CN114716562B - 一种人乳头瘤病毒58型嵌合蛋白及其用途 - Google Patents

一种人乳头瘤病毒58型嵌合蛋白及其用途 Download PDF

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CN114716562B
CN114716562B CN202110003706.3A CN202110003706A CN114716562B CN 114716562 B CN114716562 B CN 114716562B CN 202110003706 A CN202110003706 A CN 202110003706A CN 114716562 B CN114716562 B CN 114716562B
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许雪梅
王志荣
马铭饶
张婷
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Abstract

本发明涉及一种人乳头瘤病毒58型嵌合蛋白及其用途。具体地，本发明涉及一种人乳头瘤病毒嵌合蛋白，其包含HPV58型L1蛋白或HPV58型L1蛋白的突变体以及插入所述HPV58型L1蛋白或HPV58型L1蛋白的突变体的表面区的来自HPV16型L2蛋白的多肽、或由其组成，其中所述HPV58型L1蛋白的氨基酸序列如SEQ ID NO.1所示，所述HPV16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58型嵌合蛋白及其用途
技术领域
本发明涉及生物技术领域。具体地，本发明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，以及所述人乳头瘤病毒嵌合蛋白、由所述人乳头瘤病毒嵌合蛋白形成的五聚体或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，根据主要外壳蛋白L1氨基酸的同源性，已鉴定了200多型，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，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-18在世界范围内宫颈癌中的检出率分别达50-60％及～20％，HPV58及-52在我国南方宫颈癌中检出率仅次于HPV16或HPV16/-18。在亚洲，高危型HPV58的总体检出率仅次于HPV16和HPV18型，在宫颈癌、高度宫颈内膜瘤变及低度宫颈内膜瘤变标本中的检出率较高，分别为7.3％、15.5％、10.8％，均居于第三位；在中美及南美洲，HPV58在高度宫颈上皮瘤变(cervicalintraepithelial neoplasia III，CIN3)标本中的检出率分别高达11.6％和11.0％，居于第二位，在墨西哥，HPV58在宫颈癌前病变标本中的检出率甚至大于或等于HPV16。因此，在这些经济欠发达地区HPV58的感染流行率较高，其感染相关疾病造成的公共卫生负荷及经济负担也相对较重。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,L1VLP)主要诱发型别特异性中和抗体和保护反应，由L1病毒样颗粒组成的疫苗只能通过增加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L1VLP疫苗不能通过无限制的增加L1VLP的型别来扩大保护范围，因此L1VLP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 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。
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，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。但研究结果表明，多种RG-1表位肽相关疫苗的活性结果较差，如表面展示HPV 4型RG-1、HPV 6型RG-1、HPV 17型RG-1的3种16cVLP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；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18L1VLP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 et al.,PLoS One2015,10(3):e0120152]；表面展示HPV51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。
另一方面，Schellenbacher报道的16RG1-cVLP及我们课题组报道的31RG1-cVLP、33RG1-cVLP及58RG1-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16L1 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.Schellenbacheret al.,The Journal ofinvestigative dermatology 2013,133(12):2706-13；X.Chen et al.,Oncotarget 2017,8(38):63333-63344；X.Chen et al.,Human Vaccines&Immunotherapeutics 2018,14(8):2025-2033；PCT/CN2017/075402]。
上述数据提示，不同型别的RG-1表位肽的免疫原性存在差异。文献首次比较了58RG-1及6RG-1的免疫原性，发现58RG-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6RG-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。
值得注意的是，Schellenbacher和Wang的报道的HPV16 cVLP疫苗研究显示，同是将16RG-1表位肽插入16L1VLP载体的表面区，由于16RG-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16RG1-cVLP的免疫活性具有显著差异，其中最好的是在16L1的DEloop区插入16RG-1的cVLP，最差的是在16L1的h4区插入16RG-1核心序列的cVLP。另外，Chen和Boxus均报道了33RG-1的cVLP，但采用的载体不同，分别是HPV16L1VLP及18L1VLP，虽然两篇报道均选择了DE loop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，但获得的两种33RG1-cVLP诱发产生的33RG-1依赖的交叉中和抗体的活性差异十分显著，33RG1-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33RG1-18cVLP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。因此，上述数据显示，即使选用免疫原性较强的RG-1表位，但因载体不同、插入位点不同、旁侧序列不同、插入方式不同，构建获得的cVLP，其免疫活性及表达量均不相同。因此，现有的研究数据显示，RG-1多肽的型别及其长度(表位旁侧序列的差异)、L1VLP载体型别及其插入位点和插入方式(直接插入、置换插入及插入位点区引入氨基酸如连接子)，对形成的RG1-L1嵌合蛋白的表达水平、组装能力及免疫活性均具有影响，而且这种影响是不可预测的。
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HPV58 L1与HPV L2嵌合蛋白的疫苗，其即可保持或增强HPV58 L1的中和表位，又能够提供针对更多HPV型别的交叉保护。
发明内容
本发明选用了多种不同长度16RG-1表位肽，用于HPV58型嵌合五聚体或cVLP的研究，结果显示，本发明获得的HPV58嵌合五聚体或cVLP的免疫原性很强，诱发的针对载体型别HPV58的中和抗体水平与58L1VLP的相当，并可诱发针对来自不同属/亚属的多种型别HPV的广谱中和抗体。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。本发明人出人意料地发现，在野生型HPV58型L1蛋白或其突变体的表面区插入源自HPV16型L2蛋白的多肽，可提高HPV16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病毒样颗粒(VLP)或嵌合五聚体，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。
基于上述目的，本发明提供了一种人乳头瘤病毒嵌合蛋白，其骨架是HPV58型L1蛋白或HPV58型L1蛋白的突变体，所述的骨架上嵌合至少一个源自HPV16型L2蛋白的多肽。
即，在第一方面中，本发明提供了一种人乳头瘤病毒嵌合蛋白，其包含HPV58型L1蛋白或HPV58型L1蛋白的突变体以及插入所述HPV58型L1蛋白或HPV58型L1蛋白的突变体的表面区的来自HPV16型L2蛋白的多肽、或由其组成，其中所述HPV58型L1蛋白的氨基酸序列如SEQ ID NO.1所示，所述HPV16型L2蛋白的氨基酸序列如SEQ ID NO.2所示。
在本发明所述的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述的来自HPV16型L2蛋白的多肽选自SEQ ID No.2所示的HPV16型L2蛋白的aa.1-50区域内的任意连续8-33个氨基酸的片段。进一步优选地，所述的来自HPV16型L2蛋白的多肽为HPV16型L2蛋白RG-1表位肽或其突变体表位肽。
在本发明所述的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述的来自HPV16型L2蛋白的多肽的氨基酸序列如SEQ ID No.3、SEQ ID No.4、SEQ IDNo.5或SEQ ID No.6所示。
在进一步优选的实施方案中，所述的来自HPV16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。
在进一步优选的实施方案中，所述的来自HPV16型L2蛋白的多肽还可以是与SEQID No.3所示的氨基酸序列具有大于60％、优选地大于70％、大于80％、大于90％、甚至更优选大于95％序列同一性的多肽。
在本发明优选的实施方案中，本发明涉及的嵌合蛋白骨架选自HPV58型L1蛋白(例如NCBI数据库中的CAX48979.1所示序列，和SEQ ID No.1一致)或HPV58型L1蛋白突变体。HPV58型L1蛋白骨架可来自但不限于NCBI数据库中的AFS33402.1、ADK78323.1、AMY16498.1、ACJ13512.1、ADK78590.1、ADK78685.1等来自HPV58变异株的L1蛋白。优选地，所述的HPV58型L1蛋白的氨基酸序列如SEQ ID No.1所示。
在本发明所述的人乳头瘤病毒嵌合蛋白的优选的实施方案中，本发明所述的HPV58型L1蛋白的突变体与SEQ ID No.1所示的HPV58型L1蛋白相比，包含删除突变、C端截短突变和置换突变中的任何一种或多种，其中：
所述删除突变为删除N端的第2-4位氨基酸；
所述C端截短突变为C端截短25个氨基酸；
所述置换突变选自以下i)至iii)中任何一组：
i)476G、481G、492G、493G、497G、478S、487S、494S、498S、480A和495A；
ii)474G、476G、481G、492G、493G、497G、478S、487S、494S、498S、480A和495A；和
iii)476G、481G、492G、493G、497G、478S、494S、498S、480A和495A。
在本文所使用的置换突变的表示中，中间的数字代表与对照序列相比(例如，SEQID No.1所示的氨基酸序列)的氨基酸位置，数字前面的字母(如果有)代表突变前的氨基酸残基，数字后的字母代表突变后的氨基酸残基。
可选地，所述的HPV58型L1蛋白的突变体是在所述的HPV58型L1蛋白的N端截短0-8个氨基酸和/或C端截短0-25个氨基酸的所获得的蛋白。
可选地，所述的HPV58型L1蛋白的突变体是将所述的HPV58型L1蛋白氨基酸序列的删除N端第2-4位氨基酸和/或C端截短25个氨基酸的突变体。
可选地，所述的HPV58型L1蛋白的突变体是将所述的HPV58型L1蛋白氨基酸序列的删除N端第2-4位氨基酸，和将所述的HPV58型L1蛋白的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS1)。
可选地，所述的HPV58型L1蛋白的突变体是将所述的HPV58型L1蛋白氨基酸序列的删除N端第2-4位氨基酸，和将所述的HPV58型L1蛋白的氨基酸474、476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS2)。
可选地，所述的HPV58型L1蛋白的突变体是将所述的HPV58型L1蛋白氨基酸序列的删除N端第2-4位氨基酸，和将所述的HPV58型L1蛋白的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS3)。
可选地，所述的来自HPV16型L2蛋白的多肽插入所述的HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的表面区，优选插入所述的HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的DE环，更优选通过直接插入的方式插入所述的HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的氨基酸136和氨基酸137之间、或氨基酸431和432之间，或者通过非等长置换的方式插入所述的HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的氨基酸429至432区域、或氨基酸426至429区域、或氨基酸412-426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136和氨基酸137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136和氨基酸137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429至432区域的非等长置换指的是，删除SEQ ID NO.1的氨基酸430-431之后，将所选择的肽片段插入到SEQ IDNO.1的氨基酸氨基酸429至432之间。
可选地，在所述直接插入或非等长置换的方式中，所述源自HPV16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端的连接子由G(甘氨酸)P(脯氨酸)组成，C端的连接子由P(脯氨酸)组成。
可选地，在所述直接插入的方式中，所述来自HPV16型L2蛋白的多肽的氨基酸序列是SEQ ID No.6，插入位点为所述的HPV58型L1蛋白或C端截短25个氨基酸的所述HPV58型L1蛋白的突变体的氨基酸136和氨基酸137之间，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.7或SEQ ID No.8所示。
可选地，在所述非等长置换的插入方式中，所述来自HPV16型L2蛋白的多肽的氨基酸序列是SEQ ID No.6，插入位点为所述的HPV58型L1蛋白或C端截短25个氨基酸的HPV58型L1蛋白突变体的氨基酸429-432区域，删除所述HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的氨基酸430-431区域后，在氨基酸429及432之间插入SEQ ID No.6所示多肽，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.9或SEQ ID No.10所示。
可选地，在所述非等长置换的插入方式中，所述来自HPV16型L2蛋白的多肽的氨基酸序列是SEQ ID No.4或SEQ ID No.5所示，插入位点为所述HPV58型L1蛋白的N端截短突变体的氨基酸426-429区域，删除氨基酸427-428区域后，在氨基酸426及429之间插入来自HPV16型L2蛋白的多肽，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、SEQ IDNo.14、SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
可选地，在所述非等长置换的插入方式中，所述来自HPV16型L2蛋白的多肽的氨基酸序列是SEQ ID No.3所示，插入位点为所述HPV58型L1蛋白或所述HPV58型L1蛋白的突变体的氨基酸412-426区域，删除氨基酸413-425区域后，在氨基酸412及426之间插入来自HPV16型L2蛋白的多肽，获得的乳头瘤病毒嵌合蛋白氨基酸序列如SEQ ID No.18或SEQ IDNo.19所示。
可选地，所述来自HPV16型L2蛋白的多肽通过直接插入或非等长置换插入的方式插入所述HPV58型L1蛋白突变体的表面区，所述HPV58型L1蛋白突变体选自：
将SEQ ID No.1所示的氨基酸序列的删除N端第2-4位氨基酸和/或C端截短25个氨基酸的突变体；或者
将SEQ ID No.1所示氨基酸序列的删除N端第2-4位氨基酸，和将SEQ IDNo.1所示序列的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS1)；或者，
将SEQ ID No.1所示氨基酸序列的删除N端第2-4位氨基酸，和将SEQ IDNo.1所示序列的氨基酸474、476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS2)；或者，
将SEQ ID No.1所示氨基酸序列的删除N端第2-4位氨基酸，和将SEQ IDNo.1所示序列的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体(CS3)。
本发明的另一方面涉及编码上述的人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人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，所述多聚物为本发明的人乳头瘤病毒嵌合蛋白形成的五聚体或嵌合病毒样颗粒，其中所述多聚物包含本发明所述的人乳头瘤病毒嵌合蛋白，或者由本发明所述的人乳头瘤病毒嵌合蛋白形成。
本发明还提供了本发明所述的人乳头瘤病毒嵌合蛋白、人乳头瘤病毒嵌合蛋白形成的五聚体或病毒样颗粒在制备预防人乳头瘤病毒感染或感染诱发的疾病的疫苗中的用途。
本发明涉及的人乳头瘤病毒感染诱发的疾病，包括但不限于：宫颈上皮内瘤变、宫颈癌、阴唇癌、阴茎癌、阴道癌、肛门肛周癌、口咽癌、肛周生殖器尖锐湿疣、呼吸道复发性乳头瘤、皮肤疣状增生、皮肤鳞状细胞癌及基底细胞癌。在一些实施方案中，所述人乳头瘤病毒感染和选自以下的病毒有关：致癌型HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV70、HPV73；低危型HPV6、HPV11；以及皮肤型HPV2、HPV5、HPV27、HPV57。
本发明还提供了还提供了一种用于预防人乳头瘤病毒感染或感染诱发的疾病的疫苗，其包含：
1)本发明的人乳头瘤病毒嵌合蛋白、和/或本发明的人乳头瘤病毒嵌合蛋白形成的五聚体或病毒样颗粒；
2)任选地，佐剂；
3)任选地，疫苗用赋形剂或载体；
4)优选地，还包含至少一种嗜黏膜组的HPV和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。
在一些实施方案中，上述病毒样颗粒在所述疫苗中的含量分别为能诱发保护性免疫反应的有效量。
在一些实施方案中，所述佐剂为人用佐剂。优选，所述佐剂包括但不限于：铝佐剂；水包油乳剂或油包水乳剂及TLR刺激剂的佐剂组合物；氢氧化铝佐剂或磷酸铝佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物；或者MF59佐剂与聚肌苷酸-聚胞苷酸佐剂及稳定剂的组合物。
在一些实施方案中，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。
在一些实施方案中，本发明疫苗优选制备成单位剂型的形式，其中单位剂型中人乳头瘤病毒嵌合蛋白或蛋白病毒样颗粒的剂量为5μg至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至60μg每单位剂型。
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“野生型HPV58型L1蛋白”的例子包括但不限于NCBI数据库中编号为CAX48979.1的蛋白。
“HPV58型L1蛋白突变体”的基因片段指的是其与编码野生型HPV 58型L1蛋白的基因相比，在其5’端和/或3’端缺失编码1个或多个氨基酸残基的核苷酸，和/或其序列中一个或多个位点存在导致氨基酸突变的核苷酸突变，其中“野生型HPV58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：AFS33402.1、ADK78323.1、AMY16498.1、ACJ13512.1、ADK78590.1、ADK78685.1等。
根据本发明，术语“疫苗用赋形剂或载体”是指选自以下的一种或多种，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，术语“乳剂”是指由水相成分、油相成分及乳化剂按适当比例混合，经乳化后形成的非均相液体分散体系。其中水相成分包括但不限于磷酸盐缓冲液、HEPES缓冲液等缓冲系统；油相成分为可代谢脂类，包括但不限于植物油、鱼油、动物油、合成油及其他脂类成分(例如但不限于角鲨烯、生育酚)。乳化剂为适宜的表面活性剂，例如但不限于山梨醇酐三油酸酯(Span-85)、聚山梨酯80(Tween-80)。
根据本发明，术语“稳定剂”是指可与佐剂中的聚肌苷酸-聚胞苷酸结合并起到稳定作用的成分，包括但不限于抗生素(例如但不限于卡那霉素、新霉素、庆大霉素)、无机盐(例如但不限于氯化钙、氯化镁、磷酸钙)、阳离子的有机复合物(例如但不限于硬脂酸钙、葡萄糖酸钙)。
附图说明
图1A-图1B：本发明实施例5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，嵌合蛋白均可在大肠杆菌或昆虫细胞中高水平表达。
图1A：嵌合蛋白在大肠杆菌中的表达鉴定，1至5分别表示58L1DE/16dEs，58L1h4/16dEs，58L1ΔN4h4/16dE，58L1ΔN4h4/16dEs，58L1h4/16dE。
图1B：嵌合蛋白在昆虫细胞中的表达鉴定，1至8分别表示58L1ΔCDE/16dEs，58L1ΔCh4/16dEs，58L1ΔN4Ch4/16dE，58L1ΔN4Ch4/16dEs，58L1ΔCh4/16dE，58L1ΔN4h4/16dE-CS1，58L1ΔN4h4/16dE-CS2，58L1ΔN4h4/16dE-CS3。
图2A-图2E：本发明实施例6中纯化后获得的嵌合籽粒或cVLP的动态光散射分析结果。结果显示58L1ΔCDE/16dEs、58L1ΔCh4/16dEs、58L1ΔN4Ch4/16dE及58L1ΔN4Ch4/16dEs重组蛋白形成的病毒样颗粒水化动力学直径分别为90nm、114.6nm、83.5nm和93nm，颗粒组装的百分比均为100％；58L1ΔCh4/16dE重组蛋白形成的颗粒水化动力学直径为12.28nm。
图2A：58L1ΔCDE/16dEs获得的cVLP的动态光散射分析结果；
图2B：58L1ΔCh4/16dEs获得的cVLP的动态光散射分析结果；
图2C：58L1ΔN4Ch4/16dE获得的cVLP的动态光散射分析结果；
图2D：58L1ΔN4Ch4/16dEs获得的cVLP的动态光散射分析结果；
图2E：58L1ΔCh4/16dE获得的嵌合五聚体的动态光散射分析结果。
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。Bar＝100nm。
图3A：58L1ΔCDE/16dEs获得的cVLP的透射电镜观察结果；
图3B：58L1ΔCh4/16dEs获得的cVLP的透射电镜观察结果；
图3C：58L1ΔN4Ch4/16dE获得的cVLP的透射电镜观察结果；
图3D：58L1ΔN4Ch4/16dEs获得的cVLP的透射电镜观察结果。
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：嵌合蛋白的基因的合成及表达载体构建
13种嵌合蛋白，分别为：
1)嵌合蛋白58L1DE/16dEs：骨架为全长HPV58型L1蛋白(序列如SEQ IDNo.1所示)，在aa.136/137之间融合HPV16型L2蛋白的aa.19-31多肽(氨基酸序列如SEQ ID No.6所示)，嵌合蛋白58L1DE/16dEs的氨基酸序列如SEQ ID No.7所示。编码58L1DE/16dEs的多核苷酸序列经大肠杆菌密码子优化设计，其序列如SEQ ID No.20所示；
2)嵌合蛋白58L1h4/16dEs：骨架为全长HPV58型L1蛋白(序列如SEQ IDNo.1所示)，删除其aa.430-431区域，并在aa.429/432之间融合HPV16型L2蛋白的aa.19-31多肽(氨基酸序列如SEQ ID No.6所示)，嵌合蛋白58L1h4/16dEs的氨基酸序列如SEQ ID No.9所示。编码58L1h4/16dEs的多核苷酸序列经大肠杆菌密码子优化设计，其序列如SEQ ID No.22所示；
3)嵌合蛋白58L1ΔN4h4/16dE：骨架为删除N端第2-4位氨基酸的HPV58型L1蛋白(序列为SEQ ID No.1的N端删除第2-4个氨基酸)，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-38多肽(氨基酸序列如SEQ ID No.4所示)，嵌合蛋白58L1ΔN4h4/16dE的氨基酸序列如SEQ ID No.11所示。编码58L1ΔN4h4/16dE的多核苷酸序列经大肠杆菌密码子优化设计，其序列如SEQ ID No.24所示；
4)嵌合蛋白58L1ΔN4h4/16dEs：骨架为删除N端第2-4位氨基酸的HPV58型L1蛋白(序列为SEQ ID No.1的N端删除第2-4个氨基酸)，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-32多肽(氨基酸序列如SEQ ID No.5所示)，嵌合蛋白58L1ΔN4h4/16dEs的氨基酸序列如SEQ ID No.13所示。编码58L1ΔN4h4/16dEs的多核苷酸序列经大肠杆菌密码子优化设计，其序列如SEQ ID No.26所示；
5)嵌合蛋白58L1h4/16dE：骨架为全长HPV58型L1蛋白(序列如SEQ ID No.1所示)，删除其aa.413-425区域，并在aa.412/426之间融合HPV16型L2蛋白的aa.17-38多肽(氨基酸序列如SEQ ID No.3所示)，嵌合蛋白58L1h4/16dE的氨基酸序列如SEQ ID No.18所示。编码58L1h4/16dEs的多核苷酸序列经大肠杆菌密码子优化设计，其序列如SEQ ID No.31所示；
6)嵌合蛋白58L1ΔCDE/16dEs：骨架为C端截短25个氨基酸的HPV58型L1蛋白(SEQID No.1的C端截短25个氨基酸)，在aa.136/137之间融合HPV16型L2蛋白的aa.19-31多肽(氨基酸序列如SEQ ID No.6所示)，嵌合蛋白58L1ΔCDE/16dEs的氨基酸序列如SEQ IDNo.8所示。编码58L1ΔCDE/16dEs的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.21所示；
7)嵌合蛋白58L1ΔCh4/16dEs：骨架为C端截短25个氨基酸的HPV58型L1蛋白(SEQID No.1的C端截短25个氨基酸)，删除其aa.430-431区域，并在aa.429/432之间融合HPV16型L2蛋白的aa.19-31多肽(氨基酸序列如SEQ ID No.6所示)，嵌合蛋白58L1ΔCh4/16dEs的氨基酸序列如SEQ ID No.10所示。编码58L1ΔCh4/16dEs的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.23所示；
8)嵌合蛋白58L1ΔN4Ch4/16dE：骨架为删除N端第2-4位氨基酸、C端截短25个氨基酸的HPV58型L1蛋白(序列为SEQ ID No.1的N端删除第2-4个氨基酸，C端删除25个氨基酸)，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-38多肽(氨基酸序列如SEQ ID No.4所示)，58L1ΔN4Ch4/16dE的氨基酸序列如SEQ ID No.12所示。编码58L1ΔN4Ch4/16dE的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ IDNo.25所示；
9)嵌合蛋白58L1ΔN4Ch4/16dEs：骨架为删除N端第2-4位氨基酸、C端截短25个氨基酸的HPV58型L1蛋白(序列为SEQ ID No.1的N端删除第2-4个氨基酸，C端删除25个氨基酸)，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-32多肽(氨基酸序列如SEQ ID No.5所示)，嵌合蛋白58L1ΔN4Ch4/16dEs的氨基酸序列如SEQ IDNo.14所示。编码58L1ΔN4Ch4/16dEs的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.27所示；
10)嵌合蛋白58L1ΔN4h4/16dE-CS1：骨架为删除SEQ ID No.1所示氨基酸序列的N端第2-4位氨基酸，且将SEQ ID No.1所示序列的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-38多肽(氨基酸序列如SEQ IDNo.4所示)，58L1ΔN4h4/16dE-CS1的氨基酸序列如SEQ IDNo.15所示。编码58L1ΔN4h4/16dE-CS1的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.28所示；
11)嵌合蛋白58L1ΔN4h4/16dE-CS2：骨架为删除SEQ ID No.1所示氨基酸序列的N端第2-4位氨基酸，且将SEQ ID No.1所示序列的氨基酸474、476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、487、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-38多肽(氨基酸序列如SEQ ID No.4所示)，58L1ΔN4h4/16dE-CS2的氨基酸序列如SEQ ID No.16所示。编码58L1ΔN4h4/16dE-CS2的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.29所示；
12)嵌合蛋白58L1ΔN4h4/16dE-CS3：骨架为删除SEQ ID No.1所示氨基酸序列的N端第2-4位氨基酸，且将SEQ ID No.1所示序列的氨基酸476、481、492、493、497置换为甘氨酸(G)、将氨基酸478、494、498置换为丝氨酸(S)、且将氨基酸480和495置换为丙氨酸(A)的突变体，删除其aa.427-428区域，并在aa.426/429之间融合HPV16型L2蛋白的aa.18-38多肽(氨基酸序列如SEQ ID No.4所示)，58L1ΔN4h4/16dE-CS3的氨基酸序列如SEQ ID No.17所示。编码58L1ΔN4h4/16dE-CS3的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.30所示；
13)嵌合蛋白58L1ΔCh4/16dE：骨架为C端截短25个氨基酸的HPV58型L1蛋白(SEQID No.1的C端截短25个氨基酸)，删除其aa.413-425区域，并在aa.412/426之间融合HPV16型L2蛋白的aa.17-38多肽(氨基酸序列如SEQ ID No.3所示)，嵌合蛋白58L1ΔCh4/16dE的氨基酸序列如SEQ ID No.19所示。编码58L1ΔCh4/16dE的多核苷酸序列经昆虫细胞Sf9密码子优化设计，其序列如SEQ ID No.32所示。
依据大肠杆菌密码子及依据昆虫细胞密码子优化的嵌合L1基因，采用全基因合成的方式，由上海生工生物工程技术服务有限公司合成。
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EcoRI/Xba I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，分别为：
pET22b-58L1DE/16dEs；
pET22b-58L1h4/16dEs；
pET22b-58L1ΔN4h4/16dE；
pET22b-58L1ΔN4h4/16dEs；
pET22b-58L1h4/16dE；
pFastBac1-58L1ΔCDE/16dEs；
pFastBac1-58L1ΔCh4/16dEs；
pFastBac1-58L1ΔN4Ch4/16dE；
pFastBac1-58L1ΔN4Ch4/16dEs；
pFastBac1-58L1ΔN4h4/16dE-CS1；
pFastBac1-58L1ΔN4h4/16dE-CS2；
pFastBac1-58L1ΔN4h4/16dE-CS3；
pFastBac1-58L1ΔCh4/16dE。
上述酶切、连接及克隆构建的方法都是公知的，例如专利CN101293918 B。
本发明涉及的L1、L2蛋白和嵌合蛋白的氨基酸序列如下所示：
58型L1蛋白全长
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYF
SIKSPNNNKK VLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV
WACVGLEIGR GQPLGVGVSG HPYLNKFDDT ETSNRYPAQP GSDNRECLSM
DYKQTQLCLI GCKPPTGEHW GKGVACNNNA AATDCPPLEL FNSIIEDGDM
VDTGFGCMDF GTLQANKSDV PIDICNSTCK YPDYLKMASE PYGDSLFFFL
RREQMFVRHF FNRAGKLGEA VPDDLYIKGS GNTAVIQSSA FFPTPSGSIV
TSESQLFNKP YWLQRAQGHN NGICWGNQLF VTVVDTTRST NMTLCTEVTK
EGTYKNDNFK EYVRHVEEYD LQFVFQLCKI TLTAEIMTYI HTMDSNILED
WQFGLTPPPS ASLQDTYRFV TSQAITCQKT APPKEKEDPL NKYTFWEVNL
KEKFSADLDQ FPLGRKFLLQ SGLKAKPRLK RSAPTTRAPS TKRKKVKK
SEQ ID No.1
16型L2蛋白全长
MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIAEQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGT
RPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTD
TTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTFIVSTNPNTV
TSSTPIPGSRPVARLGLYSRTTQQVKVVDPAFVTTPTKLITYDNPAYEGIDVDNTLYFSSNDNSINIAPD
PDFLDIVALHRPALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHYYYDLSTIDPAEEIELQTITPSTYT
TTSHAASPTSINNGLYDIYADDFITDTSTTPVPSVPSTSLSGYIPANTTIPFGGAYNIPLVSGPDIPINI
TDQAPSLIPIVPGSPQYTIIADAGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA
SEQ ID No.2
16L2 aa.17-38
QLYKTCKQAGTCPPDIIPKVEG SEQ ID No.3
16L2 aa.18-38
LYKTCKQAGT CPPDIIPKVE G SEQ ID No.4
16L2 aa.18-32
LYKTCKQAGT CPPDI SEQ ID No.5
16L2 aa.19-31
YKTCKQAGTC PPD SEQ ID No.6
58L1DE/16dEs
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYYKTC KQAGTCPPDP AQPGSDNREC LSMDYKQTQLCLIGCKPPTGEHWGKGVACN NNAAATDCPP LELFNSIIED GDMVDTGFGC MDFGTLQANKSDVPIDICNSTCKYPDYLKM ASEPYGDSLF FFLRREQMFV RHFFNRAGKL GEAVPDDLYIKGSGNTAVIQSSAFFPTPSG SIVTSESQLF NKPYWLQRAQ GHNNGICWGN QLFVTVVDTTRSTNMTLCTEVTKEGTYKND NFKEYVRHVE EYDLQFVFQL CKITLTAEIM TYIHTMDSNILEDWQFGLTPPPSASLQDTY RFVTSQAITC QKTAPPKEKE DPLNKYTFWE VNLKEKFSADLDQFPLGRKFLLQSGLKAKP RLKRSAPTTR APSTKRKKVK K
SEQ ID No.7
58L1ΔCDE/16dEs
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYYKTC KQAGTCPPDP AQPGSDNREC LSMDYKQTQLCLIGCKPPTGEHWGKGVACN NNAAATDCPP LELFNSIIED GDMVDTGFGC MDFGTLQANKSDVPIDICNSTCKYPDYLKM ASEPYGDSLF FFLRREQMFV RHFFNRAGKL GEAVPDDLYIKGSGNTAVIQSSAFFPTPSG SIVTSESQLF NKPYWLQRAQ GHNNGICWGN QLFVTVVDTTRSTNMTLCTEVTKEGTYKND NFKEYVRHVE EYDLQFVFQL CKITLTAEIM TYIHTMDSNILEDWQFGLTPPPSASLQDTY RFVTSQAITC QKTAPPKEKE DPLNKYTFWE VNLKEKFSADLDQFPLGRKFLLQSGL SEQ ID No.8
58L1h4/16dEs
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYPAQP GSDNRECLSM DYKQTQLCLI GCKPPTGEHWGKGVACNNNAAATDCPPLEL FNSIIEDGDM VDTGFGCMDF GTLQANKSDV PIDICNSTCKYPDYLKMASEPYGDSLFFFL RREQMFVRHF FNRAGKLGEA VPDDLYIKGS GNTAVIQSSAFFPTPSGSIVTSESQLFNKP YWLQRAQGHN NGICWGNQLF VTVVDTTRST NMTLCTEVTKEGTYKNDNFKEYVRHVEEYD LQFVFQLCKI TLTAEIMTYI HTMDSNILED WQFGLTPPPSASLQDTYRFVTSQAITCQKY KTCKQAGTCP PDPPKEKEDP LNKYTFWEVN LKEKFSADLDQFPLGRKFLLQSGLKAKPRL KRSAPTTRAP STKRKKVKK
SEQ ID No.958L1ΔCh4/16dEs
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYPAQP GSDNRECLSM DYKQTQLCLI GCKPPTGEHWGKGVACNNNAAATDCPPLEL FNSIIEDGDM VDTGFGCMDF GTLQANKSDV PIDICNSTCKYPDYLKMASEPYGDSLFFFL RREQMFVRHF FNRAGKLGEA VPDDLYIKGS GNTAVIQSSAFFPTPSGSIVTSESQLFNKP YWLQRAQGHN NGICWGNQLF VTVVDTTRST NMTLCTEVTKEGTYKNDNFKEYVRHVEEYD LQFVFQLCKI TLTAEIMTYI HTMDSNILED WQFGLTPPPSASLQDTYRFVTSQAITCQKY KTCKQAGTCP PDPPKEKEDP LNKYTFWEVN LKEKFSADLDQFPLGRKFLLQSGL SEQ ID No.10
58L1ΔN4h4/16dE
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IIPKVEGPPK EKEDPLNKYT FWEVNLKEKFSADLDQFPLGRKFLLQSGLK AKPRLKRSAP TTRAPSTKRK KVKK
SEQ ID No.1158L1ΔN4Ch4/16dE
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IIPKVEGPPK EKEDPLNKYT FWEVNLKEKFSADLDQFPLGRKFLLQSGL SEQ ID No.12
58L1ΔN4h4/16dEs
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IPPKEKEDPL NKYTFWEVNL KEKFSADLDQFPLGRKFLLQSGLKAKPRLK RSAPTTRAPS TKRKKVKK
SEQ ID No.1358L1ΔN4Ch4/16dEs
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IPPKEKEDPL NKYTFWEVNL KEKFSADLDQFPLGRKFLLQSGL
SEQ ID No.14
58L1ΔN4h4/16dE-CS1
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IIPKVEGPPK EKEDPLNKYT FWEVNLKEKFSADLDQFPLGRKFLLQSGLK AGPSLAGSAP TTSAPSTGGS AVGS
SEQ ID No.15
58L1ΔN4h4/16dE-CS2
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQPLGVGVSGHPYLNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IIPKVEGPPK EKEDPLNKYT FWEVNLKEKFSADLDQFPLGRKFLLQSGLG AGPSLAGSAP TTSAPSTGGS AVGS
SEQ ID No.1658L1ΔN4h4/16dE-CS3
MRPSEATVYL PPVPVSKVVS TDEYVSRTSI YYYAGSSRLL AVGNPYFSIKSPNNNKKVLVPKVSGLQYRV FRVRLPDPNK FGFPDTSFYN PDTQRLVWAC VGLEIGRGQP LGVGVSGHPY
LNKFDDTETS NRYPAQPGSD NRECLSMDYK QTQLCLIGCK PPTGEHWGKGVACNNNAAATDCPPLELFNS IIEDGDMVDT GFGCMDFGTL QANKSDVPID ICNSTCKYPDYLKMASEPYGDSLFFFLRRE QMFVRHFFNR AGKLGEAVPD DLYIKGSGNT AVIQSSAFFPTPSGSIVTSESQLFNKPYWL QRAQGHNNGI CWGNQLFVTV VDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQF VFQLCKITLT AEIMTYIHTM DSNILEDWQF GLTPPPSASLQDTYRFVTSQAITCQKLYKT CKQAGTCPPD IIPKVEGPPK EKEDPLNKYT FWEVNLKEKFSADLDQFPLGRKFLLQSGLK AGPSLAGSAP TTRAPSTGGS AVGS
SEQ ID No.17
58L1h4/16dE
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYPAQP GSDNRECLSM DYKQTQLCLI GCKPPTGEHWGKGVACNNNAAATDCPPLEL FNSIIEDGDM VDTGFGCMDF GTLQANKSDV PIDICNSTCKYPDYLKMASEPYGDSLFFFL RREQMFVRHF FNRAGKLGEA VPDDLYIKGS GNTAVIQSSAFFPTPSGSIVTSESQLFNKP YWLQRAQGHN NGICWGNQLF VTVVDTTRST NMTLCTEVTKEGTYKNDNFKEYVRHVEEYD LQFVFQLCKI TLTAEIMTYI HTMDSNILED WQFGLTPPPSASQLYKTCKQAGTCPPDIIP KVEGTCQKTA PPKEKEDPLN KYTFWEVNLK EKFSADLDQFPLGRKFLLQSGLKAKPRLKR SAPTTRAPST KRKKVKK
SEQ ID No.18
58L1ΔCh4/16dE
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLAVGNPYFSIKSPNNNKKVLVPKVSGLQ YRVFRVRLPD PNKFGFPDTS FYNPDTQRLV WACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYPAQP GSDNRECLSM DYKQTQLCLI GCKPPTGEHWGKGVACNNNAAATDCPPLEL FNSIIEDGDM VDTGFGCMDF GTLQANKSDV PIDICNSTCKYPDYLKMASEPYGDSLFFFL RREQMFVRHF FNRAGKLGEA VPDDLYIKGS GNTAVIQSSAFFPTPSGSIVTSESQLFNKP YWLQRAQGHN NGICWGNQLF VTVVDTTRST NMTLCTEVTKEGTYKNDNFKEYVRHVEEYD LQFVFQLCKI TLTAEIMTYI HTMDSNILED WQFGLTPPPSASQLYKTCKQAGTCPPDIIP KVEGTCQKTA PPKEKEDPLN KYTFWEVNLK EKFSADLDQF PLGRKFLLQSGL
SEQ ID No.19
本发明涉及的编码嵌合蛋白的氨基酸序列如下所示：
58L1DE/16dEs nt
ATG TCC GTC TGG CGT CCG AGT GAA GCC ACG GTG TAT TTG CCA CCA GTC CCGGTT AGC AAA GTAGTG TCC ACC GAC GAG TAC GTG AGC CGC ACT TCG ATC TAC TAC TACGCA GGA AGC TCG CGC CTGTTG GCT GTA GGC AAC CCC TAT TTC AGT ATC AAA TCA CCGAAT AAT AAC AAG AAG GTT TTA GTGCCG AAG GTA TCT GGG CTG CAA TAC CGT GTG TTCCGT GTC CGC CTG CCG GAC CCA AAT AAA TTTGGA TTT CCT GAT ACT TCC TTC TAT AATCCT GAC ACC CAA CGT CTG GTA TGG GCC TGT GTT GGACTT GAA ATT GGA CGC GGT CAACCT CTG GGC GTT GGC GTA AGT GGT CAC CCA TAT CTG AAC AAGTTC GAC GAT ACT GAGACA TCC AAC CGC TAT TAT AAG ACA TGT AAG CAG GCA GGC ACG TGT CCCCCA GAT CCTGCT CAG CCC GGG TCG GAC AAC CGT GAA TGC CTT TCA ATG GAC TAC AAG CAA ACCCAACTT TGC TTA ATC GGG TGT AAG CCC CCT ACA GGC GAG CAT TGG GGG AAG GGG GTG GCGTGTAAT AAC AAT GCA GCT GCT ACT GAT TGT CCT CCC CTT GAG CTG TTC AAT AGT ATTATT GAA GACGGA GAC ATG GTA GAT ACT GGG TTT GGC TGT ATG GAC TTT GGG ACG CTTCAA GCG AAT AAG TCAGAT GTA CCC ATT GAC ATT TGC AAT TCG ACT TGT AAG TAT CCCGAC TAC CTT AAA ATG GCT TCCGAG CCA TAT GGC GAC AGC CTG TTT TTT TTC CTG CGCCGC GAA CAA ATG TTC GTT CGT CAC TTTTTC AAC CGT GCT GGG AAA CTT GGC GAG GCTGTC CCG GAC GAC TTA TAC ATT AAG GGG AGC GGAAAC ACA GCT GTC ATC CAA TCT TCCGCG TTC TTC CCG ACG CCA TCA GGG TCG ATC GTG ACC AGCGAA AGC CAG CTG TTC AATAAA CCG TAC TGG TTG CAA CGT GCT CAG GGA CAT AAT AAC GGC ATTTGT TGG GGC AACCAG CTT TTC GTA ACA GTA GTA GAC ACG ACT CGT TCA ACT AAC ATG ACG TTGTGT ACGGAG GTT ACT AAG GAA GGC ACA TAT AAG AAC GAC AAT TTT AAA GAG TAC GTA CGTCATGTC GAG GAG TAC GAT CTG CAA TTT GTC TTC CAA CTT TGC AAG ATT ACC CTT ACTGCT GAG ATCATG ACA TAT ATC CAT ACA ATG GAT TCT AAT ATT TTG GAA GAC TGG CAGTTC GGC CTT ACT CCCCCT CCT AGC GCG AGT CTT CAG GAT ACC TAT CGT TTT GTC ACAAGT CAA GCT ATC ACC TGC CAGAAG ACC GCA CCG CCG AAG GAA AAA GAA GAT CCG CTTAAT AAG TAC ACC TTC TGG GAG GTT AACTTG AAG GAA AAG TTC AGC GCC GAC TTG GATCAA TTT CCC TTA GGT CGT AAA TTC CTT TTG CAGTCT GGA TTG AAA GCT AAA CCG CGCTTA AAA CGC TCC GCT CCT ACT ACG CGT GCC CCC AGC ACGAAG CGC AAA AAG GTA AAAAAG TAA
SEQ ID No.20
58L1ΔCDE/16dEs nt
ATGAGCGTGT GGAGACCCTC CGAAGCAACC GTCTATCTCC CACCCGTCCCCGTCAGCAAAGTCGTGTCAA CCGACGAGTA CGTCAGCAGG ACCTCAATCT ACTACTACGCTGGTTCCAGTCGCTTGCTCG CCGTCGGCAA CCCCTACTTC AGTATTAAGT CCCCAAACAACAACAAGAAGGTGCTGGTCC CAAAAGTGAG CGGCCTGCAA TACCGCGTGT TCCGCGTCAGGCTGCCCGACCCAAACAAGT TCGGCTTCCC CGACACCAGC TTCTACAATC CCGACACCCAGAGGCTCGTGTGGGCCTGCG TGGGCTTGGA GATCGGCCGC GGCCAACCAC TCGGCGTCGGCGTGTCCGGCCACCCCTACC TGAACAAGTT CGACGATACC GAGACATCCA ATCGCTATTACAAGACTTGCAAGCAGGCTG GTACCTGCCC CCCTGACCCA GCCCAACCCG GCAGCGACAATCGCGAGTGTCTGAGCATGG ACTACAAGCA GACCCAGCTG TGCCTGATCG GCTGCAAGCCCCCAACCGGTGAACACTGGG GCAAGGGCGT CGCTTGCAAC AACAACGCCG CCGCCACCGACTGCCCACCCCTCGAGTTGT TCAACAGCAT CATCGAAGAC GGCGATATGG TGGACACCGGCTTCGGCTGTATGGATTTCG GCACCCTCCA AGCCAACAAG TCCGACGTCC CCATCGACATCTGCAATTCCACCTGTAAGT ACCCCGACTA CCTGAAGATG GCATCCGAGC CCTACGGCGACAGCCTCTTCTTCTTCCTCC GCAGGGAACA AATGTTCGTC CGCCATTTCT TCAACCGCGCCGGCAAGTTGGGCGAAGCCG TGCCCGACGA TTTGTACATC AAGGGCAGTG GCAACACCGCCGTCATTCAGTCCTCCGCAT TCTTCCCAAC CCCCTCCGGC AGCATCGTCA CAAGCGAGAGCCAGCTGTTCAACAAGCCCT ACTGGTTGCA AAGGGCCCAG GGCCACAATA ACGGCATCTGTTGGGGCAACCAACTGTTCG TCACAGTCGT CGACACAACC AGGTCAACCA ACATGACCCTGTGTACCGAGGTGACCAAGG AGGGCACCTA CAAGAACGAC AACTTCAAAG AGTACGTGAGGCACGTCGAGGAGTACGATC TGCAATTCGT CTTCCAATTG TGTAAGATCA CCTTGACCGCCGAAATCATGACCTACATCC ACACCATGGA CAGTAACATC CTCGAAGATT GGCAGTTCGGCCTGACCCCCCCACCCAGCG CATCCCTGCA AGATACCTAC CGCTTCGTCA CAAGTCAAGCCATCACCTGTCAGAAGACCG CTCCACCCAA GGAGAAAGAG GACCCCCTGA ACAAGTACACCTTCTGGGAAGTCAATCTGA AAGAGAAATT CAGCGCCGAC TTGGACCAAT TCCCACTCGGCAGGAAGTTCCTGCTGCAGA GCGGCTTGTA A
SEQ ID No.21
58L1h4/16dEs nt
ATGTCTGTTT GGCGTCCGTC GGAAGCCACC GTTTATCTGC CACCTGTGCC GGTGAGCAAAGTTGTCTCGA CGGATGAATA TGTGAGTCGC ACCAGCATTT ATTACTATGC AGGTTCTTCG CGTTTGTTGGCGGTGGGCAA TCCGTACTTT AGCATCAAAT CGCCTAACAA TAACAAAAAG GTTTTAGTGC CGAAAGTGAGTGGCCTGCAA TATCGTGTGT TTCGCGTCCG CCTGCCGGAT CCAAATAAGT TTGGGTTCCC TGACACCAGCTTTTATAACC CAGATACCCA GCGTCTGGTT TGGGCGTGCG TGGGTTTGGA AATTGGCCGC GGCCAACCGTTGGGTGTGGG TGTTTCTGGC CATCCTTACT TGAATAAATT TGATGACACC GAAACTTCGA ACCGTTATCCGGCCCAGCCA GGCAGCGACA ATCGGGAATG TCTGTCGATG GATTACAAAC AAACGCAGCT GTGCTTAATTGGGTGTAAGC CGCCAACCGG TGAGCACTGG GGCAAAGGCG TTGCATGTAA CAATAACGCGGCGGCCACGGATTGCCCGCC ATTGGAATTG TTTAATAGTA TCATCGAAGA TGGTGACATG GTGGATACCG GTTTTGGCTGCATGGATTTC GGCACGTTAC AGGCCAACAA AAGCGACGTG CCAATTGATA TCTGCAATTC TACCTGTAAGTACCCGGATT ATCTGAAGAT GGCGTCGGAA CCGTACGGGG ATAGCCTGTT TTTCTTTCTG CGCCGCGAGCAGATGTTCGT TCGTCATTTC TTTAACCGCG CGGGTAAATT GGGCGAGGCC GTTCCGGACG ATTTGTATATTAAAGGCTCG GGGAATACCG CAGTGATTCA GAGTAGCGCG TTTTTCCCTA CCCCATCTGG TTCGATCGTGACCAGCGAAT CGCAACTGTT TAACAAGCCG TATTGGCTGC AGCGTGCGCA GGGCCACAAT AACGGCATCTGCTGGGGTAA TCAACTGTTT GTTACGGTCG TGGACACCAC GCGGAGTACG AACATGACGC TGTGTACCGAAGTGACTAAA GAGGGTACCT ACAAGAATGA TAACTTTAAA GAGTATGTTC GCCATGTTGA AGAGTATGATTTGCAGTTTG TGTTCCAATT GTGCAAAATT ACGTTAACGG CCGAGATCAT GACTTACATT CACACCATGGATAGCAATAT TCTGGAAGAC TGGCAGTTCG GCCTGACGCC GCCACCTTCT GCCTCGCTGC AGGATACCTATCGCTTTGTG ACCAGCCAGG CGATCACGTG CCAAAAATAT AAAACCTGCA AACAGGCCGG CACCTGCCCGCCAGATCCGC CAAAGGAAAA AGAGGACCCG TTGAACAAAT ATACGTTTTG GGAAGTTAAT TTGAAGGAAAAATTTTCGGC CGATCTGGAC CAGTTCCCAC TGGGCCGTAA ATTTCTGTTA CAAAGTGGTT TGAAAGCCAAGCCGCGCTTG AAACGTAGCG CGCCAACCAC GCGGGCGCCG TCTACCAAAC GCAAGAAAGT TAAGAAATAA
SEQ ID No.22
58L1ΔCh4/16dEs nt
ATGAGCGTGT GGAGACCCTC CGAAGCAACC GTCTATCTCC CACCCGTCCC CGTCAGCAAAGTCGTGTCAA CCGACGAGTA CGTCAGCAGG ACCTCAATCT ACTACTACGC TGGTTCCAGT CGCTTGCTCGCCGTCGGCAA CCCCTACTTC AGTATTAAGT CCCCAAACAA CAACAAGAAG GTGCTGGTCC CAAAAGTGAGCGGCCTGCAA TACCGCGTGT TCCGCGTCAG GCTGCCCGAC CCAAACAAGT TCGGCTTCCC CGACACCAGCTTCTACAATC CCGACACCCA GAGGCTCGTG TGGGCCTGCG TGGGCTTGGA GATCGGCCGC GGCCAACCACTCGGCGTCGG CGTGTCCGGC CACCCCTACC TGAACAAGTT CGACGATACC GAGACATCCA ATCGCTACCCAGCCCAACCC GGCAGCGACA ATCGCGAGTG TCTGAGCATG GACTACAAGC AGACCCAGCT GTGCCTGATCGGCTGCAAGC CCCCAACCGG TGAACACTGG GGCAAGGGCG TCGCTTGCAA CAACAACGCC GCCGCCACCGACTGCCCACC CCTCGAGTTG TTCAACAGCA TCATCGAAGA CGGCGATATG GTGGACACCG GCTTCGGCTGTATGGATTTC GGCACCCTCC AAGCCAACAA GTCCGACGTC CCCATCGACA TCTGCAATTC CACCTGTAAGTACCCCGACT ACCTGAAGAT GGCATCCGAG CCCTACGGCG ACAGCCTCTT CTTCTTCCTC CGCAGGGAACAAATGTTCGT CCGCCATTTCTTCAACCGCG CCGGCAAGTT GGGCGAAGCC GTGCCCGACG ATTTGTACATCAAGGGCAGTGGCAACACCG CCGTCATTCA GTCCTCCGCA TTCTTCCCAA CCCCCTCCGGCAGCATCGTCACAAGCGAGA GCCAGCTGTT CAACAAGCCC TACTGGTTGC AAAGGGCCCAGGGCCACAATAACGGCATCT GTTGGGGCAA CCAACTGTTC GTCACAGTCG TCGACACAACCAGGTCAACCAACATGACCC TGTGTACCGA GGTGACCAAG GAGGGCACCT ACAAGAACGACAACTTCAAAGAGTACGTGA GGCACGTCGA GGAGTACGAT CTGCAATTCG TCTTCCAATTGTGTAAGATCACCTTGACCG CCGAAATCAT GACCTACATC CACACCATGG ACAGTAACATCCTCGAAGATTGGCAGTTCG GCCTGACCCC CCCACCCAGC GCATCCCTGC AAGATACCTACCGCTTCGTCACAAGTCAAG CCATCACCTG TCAGAAGTAC AAGACCTGCA AGCAGGCCGGTACCTGCCCCCCTGACCCAC CCAAGGAGAA AGAGGACCCC CTGAACAAGT ACACCTTCTGGGAAGTCAATCTGAAAGAGA AATTCAGCGC CGACTTGGAC CAATTCCCAC TCGGCAGGAAGTTCCTGCTGCAGAGCGGCT TGTAA
SEQ ID No.23
58L1ΔN4h4/16dE nt
ATGCGTCCGT CGGAAGCCAC CGTTTATCTG CCACCTGTGC CGGTGAGCAAAGTTGTCTCGACGGATGAAT ATGTGAGTCG CACCAGCATT TATTACTATG CAGGTTCTTCGCGTTTGTTGGCGGTGGGCA ATCCGTACTT TAGCATCAAA TCGCCTAACA ATAACAAAAAGGTTTTAGTGCCGAAAGTGA GTGGCCTGCA ATATCGTGTG TTTCGCGTCC GCCTGCCGGATCCAAATAAGTTTGGGTTCC CTGACACCAG CTTTTATAAC CCAGATACCC AGCGTCTGGTTTGGGCGTGCGTGGGTTTGG AAATTGGCCG CGGCCAACCG TTGGGTGTGG GTGTTTCTGGCCATCCTTACTTGAATAAAT TTGATGACAC CGAAACTTCG AACCGTTATC CGGCCCAGCCAGGCAGCGACAATCGGGAAT GTCTGTCGAT GGATTACAAA CAAACGCAGC TGTGCTTAATTGGGTGTAAGCCGCCAACCG GTGAGCACTG GGGCAAAGGC GTTGCATGTA ACAATAACGCGGCGGCCACGGATTGCCCGC CATTGGAATT GTTTAATAGT ATCATCGAAG ATGGTGACATGGTGGATACCGGTTTTGGCT GCATGGATTT CGGCACGTTA CAGGCCAACA AAAGCGACGTGCCAATTGATATCTGCAATT CTACCTGTAA GTACCCGGAT TATCTGAAGA TGGCGTCGGAACCGTACGGGGATAGCCTGT TTTTCTTTCT GCGCCGCGAG CAGATGTTCG TTCGTCATTTCTTTAACCGCGCGGGTAAAT TGGGCGAGGC CGTTCCGGAC GATTTGTATA TTAAAGGCTCGGGGAATACCGCAGTGATTC AGAGTAGCGC GTTTTTCCCT ACCCCATCTG GTTCGATCGTGACCAGCGAATCGCAACTGT TTAACAAGCC GTATTGGCTG CAGCGTGCGC AGGGCCACAATAACGGCATCTGCTGGGGTA ATCAACTGTT TGTTACGGTC GTGGACACCA CGCGGAGTACGAACATGACGCTGTGTACCG AAGTGACTAA AGAGGGTACC TACAAGAATG ATAACTTTAAAGAGTATGTTCGCCATGTTG AAGAGTATGA TTTGCAGTTT GTGTTCCAAT TGTGCAAAATTACGTTAACGGCCGAGATCA TGACTTACAT TCACACCATG GATAGCAATA TTCTGGAAGACTGGCAGTTCGGCCTGACGC CGCCACCTTC TGCCTCGCTG CAGGATACCT ATCGCTTTGTGACCAGCCAGGCGATCACGT GCCAAAAACT GTATAAAACC TGCAAACAGG CCGGCACCTGCCCGCCAGATATTATCCCGA AAGTTGAAGG TCCGCCAAAG GAAAAAGAGG ACCCGTTGAACAAATATACGTTTTGGGAAG TTAATTTGAA GGAAAAATTT TCGGCCGATC TGGACCAGTTCCCACTGGGCCGTAAATTTC TGTTACAAAG TGGTTTGAAA GCCAAGCCGC GCTTGAAACGTAGCGCGCCAACCACGCGGG CGCCGTCTAC CAAACGCAAG AAAGTTAAGA AATAA
SEQ ID No.24
58L1ΔN4Ch4/16dE nt
ATGAGACCCT CCGAAGCAAC CGTCTATCTC CCACCCGTCC CCGTCAGCAAAGTCGTGTCAACCGACGAGT ACGTCAGCAG GACCTCAATC TACTACTACG CTGGTTCCAGTCGCTTGCTCGCCGTCGGCA ACCCCTACTT CAGTATTAAG TCCCCAAACA ACAACAAGAAGGTGCTGGTCCCAAAAGTGA GCGGCCTGCA ATACCGCGTG TTCCGCGTCA GGCTGCCCGACCCAAACAAGTTCGGCTTCC CCGACACCAG CTTCTACAAT CCCGACACCC AGAGGCTCGTGTGGGCCTGCGTGGGCTTGG AGATCGGCCG CGGCCAACCA CTCGGCGTCG GCGTGTCCGGCCACCCCTACCTGAACAAGT TCGACGATAC CGAGACATCC AATCGCTACC CAGCCCAACCCGGCAGCGACAATCGCGAGT GTCTGAGCAT GGACTACAAG CAGACCCAGC TGTGCCTGATCGGCTGCAAGCCCCCAACCG GTGAACACTG GGGCAAGGGC GTCGCTTGCA ACAACAACGCCGCCGCCACCGACTGCCCAC CCCTCGAGTT GTTCAACAGC ATCATCGAAG ACGGCGATATGGTGGACACCGGCTTCGGCT GTATGGATTT CGGCACCCTC CAAGCCAACA AGTCCGACGTCCCCATCGACATCTGCAATT CCACCTGTAA GTACCCCGAC TACCTGAAGA TGGCATCCGAGCCCTACGGCGACAGCCTCT TCTTCTTCCT CCGCAGGGAA CAAATGTTCG TCCGCCATTTCTTCAACCGCGCCGGCAAGT TGGGCGAAGC CGTGCCCGAC GATTTGTACA TCAAGGGCAGTGGCAACACCGCCGTCATTC AGTCCTCCGC ATTCTTCCCA ACCCCCTCCG GCAGCATCGTCACAAGCGAGAGCCAGCTGT TCAACAAGCC CTACTGGTTG CAAAGGGCCC AGGGCCACAATAACGGCATCTGTTGGGGCA ACCAACTGTT CGTCACAGTC GTCGACACAA CCAGGTCAACCAACATGACCCTGTGTACCG AGGTGACCAA GGAGGGCACC TACAAGAACG ACAACTTCAAAGAGTACGTGAGGCACGTCG AGGAGTACGA TCTGCAATTC GTCTTCCAAT TGTGTAAGATCACCTTGACCGCCGAAATCA TGACCTACAT CCACACCATG GACAGTAACA TCCTCGAAGATTGGCAGTTCGGCCTGACCC CCCCACCCAG CGCATCCCTG CAAGATACCT ACCGCTTCGTCACAAGTCAAGCCATCACCT GTCAGAAGCT GTACAAGACC TGCAAGCAGG CCGGTACCTGCCCCCCTGACATCATCCCCA AGGTCGAAGG ACCACCCAAG GAGAAAGAGG ACCCCCTGAACAAGTACACCTTCTGGGAAG TCAATCTGAA AGAGAAATTC AGCGCCGACT TGGACCAATTCCCACTCGGCAGGAAGTTCC TGCTGCAGAG CGGCTTGTAA
SEQ ID No.2558L1ΔN4h4/16dEs
ATGCGTCCGT CGGAAGCCAC CGTTTATCTG CCACCTGTGC CGGTGAGCAAAGTTGTCTCGACGGATGAAT ATGTGAGTCG CACCAGCATT TATTACTATG CAGGTTCTTCGCGTTTGTTGGCGGTGGGCA ATCCGTACTT TAGCATCAAA TCGCCTAACA ATAACAAAAAGGTTTTAGTGCCGAAAGTGA GTGGCCTGCA ATATCGTGTG TTTCGCGTCC GCCTGCCGGATCCAAATAAGTTTGGGTTCC CTGACACCAG CTTTTATAAC CCAGATACCC AGCGTCTGGTTTGGGCGTGCGTGGGTTTGG AAATTGGCCG CGGCCAACCG TTGGGTGTGG GTGTTTCTGGCCATCCTTACTTGAATAAAT TTGATGACAC CGAAACTTCG AACCGTTATC CGGCCCAGCCAGGCAGCGACAATCGGGAAT GTCTGTCGAT GGATTACAAA CAAACGCAGC TGTGCTTAATTGGGTGTAAGCCGCCAACCG GTGAGCACTG GGGCAAAGGC GTTGCATGTA ACAATAACGCGGCGGCCACGGATTGCCCGC CATTGGAATT GTTTAATAGT ATCATCGAAG ATGGTGACATGGTGGATACCGGTTTTGGCT GCATGGATTT CGGCACGTTA CAGGCCAACA AAAGCGACGTGCCAATTGATATCTGCAATT CTACCTGTAA GTACCCGGAT TATCTGAAGA TGGCGTCGGAACCGTACGGGGATAGCCTGT TTTTCTTTCT GCGCCGCGAG CAGATGTTCG TTCGTCATTTCTTTAACCGCGCGGGTAAAT TGGGCGAGGC CGTTCCGGAC GATTTGTATA TTAAAGGCTCGGGGAATACCGCAGTGATTC AGAGTAGCGC GTTTTTCCCT ACCCCATCTG GTTCGATCGTGACCAGCGAATCGCAACTGT TTAACAAGCC GTATTGGCTG CAGCGTGCGC AGGGCCACAATAACGGCATCTGCTGGGGTA ATCAACTGTT TGTTACGGTC GTGGACACCA CGCGGAGTACGAACATGACGCTGTGTACCG AAGTGACTAA AGAGGGTACC TACAAGAATG ATAACTTTAAAGAGTATGTTCGCCATGTTG AAGAGTATGA TTTGCAGTTT GTGTTCCAAT TGTGCAAAATTACGTTAACGGCCGAGATCA TGACTTACAT TCACACCATG GATAGCAATA TTCTGGAAGACTGGCAGTTCGGCCTGACGC CGCCACCTTC TGCCTCGCTG CAGGATACCT ATCGCTTTGTGACCAGCCAGGCGATCACGT GCCAAAAACT GTATAAAACC TGCAAACAGG CCGGCACCTGCCCGCCAGATATTCCGCCAA AGGAAAAAGA GGACCCGTTG AACAAATATA CGTTTTGGGAAGTTAATTTGAAGGAAAAAT TTTCGGCCGA TCTGGACCAG TTCCCACTGG GCCGTAAATTTCTGTTACAAAGTGGTTTGA AAGCCAAGCC GCGCTTGAAA CGTAGCGCGC CAACCACGCGGGCGCCGTCTACCAAACGCA AGAAAGTTAA GAAATAA
SEQ ID No.26
58L1ΔN4Ch4/16dEs nt
ATGAGACCCT CCGAAGCAAC CGTCTATCTC CCACCCGTCC CCGTCAGCAAAGTCGTGTCAACCGACGAGT ACGTCAGCAG GACCTCAATC TACTACTACG CTGGTTCCAGTCGCTTGCTCGCCGTCGGCA ACCCCTACTT CAGTATTAAG TCCCCAAACA ACAACAAGAAGGTGCTGGTCCCAAAAGTGA GCGGCCTGCA ATACCGCGTG TTCCGCGTCA GGCTGCCCGACCCAAACAAGTTCGGCTTCC CCGACACCAG CTTCTACAAT CCCGACACCC AGAGGCTCGTGTGGGCCTGCGTGGGCTTGG AGATCGGCCG CGGCCAACCA CTCGGCGTCG GCGTGTCCGGCCACCCCTACCTGAACAAGT TCGACGATAC CGAGACATCC AATCGCTACC CAGCCCAACCCGGCAGCGACAATCGCGAGT GTCTGAGCAT GGACTACAAG CAGACCCAGC TGTGCCTGATCGGCTGCAAGCCCCCAACCG GTGAACACTG GGGCAAGGGC GTCGCTTGCA ACAACAACGCCGCCGCCACCGACTGCCCAC CCCTCGAGTT GTTCAACAGC ATCATCGAAG ACGGCGATATGGTGGACACCGGCTTCGGCT GTATGGATTT CGGCACCCTC CAAGCCAACA AGTCCGACGTCCCCATCGACATCTGCAATT CCACCTGTAA GTACCCCGAC TACCTGAAGA TGGCATCCGAGCCCTACGGCGACAGCCTCT TCTTCTTCCT CCGCAGGGAA CAAATGTTCG TCCGCCATTTCTTCAACCGCGCCGGCAAGT TGGGCGAAGC CGTGCCCGAC GATTTGTACA TCAAGGGCAGTGGCAACACCGCCGTCATTC AGTCCTCCGC ATTCTTCCCA ACCCCCTCCG GCAGCATCGTCACAAGCGAGAGCCAGCTGT TCAACAAGCC CTACTGGTTG CAAAGGGCCC AGGGCCACAATAACGGCATCTGTTGGGGCA ACCAACTGTT CGTCACAGTC GTCGACACAA CCAGGTCAACCAACATGACCCTGTGTACCG AGGTGACCAA GGAGGGCACC TACAAGAACG ACAACTTCAAAGAGTACGTGAGGCACGTCG AGGAGTACGA TCTGCAATTC GTCTTCCAAT TGTGTAAGATCACCTTGACCGCCGAAATCA TGACCTACAT CCACACCATG GACAGTAACA TCCTCGAAGATTGGCAGTTCGGCCTGACCC CCCCACCCAG CGCATCCCTG CAAGATACCT ACCGCTTCGTCACAAGTCAAGCCATCACCT GTCAGAAGCT GTACAAGACT TGCAAGCAGG CTGGTACCTGCCCCCCTGACATCCCACCCA AGGAGAAAGA GGACCCCCTG AACAAGTACA CCTTCTGGGAAGTCAATCTGAAAGAGAAAT TCAGCGCCGA CTTGGACCAA TTCCCACTCG GCAGGAAGTTCCTGCTGCAGAGCGGCTTGT AA
SEQ ID No.27
58L1ΔN4h4/16dE-CS1 nt
ATGAGACCCT CCGAAGCAAC CGTCTATCTC CCACCCGTCC CCGTCAGCAAAGTCGTGTCAACCGACGAGT ACGTCAGCAG GACCTCAATC TACTACTACG CTGGTTCCAGTCGCTTGCTCGCCGTCGGCA ACCCCTACTT CAGTATTAAG TCCCCAAACA ACAACAAGAAGGTGCTGGTCCCAAAAGTGA GCGGCCTGCA ATACCGCGTG TTCCGCGTCA GGCTGCCCGACCCAAACAAGTTCGGCTTCC CCGACACCAG CTTCTACAAT CCCGACACCC AGAGGCTCGTGTGGGCCTGCGTGGGCTTGG AGATCGGCCG CGGCCAACCA CTCGGCGTCG GCGTGTCCGGCCACCCCTACCTGAACAAGT TCGACGATAC CGAGACATCC AATCGCTACC CAGCCCAACCCGGCAGCGACAATCGCGAGT GTCTGAGCAT GGACTACAAG CAGACCCAGC TGTGCCTGATCGGCTGCAAGCCCCCAACCG GTGAACACTG GGGCAAGGGC GTCGCTTGCA ACAACAACGCCGCCGCCACCGACTGCCCAC CCCTCGAGTT GTTCAACAGC ATCATCGAAG ACGGCGATATGGTGGACACCGGCTTCGGCT GTATGGATTT CGGCACCCTC CAAGCCAACA AGTCCGACGTCCCCATCGACATCTGCAATT CCACCTGTAA GTACCCCGAC TACCTGAAGA TGGCATCCGAGCCCTACGGCGACAGCCTCT TCTTCTTCCT CCGCAGGGAA CAAATGTTCG TCCGCCATTTCTTCAACCGCGCCGGCAAGT TGGGCGAAGC CGTGCCCGAC GATTTGTACA TCAAGGGCAGTGGCAACACCGCCGTCATTC AGTCCTCCGC ATTCTTCCCA ACCCCCTCCG GCAGCATCGTCACAAGCGAGAGCCAGCTGT TCAACAAGCC CTACTGGTTG CAAAGGGCCC AGGGCCACAATAACGGCATCTGTTGGGGCA ACCAACTGTT CGTCACAGTC GTCGACACAA CCAGGTCAACCAACATGACCCTGTGTACCG AGGTGACCAA GGAGGGCACC TACAAGAACG ACAACTTCAAAGAGTACGTGAGGCACGTCG AGGAGTACGA TCTGCAATTC GTCTTCCAAT TGTGTAAGATCACCTTGACCGCCGAAATCA TGACCTACAT CCACACCATG GACAGTAACA TCCTCGAAGATTGGCAGTTCGGCCTGACCC CCCCACCCAG CGCATCCCTG CAAGATACCT ACCGCTTCGTCACAAGTCAAGCCATCACCT GTCAGAAGCT GTACAAGACC TGCAAGCAGG CCGGTACCTGCCCCCCTGACATCATCCCCA AGGTCGAAGG ACCACCCAAG GAGAAAGAGG ACCCCCTGAACAAGTACACCTTCTGGGAAG TCAATCTGAA AGAGAAATTC AGCGCCGACT TGGACCAATTCCCACTCGGCAGGAAGTTCC TGCTGCAGAG CGGCTTGAAA GCCGGCCCTT CGTTGGCCGGCTCGGCCCCTACGACCTCGG CGCCGTCGAC GGGTGGCAGC GCCGTGGGTA GCTAA
SEQ ID No.28
58L1ΔN4h4/16dE-CS2 nt
ATGAGACCCT CCGAAGCAAC CGTCTATCTC CCACCCGTCC CCGTCAGCAAAGTCGTGTCAACCGACGAGT ACGTCAGCAG GACCTCAATC TACTACTACG CTGGTTCCAGTCGCTTGCTCGCCGTCGGCA ACCCCTACTT CAGTATTAAG TCCCCAAACA ACAACAAGAAGGTGCTGGTCCCAAAAGTGA GCGGCCTGCA ATACCGCGTG TTCCGCGTCA GGCTGCCCGACCCAAACAAGTTCGGCTTCC CCGACACCAG CTTCTACAAT CCCGACACCC AGAGGCTCGTGTGGGCCTGCGTGGGCTTGG AGATCGGCCG CGGCCAACCA CTCGGCGTCG GCGTGTCCGGCCACCCCTACCTGAACAAGT TCGACGATAC CGAGACATCC AATCGCTACC CAGCCCAACCCGGCAGCGACAATCGCGAGT GTCTGAGCAT GGACTACAAG CAGACCCAGC TGTGCCTGATCGGCTGCAAGCCCCCAACCG GTGAACACTG GGGCAAGGGC GTCGCTTGCA ACAACAACGCCGCCGCCACCGACTGCCCAC CCCTCGAGTT GTTCAACAGC ATCATCGAAG ACGGCGATATGGTGGACACCGGCTTCGGCT GTATGGATTT CGGCACCCTC CAAGCCAACA AGTCCGACGTCCCCATCGACATCTGCAATT CCACCTGTAA GTACCCCGAC TACCTGAAGA TGGCATCCGAGCCCTACGGCGACAGCCTCT TCTTCTTCCT CCGCAGGGAA CAAATGTTCG TCCGCCATTTCTTCAACCGCGCCGGCAAGT TGGGCGAAGC CGTGCCCGAC GATTTGTACA TCAAGGGCAGTGGCAACACCGCCGTCATTC AGTCCTCCGC ATTCTTCCCA ACCCCCTCCG GCAGCATCGTCACAAGCGAGAGCCAGCTGT TCAACAAGCC CTACTGGTTG CAAAGGGCCC AGGGCCACAATAACGGCATCTGTTGGGGCA ACCAACTGTT CGTCACAGTC GTCGACACAA CCAGGTCAAC CAACATGACC
CTGTGTACCG AGGTGACCAA GGAGGGCACC TACAAGAACG ACAACTTCAAAGAGTACGTGAGGCACGTCG AGGAGTACGA TCTGCAATTC GTCTTCCAAT TGTGTAAGATCACCTTGACCGCCGAAATCA TGACCTACAT CCACACCATG GACAGTAACA TCCTCGAAGATTGGCAGTTCGGCCTGACCC CCCCACCCAG CGCATCCCTG CAAGATACCT ACCGCTTCGTCACAAGTCAAGCCATCACCT GTCAGAAGCT GTACAAGACC TGCAAGCAGG CCGGTACCTGCCCCCCTGACATCATCCCCA AGGTCGAAGG ACCACCCAAG GAGAAAGAGG ACCCCCTGAACAAGTACACCTTCTGGGAAG TCAATCTGAA AGAGAAATTC AGCGCCGACT TGGACCAATTCCCACTCGGCAGGAAGTTCC TGCTGCAGAG CGGCTTGGGT GCCGGCCCTT CGTTGGCCGGCTCGGCCCCTACGACCTCGG CGCCGTCGAC GGGTGGCAGC GCCGTGGGTA GCTAA
SEQ ID No.29
58L1ΔN4h4/16dE-CS3 nt
ATGAGACCCT CCGAAGCAAC CGTCTATCTC CCACCCGTCC CCGTCAGCAAAGTCGTGTCAACCGACGAGT ACGTCAGCAG GACCTCAATC TACTACTACG CTGGTTCCAGTCGCTTGCTCGCCGTCGGCA ACCCCTACTT CAGTATTAAG TCCCCAAACA ACAACAAGAAGGTGCTGGTCCCAAAAGTGA GCGGCCTGCA ATACCGCGTG TTCCGCGTCA GGCTGCCCGACCCAAACAAGTTCGGCTTCC CCGACACCAG CTTCTACAAT CCCGACACCC AGAGGCTCGTGTGGGCCTGCGTGGGCTTGG AGATCGGCCG CGGCCAACCA CTCGGCGTCG GCGTGTCCGGCCACCCCTACCTGAACAAGT TCGACGATAC CGAGACATCC AATCGCTACC CAGCCCAACCCGGCAGCGACAATCGCGAGT GTCTGAGCAT GGACTACAAG CAGACCCAGC TGTGCCTGATCGGCTGCAAGCCCCCAACCG GTGAACACTG GGGCAAGGGC GTCGCTTGCA ACAACAACGCCGCCGCCACCGACTGCCCAC CCCTCGAGTT GTTCAACAGC ATCATCGAAG ACGGCGATATGGTGGACACCGGCTTCGGCT GTATGGATTT CGGCACCCTC CAAGCCAACA AGTCCGACGTCCCCATCGACATCTGCAATT CCACCTGTAA GTACCCCGAC TACCTGAAGA TGGCATCCGAGCCCTACGGCGACAGCCTCT TCTTCTTCCT CCGCAGGGAA CAAATGTTCG TCCGCCATTTCTTCAACCGCGCCGGCAAGT TGGGCGAAGC CGTGCCCGAC GATTTGTACA TCAAGGGCAGTGGCAACACCGCCGTCATTC AGTCCTCCGC ATTCTTCCCA ACCCCCTCCG GCAGCATCGTCACAAGCGAGAGCCAGCTGT TCAACAAGCC CTACTGGTTG CAAAGGGCCC AGGGCCACAATAACGGCATCTGTTGGGGCA ACCAACTGTT CGTCACAGTC GTCGACACAA CCAGGTCAACCAACATGACCCTGTGTACCG AGGTGACCAA GGAGGGCACC TACAAGAACG ACAACTTCAAAGAGTACGTGAGGCACGTCG AGGAGTACGA TCTGCAATTC GTCTTCCAAT TGTGTAAGATCACCTTGACCGCCGAAATCA TGACCTACAT CCACACCATG GACAGTAACA TCCTCGAAGATTGGCAGTTCGGCCTGACCC CCCCACCCAG CGCATCCCTG CAAGATACCT ACCGCTTCGTCACAAGTCAAGCCATCACCT GTCAGAAGCT GTACAAGACC TGCAAGCAGG CCGGTACCTGCCCCCCTGACATCATCCCCA AGGTCGAAGG ACCACCCAAG GAGAAAGAGG ACCCCCTGAACAAGTACACCTTCTGGGAAG TCAATCTGAA AGAGAAATTC AGCGCCGACT TGGACCAATTCCCACTCGGCAGGAAGTTCC TGCTGCAGAG CGGCTTGAAA GCCGGCCCTT CGTTGGCCGGCTCGGCCCCTACGACCAGAG CGCCGTCGAC GGGTGGCAGC GCCGTGGGTA GCTAA
SEQ ID No.30
58L1h4/16dE
ATG AGC GTC TGG CGC CCT TCT GAA GCG ACT GTT TAC CTG CCG CCC GTC CCAGTA AGC AAG GTTGTG AGC ACC GAT GAA TAT GTT AGT CGT ACA TCG ATC TAT TAC TATGCT GGA AGC TCA CGT TTGCTG GCG GTA GGT AAC CCT TAT TTT TCC ATT AAA AGT CCCAAT AAT AAT AAG AAA GTT TTA GTCCCG AAG GTG TCT GGG TTG CAA TAC CGC GTA TTCCGT GTC CGT CTT CCG GAC CCG AAT AAA TTTGGC TTC CCC GAT ACT TCT TTT TAT AATCCC GAT ACT CAG CGT TTA GTA TGG GCC TGT GTT GGGCTT GAG ATC GGA CGC GGG CAACCT CTG GGG GTT GGC GTC AGC GGC CAT CCT TAT CTT AAT AAATTT GAC GAT ACG GAGACC AGT AAC CGC TAC CCG GCT CAG CCG GGG TCC GAC AAC CGT GAA TGTTTG TCC ATGGAT TAT AAG CAG ACT CAG CTG TGT TTG ATC GGT TGT AAA CCT CCT ACA GGT GAACATTGG GGC AAA GGT GTA GCC TGC AAT AAT AAC GCA GCG GCT ACC GAT TGT CCG CCT TTGGAGTTG TTC AAC TCG ATT ATC GAA GAC GGT GAT ATG GTA GAC ACT GGG TTC GGT TGCATG GAC TTTGGG ACC CTT CAA GCC AAT AAG TCC GAC GTG CCA ATC GAC ATT TGC AACTCT ACA TGC AAG TACCCT GAC TAT CTT AAG ATG GCG TCC GAA CCG TAC GGA GAT TCTTTG TTT TTT TTC CTG CGT CGTGAG CAA ATG TTC GTA CGC CAT TTC TTC AAC CGT GCTGGA AAG TTA GGA GAA GCT GTT CCT GATGAT CTG TAT ATT AAG GGA TCT GGG AAC ACAGCG GTT ATT CAA TCC TCC GCT TTC TTT CCA ACGCCA AGC GGG TCC ATT GTG ACC AGTGAA AGC CAA TTA TTT AAC AAG CCG TAC TGG TTA CAA CGTGCG CAG GGA CAC AAT AATGGA ATC TGC TGG GGC AAT CAA TTG TTC GTG ACG GTT GTT GAC ACGACC CGT TCG ACCAAT ATG ACC CTT TGC ACT GAG GTA ACT AAG GAG GGT ACG TAC AAA AAT GACAAT TTTAAG GAG TAC GTC CGT CAT GTC GAG GAG TAT GAT CTT CAG TTT GTC TTC CAG TTATGTAAA ATT ACC TTG ACC GCG GAA ATC ATG ACG TAT ATC CAC ACG ATG GAC TCA AACATC CTG GAGGAT TGG CAA TTC GGT TTG ACT CCG CCC CCG AGC GCG TCT CAA TTG TATAAG ACT TGT AAG CAGGCA GGT ACA TGC CCA CCA GAC ATC ATT CCA AAA GTT GAA GGAACG TGC CAA AAG ACG GCA CCACCG AAA GAA AAG GAG GAC CCC CTG AAC AAA TAT ACGTTT TGG GAG GTG AAT TTG AAG GAGAAA TTC TCA GCA GAC TTA GAC CAG TTT CCG CTTGGC CGC AAG TTT CTG TTG CAG TCT GGG TTGAAG GCA AAA CCG CGT CTT AAG CGT AGCGCC CCC ACG ACT CGC GCC CCC AGT ACA AAA CGT AAGAAG GTT AAA AAG
SEQ ID No.31
58L1ΔCh4/16dE nt
ATGAGCGTGT GGAGACCCTC CGAAGCAACC GTCTATCTCC CACCCGTCCCCGTCAGCAAAGTCGTGTCAA CCGACGAGTA CGTCAGCAGG ACCTCAATCT ACTACTACGCTGGTTCCAGTCGCTTGCTCG CCGTCGGCAA CCCCTACTTC AGTATTAAGT CCCCAAACAACAACAAGAAGGTGCTGGTCC CAAAAGTGAG CGGCCTGCAA TACCGCGTGT TCCGCGTCAGGCTGCCCGACCCAAACAAGT TCGGCTTCCC CGACACCAGC TTCTACAATC CCGACACCCAGAGGCTCGTGTGGGCCTGCG TGGGCTTGGA GATCGGCCGC GGCCAACCAC TCGGCGTCGGCGTGTCCGGCCACCCCTACC TGAACAAGTT CGACGATACC GAGACATCCA ATCGCTACCCAGCCCAACCCGGCAGCGACA ATCGCGAGTG TCTGAGCATG GACTACAAGC AGACCCAGCTGTGCCTGATCGGCTGCAAGC CCCCAACCGG TGAACACTGG GGCAAGGGCG TCGCTTGCAACAACAACGCCGCCGCCACCG ACTGCCCACC CCTCGAGTTG TTCAACAGCA TCATCGAAGACGGCGATATGGTGGACACCG GCTTCGGCTG TATGGATTTC GGCACCCTCC AAGCCAACAAGTCCGACGTCCCCATCGACA TCTGCAATTC CACCTGTAAG TACCCCGACT ACCTGAAGATGGCATCCGAGCCCTACGGCG ACAGCCTCTT CTTCTTCCTC CGCAGGGAAC AAATGTTCGTCCGCCATTTCTTCAACCGCG CCGGCAAGTT GGGCGAAGCC GTGCCCGACG ATTTGTACATCAAGGGCAGTGGCAACACCG CCGTCATTCA GTCCTCCGCA TTCTTCCCAA CCCCCTCCGGCAGCATCGTCACAAGCGAGA GCCAGCTGTT CAACAAGCCC TACTGGTTGC AAAGGGCCCAGGGCCACAATAACGGCATCT GTTGGGGCAA CCAACTGTTC GTCACAGTCG TCGACACAACCAGGTCAACCAACATGACCC TGTGTACCGA GGTGACCAAG GAGGGCACCT ACAAGAACGACAACTTCAAAGAGTACGTGA GGCACGTCGA GGAGTACGAT CTGCAATTCG TCTTCCAATTGTGTAAGATCACCTTGACCG CCGAAATCAT GACCTACATC CACACCATGG ACAGTAACATCCTCGAAGATTGGCAGTTCG GCCTGACCCC CCCACCTAGC GCATCCCAAC TGTACAAGACCTGCAAGCAGGCCGGTACCT GCCCCCCTGA CATCATCCCC AAGGTCGAAG GCACCTGTCAGAAGACTGCTCCACCCAAGG AGAAAGAGGA CCCCCTGAAC AAGTACACCT TCTGGGAAGTCAATCTGAAAGAGAAATTCA GCGCCGACTT GGACCAATTC CCACTCGGCA GGAAGTTCCTGCTGCAGAGCGGCTTGTAA
SEQ ID No.32
实施例2：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pFastBac1-58L1ΔCDE/16dEs、pFastBac1-58L1ΔCh4/16dEs、pFastBac1-58L1ΔN4Ch4/16dE、pFastBac1-58L1ΔN4Ch4/16dEs、pFastBac1-58L1ΔN4h4/16dE-CS1、pFastBac1-58L1ΔN4h4/16dE-CS2、pFastBac1-58L1ΔN4h4/16dE-CS3、pFastBac1-58L1ΔCh4/16dE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CN101148661 B。
实施例3：嵌合L1蛋白的基因在Sf9细胞中的表达
Sf9细胞分别接种8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4：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-58L1DE/16dEs、pET22b-58L1h4/16dEs、pET22b-58L1ΔN4h4/16dE、pET22b-58L1ΔN4h4/16dEs、pET22b-58L1h4/16dE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5：嵌合L1蛋白的表达鉴定
取实施例3及实施例4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10μl进行SDS-PAGE电泳及Western blot鉴定。结果如图1A至图1B所示，13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58L1DE/16dEs、58L1h4/16dEs、58L1ΔN4h4/16dE、58L1ΔN4h4/16dEs、58L1h4/16dE、58L1ΔN4h4/16dE-CS1、58L1ΔN4h4/16dE-CS2、58L1ΔN4h4/16dE-CS3大小约59kDa，其余5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CN101148661 B。
实施例6：嵌合L1蛋白在昆虫细胞中的表达量比较
取表达野生型HPV58L1蛋白及实施例3中表达8种嵌合L1蛋白的细胞各15×106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HPV58L1单克隆抗体包被酶标板，80ng/孔，4℃孵育过10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HPV58L1 VLP标准品也进行梯度稀释，浓度从2μg/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HPV58L1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，15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HPV58L1蛋白及58L1嵌合蛋白的浓度。
结果如表1所示，本发明的HPV58嵌合L1蛋白的表达量均高于野生型HPV58L1骨架；此外，以N端截短联合C端置换的58L1突变体为骨架的嵌合蛋白58L1ΔN4h4/16dE-CS1、58L1ΔN4h4/16dE-CS2、58L1ΔN4h4/16dE-CS1的表达量均高于HPV58L1骨架及相应的C端截短的嵌合蛋白58L1ΔN4Ch4/16dE。
表1.嵌合L1蛋白表达量分析
25
实施例7：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,180mM 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。
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2所示，其中58L1ΔCDE/16dEs、58L1ΔCh4/16dEs、58L1ΔN4Ch4/16dE、58L1ΔN4Ch4/16dEs及58L1ΔCh4/16dE的DLS分析图如图2A至2E所示。58L1h4/16dE及58L1ΔCh4/16dE的粒径仅为9.672nm及12.28nm，提示这两个嵌合蛋白未组装成VLP。
表2嵌合蛋白DLS分析
嵌合蛋白名称 水力学直径(nm) PDI
58L1DE/16dEs 88 0.149
58L1h4/16dEs 102.4 0.162
58L1ΔN4h4/16dE 87.8 0.173
58L1ΔN4h4/16dEs 95.5 0.152
58L1h4/16dE 9.672 0.185
58L1ΔCDE/16dE 90 0.155
58L1ΔCh4/16dEs 114.6 0.158
58L1ΔN4Ch4/16dE 83.5 0.188
58L1ΔN4Ch4/16dEs 93 0.188
58L1ΔCh4/16dE 12.28 0.192
58L1ΔN4h4/16dE-CS1 96.5 0.194
58L1ΔN4h4/16dE-CS2 99.2 0.187
58L1ΔN4h4/16dE-CS3 102.4 0.143
实施例8：嵌合VLP的透射电镜观察
按实施例7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，58L1h4/16dE及58L1ΔCh4/16dE形成直径约10nm的嵌合五聚体，其余大肠杆菌及昆虫细胞表达的嵌合蛋白均可组装成嵌合VLP(cVLP)。昆虫细胞表达的cVLP直径约为50nm，大小均匀，形状规则；原核表达的cVLP直径也在45-50nm之间。部分结果如图3A至3D所示。铜网制备及电镜观察的方法均是公开的，例如专利CN 101148661 B。
实施例9：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、10μgHPV58 L1 VLP、10μg或30μg嵌合五聚体，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15种HPV假病毒对免疫血清的中和抗体滴度进行检测，HPV58L1VLP免疫血清的HPV58中和抗体滴度为409600，未检测到针对其他型别的交叉中和抗体；10μg 58L1ΔCh4/16dE嵌合五聚体免疫血清的HPV58中和抗体滴度为128000，但交叉中和活性低，仅检测到了HPV16中和抗体(滴度约50)；cVLP及30μg嵌合五聚体的中和抗体检测结果如表3所示。58L1ΔCDE/16dEs cVLP诱发的针对骨架HPV58的中和抗体水平显著低于其他cVLP及HPV58L1 VLP，诱发的交叉中和抗体水平也很低。其余cVLP及嵌合五聚体免疫小鼠后，均可诱发高水平的HPV58中和抗体(滴度>105)，与HPV58L1VLP无统计学差异，并可诱发较高水平的交叉中和抗体。其中58L1ΔCh4/16dEs、58L1ΔN4Ch4/16dE、58L1ΔN4Ch4/16dEs、C端置换改造的cVLP及58L1ΔCh4/16dE五聚体免疫血清不仅中和HPV58的滴度高，还可中和其余14种检测的假病毒。特别是58L1ΔN4Ch4/16dE和58L1ΔN4Ch4/16dE-CS1 cVLP免疫血清，中和HPV16、-18、-57假病毒的滴度均在400以上。值得注意的是，C端置换改造后，58L1ΔN4h4/16dE-CS1不仅表达水平较58L1ΔN4Ch4/16dE显著提高，其免疫血清中和优势型别(HPV16、-18、-57)的抗体滴度也有所提高。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。
因此，本发明涉及的cVLP或嵌合五聚体可作为广谱HPV疫苗的候选，可与不同优势高危型别HPV的L1VLP、cVLP或嵌合籽粒联合免疫，构建成本较低的广谱疫苗，具有极大的研发价值。
表3不同cVLP或嵌合五聚体在小鼠中诱发的中和抗体滴度
*ND：血清1:10稀释时未检测到中和抗体。
序列表
<110> 中国医学科学院基础医学研究所
<120> 一种人乳头瘤病毒58型嵌合蛋白及其用途
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Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
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Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
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Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
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Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr
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Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro
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Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala
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Ile Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn
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Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala Glu
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Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser Thr His Asn
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Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val Ser Thr Asn Pro Asn
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Thr Val Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Val Ala Arg
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Leu Gly Leu Tyr Ser Arg Thr Thr Gln Gln Val Lys Val Val Asp Pro
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Ala Phe Val Thr Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala
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Tyr Glu Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Ser Ser Asn Asp
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Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val Ala
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Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile Arg Tyr Ser
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Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg Ser Gly Lys Ser Ile
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Gly Ala Lys Val His Tyr Tyr Tyr Asp Leu Ser Thr Ile Asp Pro Ala
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Glu Glu Ile Glu Leu Gln Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr
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Ser His Ala Ala Ser Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile
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Tyr Ala Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val Pro Ser
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Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser Gly Pro Asp Ile
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His Pro Ser Tyr Tyr Met Leu Arg Lys Arg Arg Lys Arg Leu Pro Tyr
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Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile
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Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile
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Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile
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Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp
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Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
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Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
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Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
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Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
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Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
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Asp Thr Glu Thr Ser Asn Arg Tyr Tyr Lys Thr Cys Lys Gln Ala Gly
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Thr Cys Pro Pro Asp Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
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Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
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Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
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Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
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Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
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Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
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Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
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Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
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Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
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Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
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Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
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Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
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Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
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Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
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Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
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Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
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Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
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Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu
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Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys
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Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
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Leu Leu Gln Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala
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Pro Thr Thr Arg Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
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<211> 486
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(484)
<223> 58L1ΔCDE/16dEs
<400> 8
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
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Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
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Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
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Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
50 55 60
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
65 70 75 80
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
85 90 95
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
115 120 125
Asp Thr Glu Thr Ser Asn Arg Tyr Tyr Lys Thr Cys Lys Gln Ala Gly
130 135 140
Thr Cys Pro Pro Asp Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
145 150 155 160
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
165 170 175
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
180 185 190
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
195 200 205
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
210 215 220
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
225 230 235 240
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
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Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
260 265 270
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
275 280 285
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
290 295 300
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
305 310 315 320
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
325 330 335
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355 360 365
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
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Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
385 390 395 400
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
405 410 415
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
420 425 430
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu
435 440 445
Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys
450 455 460
Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
465 470 475 480
Leu Leu Gln Ser Gly Leu
485
<210> 9
<211> 509
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(509)
<223> 58L1h4/16dEs
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Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
1 5 10 15
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
20 25 30
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
35 40 45
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
50 55 60
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
65 70 75 80
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
85 90 95
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
115 120 125
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
130 135 140
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
145 150 155 160
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
165 170 175
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
180 185 190
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
195 200 205
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
210 215 220
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
225 230 235 240
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
245 250 255
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
260 265 270
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
275 280 285
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly
340 345 350
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
355 360 365
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
370 375 380
Glu Ile Met Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp
385 390 395 400
Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr
405 410 415
Tyr Arg Phe Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Tyr Lys Thr
420 425 430
Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Pro Pro Lys Glu Lys Glu
435 440 445
Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
450 455 460
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
465 470 475 480
Gln Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr
485 490 495
Thr Arg Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
500 505
<210> 10
<211> 484
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(484)
<223> 58L1ΔCh4/16dEs
<400> 10
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
1 5 10 15
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
20 25 30
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
35 40 45
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
50 55 60
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
65 70 75 80
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
85 90 95
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
115 120 125
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
130 135 140
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
145 150 155 160
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
165 170 175
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
180 185 190
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
195 200 205
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
210 215 220
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
225 230 235 240
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
245 250 255
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
260 265 270
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
275 280 285
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly
340 345 350
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
355 360 365
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
370 375 380
Glu Ile Met Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp
385 390 395 400
Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr
405 410 415
Tyr Arg Phe Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Tyr Lys Thr
420 425 430
Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Pro Pro Lys Glu Lys Glu
435 440 445
Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
450 455 460
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
465 470 475 480
Gln Ser Gly Leu
<210> 11
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(514)
<223> 58L1ΔN4h4/16dE
<400> 11
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Pro
435 440 445
Pro Lys Glu Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val
450 455 460
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
465 470 475 480
Arg Lys Phe Leu Leu Gln Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys
485 490 495
Arg Ser Ala Pro Thr Thr Arg Ala Pro Ser Thr Lys Arg Lys Lys Val
500 505 510
Lys Lys
<210> 12
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(489)
<223> 58L1ΔN4Ch4/16dE
<400> 12
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Pro
435 440 445
Pro Lys Glu Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val
450 455 460
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
465 470 475 480
Arg Lys Phe Leu Leu Gln Ser Gly Leu
485
<210> 13
<211> 508
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(508)
<223> 58L1ΔN4h4/16dEs
<400> 13
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Pro Pro Lys Glu Lys Glu Asp
435 440 445
Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe
450 455 460
Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln
465 470 475 480
Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr Thr
485 490 495
Arg Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
500 505
<210> 14
<211> 483
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(483)
<223> 58L1ΔN4Ch4/16dEs
<400> 14
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Pro Pro Lys Glu Lys Glu Asp
435 440 445
Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe
450 455 460
Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln
465 470 475 480
Ser Gly Leu
<210> 15
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(514)
<223> 58L1ΔN4h4/16dE-CS1
<400> 15
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Pro
435 440 445
Pro Lys Glu Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val
450 455 460
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
465 470 475 480
Arg Lys Phe Leu Leu Gln Ser Gly Leu Lys Ala Gly Pro Ser Leu Ala
485 490 495
Gly Ser Ala Pro Thr Thr Ser Ala Pro Ser Thr Gly Gly Ser Ala Val
500 505 510
Gly Ser
<210> 16
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(514)
<223> 58L1ΔN4h4/16dE-CS2
<400> 16
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Pro
435 440 445
Pro Lys Glu Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val
450 455 460
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
465 470 475 480
Arg Lys Phe Leu Leu Gln Ser Gly Leu Gly Ala Gly Pro Ser Leu Ala
485 490 495
Gly Ser Ala Pro Thr Thr Ser Ala Pro Ser Thr Gly Gly Ser Ala Val
500 505 510
Gly Ser
<210> 17
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(514)
<223> 58L1ΔN4h4/16dE-CS3
<400> 17
Met Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr
20 25 30
Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser
35 40 45
Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys
145 150 155 160
Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn
165 170 175
Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly
195 200 205
Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser
210 215 220
Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu
260 265 270
Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe
275 280 285
Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met
370 375 380
Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Leu Tyr Lys Thr Cys Lys
420 425 430
Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Pro
435 440 445
Pro Lys Glu Lys Glu Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val
450 455 460
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
465 470 475 480
Arg Lys Phe Leu Leu Gln Ser Gly Leu Lys Ala Gly Pro Ser Leu Ala
485 490 495
Gly Ser Ala Pro Thr Thr Arg Ala Pro Ser Thr Gly Gly Ser Ala Val
500 505 510
Gly Ser
<210> 18
<211> 507
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(507)
<223> 58L1h4/16dE
<400> 18
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
1 5 10 15
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
20 25 30
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
35 40 45
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
50 55 60
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
65 70 75 80
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
85 90 95
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
115 120 125
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
130 135 140
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
145 150 155 160
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
165 170 175
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
180 185 190
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
195 200 205
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
210 215 220
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
225 230 235 240
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
245 250 255
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
260 265 270
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
275 280 285
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly
340 345 350
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
355 360 365
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
370 375 380
Glu Ile Met Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp
385 390 395 400
Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Gln Leu Tyr Lys
405 410 415
Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val
420 425 430
Glu Gly Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu Lys Glu Asp Pro
435 440 445
Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser
450 455 460
Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser
465 470 475 480
Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr Thr Arg
485 490 495
Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
500 505
<210> 19
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(482)
<223> 58L1ΔCh4/16dE
<400> 19
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
1 5 10 15
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
20 25 30
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
35 40 45
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
50 55 60
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
65 70 75 80
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
85 90 95
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
100 105 110
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
115 120 125
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
130 135 140
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
145 150 155 160
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
165 170 175
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
180 185 190
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
195 200 205
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
210 215 220
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
225 230 235 240
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
245 250 255
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
260 265 270
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
275 280 285
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly
340 345 350
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
355 360 365
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
370 375 380
Glu Ile Met Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp
385 390 395 400
Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Gln Leu Tyr Lys
405 410 415
Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val
420 425 430
Glu Gly Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu Lys Glu Asp Pro
435 440 445
Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser
450 455 460
Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser
465 470 475 480
Gly Leu
<210> 20
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1536)
<223> 58L1DE/16dEs nt
<400> 20
atgtccgtct ggcgtccgag tgaagccacg gtgtatttgc caccagtccc ggttagcaaa 60
gtagtgtcca ccgacgagta cgtgagccgc acttcgatct actactacgc aggaagctcg 120
cgcctgttgg ctgtaggcaa cccctatttc agtatcaaat caccgaataa taacaagaag 180
gttttagtgc cgaaggtatc tgggctgcaa taccgtgtgt tccgtgtccg cctgccggac 240
ccaaataaat ttggatttcc tgatacttcc ttctataatc ctgacaccca acgtctggta 300
tgggcctgtg ttggacttga aattggacgc ggtcaacctc tgggcgttgg cgtaagtggt 360
cacccatatc tgaacaagtt cgacgatact gagacatcca accgctatta taagacatgt 420
aagcaggcag gcacgtgtcc cccagatcct gctcagcccg ggtcggacaa ccgtgaatgc 480
ctttcaatgg actacaagca aacccaactt tgcttaatcg ggtgtaagcc ccctacaggc 540
gagcattggg ggaagggggt ggcgtgtaat aacaatgcag ctgctactga ttgtcctccc 600
cttgagctgt tcaatagtat tattgaagac ggagacatgg tagatactgg gtttggctgt 660
atggactttg ggacgcttca agcgaataag tcagatgtac ccattgacat ttgcaattcg 720
acttgtaagt atcccgacta ccttaaaatg gcttccgagc catatggcga cagcctgttt 780
tttttcctgc gccgcgaaca aatgttcgtt cgtcactttt tcaaccgtgc tgggaaactt 840
ggcgaggctg tcccggacga cttatacatt aaggggagcg gaaacacagc tgtcatccaa 900
tcttccgcgt tcttcccgac gccatcaggg tcgatcgtga ccagcgaaag ccagctgttc 960
aataaaccgt actggttgca acgtgctcag ggacataata acggcatttg ttggggcaac 1020
cagcttttcg taacagtagt agacacgact cgttcaacta acatgacgtt gtgtacggag 1080
gttactaagg aaggcacata taagaacgac aattttaaag agtacgtacg tcatgtcgag 1140
gagtacgatc tgcaatttgt cttccaactt tgcaagatta cccttactgc tgagatcatg 1200
acatatatcc atacaatgga ttctaatatt ttggaagact ggcagttcgg ccttactccc 1260
cctcctagcg cgagtcttca ggatacctat cgttttgtca caagtcaagc tatcacctgc 1320
cagaagaccg caccgccgaa ggaaaaagaa gatccgctta ataagtacac cttctgggag 1380
gttaacttga aggaaaagtt cagcgccgac ttggatcaat ttcccttagg tcgtaaattc 1440
cttttgcagt ctggattgaa agctaaaccg cgcttaaaac gctccgctcc tactacgcgt 1500
gcccccagca cgaagcgcaa aaaggtaaaa aagtaa 1536
<210> 21
<211> 1461
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1461)
<223> 58L1ΔCDE/16dEs nt
<400> 21
atgagcgtgt ggagaccctc cgaagcaacc gtctatctcc cacccgtccc cgtcagcaaa 60
gtcgtgtcaa ccgacgagta cgtcagcagg acctcaatct actactacgc tggttccagt 120
cgcttgctcg ccgtcggcaa cccctacttc agtattaagt ccccaaacaa caacaagaag 180
gtgctggtcc caaaagtgag cggcctgcaa taccgcgtgt tccgcgtcag gctgcccgac 240
ccaaacaagt tcggcttccc cgacaccagc ttctacaatc ccgacaccca gaggctcgtg 300
tgggcctgcg tgggcttgga gatcggccgc ggccaaccac tcggcgtcgg cgtgtccggc 360
cacccctacc tgaacaagtt cgacgatacc gagacatcca atcgctatta caagacttgc 420
aagcaggctg gtacctgccc ccctgaccca gcccaacccg gcagcgacaa tcgcgagtgt 480
ctgagcatgg actacaagca gacccagctg tgcctgatcg gctgcaagcc cccaaccggt 540
gaacactggg gcaagggcgt cgcttgcaac aacaacgccg ccgccaccga ctgcccaccc 600
ctcgagttgt tcaacagcat catcgaagac ggcgatatgg tggacaccgg cttcggctgt 660
atggatttcg gcaccctcca agccaacaag tccgacgtcc ccatcgacat ctgcaattcc 720
acctgtaagt accccgacta cctgaagatg gcatccgagc cctacggcga cagcctcttc 780
ttcttcctcc gcagggaaca aatgttcgtc cgccatttct tcaaccgcgc cggcaagttg 840
ggcgaagccg tgcccgacga tttgtacatc aagggcagtg gcaacaccgc cgtcattcag 900
tcctccgcat tcttcccaac cccctccggc agcatcgtca caagcgagag ccagctgttc 960
aacaagccct actggttgca aagggcccag ggccacaata acggcatctg ttggggcaac 1020
caactgttcg tcacagtcgt cgacacaacc aggtcaacca acatgaccct gtgtaccgag 1080
gtgaccaagg agggcaccta caagaacgac aacttcaaag agtacgtgag gcacgtcgag 1140
gagtacgatc tgcaattcgt cttccaattg tgtaagatca ccttgaccgc cgaaatcatg 1200
acctacatcc acaccatgga cagtaacatc ctcgaagatt ggcagttcgg cctgaccccc 1260
ccacccagcg catccctgca agatacctac cgcttcgtca caagtcaagc catcacctgt 1320
cagaagaccg ctccacccaa ggagaaagag gaccccctga acaagtacac cttctgggaa 1380
gtcaatctga aagagaaatt cagcgccgac ttggaccaat tcccactcgg caggaagttc 1440
ctgctgcaga gcggcttgta a 1461
<210> 22
<211> 1530
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1530)
<223> 58L1h4/16dEs nt
<400> 22
atgtctgttt ggcgtccgtc ggaagccacc gtttatctgc cacctgtgcc ggtgagcaaa 60
gttgtctcga cggatgaata tgtgagtcgc accagcattt attactatgc aggttcttcg 120
cgtttgttgg cggtgggcaa tccgtacttt agcatcaaat cgcctaacaa taacaaaaag 180
gttttagtgc cgaaagtgag tggcctgcaa tatcgtgtgt ttcgcgtccg cctgccggat 240
ccaaataagt ttgggttccc tgacaccagc ttttataacc cagataccca gcgtctggtt 300
tgggcgtgcg tgggtttgga aattggccgc ggccaaccgt tgggtgtggg tgtttctggc 360
catccttact tgaataaatt tgatgacacc gaaacttcga accgttatcc ggcccagcca 420
ggcagcgaca atcgggaatg tctgtcgatg gattacaaac aaacgcagct gtgcttaatt 480
gggtgtaagc cgccaaccgg tgagcactgg ggcaaaggcg ttgcatgtaa caataacgcg 540
gcggccacgg attgcccgcc attggaattg tttaatagta tcatcgaaga tggtgacatg 600
gtggataccg gttttggctg catggatttc ggcacgttac aggccaacaa aagcgacgtg 660
ccaattgata tctgcaattc tacctgtaag tacccggatt atctgaagat ggcgtcggaa 720
ccgtacgggg atagcctgtt tttctttctg cgccgcgagc agatgttcgt tcgtcatttc 780
tttaaccgcg cgggtaaatt gggcgaggcc gttccggacg atttgtatat taaaggctcg 840
gggaataccg cagtgattca gagtagcgcg tttttcccta ccccatctgg ttcgatcgtg 900
accagcgaat cgcaactgtt taacaagccg tattggctgc agcgtgcgca gggccacaat 960
aacggcatct gctggggtaa tcaactgttt gttacggtcg tggacaccac gcggagtacg 1020
aacatgacgc tgtgtaccga agtgactaaa gagggtacct acaagaatga taactttaaa 1080
gagtatgttc gccatgttga agagtatgat ttgcagtttg tgttccaatt gtgcaaaatt 1140
acgttaacgg ccgagatcat gacttacatt cacaccatgg atagcaatat tctggaagac 1200
tggcagttcg gcctgacgcc gccaccttct gcctcgctgc aggataccta tcgctttgtg 1260
accagccagg cgatcacgtg ccaaaaatat aaaacctgca aacaggccgg cacctgcccg 1320
ccagatccgc caaaggaaaa agaggacccg ttgaacaaat atacgttttg ggaagttaat 1380
ttgaaggaaa aattttcggc cgatctggac cagttcccac tgggccgtaa atttctgtta 1440
caaagtggtt tgaaagccaa gccgcgcttg aaacgtagcg cgccaaccac gcgggcgccg 1500
tctaccaaac gcaagaaagt taagaaataa 1530
<210> 23
<211> 1455
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1455)
<223> 58L1ΔCh4/16dEs nt
<400> 23
atgagcgtgt ggagaccctc cgaagcaacc gtctatctcc cacccgtccc cgtcagcaaa 60
gtcgtgtcaa ccgacgagta cgtcagcagg acctcaatct actactacgc tggttccagt 120
cgcttgctcg ccgtcggcaa cccctacttc agtattaagt ccccaaacaa caacaagaag 180
gtgctggtcc caaaagtgag cggcctgcaa taccgcgtgt tccgcgtcag gctgcccgac 240
ccaaacaagt tcggcttccc cgacaccagc ttctacaatc ccgacaccca gaggctcgtg 300
tgggcctgcg tgggcttgga gatcggccgc ggccaaccac tcggcgtcgg cgtgtccggc 360
cacccctacc tgaacaagtt cgacgatacc gagacatcca atcgctaccc agcccaaccc 420
ggcagcgaca atcgcgagtg tctgagcatg gactacaagc agacccagct gtgcctgatc 480
ggctgcaagc ccccaaccgg tgaacactgg ggcaagggcg tcgcttgcaa caacaacgcc 540
gccgccaccg actgcccacc cctcgagttg ttcaacagca tcatcgaaga cggcgatatg 600
gtggacaccg gcttcggctg tatggatttc ggcaccctcc aagccaacaa gtccgacgtc 660
cccatcgaca tctgcaattc cacctgtaag taccccgact acctgaagat ggcatccgag 720
ccctacggcg acagcctctt cttcttcctc cgcagggaac aaatgttcgt ccgccatttc 780
ttcaaccgcg ccggcaagtt gggcgaagcc gtgcccgacg atttgtacat caagggcagt 840
ggcaacaccg ccgtcattca gtcctccgca ttcttcccaa ccccctccgg cagcatcgtc 900
acaagcgaga gccagctgtt caacaagccc tactggttgc aaagggccca gggccacaat 960
aacggcatct gttggggcaa ccaactgttc gtcacagtcg tcgacacaac caggtcaacc 1020
aacatgaccc tgtgtaccga ggtgaccaag gagggcacct acaagaacga caacttcaaa 1080
gagtacgtga ggcacgtcga ggagtacgat ctgcaattcg tcttccaatt gtgtaagatc 1140
accttgaccg ccgaaatcat gacctacatc cacaccatgg acagtaacat cctcgaagat 1200
tggcagttcg gcctgacccc cccacccagc gcatccctgc aagataccta ccgcttcgtc 1260
acaagtcaag ccatcacctg tcagaagtac aagacctgca agcaggccgg tacctgcccc 1320
cctgacccac ccaaggagaa agaggacccc ctgaacaagt acaccttctg ggaagtcaat 1380
ctgaaagaga aattcagcgc cgacttggac caattcccac tcggcaggaa gttcctgctg 1440
cagagcggct tgtaa 1455
<210> 24
<211> 1545
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1545)
<223> 58L1ΔN4h4/16dE nt
<400> 24
atgcgtccgt cggaagccac cgtttatctg ccacctgtgc cggtgagcaa agttgtctcg 60
acggatgaat atgtgagtcg caccagcatt tattactatg caggttcttc gcgtttgttg 120
gcggtgggca atccgtactt tagcatcaaa tcgcctaaca ataacaaaaa ggttttagtg 180
ccgaaagtga gtggcctgca atatcgtgtg tttcgcgtcc gcctgccgga tccaaataag 240
tttgggttcc ctgacaccag cttttataac ccagataccc agcgtctggt ttgggcgtgc 300
gtgggtttgg aaattggccg cggccaaccg ttgggtgtgg gtgtttctgg ccatccttac 360
ttgaataaat ttgatgacac cgaaacttcg aaccgttatc cggcccagcc aggcagcgac 420
aatcgggaat gtctgtcgat ggattacaaa caaacgcagc tgtgcttaat tgggtgtaag 480
ccgccaaccg gtgagcactg gggcaaaggc gttgcatgta acaataacgc ggcggccacg 540
gattgcccgc cattggaatt gtttaatagt atcatcgaag atggtgacat ggtggatacc 600
ggttttggct gcatggattt cggcacgtta caggccaaca aaagcgacgt gccaattgat 660
atctgcaatt ctacctgtaa gtacccggat tatctgaaga tggcgtcgga accgtacggg 720
gatagcctgt ttttctttct gcgccgcgag cagatgttcg ttcgtcattt ctttaaccgc 780
gcgggtaaat tgggcgaggc cgttccggac gatttgtata ttaaaggctc ggggaatacc 840
gcagtgattc agagtagcgc gtttttccct accccatctg gttcgatcgt gaccagcgaa 900
tcgcaactgt ttaacaagcc gtattggctg cagcgtgcgc agggccacaa taacggcatc 960
tgctggggta atcaactgtt tgttacggtc gtggacacca cgcggagtac gaacatgacg 1020
ctgtgtaccg aagtgactaa agagggtacc tacaagaatg ataactttaa agagtatgtt 1080
cgccatgttg aagagtatga tttgcagttt gtgttccaat tgtgcaaaat tacgttaacg 1140
gccgagatca tgacttacat tcacaccatg gatagcaata ttctggaaga ctggcagttc 1200
ggcctgacgc cgccaccttc tgcctcgctg caggatacct atcgctttgt gaccagccag 1260
gcgatcacgt gccaaaaact gtataaaacc tgcaaacagg ccggcacctg cccgccagat 1320
attatcccga aagttgaagg tccgccaaag gaaaaagagg acccgttgaa caaatatacg 1380
ttttgggaag ttaatttgaa ggaaaaattt tcggccgatc tggaccagtt cccactgggc 1440
cgtaaatttc tgttacaaag tggtttgaaa gccaagccgc gcttgaaacg tagcgcgcca 1500
accacgcggg cgccgtctac caaacgcaag aaagttaaga aataa 1545
<210> 25
<211> 1470
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1470)
<223> 58L1ΔN4Ch4/16dE nt
<400> 25
atgagaccct ccgaagcaac cgtctatctc ccacccgtcc ccgtcagcaa agtcgtgtca 60
accgacgagt acgtcagcag gacctcaatc tactactacg ctggttccag tcgcttgctc 120
gccgtcggca acccctactt cagtattaag tccccaaaca acaacaagaa ggtgctggtc 180
ccaaaagtga gcggcctgca ataccgcgtg ttccgcgtca ggctgcccga cccaaacaag 240
ttcggcttcc ccgacaccag cttctacaat cccgacaccc agaggctcgt gtgggcctgc 300
gtgggcttgg agatcggccg cggccaacca ctcggcgtcg gcgtgtccgg ccacccctac 360
ctgaacaagt tcgacgatac cgagacatcc aatcgctacc cagcccaacc cggcagcgac 420
aatcgcgagt gtctgagcat ggactacaag cagacccagc tgtgcctgat cggctgcaag 480
cccccaaccg gtgaacactg gggcaagggc gtcgcttgca acaacaacgc cgccgccacc 540
gactgcccac ccctcgagtt gttcaacagc atcatcgaag acggcgatat ggtggacacc 600
ggcttcggct gtatggattt cggcaccctc caagccaaca agtccgacgt ccccatcgac 660
atctgcaatt ccacctgtaa gtaccccgac tacctgaaga tggcatccga gccctacggc 720
gacagcctct tcttcttcct ccgcagggaa caaatgttcg tccgccattt cttcaaccgc 780
gccggcaagt tgggcgaagc cgtgcccgac gatttgtaca tcaagggcag tggcaacacc 840
gccgtcattc agtcctccgc attcttccca accccctccg gcagcatcgt cacaagcgag 900
agccagctgt tcaacaagcc ctactggttg caaagggccc agggccacaa taacggcatc 960
tgttggggca accaactgtt cgtcacagtc gtcgacacaa ccaggtcaac caacatgacc 1020
ctgtgtaccg aggtgaccaa ggagggcacc tacaagaacg acaacttcaa agagtacgtg 1080
aggcacgtcg aggagtacga tctgcaattc gtcttccaat tgtgtaagat caccttgacc 1140
gccgaaatca tgacctacat ccacaccatg gacagtaaca tcctcgaaga ttggcagttc 1200
ggcctgaccc ccccacccag cgcatccctg caagatacct accgcttcgt cacaagtcaa 1260
gccatcacct gtcagaagct gtacaagacc tgcaagcagg ccggtacctg cccccctgac 1320
atcatcccca aggtcgaagg accacccaag gagaaagagg accccctgaa caagtacacc 1380
ttctgggaag tcaatctgaa agagaaattc agcgccgact tggaccaatt cccactcggc 1440
aggaagttcc tgctgcagag cggcttgtaa 1470
<210> 26
<211> 1527
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1527)
<223> 58L1ΔN4h4/16dEs
<400> 26
atgcgtccgt cggaagccac cgtttatctg ccacctgtgc cggtgagcaa agttgtctcg 60
acggatgaat atgtgagtcg caccagcatt tattactatg caggttcttc gcgtttgttg 120
gcggtgggca atccgtactt tagcatcaaa tcgcctaaca ataacaaaaa ggttttagtg 180
ccgaaagtga gtggcctgca atatcgtgtg tttcgcgtcc gcctgccgga tccaaataag 240
tttgggttcc ctgacaccag cttttataac ccagataccc agcgtctggt ttgggcgtgc 300
gtgggtttgg aaattggccg cggccaaccg ttgggtgtgg gtgtttctgg ccatccttac 360
ttgaataaat ttgatgacac cgaaacttcg aaccgttatc cggcccagcc aggcagcgac 420
aatcgggaat gtctgtcgat ggattacaaa caaacgcagc tgtgcttaat tgggtgtaag 480
ccgccaaccg gtgagcactg gggcaaaggc gttgcatgta acaataacgc ggcggccacg 540
gattgcccgc cattggaatt gtttaatagt atcatcgaag atggtgacat ggtggatacc 600
ggttttggct gcatggattt cggcacgtta caggccaaca aaagcgacgt gccaattgat 660
atctgcaatt ctacctgtaa gtacccggat tatctgaaga tggcgtcgga accgtacggg 720
gatagcctgt ttttctttct gcgccgcgag cagatgttcg ttcgtcattt ctttaaccgc 780
gcgggtaaat tgggcgaggc cgttccggac gatttgtata ttaaaggctc ggggaatacc 840
gcagtgattc agagtagcgc gtttttccct accccatctg gttcgatcgt gaccagcgaa 900
tcgcaactgt ttaacaagcc gtattggctg cagcgtgcgc agggccacaa taacggcatc 960
tgctggggta atcaactgtt tgttacggtc gtggacacca cgcggagtac gaacatgacg 1020
ctgtgtaccg aagtgactaa agagggtacc tacaagaatg ataactttaa agagtatgtt 1080
cgccatgttg aagagtatga tttgcagttt gtgttccaat tgtgcaaaat tacgttaacg 1140
gccgagatca tgacttacat tcacaccatg gatagcaata ttctggaaga ctggcagttc 1200
ggcctgacgc cgccaccttc tgcctcgctg caggatacct atcgctttgt gaccagccag 1260
gcgatcacgt gccaaaaact gtataaaacc tgcaaacagg ccggcacctg cccgccagat 1320
attccgccaa aggaaaaaga ggacccgttg aacaaatata cgttttggga agttaatttg 1380
aaggaaaaat tttcggccga tctggaccag ttcccactgg gccgtaaatt tctgttacaa 1440
agtggtttga aagccaagcc gcgcttgaaa cgtagcgcgc caaccacgcg ggcgccgtct 1500
accaaacgca agaaagttaa gaaataa 1527
<210> 27
<211> 1452
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1452)
<223> 58L1ΔN4Ch4/16dEs nt
<400> 27
atgagaccct ccgaagcaac cgtctatctc ccacccgtcc ccgtcagcaa agtcgtgtca 60
accgacgagt acgtcagcag gacctcaatc tactactacg ctggttccag tcgcttgctc 120
gccgtcggca acccctactt cagtattaag tccccaaaca acaacaagaa ggtgctggtc 180
ccaaaagtga gcggcctgca ataccgcgtg ttccgcgtca ggctgcccga cccaaacaag 240
ttcggcttcc ccgacaccag cttctacaat cccgacaccc agaggctcgt gtgggcctgc 300
gtgggcttgg agatcggccg cggccaacca ctcggcgtcg gcgtgtccgg ccacccctac 360
ctgaacaagt tcgacgatac cgagacatcc aatcgctacc cagcccaacc cggcagcgac 420
aatcgcgagt gtctgagcat ggactacaag cagacccagc tgtgcctgat cggctgcaag 480
cccccaaccg gtgaacactg gggcaagggc gtcgcttgca acaacaacgc cgccgccacc 540
gactgcccac ccctcgagtt gttcaacagc atcatcgaag acggcgatat ggtggacacc 600
ggcttcggct gtatggattt cggcaccctc caagccaaca agtccgacgt ccccatcgac 660
atctgcaatt ccacctgtaa gtaccccgac tacctgaaga tggcatccga gccctacggc 720
gacagcctct tcttcttcct ccgcagggaa caaatgttcg tccgccattt cttcaaccgc 780
gccggcaagt tgggcgaagc cgtgcccgac gatttgtaca tcaagggcag tggcaacacc 840
gccgtcattc agtcctccgc attcttccca accccctccg gcagcatcgt cacaagcgag 900
agccagctgt tcaacaagcc ctactggttg caaagggccc agggccacaa taacggcatc 960
tgttggggca accaactgtt cgtcacagtc gtcgacacaa ccaggtcaac caacatgacc 1020
ctgtgtaccg aggtgaccaa ggagggcacc tacaagaacg acaacttcaa agagtacgtg 1080
aggcacgtcg aggagtacga tctgcaattc gtcttccaat tgtgtaagat caccttgacc 1140
gccgaaatca tgacctacat ccacaccatg gacagtaaca tcctcgaaga ttggcagttc 1200
ggcctgaccc ccccacccag cgcatccctg caagatacct accgcttcgt cacaagtcaa 1260
gccatcacct gtcagaagct gtacaagact tgcaagcagg ctggtacctg cccccctgac 1320
atcccaccca aggagaaaga ggaccccctg aacaagtaca ccttctggga agtcaatctg 1380
aaagagaaat tcagcgccga cttggaccaa ttcccactcg gcaggaagtt cctgctgcag 1440
agcggcttgt aa 1452
<210> 28
<211> 1545
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1545)
<223> 58L1ΔN4h4/16dE-CS1 nt
<400> 28
atgagaccct ccgaagcaac cgtctatctc ccacccgtcc ccgtcagcaa agtcgtgtca 60
accgacgagt acgtcagcag gacctcaatc tactactacg ctggttccag tcgcttgctc 120
gccgtcggca acccctactt cagtattaag tccccaaaca acaacaagaa ggtgctggtc 180
ccaaaagtga gcggcctgca ataccgcgtg ttccgcgtca ggctgcccga cccaaacaag 240
ttcggcttcc ccgacaccag cttctacaat cccgacaccc agaggctcgt gtgggcctgc 300
gtgggcttgg agatcggccg cggccaacca ctcggcgtcg gcgtgtccgg ccacccctac 360
ctgaacaagt tcgacgatac cgagacatcc aatcgctacc cagcccaacc cggcagcgac 420
aatcgcgagt gtctgagcat ggactacaag cagacccagc tgtgcctgat cggctgcaag 480
cccccaaccg gtgaacactg gggcaagggc gtcgcttgca acaacaacgc cgccgccacc 540
gactgcccac ccctcgagtt gttcaacagc atcatcgaag acggcgatat ggtggacacc 600
ggcttcggct gtatggattt cggcaccctc caagccaaca agtccgacgt ccccatcgac 660
atctgcaatt ccacctgtaa gtaccccgac tacctgaaga tggcatccga gccctacggc 720
gacagcctct tcttcttcct ccgcagggaa caaatgttcg tccgccattt cttcaaccgc 780
gccggcaagt tgggcgaagc cgtgcccgac gatttgtaca tcaagggcag tggcaacacc 840
gccgtcattc agtcctccgc attcttccca accccctccg gcagcatcgt cacaagcgag 900
agccagctgt tcaacaagcc ctactggttg caaagggccc agggccacaa taacggcatc 960
tgttggggca accaactgtt cgtcacagtc gtcgacacaa ccaggtcaac caacatgacc 1020
ctgtgtaccg aggtgaccaa ggagggcacc tacaagaacg acaacttcaa agagtacgtg 1080
aggcacgtcg aggagtacga tctgcaattc gtcttccaat tgtgtaagat caccttgacc 1140
gccgaaatca tgacctacat ccacaccatg gacagtaaca tcctcgaaga ttggcagttc 1200
ggcctgaccc ccccacccag cgcatccctg caagatacct accgcttcgt cacaagtcaa 1260
gccatcacct gtcagaagct gtacaagacc tgcaagcagg ccggtacctg cccccctgac 1320
atcatcccca aggtcgaagg accacccaag gagaaagagg accccctgaa caagtacacc 1380
ttctgggaag tcaatctgaa agagaaattc agcgccgact tggaccaatt cccactcggc 1440
aggaagttcc tgctgcagag cggcttgaaa gccggccctt cgttggccgg ctcggcccct 1500
acgacctcgg cgccgtcgac gggtggcagc gccgtgggta gctaa 1545
<210> 29
<211> 1545
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1545)
<223> 58L1ΔN4h4/16dE-CS2 nt
<400> 29
atgagaccct ccgaagcaac cgtctatctc ccacccgtcc ccgtcagcaa agtcgtgtca 60
accgacgagt acgtcagcag gacctcaatc tactactacg ctggttccag tcgcttgctc 120
gccgtcggca acccctactt cagtattaag tccccaaaca acaacaagaa ggtgctggtc 180
ccaaaagtga gcggcctgca ataccgcgtg ttccgcgtca ggctgcccga cccaaacaag 240
ttcggcttcc ccgacaccag cttctacaat cccgacaccc agaggctcgt gtgggcctgc 300
gtgggcttgg agatcggccg cggccaacca ctcggcgtcg gcgtgtccgg ccacccctac 360
ctgaacaagt tcgacgatac cgagacatcc aatcgctacc cagcccaacc cggcagcgac 420
aatcgcgagt gtctgagcat ggactacaag cagacccagc tgtgcctgat cggctgcaag 480
cccccaaccg gtgaacactg gggcaagggc gtcgcttgca acaacaacgc cgccgccacc 540
gactgcccac ccctcgagtt gttcaacagc atcatcgaag acggcgatat ggtggacacc 600
ggcttcggct gtatggattt cggcaccctc caagccaaca agtccgacgt ccccatcgac 660
atctgcaatt ccacctgtaa gtaccccgac tacctgaaga tggcatccga gccctacggc 720
gacagcctct tcttcttcct ccgcagggaa caaatgttcg tccgccattt cttcaaccgc 780
gccggcaagt tgggcgaagc cgtgcccgac gatttgtaca tcaagggcag tggcaacacc 840
gccgtcattc agtcctccgc attcttccca accccctccg gcagcatcgt cacaagcgag 900
agccagctgt tcaacaagcc ctactggttg caaagggccc agggccacaa taacggcatc 960
tgttggggca accaactgtt cgtcacagtc gtcgacacaa ccaggtcaac caacatgacc 1020
ctgtgtaccg aggtgaccaa ggagggcacc tacaagaacg acaacttcaa agagtacgtg 1080
aggcacgtcg aggagtacga tctgcaattc gtcttccaat tgtgtaagat caccttgacc 1140
gccgaaatca tgacctacat ccacaccatg gacagtaaca tcctcgaaga ttggcagttc 1200
ggcctgaccc ccccacccag cgcatccctg caagatacct accgcttcgt cacaagtcaa 1260
gccatcacct gtcagaagct gtacaagacc tgcaagcagg ccggtacctg cccccctgac 1320
atcatcccca aggtcgaagg accacccaag gagaaagagg accccctgaa caagtacacc 1380
ttctgggaag tcaatctgaa agagaaattc agcgccgact tggaccaatt cccactcggc 1440
aggaagttcc tgctgcagag cggcttgggt gccggccctt cgttggccgg ctcggcccct 1500
acgacctcgg cgccgtcgac gggtggcagc gccgtgggta gctaa 1545
<210> 30
<211> 1545
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1545)
<223> 58L1ΔN4h4/16dE-CS3 nt
<400> 30
atgagaccct ccgaagcaac cgtctatctc ccacccgtcc ccgtcagcaa agtcgtgtca 60
accgacgagt acgtcagcag gacctcaatc tactactacg ctggttccag tcgcttgctc 120
gccgtcggca acccctactt cagtattaag tccccaaaca acaacaagaa ggtgctggtc 180
ccaaaagtga gcggcctgca ataccgcgtg ttccgcgtca ggctgcccga cccaaacaag 240
ttcggcttcc ccgacaccag cttctacaat cccgacaccc agaggctcgt gtgggcctgc 300
gtgggcttgg agatcggccg cggccaacca ctcggcgtcg gcgtgtccgg ccacccctac 360
ctgaacaagt tcgacgatac cgagacatcc aatcgctacc cagcccaacc cggcagcgac 420
aatcgcgagt gtctgagcat ggactacaag cagacccagc tgtgcctgat cggctgcaag 480
cccccaaccg gtgaacactg gggcaagggc gtcgcttgca acaacaacgc cgccgccacc 540
gactgcccac ccctcgagtt gttcaacagc atcatcgaag acggcgatat ggtggacacc 600
ggcttcggct gtatggattt cggcaccctc caagccaaca agtccgacgt ccccatcgac 660
atctgcaatt ccacctgtaa gtaccccgac tacctgaaga tggcatccga gccctacggc 720
gacagcctct tcttcttcct ccgcagggaa caaatgttcg tccgccattt cttcaaccgc 780
gccggcaagt tgggcgaagc cgtgcccgac gatttgtaca tcaagggcag tggcaacacc 840
gccgtcattc agtcctccgc attcttccca accccctccg gcagcatcgt cacaagcgag 900
agccagctgt tcaacaagcc ctactggttg caaagggccc agggccacaa taacggcatc 960
tgttggggca accaactgtt cgtcacagtc gtcgacacaa ccaggtcaac caacatgacc 1020
ctgtgtaccg aggtgaccaa ggagggcacc tacaagaacg acaacttcaa agagtacgtg 1080
aggcacgtcg aggagtacga tctgcaattc gtcttccaat tgtgtaagat caccttgacc 1140
gccgaaatca tgacctacat ccacaccatg gacagtaaca tcctcgaaga ttggcagttc 1200
ggcctgaccc ccccacccag cgcatccctg caagatacct accgcttcgt cacaagtcaa 1260
gccatcacct gtcagaagct gtacaagacc tgcaagcagg ccggtacctg cccccctgac 1320
atcatcccca aggtcgaagg accacccaag gagaaagagg accccctgaa caagtacacc 1380
ttctgggaag tcaatctgaa agagaaattc agcgccgact tggaccaatt cccactcggc 1440
aggaagttcc tgctgcagag cggcttgaaa gccggccctt cgttggccgg ctcggcccct 1500
acgaccagag cgccgtcgac gggtggcagc gccgtgggta gctaa 1545
<210> 31
<211> 1521
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1521)
<223> 58L1h4/16dE
<400> 31
atgagcgtct ggcgcccttc tgaagcgact gtttacctgc cgcccgtccc agtaagcaag 60
gttgtgagca ccgatgaata tgttagtcgt acatcgatct attactatgc tggaagctca 120
cgtttgctgg cggtaggtaa cccttatttt tccattaaaa gtcccaataa taataagaaa 180
gttttagtcc cgaaggtgtc tgggttgcaa taccgcgtat tccgtgtccg tcttccggac 240
ccgaataaat ttggcttccc cgatacttct ttttataatc ccgatactca gcgtttagta 300
tgggcctgtg ttgggcttga gatcggacgc gggcaacctc tgggggttgg cgtcagcggc 360
catccttatc ttaataaatt tgacgatacg gagaccagta accgctaccc ggctcagccg 420
gggtccgaca accgtgaatg tttgtccatg gattataagc agactcagct gtgtttgatc 480
ggttgtaaac ctcctacagg tgaacattgg ggcaaaggtg tagcctgcaa taataacgca 540
gcggctaccg attgtccgcc tttggagttg ttcaactcga ttatcgaaga cggtgatatg 600
gtagacactg ggttcggttg catggacttt gggacccttc aagccaataa gtccgacgtg 660
ccaatcgaca tttgcaactc tacatgcaag taccctgact atcttaagat ggcgtccgaa 720
ccgtacggag attctttgtt ttttttcctg cgtcgtgagc aaatgttcgt acgccatttc 780
ttcaaccgtg ctggaaagtt aggagaagct gttcctgatg atctgtatat taagggatct 840
gggaacacag cggttattca atcctccgct ttctttccaa cgccaagcgg gtccattgtg 900
accagtgaaa gccaattatt taacaagccg tactggttac aacgtgcgca gggacacaat 960
aatggaatct gctggggcaa tcaattgttc gtgacggttg ttgacacgac ccgttcgacc 1020
aatatgaccc tttgcactga ggtaactaag gagggtacgt acaaaaatga caattttaag 1080
gagtacgtcc gtcatgtcga ggagtatgat cttcagtttg tcttccagtt atgtaaaatt 1140
accttgaccg cggaaatcat gacgtatatc cacacgatgg actcaaacat cctggaggat 1200
tggcaattcg gtttgactcc gcccccgagc gcgtctcaat tgtataagac ttgtaagcag 1260
gcaggtacat gcccaccaga catcattcca aaagttgaag gaacgtgcca aaagacggca 1320
ccaccgaaag aaaaggagga ccccctgaac aaatatacgt tttgggaggt gaatttgaag 1380
gagaaattct cagcagactt agaccagttt ccgcttggcc gcaagtttct gttgcagtct 1440
gggttgaagg caaaaccgcg tcttaagcgt agcgccccca cgactcgcgc ccccagtaca 1500
aaacgtaaga aggttaaaaa g 1521
<210> 32
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> gene
<222> (1)..(1449)
<223> 58L1ΔCh4/16dE nt
<400> 32
atgagcgtgt ggagaccctc cgaagcaacc gtctatctcc cacccgtccc cgtcagcaaa 60
gtcgtgtcaa ccgacgagta cgtcagcagg acctcaatct actactacgc tggttccagt 120
cgcttgctcg ccgtcggcaa cccctacttc agtattaagt ccccaaacaa caacaagaag 180
gtgctggtcc caaaagtgag cggcctgcaa taccgcgtgt tccgcgtcag gctgcccgac 240
ccaaacaagt tcggcttccc cgacaccagc ttctacaatc ccgacaccca gaggctcgtg 300
tgggcctgcg tgggcttgga gatcggccgc ggccaaccac tcggcgtcgg cgtgtccggc 360
cacccctacc tgaacaagtt cgacgatacc gagacatcca atcgctaccc agcccaaccc 420
ggcagcgaca atcgcgagtg tctgagcatg gactacaagc agacccagct gtgcctgatc 480
ggctgcaagc ccccaaccgg tgaacactgg ggcaagggcg tcgcttgcaa caacaacgcc 540
gccgccaccg actgcccacc cctcgagttg ttcaacagca tcatcgaaga cggcgatatg 600
gtggacaccg gcttcggctg tatggatttc ggcaccctcc aagccaacaa gtccgacgtc 660
cccatcgaca tctgcaattc cacctgtaag taccccgact acctgaagat ggcatccgag 720
ccctacggcg acagcctctt cttcttcctc cgcagggaac aaatgttcgt ccgccatttc 780
ttcaaccgcg ccggcaagtt gggcgaagcc gtgcccgacg atttgtacat caagggcagt 840
ggcaacaccg ccgtcattca gtcctccgca ttcttcccaa ccccctccgg cagcatcgtc 900
acaagcgaga gccagctgtt caacaagccc tactggttgc aaagggccca gggccacaat 960
aacggcatct gttggggcaa ccaactgttc gtcacagtcg tcgacacaac caggtcaacc 1020
aacatgaccc tgtgtaccga ggtgaccaag gagggcacct acaagaacga caacttcaaa 1080
gagtacgtga ggcacgtcga ggagtacgat ctgcaattcg tcttccaatt gtgtaagatc 1140
accttgaccg ccgaaatcat gacctacatc cacaccatgg acagtaacat cctcgaagat 1200
tggcagttcg gcctgacccc cccacctagc gcatcccaac tgtacaagac ctgcaagcag 1260
gccggtacct gcccccctga catcatcccc aaggtcgaag gcacctgtca gaagactgct 1320
ccacccaagg agaaagagga ccccctgaac aagtacacct tctgggaagt caatctgaaa 1380
gagaaattca gcgccgactt ggaccaattc ccactcggca ggaagttcct gctgcagagc 1440
ggcttgtaa 1449

Claims (13)

1.一种人乳头瘤病毒嵌合蛋白，其由HPV58型L1蛋白或HPV58型L1蛋白的突变体以及插入所述HPV58型L1蛋白或HPV58型L1蛋白的突变体的表面区的来自HPV16型L2蛋白的多肽组成，其中所述HPV58型L1蛋白的氨基酸序列如SEQ ID No.1所示，所述HPV16型L2蛋白的氨基酸序列如SEQ ID No.2所示；
其中所述人乳头瘤病毒嵌合蛋白的氨基酸序列如SEQ ID No.10、12、15、19中任一项所示。
2.一种多核苷酸，其编码权利要求1所述的人乳头瘤病毒嵌合蛋白。
3.根据权利要求2所述的多核苷酸，其中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。
4.根据权利要求2所述的多核苷酸，其中所述多核苷酸的序列如SEQ ID No.23、25、28、32中任一项所示。
5.一种载体，其包含如权利要求2至4中任一项所述的多核苷酸。
6.一种细胞，其包含如权利要求5所述的载体。
7.一种多聚物，该多聚物为嵌合五聚体或嵌合病毒样颗粒，其由权利要求1所述的人乳头瘤病毒嵌合蛋白所形成。
8.如权利要求1所述的人乳头瘤病毒嵌合蛋白或如权利要求7所述的多聚物在制备用于预防受试者中人乳头瘤病毒感染和/或人乳头瘤病毒感染诱发的疾病的疫苗中的用途，其中所述人乳头瘤病毒感染为一种或多种选自以下人乳头瘤病毒型别的感染：HPV16、HPV18、HPV33、HPV35、HPV39、HPV45、HPV52、HPV58、HPV68、HPV6、HPV11、HPV5和HPV57。
9.根据权利要求8所述的用途，其中所述人乳头瘤病毒感染诱发的疾病选自宫颈上皮内瘤变、宫颈癌、阴唇癌、阴茎癌、阴道癌、肛门肛周癌、口咽癌、肛周生殖器尖锐湿疣、呼吸道复发性乳头瘤、皮肤疣状增生、皮肤鳞状细胞癌及基底细胞癌。
10.根据权利要求8所述的用途，其中所述受试者为人受试者。
11.一种用于预防人乳头瘤病毒感染和/或人乳头瘤病毒感染诱发的疾病的疫苗，其包含权利要求1所述的人乳头瘤病毒嵌合蛋白或如权利要求7所述的多聚物，佐剂以及疫苗用赋形剂或载体。
12.根据权利要求11所述的用于预防人乳头瘤病毒感染和/或人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。
13.根据权利要求11或12所述的用于预防人乳头瘤病毒感染和/或人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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