CN102482327B - 脂质化肿瘤相关抗原及其免疫治疗的组成物及方法 - Google Patents
脂质化肿瘤相关抗原及其免疫治疗的组成物及方法 Download PDFInfo
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Abstract
本发明揭示了一种新颖多肽及融合蛋白质，所述融合蛋白质包含具有脂质化序列的第一片段，及具有肿瘤相关抗原序列的第二片段。该融合蛋白质可被脂质化并表现于细菌细胞中。一旦经投药予个体后，所述融合蛋白质可诱发对抗癌症的免疫反应。
Description
技术领域
本发明关于多肽及脂质化融合蛋白质。本发明还揭示与这些多肽及融合蛋白质相关于免疫治疗的组成物及方法。
背景技术
相关申请案
本发明对于2009年6月22日提出申请的美国临时申请案No.61/219,301主张优先权；该先申请案的内容皆以引用方式纳入本文做为参考。
发明背景
病毒感染会引起各种病症，包括癌症。举例而言，人类乳头状瘤病毒(HPVs)感染会造成数种癌症发展，尤其是成为世界妇女第二大癌症死因的子宫颈癌，且其已经决定性证实HPV为子宫颈癌的病原体(Schwarz，T.F.，Expert RevVaccines 7：1465-73，2008)。据估计，每年有大约493,000件新的子宫颈癌个案被诊断出来(Parkin，D.M.等人，CA Cancer J Clin 55：74-108，2005)。已研发用于预防引起癌症的病毒感染、治疗已存在癌症、或防止于高危险群个体内发展成癌症的疫苗。然而，其中有许多并不具有功效。故仍需要有效的疫苗与相关的试剂。
发明内容
本发明关于新颖多肽、脂质化融合蛋白质、免疫治疗的组成物及方法。
据此，本发明的一方面关于一种具有SEQ ID NO：2所述序列的单离的多肽(其序列列示于图2B)。本发明还关于一种具有编码该多肽的序列，或其互补序列的单离的核酸。该核酸的实例包括SEQ ID NO：13所述的序列(如下所示)，及其中一或多个密码子被其他编码相同残基的密码子取代的简并性变体(degenerate variants)。
ATGCATGGCGATACCCCGACCCTGCATGAATATATGCTGGATCTGCAGCCGG
AAACCACCGATCTGTATGGCTATCAGCAGCTGAACGATAGCAGCGAAGAA
GAAGATGAAATTGATGGCCCGGCGGGCCAGGCGGAACCGGATCGCGCGCA
TTAIAACATTGTGACCTTTGCGAGCAAAGCGGATAGCACCCTGCGCCTGAG
CGTGCAGAGCACCCATGTGGATATTCGCACCCTGGAAGATCTGCTGATGGG
CACCCTGGGCATTGTGGCGCCGATTGCGAGCCAGAAACCG (SEQ IDNO.：13)。
本发明还关于一种单离的融合蛋白，其包含具有脂质化序列的第一片段，及具有肿瘤相关抗原序列的第二片段。于该融合蛋白中，第一片段位于第二片段的N-端。于一项实例，该融合蛋白为脂质化。脂质化序列包括：Met Lys Lys LeuLeu Ile Ala Ala Met Met Ala Ala Ala Leu Ala Ala Cys Ser Gln Glu Ala Lys Gln GluVal Lys Glu Ala Val Gln Ala Val Glu Ser Asp Val Lys Asp Thr Ala(SEQ ID NO：12)。肿瘤相关抗原可为衍生自一种与人类慢性疾病或癌症(例如子宫颈癌)关连的病毒的病毒抗原。于一项实例，该病毒抗原衍生自人类疱疹病毒第四型(EBV)、人类乳头状瘤病毒(HPV)、C型肝炎病毒(HCV)、B型肝炎病毒(HBV)或巨细胞病毒(CMV)。于一项具体态样，该病毒抗原为去活化HPV致癌蛋白E5、E6或E7。于一项较佳具体态样，该病毒抗原为去活化HPV 16 E7致癌蛋白，例如SEQ IDNO：1或2。融合蛋白的实例包括，于下述实施例中所描述的rlipo-E7m融合蛋白。以下列示rlipo-E7m融合蛋白的胺基酸序列，及编码此融合蛋白的核酸序列(SEQ ID NOs：14及15)：
MET LYS LYS LEU LEU ILE ALA ALA MET MET ALA ALA ALA LEU ALA
ALA CYS SER GLN GLU ALA LYS GLN GLU VAL LYS GLU ALA VAL GLN
ALA VAL GLU SER ASP VAL LYS ASP THR ALA GLY SER MET HIS GLY ASP
THR PRO THR LEU HIS GLU TYR MET LEU ASP LEU GLN PRO GLU THR
THR ASP LEU TYR GLY TYR GLN GLN LEU ASN ASP SER SER GLU GLU
GLU ASP GLU ILE ASP GLY PRO ALA GLY GLN ALA GLU PRO ASP ARG ALA
HIS TYR ASN ILE VAL THR PHE ALA SER LYS ALA ASP SER THR LEU ARG
LEU SER VAL GLN SER THR HIS VAL ASP ILE ARG THR LEU GLU ASP LEU
LEU MET GLY THR LEU GLY ILE VAL ALA PRO ILE ALA SER GLN LYS PRO(SEQ ID NO：14)。
ATGAAAAAACTGCTGATTGCGGCGATGATGGCGGCGGCGCTGGCGGCGTG
CAGCCAGGAAGCGAAACAGGAAGTGAAAGAAGCGGTGCAGGCGGTGGA
AAGCGATGTGAAAGATACCGCGGGATCCATGCATGGCGATACCCCGACCCT
GCATGAATATATGCTGGATCTGCAGCCGGAAACCACCGATCTGTATGGCTAT
CAGCAGCTGAACGATAGCAGCGAAGAAGAAGATGAAATTGATGGCCCGGC
GGGCCAGGCGGAACCGGATCGTGCGCATTATAACATTGTGACCTTTGCGAG
CAAAGCGGATAGCACCCTGCGTCTGAGCGTGCAGAGCACCCATGTGGATA
TTCGTACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATTGTGGCGCCGA
TTGCGAGCCAGAAACCG(SEQ ID NO：15)。
本发明还关于一种单离的核酸，其含有编码上述融合蛋白的核酸序列，或该核酸的互补序列。该核酸的实例包括SEQ ID NO：15，及其中一或多个密码子被其他编码相同残基的密码子取代的简并性变体。
核酸是指一种DNA分子(例如cDNA或基因组DNA)、RNA分子(例如mRNA)、或是DNA或RNA类似物。DNA或RNA类似物可自核苷酸类似物合成得。核酸分子可为单股或双股，但较佳为双股DNA。“单离的核酸”是一种其构造不与任何天然核酸的，或天然基因组核酸的任何片段的构造相同的核酸。因此该术语含括(例如)(a)具有天然基因组DNA分子的部分序列的DNA，但其编码序列两端所衔接的片段并非该分子部分序列存在生物体基因组中的天然状态下的两端所衔接的片段；(b)以一种使所成的分子不与任何天然载体或基因组DNA相同的方式并入一载体中，或并入一原核或真核生物的基因组DNA中的核酸；(c)一种分离的分子例如cDNA、基因组片段、经由聚合酶链反应(PCR)制得的片段或限制酶切割片段；及(d)其为杂合基因(也即编码融合蛋白的基因)的一部份的重组核苷酸序列。上述核酸可用于表现本发明的多肽或蛋白质。为此目的，习于该项技艺的人可将该核酸操作性连接至适宜调节序列，以产生表现载体。
载体是指一种能够传送另一已与其连接的核酸的核酸分子。载体能自我复制或整合到宿主DNA中。载体的实例包括质体、黏质体或病毒载体。本发明的载体包括，以适于在宿主细胞中表现该核酸的形式存在的核酸。较佳地该载体包括一或多种调节序列，其经操作性连接至欲被表现的核酸。调节序列包括启动子、增效子及其他表现调控组件(例如，T7启动子、花椰菜镶嵌病毒35S启动子序列或聚腺化讯号序列)。调节序列包括这些直接固有性表现一核酸序列的序列，以及组织特异性调节与/或可诱导序列。表现载体的设计可取决于，诸如所选择欲转形的宿主细胞、所希望的蛋白质表现程度等因素。表现载体可引导入宿主细胞中，用以制造本发明的多肽或融合蛋白。
本发明的范围还包含一种含有前述核酸的宿主细胞。实例包括大肠杆菌细胞、昆虫细胞(例如使用杆状病毒表现载体)、植物细胞、酵母细胞或哺乳动物细胞。参见(例如)Goeddel，(1990)基因表现技术：酵素学方法185，学院出版，圣地亚哥，CA。
为制造本发明的融合蛋白/多肽，习于该项技艺的人可将宿主细胞培养于允许由本发明核酸编码的融合蛋白/多肽表现的条件下，及将该融合蛋白/多肽自所培养细胞或培养基纯化出来。或者，本发明的核酸可于活体外，例如使用T7启动子调节序列与T7聚合酶，于得自(例如)大肠杆菌的细胞溶解产物中进行转录及转译作用。脂质化融合蛋白可包括(从N-端至C-端)Ag473的D1片段与标靶蛋白，例如抗原性病毒蛋白。
于另一方面，本发明关于一种包含前述多肽或融合蛋白的免疫原组成物。免疫原组成物可与或不与医药上可接受的佐剂调配。
于又一方面，本发明关于一种诱发对于病毒(例如HPV)的免疫反应的方法。该方法包括将有效量的上述免疫原组成物投药予有其需要的个体的步骤。“个体”是指人类及非人类动物。非人类动物包括所有脊椎动物，例如哺乳动物如非人类灵长类(尤其是高等灵长类)、狗、啮齿类(例如小鼠或大鼠)、天竺鼠、猫及非哺乳动物，例如鸟类、两栖类等。于一项较佳具体态样，该个体为人类。于另一项具体态样，该个体为实验动物或适宜做为疾病模式的动物。
下述所提供的图式与详细说明例举本发明之一或多项具体态样的详细内容。本发明的其他特色、目的及优点方面将由下列说明与图示，以及由申请专利范围彰显出。
附图说明
图1A与1B为野生型HPV16 E7蛋白(1A；SEQ ID NO：1)及去活化E7蛋白E7m(1B；SEQ ID NO：2)的胺基酸序列。
图2A与2B为显示重组E7m(rE7m；2A)与重组脂蛋白lipo-E7m(rlipo-E7m；2B)，经由IMAC纯化及以15％SDS-PAGE于还原条件下进行侦测并以考马斯蓝(Coomassie Blue)染色(左图)，或使用抗-(His)6抗体进行免疫转渍侦测(右图)而得到的图片。(A)列1：经IPTG诱导后的细胞溶解产物；列2：于IPTG诱导前的细胞溶解产物；列3：经诱导细胞的可溶部份；列4：经纯化的rE7m；列5-8：免疫转渍；(B)列1：经IPTG诱导后的细胞溶解产物；列2：于IPTG诱导前的细胞溶解产物；列3：经纯化的rlipo-E7m；列4-6：免疫转渍。
图3A与3B为鉴定完整rlipo-E7m蛋白及其N-端片段的图表。
图4为显示于C57BL/6小鼠中由rlipo-E7m诱发的肿瘤-特异性细胞毒性T淋巴细胞(CTL)反应。
图5A与5B为显示rlipo-E7m于保护小鼠对抗肿瘤的功效，相较于rE7m。
具体实施方式
本发明(至少一部份)为基于意外发现一种新颖的肿瘤相关抗原变体，及肿瘤相关抗原与脂质化序列的融合蛋白。该融合蛋白(意外地)可被脂质化，并以高产量表现于诸如大肠杆菌等细菌细胞中。其一旦经投药予个体后，二者皆会诱发对抗肿瘤的免疫反应(例如，细胞毒性T淋巴细胞或抗体)。
本发明特征在于一种对抗病毒感染，及/或所相关的其他病症(例如，癌症)的免疫原组成物，例如疫苗。如前所述，免疫原组成物可含有重组型融合蛋白。该融合蛋白具有一段含脂质化序列的第一片段，及一段有肿瘤相关抗原的序列的第二片段。
肿瘤相关抗原可为一种衍生自与人类慢性疾病或癌症(例如子宫颈癌)关连的病毒的病毒抗原。于一项实例，该病毒抗原衍生自人类疱疹病毒第四型(EBV)、人类乳头状瘤病毒(HPV)、C型肝炎病毒(HCV)、B型肝炎病毒(HBV)或巨细胞病毒(CMV)。以下列示例举性病毒抗原的序列。有数种蛋白质及其抗原性片段可用于制备本发明的免疫原组成物。
人类疱疹病毒第四型(Epstein-Barr病毒)，潜伏性膜蛋白-1(LMPl)：
LeuSerSerSerIleGlyLeuAlaLeuLeuLeuLeuLeuLeuAlaLeuLeuPheTrpLeu
TyrIleValMetSerAspTrpThrGlyGlyAlaLeuLeuValLeuTyrSerPheAlaLeu
MetLeuIleIleIleIleLeuIleIlePheIlePheArgArgAspLeuLeuCysProLeu
GlyGlyLeuGlyLeuLeuLeuLeuMetIleThrLeuLeuLeuIleAlaLeuTrpAsnLeu
HisGlyGlnAlaLeuTyrLeuGlyIleValLeuPheIlePheGlyCysLeuLeuValLeu
GlyLeuTrpIleTyrPheLeuGluIleLeuTrpArgLeuGlyAlaThrLeuTrpGlnLeu
LeuAlaPheIleLeuAlaPhePheLeuAlaIleIleLeuLeuIleIleAlaLeuTyrLeu
GlnGlnAsnTrpTrpThrLeuLeuValAspLeuLeuTrpLeuLeuLeuPheMetAlaIle
LeuIleTrpMetTyrTyrHisGlyProArgHisThrAspGluHisHisHisAspAspSer
LeuProHisProGlnGlnAlaThrAspAspSerSerHisGluSerAspSerAsnSerAsn
GluGlyArgHisHisLeuLeuValSerGlyAlaGlyAspGlyProProLeuCysSerGln
AsnLeuGlyAlaProGlyGlyGlyProAspAsnGlyProGlnAspProAspAsnThrAsp
AspAsnGlyProGlnAspProAspAsnThrAspAspAsnGlyProGlnAspProAspAsn
ThrAspAspAsnGlyProGlnAspProAspAsnThrAspAspAsnGlyProGlnAspPro
AspAsnThrAspAspAsnGlyProGlnAspProAspAsnThrAspAspAsnGlyProGln
AspProAspAsnThrAspAspAsnGlyProHisAspProLeuProHisAsnProSerAsp
SerAlaGlyAsnAspGlyGlyProProAsnLeuThrGluGluValAlaAsnLysGlyGly
AspArgGlyProProSerMetThrAspGlyGlyGlyGlyAspProHisLeuProThrLeu
LeuLeuGlyThrSerGlySerGlyGlyAspAspAspAspProHisGlyProValGlnLeu
SerTyrTyrAsp
人类疱疹病毒第四型(Epstein-Barr病毒)，终端蛋白LMP-2A：
MetGlySerLeuGluMetValProMetGlyAlaGlyProProSerProGlyGlyAspPro
AspGlyAspAspGlyGlyAsnAsnSerGlnTyrProSerAlaSerGlySerSerGlyAsn
ThrProThrProProAsnAspGluGluArgGluSerAsnGluGluProProProProTyr
GluAspProTyrTrpGlyAsnGlyAspArgHisSerAspTyrGlnProLeuGlyThrGln
AspGlnSerLeuTyrLeuGlyLeuGlnHisAspGlyAsnAspGlyLeuProProProPro
TyrSerProArgAspAspSerSerGlnHisIleTyrGluGluAlaGlyArgGlySerMet
AsnProValCysLeuProValIleValAlaProTyrLeuPheTrpLeuAlaAlaIleAla
AlaSerCysPheThrAlaSerValSerThrValValThrAlaThrGlyLeuAlaLeuSer
LeuLeuLeuLeuAlaAlaValAlaSerSerTyrAlaAlaAlaGlnArgLysLeuLeuThr
ProValThrValLeuThrAlaValValThrPhePheAlaIleCysLeuThrTrpArgIle
GluAspProProPheAsnSerLeuLeuPheAlaLeuLeuAlaAlaAlaGlyGlyLeuGln
GlyIleTyrValLeuValMetLeuValLeuLeuIleLeuAlaTyrArgArgArgTrpArg
ArgLeuThrValCysGlyGlyIleMetPheLeuAlaCysValLeuValLeuIleValAsp
AlaValLeuGlnLeuSerProLeuLeuGlyAlaValThrValValSerMetThrLeuLeu
LeuLeuAlaPheValLeuTrpLeuSerSerProGlyGlyLeuGlyThrLeuGlyAlaAla
LeuLeuThrLeuAlaAlaAlaLeuAlaLeuLeuAlaSerLeuIleLeuGlyThrLeuAsn
LeuThrThrMetPheLeuLeuMetLeuLeuTrpThrLeuValValLeuLeuIleCysSer
SerCysSerSerCysProLeuSerLysIleLeuLeuAlaArgLeuPheLeuTyrAlaLeu
AlaLeuLeuLeuLeuAlaSerAlaLeuIleAlaGlyGlySerIleLeuGlnThrAsnPhe
LysSerLeuSerSerThrGluPheIleProAsnLeuPheCysMetLeuLeuLeuIleVal
AlaGlyIleLeuPheIleLeuAlaIleLeuThrGluTrpGlySerGlyAsnArgThrTyr
GlyProValPheMetCysLeuGlyGlyLeuLeuThrMetValAlaGlyAlaValTrpLeu
ThrValMetThrAsnThrLeuLeuSerAlaTrpIleLeuThrAlaGlyPheLeuIlePhe
LeuIleGlyPheAlaLeuPheGlyValIleArgCysCysArgTyrCysCysTyrTyrCys
LeuThrLeuGluSerGluGluArgProProThrProTyrArgAsnThrVal
人类疱疹病毒第四型(Epstein-Barr病毒)，终端蛋白LMP-2B：
MetAsnProValCysLeuProValIleValAlaProTyrLeuPheTrpLeuAlaAlaIle
AlaAlaSerCysPheThrAlaSerValSerThrValValThrAlaThrGlyLeuAlaLeu
SerLeuLeuLeuLeuAlaAlaValAlaSerSerTyrAlaAlaAlaGlnArgLysLeuLeu
ThrProValThrValLeuThrAlaValValThrPhePheAlaIleCysLeuThrTrpArg
IleGluAspProProPheAsnSerLeuLeuPheAlaLeuLeuAlaAlaAlaGlyGlyLeu
GlnGlyIleTyrValLeuValMetLeuValLeuLeuIleLeuAlaTyrArgArgArgTrp
ArgArgLeuThrValCysGlyGlyIleMetPheLeuAlaCysValLeuValLeuIleVal
AspAlaValLeuGlnLeuSerProLeuLeuGlyAlaValThrValValSerMetThrLeu
LeuLeuLeuAlaPheValLeuTrpLeuSerSerProGlyGlyLeuGlyThrLeuGlyAla
AlaLeuLeuThrLeuAlaAlaAlaLeuAlaLeuLeuAlaSerLeuIleLeuGlyThrLeu
AsnLeuThrThrMetPheLeuLeuMetLeuLeuTrpThrLeuValValLeuLeuIleCys
SerSerCysSerSerCysProLeuSerLysIleLeuLeuAlaArgLeuPheLeuTyrAla
LeuAlaLeuLeuLeuLeuAlaSerAlaLeuIleAlaGlyGlySerIleLeuGlnThrAsn
PheLysSerLeuSerSerThrGluPheIleProAsnLeuPheCysMetLeuLeuLeuIle
ValAlaGlyIleLeuPheIleLeuAlaIleLeuThrGluTrpGlySerGlyAsnArgThr
TyrGlyProValPheMetCysLeuGlyGlyLeuLeuThrMetValAlaGlyAlaValTrp
LeuThrValMetSerAsnThrLeuLeuSerAlaTrpIleLeuThrAlaGlyPheLeuIle
PheLeuIleGlyPheAlaLeuPheGlyValIleArgCysCysArgTyrCysCysTyrTyr
CysLeuThrLeuGluSerGluGluArgProProThrProTyrArgAsnThrVal
人类疱疹病毒第四型(Epstein-Barr病毒)，外膜醣蛋白gp350：
MetGluAlaAlaLeuLeuValCysGlnTyrThrIleGlnSerLeuIleGlnLeuThrArg
AspAspProGlyPhePheAsnValGluIleLeuGluPheProPheTyrProAlaCysAsn
ValCysThrAlaAspValAsnAlaThrIleAsnPheAspValGlyGlyLysLysHisLys
LeuAsnLeuAspPheGlyLeuLeuThrProHisThrLysAlaValTyrGlnProArgGly
AlaPheGlyGlySerGluAsnAlaThrAsnLeuPheLeuLeuGluLeuLeuGlyAlaGly
GluLeuAlaLeuThrMetArgSerLysLysLeuProIleAsnIleThrThrGlyGluGlu
GlnGlnValSerLeuGluSerValAspValTyrPheGlnAspValPheGlyThrMetTrp
CysHisHisAlaGluMetGlnAsnProValTyrLeuIleProGluThrValProTyrIle
LysTrpAspAsnCysAsnSerThrAsnIleThrAlaValValArgAlaGlnGlyLeuAsp
ValThrLeuProLeuSerLeuProThrSerAlaGlnAspSerAsnPheSerValLysThr
GluMetLeuGlyAsnGluIleAspIleGluCysIleMetGluAspGlyGluIleSerGln
ValLeuProGlyAspAsnLysPheAsnIleThrCysSerGlyTyrGluSerHisValPro
SerGlyGlyIleLeuThrSerThrSerProValAlaThrProIleProGlyThrGlyTyr
AlaTyrSerLeuArgLeuThrProArgProValSerArgPheLeuGlyAsnAsnSerIle
LeuTyrValPheTyrSerGlyAsnGlyProLysAlaSerGlyGlyAspTyrCysIleGln
SerAsnIleValPheSerAspGluIleProAlaSerGlnAspMetProThrAsnThrThr
AspIleThrTyrValGlyAspAsnAlaThrTyrSerValProMetValThrSerGluAsp
AlaAsnSerProAsnValThrValThrAlaPheTrpAlaTrpProAsnAsnThrGluThr
AspPheLysCysLysTrpThrLeuThrSerGlyThrProSerGlyCysGluAsnIleSer
GlyAlaPheAlaSerAsnArgThrPheAspIleThrValSerGlyLeuGlyThrAlaPro
LysThrLeuIleIleThrArgThrAlaThrAsnAlaThrThrThrThrHisLysValIle
PheSerLysAlaProGluSerThrThrThrSerProThrLeuAsnThrThrGlyPheAla
AlaProAsnThrThrThrGlyLeuProSerSerThrHisValProThrAsnLeuThrAla
ProAlaSerThrGlyProThrValSerThrAlaAspValThrSerProThrProAlaGly
ThrThrSerGlyAlaSerProValThrProSerProSerProArgAspAsnGlyThrGlu
SerLysAlaProAspMetThrSerProThrSerAlaValThrThrProThrProAsnAla
ThrSerProThrProAlaValThrThrProThrProAsnAlaThrSerProThrLeuGly
LysThrSerProThrSerAlaValThrThrProThrProAsnAlaThrSerProThrPro
AlaValThrThrProThrProAsnAlaThrIleProThrLeuGlyLysThrSerProThr
SerAlaValThrThrProThrProAsnAlaThrSerProThrValGlyGluThrSerPro
GlnAlaAsnThrThrAsnHisThrLeuGlyGlyThrSerSerThrProValValThrSer
ProProLysAsnAlaThrSerAlaValThrThrGlyGlnHisAsnIleThrSerSerSer
ThrSerSerMetSerLeuArgProSerSerIleSerGluThrLeuSerProSerThrSer
AspAsnSerThrSerHisMetProLeuLeuThrSerAlaHisProThrGlyGlyGluAsn
IleThrGlnValThrProAlaSerThrSerThrHisHisValSerThrSerSerProAla
ProArgProGlyThrThrSerGlnAlaSerGlyProGlyAsnSerSerThrSerThrLys
ProGlyGluValAsnValThrLysGlyThrProProLysAsnAlaThrSerProGlnAla
ProSerGlyGlnLysThrAlaValProThrValThrSerThrGlyGlyLysAlaAsnSer
ThrThrGlyGlyLysHisThrThrGlyHisGlyAlaArgThrSerThrGluProThrThr
AspTyrGlyGlyAspSerThrThrProArgThrArgTyrAsnAlaThrThrTyrLeuPro
ProSerThrSerSerLysLeuArgProArgTrpThrPheThrSerProProValThrThr
AlaGlnAlaThrValProValProProThrSerGlnProArgPheSerAsnLeuSerMet
LeuValLeuGlnTrpAlaSerLeuAlaValLeuThrLeuLeuLeuLeuLeuValMetAla
AspCysAlaPheArgArgAsnLeuSerThrSerHisThrTyrThrThrProProTyrAsp
AspAlaGluThrTyrVal
人类疱疹病毒第四型(Epstein-Barr病毒)，EBNA-2核蛋白：
MetProThrTyrTyrLeuAlaLeuHisGlyGlyGlnSerTyrAsnLeuIleValAspThr
AspMetSerGlyAsnProSerLeuSerValIleProThrAsnProTyrGlnGluGlnLeu
SerAsnAsnProLeuIleGlnLeuGlnIleValValGlyGluAsnThrGlyAlaProAla
ProProGlnProProProProProProProProProProProGluArgArgAspAlaTrp
ThrGlnGluProLeuProLeuAspMetAsnProLeuGlySerAspAlaSerGlnGlyPro
LeuAlaSerSerIleArgMetLeuCysMetAlaGlnTyrLeuLeuArgAsnAlaArgGly
GlnGlnGlyLeuLeuArgProLeuGlyProGlnThrArgSerGlnValThrLeuGluArg
GlnProValHisAsnProArgGlnGluAlaProIleIleLeuLeuGlnSerProAlaPro
ProArgPheThrProValProMetValAlaLeuGlyHisThrLeuGlnProThrProPro
ProArgProThrLeuProGlnProArgIleProLeuIleIleProProArgHisThrAsn
GlnProAlaThrThrProProThrAlaProGlnArgLeuThrLeuGlyHisGlnLeuSer
LeuProProHisProProProHisGlnSerThrProHisCysSerSerAspSerThrGly
LeuProProProProThrSerTyrSerIleProSerMetThrLeuSerProGluProLeu
ProProProAlaAlaProAlaHisProLeuProGlyValIleTyrAspGlnGlnAlaLeu
ProProThrProGlyProProTrpTrpProProValArgAspProThrProThrThrGln
ThrProProThrAsnThrLysGlnGlyProAspGlnGlyGlnGlyArgGlyArgTrpArg
GlyArgGlyArgSerLysGlyArgGlyArgMetHisLysLeuProGluProArgArgPro
GlyProAspThrSerSerProSerMetProGlnLeuSerProValValSerLeuHisGln
GlyGlnGlyProGluAsnSerProThrProGlyProSerThrAlaGlyProValCysArg
ValThrProSerAlaThrProAspIleSerProIleHisGluProGluSerSerAspSer
GluGluProProPheLeuPheProSerAspTrpTyrProProThrLeuGluProAlaGlu
LeuAspGluSerTrpGluGlyIlePheGluThrThrGluSerHisSerSerAspGluGlu
AsnValGlyGlyProSerLysArgProArgThrSerThrGln
人类疱疹病毒第四型(Epstein-Barr病毒)，EBNA-3C核蛋白：
MetGluSerPheGluGlyGlnGlyAspSerArgGlnSerProAspAsnGluArgGlyAsp
AsnValGlnThrThrGlyGluHisAspGlnAspProGlyProGlyProProSerSerGly
AlaSerGluArgLeuValProGluGluSerTyrSerArgAspGlnGlnProTrpGlyGln
SerArgGlyAspGluAsnArgGlyTrpMetGlnArgIleArgArgArgArgArgArgArg
AlaAlaLeuSerGlyHisLeuLeuAspThrGluAspAsnValProProTrpLeuProPro
HisAspIleThrProTyrThrAlaArgAsnIleArgAspAlaAlaCysArgAlaValLys
GlnSerHisLeuGlnAlaLeuSerAsnLeuIleLeuAspSerGlyLeuAspThrGlnHis
IleLeuCysPheValMetAlaAlaArgGlnArgLeuGlnAspIleArgArgGlyProLeu
ValAlaGluGlyGlyValGlyTrpArgHisTrpLeuLeuThrSerProSerGlnSerTrp
ProMetGlyTyrArgThrAlaThrLeuArgThrLeuThrProValProAsnArgValGly
AlaAspSerIleMetLeuThrAlaThrPheGlyCysGlnAsnAlaAlaArgThrLeuAsn
ThrPheSerAlaThrValTrpThrProProHisAlaGlyProArgGluGlnGluArgTyr
AlaArgGluAlaGluValArgPheLeuArgGlyLysTrpGlnArgArgTyrArgArgIle
TyrAspLeuIleGluLeuCysGlySerLeuHisHisIleTrpGlnAsnLeuLeuGlnThr
GluGluAsnLeuLeuAspPheValArgPheMetGlyValMetSerSerCysAsnAsnPro
AlaValAsnTyrTrpPheHisLysThrIleGlyAsnPheLysProTyrTyrProTrpAsn
AlaProProAsnGluAsnProTyrHisAlaArgArgGlyIleLysGluHisValIleGln
AsnAlaPheArgLysAlaGlnIleGlnGlyLeuSerMetLeuAlaThrGlyGlyGluPro
ArgGlyAspAlaThrSerGluThrSerSerAspGluAspThrGlyArgGlnGlySerAsp
ValGluLeuGluSerSerAspAspGluLeuProTyrIleAspProAsnMetGluProVal
GlnGlnArgProValMetPheValSerArgValProAlaLysLysProArgLysLeuPro
TrpProThrProLysThrHisProValLysArgThrAsnValLysThrSerAspArgSer
AspLysAlaGluAlaGlnSerThrProGluArgProGlyProSerGluGlnSerSerVal
ThrValGluProAlaHisProThrProValGluMetProMetValIleLeuHisGlnPro
ProProValProLysProValProValLysProThrProProProSerArgArgArgArg
GlyAlaCysValValTyrAspAspAspValIleGluValIleAspValGluThrThrGlu
AspSerSerSerValSerGlnProAsnLysProHisArgLysHisGlnAspGlyPheGln
ArgSerGlyArgArgGlnLysArgAlaAlaProProThrValSerProSerAspThrGly
ProProAlaValGlyProProAlaAlaGlyProProAlaAlaGlyProProAlaAlaGly
ProProAlaAlaGlyProProAlaAlaGlyProProAlaAlaGlyProArgIleLeuAla
ProLeuSerAlaGlyProProAlaAlaGlyProHisIleValThrProProSerAlaArg
ProArgIleMetAlaProProValValArgMetPheMetArgGluArgGlnLeuProGln
SerThrGlyArgLysProGlnCysPheTrpGluMetArgAlaGlyArgGluIleThrGln
MetGlnGlnGluProSerSerHisLeuGlnSerAlaThrGlnProThrThrProArgPro
SerTrpAlaProSerValCysAlaLeuSerValMetAspAlaGlyLysAlaGlnProIle
GluSerSerHisLeuSerSerMetSerProThrGlnProIleSerHisGluGluGlnPro
ArgTyrGluAspProAspAlaProLeuAspLeuSerLeuHisProAspValAlaAlaGln
ProAlaProGlnAlaProTyrGlnGlyTyrGlnGluProProAlaProGlnAlaProTyr
GlnGlyTyrGlnGluProProProProGlnAlaProTyrGlnGlyTyrGlnGluProPro
AlaHisGlyLeuGlnSerSerSerTyrProGlyTyrAlaGlyProTrpThrProArgSer
GlnHisProCysTyrArgHisProTrpAlaProTrpSerGlnAspProValHisGlyHis
ThrGlnGlyProTrpAspProArgAlaProHisLeuProProGlnTrpAspGlySerAla
GlyHisGlyGlnAspGlnValSerGlnPheProHisLeuGlnSerGluThrGlyProPro
ArgLeuGlnLeuSerLeuValProLeuValSerSerSerAlaProSerTrpSerSerPro
GlnProArgAlaProIleArgProIleProThrArgPheProProProProMetProLeu
GlnAspSerMetAlaValGlyCysAspSerSerGlyThrAlaCysProSerMetProPhe
AlaSerAspTyrSerGlnGlyAlaPheThrProLeuAspIleAsnAlaThrThrProLys
ArgProArgValGluGluSerSerHisGlyProAlaArgCysSerGlnAlaThrAlaGlu
AlaGlnGluIleLeuSerAspAsnSerGluIleSerValPheProLysAspAlaLysGln
ThrAspTyrAspAlaSerThrGluSerGluLeuAsp
NS3(C型肝炎病毒)：
AlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeuLeuGlyCysIleIleThrGly
LeuThrGlyArgAspLysAsnGlnValGluGlyGluValGlnIleValSerThrAlaAla
GlnThrPheLeuAlaThrCysIleAsnGlyValCysTrpThrValTyrHisGlyAlaGly
ThrArgThrIleAlaSerSerLysGlyProValIleGlnMetTyrThrAsnValAspGln
AspLeuValGlyTrpProAlaProGlnGlyAlaArgSerLeuThrProCysThrCysGly
SerSerAspLeuTyrLeuValThrArgHisAlaAspValIleProValArgArgArgGly
AspGlyArgGlySerLeuLeuSerProArgProIleSerTyrLeuLysGlySerSerGly
GlyProLeuLeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaValCysThr
ArgGlyValAlaLysAlaValAspPheIleProValGluGlyLeuGluThrThrMetArg
SerProValPheSerAspAsnSerSerProProAlaValProGlnSerTyrGlnValAla
HisLeuHisAlaProThrGlySerGlyLysSerThrLysValProAlaAlaTyrAlaAla
GlnGlyTyrLysValLeuValLeuAsnProSerValAlaAlaThrLeuGlyPheGlyAla
TyrMetSerLysAlaHisGlyIleAspProAsnIleArgThrGlyValArgThrIleThr
ThrGlySerProIleThrTyrSerThrTyrGlyLysPheLeuAlaAspGlyGlyCysSer
GlySerAlaTyrAspIleIleIleCysAspGluCysHisSerThrAspAlaThrSerIle
LeuGlyIleGlyThrValLeuAspGlnAlaGluThrAlaGlyAlaArgLeuThrValLeu
AlaThrAlaThrProProGlySerValThrValProHisProAsnIleGluGluValAla
LeuSerThrThrGlyGluIleProPheTyrGlyLysAlaIleProLeuGluAlaIleLys
GlyGlyArgHisLeuIlePheCysHisSerLysLysLysCysAspGluLeuAlaAlaLys
LeuValAlaLeuGlyValAsnAlaValAlaTyrTyrArgGlyLeuAspValSerValIle
ProAlaSerGlyAspValValValValAlaThrAspAlaLeuMetThrGlyPheThrGly
AspPheAspSerValIleAspCysAsnThrCysValThrGlnThrValAspPheSerLeu
AspProThrPheThrIleGluThrThrThrLeuProGlnAspAlaValSerArgThrGln
ArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArgPheValThrProGlyGlu
ArgProSerGlyMetPheAspSerSerValLeuCysGluCysTyrAspAlaGlyCysAla
TrpTyrGluLeuThrProAlaGluThrThrValArgLeuArgAlaTyrMetAsnThrPro
GlyLeuProValCysGlnAspHisLeuGluPheTrpGluGlyValPheThrGlyLeuThr
HisIleAspAlaHisPheLeuSerGlnThrLysGlnSerGlyGluAsnLeuProTyrLeu
ValAlaTyrGlnAlaThrValCysAlaArgAlaGlnAlaProProProSerTrpAspGln
MetTrpLysCysLeuIleArgLeuLysProThrLeuHisGlyProThrProLeuLeuTyr
ArgLeuGlyAlaValGlnAsnGluIleThrLeuThrHisProIleThrLysTyrIleMet
ThrCysMetSerAlaAspLeuGluValValThr
NS5B(C型肝炎病毒)：
GluValTyrGlnCysCysAspLeuGluProGluAlaArgLysValIleSerAlaLeuThr
GluArgLeuTyrValGlyGlyProMetTyrAsnSerArgGlyAspLeuCysGlyThrArg
ArgCysArgAlaSerGlyValPheThrThrSerPheGlyAsnThrLeuThrCysTyrLeu
LysAlaSerAlaAlaIleArgAlaAlaGlyLeuLysAspCysThrMetLeuValCysGly
AspAspLeuValValIleAlaGluSerAspGlyValGluGluAspLysArgAlaLeuGly
AlaPheThrGluAlaMetThrArgTyrSerAlaProProGlyAspAlaProGlnProAla
TyrAspLeuGluLeuIleThrSerCysSerSerAsnValSerValAlaHisAspGlyThr
GlyLysArgValTyrTyrLeuThrArgAspProGluThrProLeuAlaArgAlaAlaTrp
GluThrAlaArgHisThrProValAsnSerTrpLeuGlyAsnIleIleIleTyrAlaPro
ThrIleTrpValArgMetValLeuMetThrHisPhePheSerIleLeuGlnSerGlnGlu
AlaLeuGluLysAlaLeuAspPheAspMetTyrGlyValThrTyrSerIleThr
E1(C型肝炎病毒)：
TyrGlnValArgAsnSerSerGlyLeuTyrHisValThrAsnAspCysProAsnSerSer
IleValTyrGluThrAlaAspIleLeuHisSerProGlyCysValProCysValArgGlu
GlyAsnThrSerLysCysTrpValAlaValAlaProThrValAlaThrLysAspGlyLys
LeuProThrThrGlnLeuArgHisIleAspLeuLeuValGlyAlaThrLeuCysSerAla
LeuTyrValGlyAspLeuCysGlySerValPheLeuValSerGlnLeuPheThrPheSer
ProArgHisTrpThrThrGlnAspCysAsnCysSerIleTyrProGlyHisValThrGly
HisArgMetAlaTrpAspMetMetMetAsnTrpSerProThrThrAlaLeuValValAla
GlnLeuLeuArgValProGlnAlaIleLeuAspMetIleAlaGlyAlaHisTrpGlyVal
LeuAlaGlyIleAlaTyrPheSerMetValGlyAsnTrpAlaLysValLeuValValLeu
LeuLeuPheAlaGlyValAspAla
E2(C型肝炎病毒)：
HisTrpGlyValMetPheGlyLeuAlaTyrPheSerMetGlnGlyAlaTrpAlaLysVal
IleValIleLeuLeuLeuThrAlaGlyValAspAlaSerSerHisAsnThrArgThrVal
GlyGlyGlnIleAlaArgGlnLeuGlnProPheThrArgLeuPheSerValGlyProAsn
GlnAsnIleGlnLeuIleAsnThrAsnGlySerTrpHis
HBsAg(B型肝炎病毒)：
SerThrThrSerThrGlyProCysArgThrCysMetThrThrAlaGlnGlyThrSerMet
TyrProSerCysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro
SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe
pre-S1蛋白(B型肝炎病毒)：
MetGlyGlnAsnLeuSerValSerAsnProLeuGlyPhePheProGluHisGlnLeuAsp
ProLeuPheLysAlaAsnSerAsnAsnProAspTrpAspPheAsnProAsnLysAspAsn
TrpProGluAlaThrGlnValGlyValGlyAlaPheGlyProGlyPheThrProProHis
GlyGlyLeuLeuGlyTrpSerSerGlnAlaGlnGlyAlaIleThrThrLeuProAlaVal
ProProSerAlaAlaThrAsnArg
pre-S2蛋白(B型肝炎病毒)：
IleSerSerIlePheSerArgIleGlyAspProAlaLeuAsnMetGluAsnIleThrSer
GlyPheLeuGlyProLeuLeuValLeuGlnAlaGlyPhePheLeuLeuThrArgIleLeu
ThrIleProGlnSerLeuAspSerTrpTrpThrSerLeuAsnPheLeuGlyGlyThrThr
ValCysLeuGlyGlnAsnSerGlnSerProThrSerAsnHisSerProThrSerCysPro
ProThrCysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPheIle
LeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGlyMetLeuProVal
CysProLeuIleProGlySerSerThrThrSerThrGlyProCysArgThrCysThrThr
ProAlaGlnGlyThrSerMetTyrProSerCysCysCysThrLysProSerAspGlyAsn
CysThrCysIleProIleProSerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAla
SerAlaArgPheSerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeu
SerProThrValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSerLeuTyr
外膜醣蛋白B，gB(人类巨细胞病毒CMV)：
TyrAsnLysProIleAlaAlaArgPheMetGlyAspValLeuGlyLeuAlaSerCysVal
ThrIleAsnGlnThrSerValLysValLeuArgAspMetAsnValLysGluSerProGly
ArgCysTyrSerArgProValValIlePheAsnPheValAsnSerSerTyrValGlnTyr
GlyGlnLeuGlyGluAspAsnGluIleLeuLeuGlyAsnHisArgThrGluGluCysGln
PheProSerLeuLysIlePheIleAlaGlyAsnSerAlaTyrGluTyrValAspTyrLeu
PheLysArgMetIleAspLeuSerSerIleTyrThrValAspSerMetIleAlaLeuAsp
IleAspProLeuGluAsnThrAspPheArgValLeuGluLeuTyrSerGlnLysGluLeu
ArgSerSerAsnValPheAspLeuGluGluIleMetArgGluPheAsnSerTyrLysGln
ArgValLysTyrValGluAspLysValValAspProLeuProPro
pp65(人类疱疹病毒第五型与人类巨细胞病毒CMV)：
MetAlaSerValLeuGlyProIleSerGlyHisValLeuLysAlaValPheSerArgGly
AspThrProValLeuProHisGluThrArgLeuLeuGlnThrGlyIleHisValArgVal
SerGlnProSerLeuIleLeuValSerGlnTyrThrProAspSerThrProCysHisArg
GlyAspAsnGlnLeuGlnValGlnHisThrTyrPheThrGlySerGluValGluAsnVal
SerValAsnValHisAsnProThrGlyArgSerIleCysProSerGlnGluProMetSer
IleTyrValTyrAlaLeuProLeuLysMetLeuAsnIleProSerIleAsnValHisHis
TyrProSerAlaAlaGluArgLysHisArgHisLeuProValAlaAspAlaValIleHis
AlaSerGlyLysGlnMetTrpGlnAlaArgLeuThrValSerGlyLeuAlaTrpThrArg
GlnGlnAsnGlnTrpLysGluProAspValTyrTyrThrSerAlaPheValPheProThr
LysAspValAlaLeuArgHisValValCysAlaHisGluLeuValCysSerMetGluAsn
ThrArgAlaThrLysMetGlnValIleGlyAspGlnTyrValLysValTyrLeuGluSer
PheCysGluAspValProSerGlyLysLeuPheMetHisValThrLeuGlySerAspVal
GluGluAspLeuThrMetThrArgAsnProGlnProPheMetArgProHisGluArgAsn
GlyPheThrValLeuCysProLysAsnMetIleIleLysProGlyLysIleSerHisIle
MetLeuAspValAlaPheThrSerHisGluHisPheGlyLeuLeuCysProLysSerIle
ProGlyLeuSerIleSerGlyAsnLeuLeuMetAsnGlyGlnGlnIlePheLeuGluVal
GlnAlaIleArgGluThrValGluLeuArgGlnTyrAspProValAlaAlaLeuPhePhe
PheAspIleAspLeuLeuLeuGlnArgGlyProGlnTyrSerGluHisProThrPheThr
SerGlnTyrArgIleGlnGlyLysLeuGluTyrArgHisThrTrpAspArgHisAspGlu
GlyAlaAlaGlnGlyAspAspAspValTrpThrSerGlySerAspSerAspGluGluLeu
ValThrThrGluArgLysThrProArgValThrGlyGlyGlyAlaMetAlaGlyAlaSer
ThrSerAlaGlyArgLysArgLysSerAlaSerSerAlaThrAlaCysThrAlaGlyVal
MetThrArgGlyArgLeuLysAlaGluSerThrValAlaProGluGluAspThrAspGlu
AspSerAspAsnGluIleHisAsnProAlaValPheThrTrpProProTrpGlnAlaGly
IleLeuAlaArgAsnLeuValProMetValAlaThrValGlnGlyGlnAsnLeuLysTyr
GlnGluPhePheTrpAspAlaAsnAspIleTyrArgIlePheAlaGluLeuGluGlyVal
TrpGlnProAlaAlaGlnProLysArgArgArgHisArgGlnAspAlaLeuProGlyPro
CysIleAlaSerThrProLysLysHisArgGly
IE-1蛋白(人类疱疹病毒第五型，与人类巨细胞病毒CMV)：
MetGluSerSerAlaLysArgLysMetAspProAspAsnProAspGluGlyProSerSer
LysValProArgProGluThrProValThrLysAlaThrThrPheLeuGlnThrMetLeu
ArgLysGluValAsnSerGlnLeuSerLeuGlyAspProLeuPheProGluLeuAlaGlu
GluSerLeuLysThrPheGluGlnValThrGluAspCysAsnGluAsnProGluLysAsp
ValLeuAlaGluLeuValLysGlnIleLysValArgValAspMetValArgHisArgIle
LysGluHisMetLeuLysLysTyrAlaGlnThrGluGluLysPheThrGlyAlaPheAsn
MetMetGlyGlyCysLeuGlnAsnAlaLeuAspIleLeuAspLysValHisGluProPhe
GluGluMetLysCysIleGlyLeuThrMetGlnSerMetTyrGluAsnTyrIleValPro
GluAspLysArgGluMetTrpMetAlaCysIleLysGluLeuHisAspValSerLysGly
AlaAlaAsnLysLeuGlyGlyAlaLeuLysAlaLysAlaArgAlaLysLysAspGluLeu
ArgArgLysMetMetTyrMetCysTyrArgAsnIleGluPhePheThrLysAsnSerAla
PheProLysThrThrAsnGlyCysSerGlnAlaMetAlaAlaLeuGlnAsnLeuProGln
CysSerProAspGluIleMetSerTyrAlaGlnLysIlePheLysIleLeuAspGluGlu
ArgAspLysValLeuThrHisIleAspHisIlePheMetAspIleLeuThrThrCysVal
GluThrMetCysAsnGluTyrLysValThrSerAspAlaCysMetMetThrMetTyrGly
GlyIleSerLeuLeuSerGluPheCysArgValLeuCysCysTyrValLeuGluGluThr
SerValMetLeuAlaLysArgProLeuIleThrLysProGluValIleSerValMetLys
ArgArgIleGluGluIleCysMetLysValPheAlaGlnTyrIleLeuGlyAlaAspPro
LeuArgValCysSerProSerValAspAspLeuArgAlaIleAlaGluGluSerAspGlu
GluAspAlaIleAlaAlaTyrThrLeuAlaThrAlaGlyAlaSerSerSerAspSerLeu
ValSerProProGluSerProValProAlaThrIleProLeuSerSerValIleValAla
GluAsnSerAspGlnGluGluSerGluGlnSerAspGluGluGlnGluGluGlyAlaGln
GluGluArgGluAspThrValSerValLysSerGluProValSerGluIleGluGluVal
AlaSerGluLysGluGluAspGlyAlaGluGluProThrThrSerGlyGlyLysSerThr
HisProMetValThrArgSerLysAlaAspGln
IE-2蛋白(人类疱疹病毒第五型，与人类巨细胞病毒CMV)：
MetLysProValLeuValLeuAlaIleLeuAlaValLeuPheLeuArgLeuAlaAspSer
ValProArgProLeuAspValValValSerGluIleArgSerAlaHisPheArgValGlu
GluAsnGlnCysTrpPheHisMetGlyMetLeuTyrPheLysGlyArgMetSerGlyAsn
PheThrGluLysHisPheValAsnValGlyIleValSerGlnSerTyrMetAspArgLeu
GlnValSerGlyGluGlnTyrHisHisAspGluArgGlyAlaTyrPheGluTrpAsnIle
前述任一种具有Ag473的N-端部分的融合蛋白，可以令人意外的高产量于大肠杆菌表现系统中制得。还意外发现，该重组型融合蛋白可诱发对抗该抗原及相关病症(例如癌症)的免疫反应。
上述的Ag473为一种脑膜炎双球菌Neisseria Mengitidis的脂蛋白，其是由四个功能域(SP及功能域1-3)所组成。以下列示其具有四个已确定功能域的胺基酸序列：
Met Lys Lys Leu Leu Ile Ala Ala Met Met Ala Ala Ala Leu Ala Ala Cys (SEQ ID NO：7)
SP：SEQ ID NO：8中的胺基酸残基1-17(以下划线示出)
功能域1：SEQ ID NO：9中的胺基酸残基18-40(以高亮标示)
功能域2：SEQ ID NO：10中的胺基酸残基41-71(以粗体标示)
功能域3：SEQ ID NO：11中的胺基酸残基72-121(以斜体标示)
术语“脂质化序列”如用于本文意指一种非天然胺基酸序列，其(a)包括与Ag473的SP具有同一性至少达80％(85％、90％、95％或99％)的第一片段，及与Ag473的功能域1具有同一性至少达80％(85％、90％、95％或99％)的第二片段，第一片段位于该脂质化序列的N-端；且(b)有助于在大肠杆菌中将在其N-端带有该脂质化序列的多肽脂质化。于脂质化序列中，第一片段直接或经由连接肽而连接至第二片段。较佳地，此序列具有最大长度为40-100(例如，40-80)个胺基酸。于一项实例，该脂质化序列包括SP与功能域1，也即SEQ IDNO：7的胺基酸1-40(SEQ ID NO：12)。脂质化序列的另外实例包括，SEQ ID NO：7的包括有SEQ ID NO：7胺基酸1-40的任何其他片段，例如1-41、1-45、1-50、1-60、1-80、1-100及1-121。
如用于本文，两种胺基酸序列的“百分比同源性”是使用经描述于Karlin与Altschul，Proc，Natl.Acad.Sci.USA 87：2264-2268，1990的算法(其修改描述于Karlin与Altschul，Proc，Natl.Acad.Sci.USA 90：5873-5877，1993)而测定得。此种算法被纳入Altschul等人，J.Mol.Biol.215：403-410，1990所述的NBLAST及XBLAST程序中。BLAST蛋白质搜寻是以XBLAST程序来执行，分数＝50，字长＝3，而获得相对于参考多肽的序列同源性。为达比较目的而得到具有空隙的比对，遂使用如Altschul等人，于Nucleic Acids Res.25：3389-3402，1997所述的具空隙BLAST(Gapped BLAST)。当利用BLAST与具空隙BLAST程序时，是使用这些各别程序(例如，与)的不履行参数。参见，www.ncbi.nlm.nih.gov。
本发明的多肽或融合蛋白可呈合成型多肽，或呈重组型多肽的形式获得。关于重组型多肽的制备，可将编码它的核酸与另一编码融合伙伴，例如谷胱甘肽-S-转移酶(GST)、6x-His表位标签或M13基因3蛋白的核酸连接。所成的融合核酸于适宜宿主细胞中表现，得一种能藉由该项技艺已知的方法单离的融合蛋白。单离的融合蛋白可进一步(例如)以酵素分解进行处理，去除融合伙伴而得到本发明的重组型多肽。
异源多肽、核酸或基因是指其源自外来物种，或(若来自同一物种)实质上已从其原始形式修饰过的。两种经融合的功能域或序列，若它们在天然蛋白质或核酸中彼此不相邻，则其对彼此而言为异源的。术语“重组型”当用于引述(例如)细胞，或核酸、蛋白质或载体时，意指该细胞、核酸、蛋白质或载体已经由导入异源核酸或蛋白质，或藉由改变天然核酸或蛋白质而受修饰，或指该细胞是衍生自经由此类修饰的细胞。因此，举例而言，重组型细胞可表现未存在于该细胞的天然形式中的基因，或表现天然基因的被正常或异常表现、表现不足或不被表现的第二复本。
于本发明，是将前述所提及的脂质化序列连接至肿瘤相关抗原，例如衍生自HPV的病毒性抗原(例如，HPV E7)而形成一种融合蛋白质，当其藉由习知重组技术在大肠杆菌中进行表现时，是呈脂质化形式。以下描述一种实例。将编码脂质化序列的DNA片段，与编码E7m的DNA片段，插入一种表现载体(其较佳地带有强启动子，例如T7、T5、T3或SP6)中，而构筑得表现质体。强启动子可为可诱导性，例如可被异丙基β-D-硫代半乳糖苷(IPTG)诱导。接着将该表现质体导入大肠杆菌宿主菌株中，并将阳性转形株培养于适于蛋白质表现的条件下。较佳地，该大肠杆菌宿主菌株对于因过度表现外源性蛋白质所诱导的毒性作用具有抗性。此类大肠杆菌株可经由美国专利案号6,361,966中所描述的方法鉴定/产生。此等大肠杆菌株的实例包括(但不限定于)C43(DE3)(ECCCB96070445)、C41(DE3)(ECCC B96070444)、C0214(DE3)、DK8(DE3)S(NCIMB40885)及C2014(DE3)(NCIMB 40884)。
较佳地，为将经此所表现得到的融合蛋白质从大肠杆菌宿主细胞分离出，并且经由该项技艺已知的方法，例如以抗-脂蛋白抗体进行的免疫转渍法，或质谱分析法确认其脂质化状态。
本发明的多肽或融合蛋白可用于制备免疫原组成物(例如，疫苗)，其供于易受病毒感染的个体(例如，人类个体)中产生对抗(例如)HPV的抗体。此类组成物可以(例如)下述的方法，或藉由该项技艺中已知的任何其他相当方法制备得到。
本发明的多肽或融合蛋白可与医药上可接受的载体，例如磷酸盐缓冲食盐水、碳酸氢盐溶液或佐剂混合，而制得一种医药组成物。载体必须为“可接受的”意指，其可与组合物中的活性成份兼容，且较佳地，能够使活性成份安定，而不会对受治疗个体产生有害影响。载体是以投药模式与途径，以及标准制药操作为基础而进行选择。适宜的医药载体与稀释剂，及供它们使用的必需品，经描述于雷明顿氏制药科学(Remington′s Pharmaceutical Sciences)中。于一项实例，是将融合蛋白质与佐剂混合而形成可用于免疫调节的组成物。此组成物可制备呈可注射液，呈液态溶液或乳液。参见，美国专利案4,601,903、4,599,231、4,599,230及4,596,792。
“佐剂”意指添加至免疫原组成物(例如，疫苗)的物质，其虽然本身不具有特异性抗原功效，但能刺激免疫系统并增加对该免疫原组成物的免疫反应。佐剂的实例包括(但不限定于)明矾沉淀物、弗氏完全佐剂、弗氏不完全佐剂、单磷氧基-脂质A/海藻糖二霉菌酸酯佐剂、含有棒状杆菌(Corynebacteriumparvum)与tRNA的油包水乳液、及其他可藉由模拟特定组演化固有分子而执行增加免疫反应的任务的物质，包括脂质体、脂多醣(LPS)、对应抗原的分子笼(molecular cage)、细菌细胞壁组成、被细胞吞噬的核酸例如双股RNA、单股DNA及含有未甲基化CpG二核苷酸的DNA。其他实例包括霍乱毒素、大肠杆菌热不安定性内毒素、脂质体、免疫刺激复合体(ISCOM)、免疫刺激性序列寡脱氧核苷酸及氢氧化铝。组成物还可包括有助于活体内递送的聚合物。参见Audran R.等人，疫苗21：1250-5，2003；及Denis-Mize等人Cell Immunol.，225：12-20，2003。
有效量的前述组成物可以非经肠道方式，例如藉由皮下注射或肌肉内注射进行投药。或者，可希望呈其他投药形式，包括栓剂与口服调配物。对于栓剂，可包括黏着剂与载体，例如聚烷二醇类或三酸甘油酯类。口服调配物可包括正常使用的赋形剂，例如制药级糖精、纤维素、碳酸镁等类。此等组成物采取溶液、悬浮液、片剂、丸剂、胶囊、持续释放调配物或粉末的形式。“有效量”意指该组成物可于个体的组织系统，或于个体中引起生物学或医学反应的量，它是由研究人员、医师或其他临床医疗人员探究得。
前述的多肽或及融合蛋白可用于制备免疫原组成物(例如，疫苗)，以供于易受病毒感染的个体中产生对抗病毒的抗体。疫苗可以与其剂量调配物兼容的方式，及以治疗上有效、具保护性且可产生免疫的量进行投药。欲投药的量视受治疗个体而定，包括(例如)该个体的免疫系统合成抗体，及(若需要)产生细胞-介导的免疫反应的能力。活性成分所需要投药的实际剂量，取决于开业医师的专业判断。然而，熟悉该项技艺的人可容易地测定得适宜的剂量范围，且其层级可为数微克的本发明多肽。对于起始投药及追加剂量的适当用药疗程也可有所差异，但可包括起始投药随后施予后续的投药。疫苗的剂量可视投药途径而定，且依据宿主的大小而变化。
术语“免疫反应”或“免疫原反应”是指于一个体中，其免疫系统在对某一种抗原反应时所产生的任何反应。脊椎动物的免疫反应实例包括(但不限定于)制造抗体、诱导细胞介导的免疫作用及活化补体。对于由同一抗原的后续刺激产生的免疫反应(也称为次级免疫反应)，较于初级免疫中所发生的情况快速。术语“免疫原性”是指，于宿主动物中产生对抗一或多种抗原的免疫反应的能力。此免疫反应是构成，由对抗特定感染性生物体所引发的保护性免疫作用的基础。
“抗原”是指一种含有一或多个会刺激宿主的免疫系统制造体液，及/或细胞抗原专一性反应的抗原表位的分子。术语“抗原”可与“免疫原”互换使用。与适当细胞接触后的结果，抗原会诱发一种敏感或免疫反应的状态，并以可论证的方式与受敏化个体的抗体或免疫细胞，于活体内或活体外进行反应。抗体可被生物个体内的抗体专一地辨识及结合。与主要组织兼容性复合体(MHC)缔合的抗原，也可被位于T淋巴细胞(T-细胞)表面上的受体辨识及结合，而导致T-细胞活化。术语“抗原表位”用于本文是指于抗原上，一特定抗原分子或T-细胞受体与其结合的部位。该术语可与“抗原决定基”或“抗原决定部位”互换使用。
“抗体”是指一种具有特定胺基酸序列，且仅和一种或一群密切相关的抗原结合的免疫球蛋白分子，或免疫球蛋白分子的至少一具有免疫活性的部份。抗体的实例包括IgG、IgM、IgA、IgD及IgE。免疫球蛋白分子的具有免疫活性的部份实例包括Fab及F(ab)′2，其可藉由将抗体以诸如胃蛋白酶的酵素处理而产生。抗体可为单株抗体或多株抗体。术语“单株抗体”是指一群仅含有一种抗原结合部位，且能够与特别抗原表位进行免疫反应的抗体分子族群。术语“多株抗体”是指一群含有一种以上抗原结合部位，且能够与多肽上一个以上抗原表位进行免疫反应的抗体分子族群。
可藉由该项技艺已知的方法鉴定出容易受病毒感染的个体，并投药予本发明的组成物。组成物的剂量取决于(例如)特别的多肽/蛋白质、是否有佐剂共同投药、及共同投药的佐剂类型、投药形式与频数等，其可由熟习该项技艺的人测定得。若需要可进行重复投药，其可由熟习该项技艺的人决定。举例而言，在第一次免疫后4至8周可给予追加剂量，且于8至12周可给予第二次追加(使用相同的调配物)。可从个体采集血清或T细胞，以测试由对抗该病毒的组成物所引起的免疫反应。分析对抗某一种蛋白质或感染的抗体或细胞毒性T细胞的方法，为该项技艺已熟知。若需要可给与额外的追加免疫。藉由改变多肽/蛋白质的用量、组成物的剂量及投药的频数，而可将免疫程序最适化以引起最大免疫反应。于大规模投药前，可希望进行功效测试。于功效测试中，可将非人类个体(例如，小鼠、大鼠、兔子、马、猪、牛或猴子)经由口服或非经肠道途径，投药予本发明的组成物。在最初投药之后，或在视需要而追加投药后，可将测试个体与对照组个体(接受伪装投药)皆以病毒进行攻毒，以测试该组成物的有效性。
可使用各种抗原。于某些具体态样，抗原可为一种肿瘤相关抗原、癌症抗原或肿瘤抗原。术语癌症抗原与肿瘤抗原可互换使用，且是指一种由癌细胞差异表现的抗原。于是，可将癌症抗原利用于差异靶定一种对抗癌细胞的免疫反应。因此癌症抗原可能刺激肿瘤-特异性免疫反应。某些癌症抗原是由正常细胞所编码，虽然不是必然会被表现。有些此等抗原的特征可能是，其在正常细胞中正常状态下为缄默的(也即，不被表现)，有些只在分化的某些阶段被表现，而有些是暂时被表现(例如，胚胎与胎儿抗原)。其他癌症抗原可由突变型细胞基因，例如致癌基因(例如经活化的ras致癌基因)、抑制因子基因(例如突变型p53)，或因内部缺失或染色体异位产生的融合蛋白。又，其他癌症抗原可由病毒基因，例如RNA及DNA肿瘤病毒所携带的基因编码。
肿瘤抗原的实例包括MAGE、MART-1/Melan-A、gp100、二肽基肽酶IV(DPPUV)、腺苷脱胺酶结合蛋白(ADAbp)、亲环蛋白(cyclophilin)b、结直肠相关抗原(CRC)-C017-1A/GA733、癌胚性抗原(CEA)及其抗原性表位CAP-1与CAP-2、etv6、aml1、前列腺专一性抗原(PSA)及其抗原性表位PSA-1、PSA-2与PSA-3、前列腺专一性膜抗原(PSMA)、T-细胞受体/CD3-.zeta.链、肿瘤抗原的MAGE-家族(例如MAGE-A1、MAGE-A2、MAGE-A3、MAGE-A4、MAGE-A5、MAGE-A6、MAGE-A7、MAGE-A8、MAGE-A9、MAGE-A10、MAGE-A11、MAGE-A12、MAGE-Xp2(MAGE-B2)、MAGE-Xp3(MAGE-B3)、MAGE-Xp4(MAGE-B4)、MAGE-C1、MAGE-C2、MAGE-C3、MAGE-C4、MAGE-C5)、肿瘤抗原的GAGE-家族(例如GAGE-1、GAGE-2、GAGE-3、GAGE-4、GAGE-5、GAGE-6、GAGE-7、GAGE-8、GAGE-9)、BAGE、RAGE、LAGE-1、NAG、GnT-V、MUM-1、CDK4、酪胺酸酶、p53、MUC家族、HER2/neu、p21ras、RCAS1、α-胎蛋白、E-钙黏着蛋白、α-连环蛋白、β-连环蛋白、γ连环蛋白、p120ctn、PRAME、NY-ESO-1、cdc27、腺肿瘤性大肠息肉蛋白(APC)、胞衬蛋白(fodrin)、连接蛋白(Connexin)37、Ig-个体遗传型、p15、gp75、GM2与GD2神经节苷脂、病毒产物例如人类乳头状瘤病毒蛋白、肿瘤抗原的Smad家族、Imp-1、P1A、EBV-编码的核抗原(EBNA)-1、脑肝糖磷酸化酶、SSX-1、SSX-2(HOM-MEL-40)、SSX-3、SSX-4、SSX-5、SCP-1与CT-7及c-erbB-2。
癌症或肿瘤及与此类肿瘤(非排他地)相关的特定肿瘤抗原，包括急性淋巴性白血症(etv6、aml1、亲环蛋白b)、细胞淋巴瘤(Ig-个体遗传型)、神经胶瘤(E-钙黏着蛋白、α-连环蛋白、β-连环蛋白、γ-连环蛋白、p120ctn)、膀胱癌(p21ras)、胆管癌(p21ras)、乳癌(MUC家族、HER2/neu、c-erbB-2)、子宫颈癌(p53、p21ras)、结肠癌(p21ras、HER2/neu、c-erbB-2、MUC家族)、结直肠癌(结直肠相关抗原(CRC)-C017-1A/GA733、APC)、绒毛膜癌(CEA)、上皮细胞癌(亲环蛋白b)、胃癌(HER2/neu、c-erbB-2、ga733醣蛋白)、肝细胞癌(α-胎蛋白)、哈金氏淋巴瘤(Imp-1、EBNA-1)、肺癌(CEA、MAGE-3、NY-ESO-1)、淋巴细胞衍生的白血病(亲环蛋白b)、黑色素瘤(p5蛋白、gp75、癌胚胎抗原、GM2与GD2神经节苷脂、Melan-A/MART-1、cdc27、MAGE-3、p21ras、gp100)、骨髓瘤(MUC家族、p21ras)、非小细胞肺癌(HER2/neu、c-erbB-2)、鼻咽癌(Imp-1、EBNA-1)、卵巢癌(MUC家族、HER2/neu、c-erbB-2)、前列腺癌(前列腺专一性抗原(PSA)及其抗原性表位PSA-1、PSA-2与PSA-3、PSMA、HER2/neu、c-erbB-2、ga733醣蛋白)、肾癌(HER2/neu、c-erbB-2)、子宫颈与食道的鳞状细胞癌(NY-ESO-1)、与T细胞白血病(HTLV-1抗原表位)，及病毒性产物或蛋白质。
如前所述，病毒感染会引起各种病症，包括癌症。引起癌症的病毒实例包括EBV、HPV、HCV、HBV及CMV。其中，HPV已知会引起包括疣(例如生殖器疣)、子宫颈细胞分化不良及癌症(例如，子宫颈癌)。于是，本发明的范围包含一种对抗感染及相关病症的免疫原组成物。可利用许多种HPV抗原。与其致癌能力有关连的HPV基因型归类为高危险型，包括16、18、31、33、35、39、45、51、52、56、58、59、68、73及82型(Munoz等人，N Engl J Med 348：518-27，2003)。其中以HPV16与HPV18特别有用，因为它们为在侵入性子宫颈癌中具有高流行率的两型(Bosch等人，J Natl Cancer Inst 87：796-802，1995)。于50％子宫颈癌中有侦测到HPV16，而HPV18与18％子宫颈癌有关。于腺癌及鳞状腺癌中，HPV流行率(76.4％)明显较其于鳞状细胞癌中的(87.3％)低，且HPV18为占优势型(～40％)(Clifford等人，Br J Cancer 89：101-5，2003)。在治疗罹患HPV-相关肿瘤的患者方面，已针对疫苗疗法进行数种临床试验。其中大多数是以E7或E7/E6致癌蛋白为基础，因为在前恶性癌及恶性癌中一致地表现E6与E7。而且，有许多报导指出细胞介导的免疫反应对此等患者的重要性(Moscicki，JAdolesc Health 43：S26-40，2008)。
如以下的实施例所述，可将去活性HPV致癌蛋白(例如去活性E7(E7m))与Ag473的D1融合，而制得重组型脂质-免疫原(rlipo-E7m)。于TC-1小鼠模式中，lipo-E7m可保护该小鼠对抗肿瘤细胞的攻毒。此等结果清楚证明，lipo-E7m可用做为一种对抗HPV-相关肿瘤的有效疫苗，且暗示脂质-免疫原策略可应用于诸如E7与E6等HPV致癌蛋白对抗HPV-相关肿瘤。
下述的特别实施例仅供做为说明范例，而非用以限制本发明揭示的其他部分。无需进行过度实验，相信熟悉该项技艺具有通常知识的人可基于本说明书的叙述，将本发明利用至其最完整的程度。本文中所引用的公开文献，皆完整地以参考方式纳入本文。此外，下文中所推论的任何机制不会限制本发明的申请专利范围。
实施例
脂质化融合蛋白rlipo-E7m的表现
使用以重迭引子进行的组合PCR方法获得E7m基因。然后将组合PCR的产物藉由习知PCR扩增(Dillon PJ，Rosen CA.Biotechniques 1990；9：298，300)。用于此步骤的前向引子(5’-GGAATTCCATATGCACGGCGATACCCCGACCCTGC-3’(SEQ ID NO：3))包括一个Nde I 切位(以下划线标示)，而反向引子(5’-AGAGCCGCTCGAGCGGTTTCTGGCTCGCAATCGG-3’(SEQ ID NO：4))包括一个Xho I切位(以下划线标示)。将PCR产物利用Nde I与Xho I切位选殖入表现载体pET-22b(+)(NOVAGEN，Madison，WI)中，而制得pE7m质体。结果，该重组蛋白的C-端含有额外的六组胺酸标签(HisTag)。
将表现质体pE7m转形至大肠杆菌株BL21Star(DE3)(INVITROGEN，USA)中，以供进行蛋白质表现。将经转形的细胞于37℃下培养过夜，然后以1mM的IPTG进行诱导3小时。为获得用于表现脂质化免疫原的质体，我们遂将能够被转形至CD43(DE3)菌株中，用以表现重组型脂蛋白rlipo-D1E3(Chen et al.，Vaccine 2009；27：1400-9)的质体pD1E3加以修改。用于此步骤的前向引子(5’-CGCGGATCCATGCACGGCGATACCCCGACCCT-3’(SEQ ID NO：5))包括一个Bam HI 切位(以下划线标示)，而反向引子(5’-AGAGCCGCTCGAGCGGTTTCTGGCTCGCAATCGG-3’(SEQ ID NO：6))包括一个Xho I切位(以下划线标示)。利用Bam HI与Xho I切位，将PCR产物选殖入pD1E3中，而制得plipo-E7m质体。结果，该重组蛋白的C-端含有额外的六组胺酸标签(HisTag)。将表现质体plipo-E7m转形至大肠杆菌株C43(DE3)(INVITROGEN，Carlsbad，CA)中，以供进行脂质-蛋白表现。将经转形的细胞于37℃下培养过夜，然后以1mM的IPTG进行诱导3小时。
rlipo-E7m的特征化
藉由如下述的固定化金属亲合性层析术(IMAC)，将自pE7m表现的重组型E7m(rE7m)与自plipoE7m表现的重组型rlipo-E7m从C43(DE3)细胞分离出。藉由离心(8000xg，20分钟)自4-升细胞培养物采收大肠杆菌细胞，并将藉此收集得到的细胞沉淀再悬浮于100ml含有50mM Tris(pH 8.0)的均质化缓冲液(homogenization buffer)中。然后使用French Press(Constant Systems，Daventry，UK)于27Kpsi.下，将大肠杆菌细胞在有清洁剂存在下打破，并将因此所得的细胞溶解产物于80,000xg下离心60分钟。收集沉淀物，并使用萃取缓冲液(1％TRITON X-100/50mM Tris(PH8.9))使其溶解。于80,000xg下离心40分钟后，将上清液与5ml Ni-NTA树脂(Qiagen，圣地亚哥，CA，USA)于冷房下培育过夜。将经过培育的样本与树脂浆液加样至管柱(1.6cm i.d.x 2.7cm)上。首先将该管柱以50mL萃取缓冲液冲洗。待以缓冲液(1％TRITON X-100，50mM Tris(PH8.9))溶析出重组蛋白质后，将它们并藉由SDS-PAGE与免疫转渍法进行特征化。结果指出，分离得的重组型lipo-E7m具有高纯度(如图2所示)。使用与铜离子偶合的IMAC，并以1000mL溶析缓冲液与300mL清洗缓冲液(100mM咪唑，1％TRITON X-100，50mM Tris(PH8.9))充分冲洗，以去除脂多醣(LPS)。于所得制剂中的残留量少于30EU/mg。
然后将融合蛋白质lipo-E7m依如下所述进行质谱术(MS)分析。特别是，将纯化的rlipo-E7m使用手动撷取模式，灌流入WATERS SYNAPT HDMS质谱仪中。使用最大熵数算法进行15重复次计算，而计算得分子质量。为鉴定rlipo-E7m的N-端片段，首先将rlipo-E7m对5mM碳酸氢铵(于pH8.5)进行透析，然后以胰蛋白酶(Promega Co.，Madison，WI)，于lipo-E7m：胰蛋白酶为50∶1(wt/wt)的比例下，于25mM碳酸氢铵(pH8.5)中于室温下处理5分钟。藉由添加甲酸(终浓度为1.2％)终止该酵素反应。使用ZIPTIP(MILLIPORE)将反应混合物进一步研磨。将一微升经胰蛋白酶分解的蛋白质与1μlα-氰基-4-羟基肉桂酸(Sigma)溶于乙腈/0.1％三氟乙酸(1∶3，vol/vol)的饱和溶液混合。将一微升混合物置于基质辅助雷射脱附游离/飞行时间(MALDI-TOF)质谱仪(Burker)的标的平盘上进行分析。从MALDI-TOF分析得到的结果指出，部份胰蛋白酶分解作用产物相当于lipo-E7m的N-端片段，且此等肽类是经脂质化。
如图3A中所示，结果证明纯化的rlipo-E7m具有至少五个不同修饰，且各修饰相差14amu。藉由最大熵数算法(MaxEnt1)测得分子质量(以道耳顿为单位)。已证明有五个分别位于15384.5、15400、15412.5、15426及15439的主要高峰。如图3B所示，为经由Bruker AutoFlex III质谱仪进行分析的10分钟-分解样本。MALDI-TOF MS光谱也证明，有五个分别位于m/z 1452、1466、1480、1492及1506的高峰，其为脂质化衍生物的特征。
上述结果实属意外，因为大肠杆菌一般不适合用于制造经修饰的蛋白质，特别是脂质化蛋白质。
细胞毒性T淋巴细胞分析
为研究rlipo-E7m致免作用是否会于活体内诱导细胞毒性T淋巴细胞，遂将C57BL/6小鼠以rE7m或rlipo-E7m蛋白进行免疫，并使用如下述的铬释出分析法来分析细胞毒性T淋巴细胞活性。
简述之，将C57BL/6小鼠以PBS、rE7m(30mg)或rlipo-E7m(30mg)，于第0天及第14天经皮下进行免疫接种。于第21天，收集各组小鼠的脾脏细胞，并以溶于10％FBS RPMI的1mg/ml RAH于10U/ml IL-2存在下进行再刺激5天供做为效应细胞。于进行最后一次免疫后七天，将红血球-耗尽的脾脏细胞(1x106细胞/mL)与1mg/mL的HPV16 E7-衍生肽RAH(RAHYSIVTF)，及TC-1 10U/mL重组型人类IL-2于24-孔平盘中进行活体外培养5天。将细胞(5x105)以100mCi的51Cr(Na2 51CrO4；PERKINELMER，Waltham，MA，USA)于下进行标定小时，以供做为标靶细胞。将经肽刺激的脾脏细胞与经标定的标靶细胞，以各种如所指定的效应细胞-对-标靶细胞比例混合。经于37℃下培育4小时后，使用加玛计数器(PERKINELMER)测量上清液的放射活性。使用下列公式计算比溶解的百分比值：100×〔(实验释出量-自发释出量)/(最大释出量-自发释出量)〕。
如图4所示，于100及50的E/T比例下，rlipo-E7m能对E7-表现的肿瘤(TC-1细胞)诱发rE7m较强的杀死效应。此等结果指出，rlipo-E7m致免作用可于活体内诱导CTL反应。
活体内肿瘤保护实验
关于活体内肿瘤保护实验，是将C57BL/6小鼠(每组六只)以30□g rE7m或rlipo-E7m进行免疫。以与用于第一次疫苗接种相同的程序，对小鼠施予两次追加免疫。于最后一次疫苗接种后两周，将小鼠以2x105TC-1肿瘤细胞/小鼠，经皮下注射于右大腿内进行攻毒，并以视诊及触诊每周进行检测两次。将另一动物模式首先注射以2x105TC-1肿瘤细胞/小鼠。在第七天，将30□g rE7m或rlipo-E7m投药予C57BL/6小鼠(每组六只)。以视诊及触诊对此等小鼠每周进行检测两次。
如图5A所示，小鼠是以免疫原进行疫苗接种，然后使用TC-1肿瘤细胞攻毒。待以rlipo-E7m疫苗接种，100％的小鼠在肿瘤接种后历经60天仍保持无肿瘤产生，而这些注射以PBS的小鼠于20天内即发展出肿瘤，且67％的小鼠在以rE7m疫苗接种后有发展出肿瘤。图5B证明，于注射以肿瘤细胞的小鼠在投药予免疫原之前，rlipo-E7m仍显示具有较其非脂质化对应物(rE7m)更为优良的功效。特别是，83％接受rlipo-E7m的小鼠仍保持无肿瘤产生，而所有注射以PBS的小鼠在20天内皆发展出肿瘤，且仅有18％接受rE7m的小鼠仍保持无肿瘤产生。此等结果证明，于该二种小鼠模式中，去活化-E7的功效在其呈现脂质化形式时有显著被增加。经rlipo-E7m疫苗接种的小鼠，当相较于以rE7m疫苗接种的小鼠，可提供显著的对抗TC-1细胞攻毒的保护作用。
其他具体态样
本说明书中所揭示的全部特征可以任何组合方式组合。本说明书中所揭示的各别特征可由依相同、相等或类似目的的替代特征取代。因此，除非另行清楚地指示，所揭示的各特征仅为一系列同等物或类似特征的实例。前述的说明已描述本发明的许多较佳实施例。习于该项技艺人士应了解，在未偏离本发明的精神及范围下可进行各种改变与修饰，以使其适于各种不同用途与状况。因此，于申请专利范围内也包含其他具体态样。
Claims (19)
1.一种单离的多肽，其特征在于，是由SEQ ID NO:2的序列组成。
2.一种单离的核酸，其特征在于，是由编码权利要求1所述的多肽的序列、或其互补序列组成。
3.根据权利要求2所述的核酸，其特征在于，核酸是由SEQ ID NO:13的序列组成。
4.一种表现载体，其特征在于，包含权利要求2所述的核酸。
5.一种宿主细胞，其特征在于，包含权利要求2所述的核酸。
6.根据权利要求5所述的宿主细胞，其特征在于，为大肠杆菌。
7.一种免疫原组成物，其特征在于，包含权利要求1所述的多肽。
8.根据权利要求7所述的免疫原组成物，其特征在于，该组成物进一步包含医药上可接受的佐剂。
9.一种制造权利要求1所述的多肽的方法，其特征在于，包含
将包含权利要求2所述的核酸的宿主细胞培养于允许由该核酸编码的多肽表现的条件下，及
将该多肽自所培养的细胞或培养基纯化出。
10.一种单离的融合蛋白质，其特征在于，该融合蛋白质是由SEQ ID NO.12的序列组成的脂质化序列的第一片段，及
权利要求1所述的多肽的序列的第二片段组成，
其中于该融合蛋白质中该第一片段是位于该第二片段的N-端。
11.根据权利要求10所述的融合蛋白质，其特征在于，该融合蛋白质为脂质化。
12.一种单离的核酸，其特征在于，是由编码权利要求10所述的融合蛋白质的序列组成，或其互补序列。
13.根据权利要求12所述的核酸，其特征在于，核酸是由SEQ ID NO:15的序列组成。
14.一种表现载体，其特征在于，包含权利要求12所述的核酸。
15.一种宿主细胞，其特征在于，包含权利要求12所述的核酸。
16.根据权利要求15所述的宿主细胞，其特征在于，为大肠杆菌。
17.一种免疫原组成物，其特征在于，包含权利要求10所述的融合蛋白质。
18.根据权利要求17所述的免疫原组成物，其特征在于，该组成物进一步包含医药上可接受的佐剂。
19.一种制造权利要求10所述的融合蛋白质的方法，其特征在于，包含
将包含权利要求12所述的核酸的宿主细胞培养于允许由该核酸编码的融合蛋白质表现的条件下，及
将该融合蛋白质自所培养细胞或培养基纯化出。
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| WO2010148496A1 (en) | 2010-12-29 |
| US8658176B2 (en) | 2014-02-25 |
| EP2445927A4 (en) | 2012-12-19 |
| TWI409275B (zh) | 2013-09-21 |
| CN102482327A (zh) | 2012-05-30 |
| HK1171457A1 (zh) | 2013-03-28 |
| WO2010148496A8 (en) | 2011-03-03 |
| EP2445927B1 (en) | 2014-04-23 |
| EP2445927A1 (en) | 2012-05-02 |
| TW201103946A (en) | 2011-02-01 |
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