES， pET22b-18L1DE137-138/59dE，pET22b-18L1h4432-433/59dE， pET22b-18L1h4434-435/59dE，pET22b-18L1DE134-135/59dES， pET22b-18L1DE134-135/59dE，pET22b-18L1DE131-138/59dE， pET22b-18L1DE121-124/59dE，pET22b-18L1h4431-433/59dE， pET22b-18L1h4432-435/59dE，pET22b-18L1DE137-138/59dES-mut1， pET22b-18L1DE137-138/59dES-mut2，pET22b-18L1DE137-138/59dES-mut3， pET22b-18L1DE137-138/59dES-mut4，pET22b-18L1DE137-138/59dES-mut5，pET22b-18L1DE137-138/59dES-mut6转化大肠杆菌BL21(DE3)。
挑单克隆接种到3ml含氨苄青霉素的LB培养基中，37℃培养过夜。将过夜培养的菌液按1：100的比例加入LB培养基中，37℃培养3h左右，待OD600达到0.8-1.0之间，加IPTG至终浓度0.5μM，16℃培养约12h，收取菌液。
实施例6：嵌合L1蛋白的表达鉴定
取实施例4及实施例5中所述表达不同嵌合L1蛋白的细胞各1×106个，重悬于200μl PBS溶液中，加入6×Loading Buffer 50μl，75℃变性8分钟，分别取10 μl进行SDS-PAGE电泳及Western blot鉴定。结果如图1A-图1B所示，26种嵌合L1蛋白均可在昆虫细胞或原核表达系统中高水平表达，其中 18L1DE137-138/59dES，18L1DE137-138/59dE，18L1h4432-433/59dE，18L1h4434-435/59dE，18L1DE134-135/59dES，18L1DE134-135/59dE， 18L1DE131-138/59dE，18L1DE121-124/59dE，18L1h4431-433/59dE， 18L1h4432-435/59dE，18L1DE137-138/59dES-mut1，18L1DE137-138/59dES-mut2， 18L1DE137-138/59dES-mut3，18L1DE137-138/59dES-mut4，18L1DE137-138/59dES-mut5，18L1DE137-138/59dES-mut6大小约59kDa，其余10种蛋白大小约55kDa。SDS-PAGE电泳及Western blot鉴定的方法是公开的，例如专利 CN101148661B。
实施例7：嵌合L1蛋白在昆虫细胞中的表达量比较
取C端截短32个氨基酸的18L1骨架蛋白的表达细胞以及实施例4中所述的以C端截短32个氨基酸的18L1为骨架或以6种18L1突变体为骨架的嵌合L1蛋白表达细胞各1×106个，重悬于200μl PBS溶液中，采用超声破碎法(宁波新芝超声破碎仪，2#探头，100W，超声5s，间隔7s，总时间3min)破碎细胞， 12000rpm高速离心10分钟。收取裂解上清，采用夹心ELISA法检测上清中的L1 含量，该方法是公知的，例如专利CN104513826A。
使用本发明人制备的HPV18 L1单克隆抗体包被酶标板，80ng/孔，4℃孵育过夜；使用5％BSA-PBST室温封闭2h，再用PBST洗板3次。用PBS将裂解上清进行连续2倍稀释，并且将HPV18L1 VLP标准品也进行梯度稀释，浓度从2μ g/ml-0.0625μg/ml，分别加入酶标板，每孔100μl，37℃孵育1h。用PBST洗板3次，加入1：3000稀释的HPV18L1兔多抗，每孔100μl，37℃孵育1h。用 PBST洗板3次，加入1：3000稀释的HRP标记的山羊抗小鼠IgG(1：3000稀释，中杉金桥公司)，37℃孵育45分钟。用PBST洗板5次，每孔加入100μl OPD底物(Sigma公司)，37℃显色5分钟，用50μl 2M硫酸终止反应，在490nm处测定吸光值。依据标准曲线计算裂解上清中HPV18L1蛋白及18L1嵌合蛋白的浓度。
结果如表3所示，本发明的18L1ΔCDE134-135/59dES、 18L1ΔCDE137-138/59dES、18L1ΔCh4431-433/59dE及18L1ΔCh4432-435/59dE的表达量很高，与HPV18L1骨架的相当；此外，以C端氨基酸置换的18L1突变体为骨架的嵌合蛋白18L1DE137-138/59dES-mut1、18L1DE137-138/59dES-mut4、 18L1DE137-138/59dES-mut5的表达量均高于HPV18L1骨架及相应的C端截短的嵌合蛋白。
表3.嵌合L1蛋白表达量分析
Figure BDA0002881944270000351
Figure BDA0002881944270000361
实施例8：嵌合L1蛋白的纯化及动态光散射粒径分析
取嵌合L1的细胞发酵液适量，使用10ml PBS重悬细胞，加PMSF至终浓度 1mg/ml，超声破碎(宁波新芝超声破碎仪，6#探头，200W，超声5s，间隔7s，总时间10min)，取破碎上清进行纯化，纯化步骤在室温进行。在裂解液中加入 4％β-巯基乙醇(w/w)对VLP进行解聚，然后使用0.22μm滤器过滤样品，依次使用DMAE阴离子交换层析或CM阳离子交换层析(20mMTris,180mM NaCl,4％β-ME,pH7.9洗脱)、TMAE阴离子交换层析或Q阳离子交换层析(20mMTris,180 mM NaCl,4％β-ME,pH7.9洗脱)及羟基磷灰石层析(100mM NaH2PO4，30mM NaCl，4％β-ME，pH 6.0洗脱)纯化。纯化产物采用Planova超滤系统进行浓缩，并更换缓冲液(20mMNaH2PO4，500mM NaCl，pH6.0)促使VLP组装。以上纯化方法均是公开的，例如专利CN101293918B、CN1976718A等。
嵌合蛋白纯品组装过程中发现，18L1h4431-433/59dE、18L1h4432-435/59dE、 18L1ΔCh4431-433/59dE及18L1ΔCh4432-435/59dE严重凝集，其他嵌合蛋白组装后未观察到凝集现象。取组装后的嵌合蛋白溶液进行DLS粒径分析(Zetasizer Nano ZS 90动态光散射仪，Malvern公司)，结果如表4所示，其中18L1ΔCDE134-135/59dE、 18L1ΔCDE134-135/59dES、18L1ΔCDE137-138/59dE、18L1ΔCDE137-138/59dES的DLS 分析图如图2A至图2D所示。
表4.嵌合L1蛋白DLS分析
Figure BDA0002881944270000362
Figure BDA0002881944270000371
实施例9：嵌合VLP的透射电镜观察
按实施例8所述的层析纯化方法，分别纯化嵌合蛋白，使用组装后嵌合制备铜网，并用1％醋酸铀进行染色，充分干燥后使用JEM-1400电镜(奥林巴斯)进行观察。结果显示，大肠杆菌及昆虫细胞表达的嵌合蛋白均可组装成直径约为 50nm的cVLP。其中18L1ΔCDE134-135/59dE、18L1ΔCDE134-135/59dES、 18L1ΔCDE137-138/59dE、18L1ΔCDE137-138/59dEScVLP的电镜图片如图3A至图3D 所示。铜网制备及电镜观察的方法均是公开的，例如专利CN101148661 B。
实施例10：嵌合VLP的小鼠免疫及中和抗体滴度测定
取4-6周龄的BALB/c小鼠，随机分组，每组5只，用10μg cVLP联合 Al(OH)3 50μg及MPL佐剂5μg免疫小鼠。皮下注射，于第0，4，7，10周免疫，共4次。第4次免疫后2周尾静脉采血，分离血清。
使用24种HPV假病毒对免疫血清的中和抗体滴度进行检测，结果显示大肠杆菌及昆虫细胞表达体系生产的各种cVLP免疫小鼠后，诱发的交叉中和抗体水平及中和范围各有不同。其中，如表5所示，昆虫细胞表达的 18L1ΔCDE134-135/59dES及18L1ΔCDE137-138/59dEScVLP抗血清可中和至少23个型别的假病毒，18L1ΔCDE134-135/59dE及18L1ΔCDE137-138/59dEcVLP免疫血清可中和至少19个型别的假病毒。值得一提的是，18L1ΔCDE134-135/59dES及18L1ΔCDE137-138/59dES cVLP抗血清可中和所有的6种检测用的α7-HPV，特别是 18L1ΔCDE137-138/59dES cVLP，交叉中和α7-HPV的抗体滴度都在250以上，是目前报道的交叉中和α7-HPV能力最强的cVLP。
此外，本发明中采用C端截短32个氨基酸的18L1突变体构建的cVLP，采用上述策略免疫小鼠后，也可诱发的高水平的中和抗体，其中 18L1DE137-138/59dES-mut4诱发的各型中和抗体水平均与18L1ΔCDE137-138/59dES的相当，值得注意的是，18L1DE137-138/59dES-mut4免疫血清的HPV39和HPV59中和抗体滴度均大于103(如表5、图4所示)。
假病毒制备及假病毒中和实验的方法均是公开的，例如专利CN 104418942A。
表5不同cVLP在小鼠中诱发的中和抗体滴度
Figure BDA0002881944270000381
Figure BDA0002881944270000391
*ND表示在最低稀释度下未检测到中和抗体。
序列表
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Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 9
<211> 527
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(527)
<223> 18L1DE137-138/59dE
<400> 9
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asp Leu Tyr Lys Thr Cys Lys
130 135 140
Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Asn Val Ser
145 150 155 160
Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu
165 170 175
Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly
180 185 190
Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu
195 200 205
Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly
210 215 220
Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val
225 230 235 240
Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln
245 250 255
Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg
260 265 270
Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly
275 280 285
Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala
290 295 300
Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val
305 310 315 320
Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala
325 330 335
Gln Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr
340 345 350
Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr
355 360 365
Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr
370 375 380
Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys
385 390 395 400
Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn
405 410 415
Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr
420 425 430
Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr
435 440 445
Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg
485 490 495
Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr
500 505 510
Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 10
<211> 495
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(495)
<223> 18L1ΔCDE137-138/59dE
<400> 10
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asp Leu Tyr Lys Thr Cys Lys
130 135 140
Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Asn Val Ser
145 150 155 160
Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu
165 170 175
Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly
180 185 190
Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu
195 200 205
Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly
210 215 220
Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val
225 230 235 240
Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln
245 250 255
Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg
260 265 270
Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly
275 280 285
Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala
290 295 300
Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val
305 310 315 320
Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala
325 330 335
Gln Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr
340 345 350
Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr
355 360 365
Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr
370 375 380
Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys
385 390 395 400
Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn
405 410 415
Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr
420 425 430
Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr
435 440 445
Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 11
<211> 528
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(528)
<223> 18L1h4432-433/59dE
<400> 11
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala
500 505 510
Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 12
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(496)
<223> 18L1ΔCh4432-433/59dE
<400> 12
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 13
<211> 528
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(528)
<223> 18L1h4434-435/59dE
<400> 13
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Ala Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp
435 440 445
Val Ile Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala
500 505 510
Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 14
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(496)
<223> 18L1ΔCh4434-435/59dE
<400> 14
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Ala Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp
435 440 445
Val Ile Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 15
<211> 526
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1DE134-135/59dES
<400> 15
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala
130 135 140
Gly Thr Cys Pro Ser Asp Val Ile Pro Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 16
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCDE134-135/59dES
<400> 16
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala
130 135 140
Gly Thr Cys Pro Ser Asp Val Ile Pro Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 17
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(532)
<223> 18L1DE134-135/59dE
<400> 17
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Asp Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Pro Ala
145 150 155 160
Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr
165 170 175
Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu
180 185 190
His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly
195 200 205
Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp
210 215 220
Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp
225 230 235 240
Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr
245 250 255
Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe
260 265 270
Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg
275 280 285
Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly
290 295 300
Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro
305 310 315 320
Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr
325 330 335
Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His Asn
340 345 350
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr
355 360 365
Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr
370 375 380
Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe
385 390 395 400
Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr
405 410 415
Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val
420 425 430
Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln
435 440 445
Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys
450 455 460
Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys
465 470 475 480
Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val
485 490 495
Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser
500 505 510
Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val
515 520 525
Arg Ala Arg Lys
530
<210> 18
<211> 500
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(500)
<223> 18L1ΔCDE134-135/59dE
<400> 18
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Asp Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Pro Ala
145 150 155 160
Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr
165 170 175
Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu
180 185 190
His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly
195 200 205
Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp
210 215 220
Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp
225 230 235 240
Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr
245 250 255
Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe
260 265 270
Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg
275 280 285
Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly
290 295 300
Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro
305 310 315 320
Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr
325 330 335
Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His Asn
340 345 350
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr
355 360 365
Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr
370 375 380
Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe
385 390 395 400
Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr
405 410 415
Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val
420 425 430
Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln
435 440 445
Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys
450 455 460
Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys
465 470 475 480
Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val
485 490 495
Gln Ala Gly Leu
500
<210> 19
<211> 524
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(524)
<223> 18L1DE131-138/59dE
<400> 19
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys
130 135 140
Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Val Ser Glu Asp Val
145 150 155 160
Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu
165 170 175
Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys
180 185 190
Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys
195 200 205
Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala
210 215 220
Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp
225 230 235 240
Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala
245 250 255
Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu
260 265 270
Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val
275 280 285
Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly
290 295 300
Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp
305 310 315 320
Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His
325 330 335
Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp
340 345 350
Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro
355 360 365
Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His
370 375 380
Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr
385 390 395 400
Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile
405 410 415
Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu
420 425 430
Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys
435 440 445
Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe
450 455 460
Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr
465 470 475 480
Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro
485 490 495
Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser
500 505 510
Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520
<210> 20
<211> 492
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(492)
<400> 20
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys
130 135 140
Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Val Ser Glu Asp Val
145 150 155 160
Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu
165 170 175
Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys
180 185 190
Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys
195 200 205
Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala
210 215 220
Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp
225 230 235 240
Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala
245 250 255
Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu
260 265 270
Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val
275 280 285
Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly
290 295 300
Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp
305 310 315 320
Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His
325 330 335
Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp
340 345 350
Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro
355 360 365
Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His
370 375 380
Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr
385 390 395 400
Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile
405 410 415
Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu
420 425 430
Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys
435 440 445
Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe
450 455 460
Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr
465 470 475 480
Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 21
<211> 526
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1DE121-124/59dE
<400> 21
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Lys Thr Cys Lys Gln
115 120 125
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Lys
130 135 140
Leu Asp Asp Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 22
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCDE121-124/59dE
<400> 22
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Lys Thr Cys Lys Gln
115 120 125
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Lys
130 135 140
Leu Asp Asp Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 23
<211> 527
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(527)
<223> 18L1h4431-433/59dE
<400> 23
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Leu
420 425 430
Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn
435 440 445
Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg
485 490 495
Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr
500 505 510
Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 24
<211> 495
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(495)
<223> 18L1ΔCh4431-433/59dE
<400> 24
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Leu
420 425 430
Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn
435 440 445
Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 25
<211> 526
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1h4432-435/59dE
<400> 25
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 26
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCh4432-435/59dE
<400> 26
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 27
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut1
<400> 27
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 28
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut2
<400> 28
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 29
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut3
<400> 29
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Gly Val Asp Ala Gly Lys
515 520
<210> 30
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut4
<400> 30
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Asp Ser Val Gly Val Asp Ala Gly Lys
515 520
<210> 31
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut5
<400> 31
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Lys Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 32
<211> 523
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut6
<400> 32
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Lys Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 33
<211> 1477
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1477)
<223> 18L1ΔCDE137-138/59dES nt
<400> 33
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctctaat 1477
<210> 34
<211> 1489
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1489)
<223> 18L1ΔCDE137-138/59dE nt
<400> 34
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cgacctgtac 420
aagacctgca agcaggccgg tacctgcccc tccgacgtca tcaacaaggt caacgtcagc 480
gaagatgtcc gcgataacgt cagcgtggac tacaaacaaa cccaactgtg catcctcggt 540
tgcgcacccg ccatcggcga gcattgggcc aagggtaccg cctgcaagag caggcccctg 600
agccaaggtg actgtccacc cctggagttg aagaataccg tcctcgagga cggcgacatg 660
gtggacaccg gctacggcgc aatggatttc tccaccctgc aggacaccaa gtgcgaagtg 720
cccctcgaca tctgccaaag catctgcaag taccccgact acctgcagat gagcgccgac 780
ccctacggcg actccatgtt cttctgtctg agaagggaac aattgttcgc ccgccacttc 840
tggaaccgcg ccggcaccat gggcgatacc gtcccccagt ccctgtacat caagggtacc 900
ggcatgaggg ccagccccgg ttcatgcgtc tacagcccaa gcccctccgg tagcatcgtc 960
acaagcgatt cccaactctt caacaagccc tactggctgc acaaagccca aggccacaat 1020
aacggcgtct gttggcacaa ccagctgttc gtcaccgtcg tggacacaac caggtccaca 1080
aacctgacca tctgcgccag cacccaaagc cccgtgcccg gccagtacga cgccacaaag 1140
ttcaaacaat actcacgcca cgtcgaagag tacgacctcc aattcatctt ccaactctgc 1200
accatcaccc tgaccgccga cgtcatgtcc tacatccact ccatgaactc atccatcctg 1260
gaagactgga atttcggcgt cccaccaccc cccaccacct ccctcgtcga cacctacagg 1320
ttcgtgcaga gcgtcgccat cacatgccag aaagacgccg cccccgccga gaacaaagac 1380
ccatacgaca aactgaaatt ctggaacgtc gacctgaaag agaaattcag cctggatctg 1440
gaccagtacc cattgggcag gaagttcctc gtccaggcgg gtctctaat 1489
<210> 35
<211> 1486
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1486)
<223> 18L1ΔCDE134-135/59dES nt
<400> 35
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg caggaccact gtacaagacc 420
tgcaagcagg ccggtacctg cccctccgac gtcatcccag caaccagcaa cgtcagcgaa 480
gatgtccgcg ataacgtcag cgtggactac aaacaaaccc aactgtgcat cctcggttgc 540
gcacccgcca tcggcgagca ttgggccaag ggtaccgcct gcaagagcag gcccctgagc 600
caaggtgact gtccacccct ggagttgaag aataccgtcc tcgaggacgg cgacatggtg 660
gacaccggct acggcgcaat ggatttctcc accctgcagg acaccaagtg cgaagtgccc 720
ctcgacatct gccaaagcat ctgcaagtac cccgactacc tgcagatgag cgccgacccc 780
tacggcgact ccatgttctt ctgtctgaga agggaacaat tgttcgcccg ccacttctgg 840
aaccgcgccg gcaccatggg cgataccgtc ccccagtccc tgtacatcaa gggtaccggc 900
atgagggcca gccccggttc atgcgtctac agcccaagcc cctccggtag catcgtcaca 960
agcgattccc aactcttcaa caagccctac tggctgcaca aagcccaagg ccacaataac 1020
ggcgtctgtt ggcacaacca gctgttcgtc accgtcgtgg acacaaccag gtccacaaac 1080
ctgaccatct gcgccagcac ccaaagcccc gtgcccggcc agtacgacgc cacaaagttc 1140
aaacaatact cacgccacgt cgaagagtac gacctccaat tcatcttcca actctgcacc 1200
atcaccctga ccgccgacgt catgtcctac atccactcca tgaactcatc catcctggaa 1260
gactggaatt tcggcgtccc accacccccc accacctccc tcgtcgacac ctacaggttc 1320
gtgcagagcg tcgccatcac atgccagaaa gacgccgccc ccgccgagaa caaagaccca 1380
tacgacaaac tgaaattctg gaacgtcgac ctgaaagaga aattcagcct ggatctggac 1440
cagtacccat tgggcaggaa gttcctcgtc caggcgggtc tctaat 1486
<210> 36
<211> 1504
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1504)
<223> 18L1ΔCDE134-135/59dE nt
<400> 36
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg caggaccaga cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acaaggtcga aggaccagca 480
accagcaacg tcagcgaaga tgtccgcgat aacgtcagcg tggactacaa acaaacccaa 540
ctgtgcatcc tcggttgcgc acccgccatc ggcgagcatt gggccaaggg taccgcctgc 600
aagagcaggc ccctgagcca aggtgactgt ccacccctgg agttgaagaa taccgtcctc 660
gaggacggcg acatggtgga caccggctac ggcgcaatgg atttctccac cctgcaggac 720
accaagtgcg aagtgcccct cgacatctgc caaagcatct gcaagtaccc cgactacctg 780
cagatgagcg ccgaccccta cggcgactcc atgttcttct gtctgagaag ggaacaattg 840
ttcgcccgcc acttctggaa ccgcgccggc accatgggcg ataccgtccc ccagtccctg 900
tacatcaagg gtaccggcat gagggccagc cccggttcat gcgtctacag cccaagcccc 960
tccggtagca tcgtcacaag cgattcccaa ctcttcaaca agccctactg gctgcacaaa 1020
gcccaaggcc acaataacgg cgtctgttgg cacaaccagc tgttcgtcac cgtcgtggac 1080
acaaccaggt ccacaaacct gaccatctgc gccagcaccc aaagccccgt gcccggccag 1140
tacgacgcca caaagttcaa acaatactca cgccacgtcg aagagtacga cctccaattc 1200
atcttccaac tctgcaccat caccctgacc gccgacgtca tgtcctacat ccactccatg 1260
aactcatcca tcctggaaga ctggaatttc ggtgtcccac caccccccac cacctccctc 1320
gtcgacacct acaggttcgt gcagagcgtc gccatcacat gccagaaaga cgccgccccc 1380
gccgagaaca aagacccata cgacaaactg aaattctgga acgtcgacct gaaagagaaa 1440
ttcagcctgg atctggacca gtacccattg ggcaggaagt tcctcgtcca ggcgggtctc 1500
taat 1504
<210> 37
<211> 1480
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1480)
<223> 18L1ΔCDE131-138/59dE nt
<400> 37
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tccggtcccc tgtacaagac ctgcaagcag 420
gccggtacct gcccctccga cgtcatcaac aaggtcgaag gaaacgtcag cgaagatgtc 480
cgcgataacg tcagcgtgga ctacaaacaa acccaactgt gcatcctcgg ttgcgcaccc 540
gccatcggcg agcattgggc caagggtacc gcctgcaaga gcaggcccct gagccaaggt 600
gactgtccac ccctggagtt gaagaatacc gtcctcgagg acggcgacat ggtggacacc 660
ggctacggcg caatggattt ctccaccctg caggacacca agtgcgaagt gcccctcgac 720
atctgccaaa gcatctgcaa gtaccccgac tacctgcaga tgagcgccga cccctacggc 780
gactccatgt tcttctgtct gagaagggaa caattgttcg cccgccactt ctggaaccgc 840
gccggcacca tgggcgatac cgtcccccag tccctgtaca tcaagggtac cggcatgagg 900
gccagccccg gttcatgcgt ctacagccca agcccctccg gtagcatcgt cacaagcgat 960
tcccaactct tcaacaagcc ctactggctg cacaaagccc aaggccacaa taacggcgtc 1020
tgttggcaca accagctgtt cgtcaccgtc gtggacacaa ccaggtccac aaacctgacc 1080
atctgcgcca gcacccaaag ccccgtgccc ggccagtacg acgccacaaa gttcaaacaa 1140
tactcacgcc acgtcgaaga gtacgacctc caattcatct tccaactctg caccatcacc 1200
ctgaccgccg acgtcatgtc ctacatccac tccatgaact catccatcct ggaagactgg 1260
aatttcggcg tcccaccacc ccccaccacc tccctcgtcg acacctacag gttcgtgcag 1320
agcgtcgcca tcacatgcca gaaagacgcc gcccccgccg agaacaaaga cccatacgac 1380
aaactgaaat tctggaacgt cgacctgaaa gagaaattca gcctggatct ggaccagtac 1440
ccattgggca ggaagttcct cgtccaggcg ggtctctaat 1480
<210> 38
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut1 nt
<400> 38
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 39
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut2 nt
<400> 39
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 40
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut3 nt
<400> 40
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gggcgtggac 1560
gctggcaaat aat 1573
<210> 41
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut4 nt
<400> 41
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccgacagcgt gggcgtggac 1560
gctggcaaat aat 1573
<210> 42
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut5 nt
<400> 42
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggca aaccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 43
<211> 1573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> 外显子
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut6 nt
<400> 43
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggca aaccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 44
<211> 23
<212> PRT
<213> HPV 35
<400> 44
Thr Gln Leu Tyr Arg Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Pro Lys Val Glu Gly
20
<210> 45
<211> 23
<212> PRT
<213> HPV 39
<400> 45
Ser Thr Leu Tyr Arg Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Val Asp Lys Val Glu Gly
20
<210> 46
<211> 23
<212> PRT
<213> HPV 51
<400> 46
Thr Gln Leu Tyr Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Val Asn Lys Val Glu Gly
20
<210> 47
<211> 23
<212> PRT
<213> HPV 53
<400> 47
Thr Gln Leu Tyr Gln Thr Cys Lys Gln Ser Gly Thr Cys Pro Glu Asp
1 5 10 15
Val Ile Asn Lys Ile Glu His
20
<210> 48
<211> 23
<212> PRT
<213> HPV 56
<400> 48
Thr Gln Leu Tyr Lys Thr Cys Lys Leu Ser Gly Thr Cys Pro Glu Asp
1 5 10 15
Val Val Asn Lys Ile Glu Gln
20
<210> 49
<211> 23
<212> PRT
<213> HPV 59
<400> 49
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
1 5 10 15
Asn Lys Val Glu Gly Thr Thr
20
<210> 50
<211> 23
<212> PRT
<213> HPV 68
<400> 50
Ser Thr Leu Tyr Lys Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Asn Lys Val Glu Gly
20
<210> 51
<211> 23
<212> PRT
<213> HPV 82
<400> 51
Thr Gln Leu Tyr Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Pro Lys Val Lys Gly
20

Claims (10)

1.一种人乳头瘤病毒嵌合蛋白，其包含HPV18型L1蛋白或HPV18型L1蛋白的突变体以及插入所述HPV18型L1蛋白或HPV18型L1蛋白的突变体的表面区的来自HPV59型L2蛋白的多肽、或由其组成，其中所述HPV18型L1蛋白的氨基酸序列如SEQ ID NO.1所示，所述HPV59型L2蛋白的氨基酸序列如SEQ ID NO.2所示，优选地，所述HPV18型L1蛋白的突变体选自以下任一种：
将SEQ ID No.1所示的氨基酸序列的C端截短32个氨基酸的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、504、506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、499、504、506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、484、496、499、502、506置换为甘氨酸(G)、将氨基酸485、500置换为丝氨酸(S)且将氨基酸504置换为天冬氨酸(D)的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、478、485、496、502、506置换为甘氨酸(G)、将氨基酸486、500置换为丝氨酸(S)且将氨基酸499、504置换为天冬氨酸(D)的突变体；
将SEQ ID No.1所示氨基酸序列的氨基酸477、484、496、499、504、506置换为甘氨酸(G)且将氨基酸485、500、502置换为丝氨酸(S)的突变体；和
将SEQ ID No.1所示氨基酸序列的氨基酸477、485、496、499、504、506置换为甘氨酸(G)且将氨基酸486、500、502置换为丝氨酸(S)的突变体；
更优选地，所述来自HPV59型L2蛋白的多肽选自SEQ ID No.3、SEQ ID No.4、SEQ IDNo.5或SEQ ID No.6中任一项所示的多肽；
进一步优选地，
所述来自HPV59型L2蛋白的多肽插入所述HPV18型L1蛋白或HPV18型L1蛋白的突变体的表面区，所述表面区为DE环和/或h4区，优选地，所述来自HPV59型L2蛋白的多肽通过直接插入的方式插入所述HPV18型L1蛋白或HPV18型L1蛋白的突变体的氨基酸137和138之间、氨基酸432和433之间、或氨基酸434和435之间，或者，所述来自HPV59型L2蛋白的多肽通过非等长置换的方式插入所述HPV18型L1蛋白或HPV18型L1蛋白的突变体的氨基酸121至124区域、或氨基酸131至138区域、或氨基酸431-433区域、或氨基酸432-435区域；
更优选地，
其中所述来自HPV59型L2蛋白的多肽在其N端和/或C端包含1至3个氨基酸残基的连接子，所述连接子包含一种或多种选自以下的氨基酸：甘氨酸、丝氨酸、丙氨酸和脯氨酸，优选地，在N端的连接子由甘氨酸-脯氨酸组成，并且在C端的连接子由脯氨酸组成。
2.根据权利要求1所述的人乳头瘤病毒嵌合蛋白，其中所述人乳头瘤病毒嵌合蛋白的氨基酸序列如SEQ ID NO：7-32中的任一项所示。
3.一种多核苷酸，其编码权利要求1或2所述的人乳头瘤病毒嵌合蛋白，优选地，所述多核苷酸的序列采用大肠杆菌密码子进行全基因优化或采用昆虫细胞密码子进行全基因优化，更优选地，所述多核苷酸的序列如SEQ ID No.33、SEQ ID No.34、SEQ ID No.35、SEQ IDNo.36、SEQ ID No.37、SEQ ID No.38、SEQ ID No.39、SEQ ID No.40、SEQ ID No.41、SEQ IDNo.42和SEQ ID No.43中任一项所示。
4.一种载体，其包含如权利要求3所述的多核苷酸。
5.一种细胞，其包含如权利要求4所述的载体。
6.一种多聚物，该多聚物为嵌合五聚体或嵌合病毒样颗粒，其含有权利要求1或2所述的人乳头瘤病毒嵌合蛋白，或者由权利要求1或2所述的人乳头瘤病毒嵌合蛋白所形成。
7.如权利要求1或2所述的人乳头瘤病毒嵌合蛋白或如权利要求6所述的多聚物在制备预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗中的用途，优选地，乳头瘤病毒感染诱发的疾病选自宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌和口腔癌；
优选地，所述乳头瘤病毒感染为一种或多种选自以下乳头瘤病毒型别的感染：HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV70、HPV73；HPV6、HPV11、HPV2、HPV5、HPV27和HPV57。
8.一种用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，其包含权利要求1或2所述的人乳头瘤病毒嵌合蛋白或如权利要求6所述的多聚物、佐剂、以及疫苗用赋形剂或载体。
9.根据权利要求8所述的用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。
10.根据权利要求8或9所述的用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，其中所述佐剂为人用佐剂。
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