CN101668770A - 疫苗 - Google Patents
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- CN101668770A CN101668770A CN200880007888A CN200880007888A CN101668770A CN 101668770 A CN101668770 A CN 101668770A CN 200880007888 A CN200880007888 A CN 200880007888A CN 200880007888 A CN200880007888 A CN 200880007888A CN 101668770 A CN101668770 A CN 101668770A
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Abstract
本发明涉及包括与提供T辅助表位的免疫融合伴侣，例如B型流感嗜血杆菌的蛋白质D，连接的来源自所谓肿瘤排斥抗原PRAME(也称作DAGE)的抗原的融合蛋白，包括蛋白质D片段的融合伴侣蛋白、其制备和配制疫苗的方法及其在治疗多种癌症中的用途。
Description
本发明涉及包括与提供T辅助表位的免疫融合伴侣，例如B型流感嗜血杆菌(Haemophilus influenzae)的蛋白D，连接的来源自被称作肿瘤排斥抗原PRAME(也称作DAGE)的抗原的融合蛋白，其制备和配制疫苗的方法及其在治疗多种癌症中的用途，所述癌症包括但不限于黑色素瘤、乳腺癌、膀胱癌、肺癌例如NSCLC、肉瘤、卵巢癌、头颈部癌、肾癌、结肠直肠癌、多发性骨髓瘤、白血病(包括急性白血病)和食道癌。
在进一步的实施方案中，本发明涉及包括蛋白质D衍生物的融合伴侣蛋白及其制备方法。
在不同组的肿瘤相关抗原中，癌症睾丸抗原是免疫疗法所感兴趣的，其归因于它们广泛的肿瘤特异表达以及这些抗原通常在健康细胞中不表达这一事实。至今已描述了多于50种癌症/睾丸抗原，对它们中的许多已鉴定出T淋巴细胞识别表位。PRAME是一种癌症睾丸抗原并正作为潜在的免疫疗法进行研究。
在免疫疗法中通常以疫苗将癌症抗原引入患者中，所述疫苗例如包含蛋白质或其免疫原性片段作为抗原，或包含编码该蛋白的DNA或包含所述DNA的载体作为抗原，其可刺激患者的免疫系统攻击表达相同抗原的肿瘤。
如果适当应答被刺激，T淋巴细胞(T细胞)直接攻击抗原并提供对免疫应答的控制。特异于一种抗原类型的B细胞和T细胞发育。当免疫系统暴露于不同的抗原时，形成不同的B细胞和T细胞。当淋巴细胞发育时，它们通常学习将身体的自身组织(自身的)识别为不同于通常不存在于身体中的组织和颗粒(非自身的)。一旦形成B细胞和T细胞，这些细胞中的一些将增殖并向免疫系统提供“记忆”。这使得免疫系统再一次暴露于相同抗原时可更快更有效地应答。
一些实验似乎表明癌症睾丸抗原可刺激免疫系统中的记忆机制。
一些人推断PRAME参与细胞死亡和细胞周期。一些研究组已显示其表达于黑色素瘤和多种肿瘤包括肺、肾和头颈部肿瘤。有趣的是，它也表达于40-60％的白血病中，例如急性淋巴样白血病和急性髓细胞样白血病，参见，例如，Exp Hematol.2000Dec；28(12)：1413-22。与不过度表达该蛋白质的患者相比，已观察到患者中PRAME的过表达与更高存活率和更低复发率相关。
该抗原及其制备描述于美国专利号5830753中。PRAME以下列登录号存在于注释的人类基因数据库H-Inv DB：U65011.1、BC022008.1、AK129783.1、BC014974.2、CR608334.1、AF025440.1、CR591755.1、BC039731.1、CR623010.1、CR611321.1、CR618501.1、CR604772.1、CR456549.1和CR620272.1。
蛋白质D是革兰氏阴性细菌B型流感嗜血杆菌的表面蛋白。可从WO 91/18926获得来源自蛋白质D的免疫融合伴侣的信息。
有时制备一部分抗原和异源融合伴侣的融合蛋白以增强该抗原的免疫原性和/或辅助以适当的量和/或纯度生产所述蛋白质，参见例如WO 99/40188，其描述了MAGE和例如革兰氏阴性细菌B型流感嗜血杆菌的表面蛋白-蛋白质D的融合蛋白。重组制备了该融合蛋白并可将蛋白质D分泌序列并入融合蛋白以潜在辅助终产物的分泌和增溶。
附图和构建体/序列简述
| 图1 | 构建体3和4的SDS-page分析 |
| 图2 | 构建体3a和4a的SDS-page分析 |
| 图3 | CD4应答(AS01B佐剂) |
| 图4 | CD8应答(AS01B佐剂-20070499) |
| 图5 | CD4应答(AS15佐剂) |
| 图6 | CD8应答(AS15佐剂) |
| 图7 | 本发明构建体实施例的标记氨基酸序列 |
| 图8 | LipoD-MAGE3-His(SEQ ID NO：43)和pD1/3-PRAME-His(SEQ ID NO：10)之间的比对 |
| 图9 | 流感嗜血杆菌的原始蛋白质D(SEQ ID NO：45)和LipoD-MAGE3-His(SEQ ID NO：43)的共享序列之间的比对 |
| 图10 | 流感嗜血杆菌的原始蛋白质D(SEQ ID NO：41)、pD-MAGE3-His(SEQ ID NO：45)和不包括氨基酸2-D和3-P的pD1/3-PRAME-His的构建体(SEQ IDNO：44)的共享序列之间的比对 |
| 图11 | pET21载体中含有或不含分泌信号(SS)和组氨酸-尾(组氨酸)的pD1/3-PRAME的SDS-page分析 |
| 图12 | 蛋白质D1/3MAGE-A3-His(SEQ ID NO：44)的序列 |
| 构建体1(实施例1) | MDP-20-127-蛋白质D-PRAME-TSGHHHHHH(质粒TCMP14)(核苷酸序列SEQ ID NO：1，氨基酸序列SEQ IDNO：2) |
| 构建体2(实施例2) | MDP-20-127-蛋白质D-PRAME-无组氨酸尾(质粒TCMP14)(核苷酸序列SEQ ID NO：3，氨基酸序列SEQ IDNO：4) |
| 构建体3(实施例3) | MDP-20-127-蛋白质D-PRAME-LEHHHHHH(质粒pET21)(核苷酸序列SEQ ID NO：5，氨基酸序列SEQ IDNO：6) |
| 构建体4(实施例4) | MDP-20-127-蛋白质D-PRAME-无组氨酸尾(质粒pET21)(核苷酸序列SEQ ID NO：7，氨基酸序列SEQ IDNO：8) |
| 构建体3a(实施例3a) | MDP-20-127-蛋白质D-PRAME-HHHHHH(质粒pET26)(核苷酸序列SEQ ID NO：9，氨基酸序列SEQ IDNO：10) |
| 构建体4a(实施例4a) | MDP-20-127-蛋白质D-PRAME-无组氨酸尾(质粒pET26)(核苷酸序列SEQ ID NO：11，氨基酸序列SEQ IDNO：12) |
| 序列1(SEQ ID NO：1) | 为实施例1的DNA序列 |
| 序列2(SEQ ID NO：2) | 为实施例1的氨基酸序列 |
| 序列3 | 为实施例2的DNA序列 |
| (SEQ ID NO：3) | |
| 序列4(SEQ ID NO：4) | 为实施例2的氨基酸序列 |
| 序列5(SEQ ID NO：5) | 为实施例3的DNA序列 |
| 序列6(SEQ ID NO：6) | 为实施例3的氨酸酸序列 |
| 序列7(SEQ ID NO：7) | 为实施例4的DNA序列 |
| 序列8(SEQ ID NO：8) | 为实施例4的氨基酸序列 |
| 序列9(SEQ ID NO：9) | 为实施例3a的密码子优化DNA序列 |
| 序列10(SEQ ID NO：10) | 为实施例3a的氨基酸序列 |
| 序列11(SEQ ID NO：11) | 为实施例4a的密码子优化DNA序列 |
| 序列12(SEQ ID NO：12) | 为实施例4a的氨基酸序列 |
| SEQ ID NO：13 | VLDGLDVLL(PRA100-108) |
| SEQ ID NO：14 | SLYSFPEPEA(PRA142-151) |
| SEQ ID NO：15 | ALYVDSLFFL(PRA300-309) |
| SEQ ID NO：16 | LYVDSLFFL(PRA301-309) |
| SEQ ID NO：17 | SLLQHLIGL(PRA425-433) |
| SEQ ID NO：18至35 | 用于本发明实施例的寡核苷酸 |
| SEQ ID NO：36 | TCC ATG ACG TTC CTG ACG TT(CpG 1826；) |
| SEQ ID NO：37 | TCT CCC AGC GTG CGC CAT (CpG 1758) |
| SEQ ID NO：38 | ACC GAT GAC GTC GCC GGT GAC GGC ACCACG TCG TCG TTT TGT CGT TTT GTC GTT(CpG 2006) |
| SEQ ID NO：39 | TCC ATG ACG TTC CTG ATG CT(CpG 1668) |
| SEQ ID NO：40 | TCG ACG TTT TCG GCG CGC GCC G(CpG 5456) |
| SEQ ID NO：41 | 流感嗜血杆菌蛋白质D的氨基酸1至127 |
| SEQ ID NO：42 | 1-Met；2-Asp；3-Pro；其后为蛋白质D的氨基酸20至127 |
| SEQ ID NO：43 | 蛋白质D1/3-MAGE-A3-His的氨基酸序列 |
| SEQ ID NO：44 | 用于pD1/3-PRAME-His序列的流感嗜血杆菌蛋白质D的氨基酸 |
| SEQ ID NO：45 | 用于pD1/3-MAGE-His序列的流感嗜血杆菌蛋白质D的氨基酸 |
发明概述
本发明提供融合蛋白，其包括：
a)PRAME或其免疫原性片段，和
b)来源自蛋白质D的异源融合伴侣，
其中所述融合蛋白不包括蛋白质D的分泌序列(信号序列)。
本发明进一步提供如本文所述来源自蛋白质D的融合伴侣蛋白，其中所述融合伴侣蛋白不包括蛋白质D的分泌序列或信号序列。
本发明进一步提供如本文所述的融合蛋白和抗原或其片段。
本发明进一步提供来源自蛋白质D的融合伴侣蛋白，其中所述融合伴侣蛋白包括或由蛋白质D的氨基酸20至127组成。在本发明的一个实施方案中，如本文所述的蛋白质D融合伴侣蛋白的一个或更多个氨基酸可被缺失或经替换被取代。这些氨基酸可利用本文定义的保守替换而替换，或可使用其他氨基酸。在一个实施方案中，可替换1、2、3、4、5、6、7、8、9或更多个氨基酸。
如本文所述的蛋白质D融合伴侣蛋白与野生型蛋白质D相比可附加地或可选择性地包含氨基酸序列内部的缺失或插入。在一个实施方案中，可插入或缺失1、2、3、4、5、6、7、8、9或更多个氨基酸。
在本申请上下文下，术语蛋白质D的“分泌序列”或“信号序列”或“分泌信号”意指天然蛋白质的大约氨基酸1至16、17、18或19。在一个实施方案中，蛋白质D的分泌或信号序列或分泌信号指蛋白质D的N-末端19个氨基酸。术语“分泌序列”或“信号序列”或“分泌信号”在本说明书中可交替使用。
本发明融合伴侣蛋白可包括剩余的完整长度蛋白质D蛋白，或可包括大约剩余的N-末端三分之一的蛋白质D。举例而言，剩余的N-末端三分之一的蛋白质D可包括大致或大约蛋白质D的氨基酸20至127。在一个实施方案中，用于本发明的蛋白质D序列包括蛋白质D的氨基酸20至127。在另一个实施方案中，本发明包括或由任何以下序列组成，这些序列起始于蛋白质D序列的下列任何氨基酸：17、18、19、20、21或22；终止于蛋白质D序列的下列任何氨基酸：125、126、127、128、129、130、131、132、133、134、135、136、137、138、139或140。
上下文中“剩余的”指不包含如本文所述的分泌或信号序列的蛋白质D蛋白的序列。
在本发明的一个实施方案中(其中所述融合蛋白包括PRAME或其免疫原性片段)，本发明的蛋白质D衍生物包括该蛋白质的约起始1/3，更具体而言是氨基酸20至127。在本发明的可选择性实施方案中(其中所述融合蛋白包括PRAME或其免疫原性片段)，蛋白质D包括该蛋白的约起始1/3，其中使用蛋白质D的N-末端109个氨基酸。在本发明的一个实施方案中，所述蛋白质D部分不包括该蛋白质的分泌序列。在本发明的一个实施方案中所述蛋白质D衍生物是没有脂质化的。
在一个实施方案中，本发明提供如本文所述的蛋白质D构建体作为融合伴侣蛋白。该蛋白质D构建体可为附加包括如本文所述的PRAME或MAGE-A3构建体的构建体的融合伴侣蛋白或可为附加包括另一种癌症抗原或任何其他抗原的构建体的融合伴侣蛋白。
对于包括PRAME或其免疫原性片段和蛋白质D的融合蛋白，或对于包括蛋白质D的融合蛋白，或对于包括蛋白质D的融合伴侣蛋白质而言，分泌序列(或信号序列)的存在可对所生产融合蛋白的量产生不利影响。
PRAME
一方面本发明的融合蛋白包括如本文所述的融合伴侣蛋白和PRAME抗原或其免疫原性片段。通常PRAME蛋白具有509个氨基酸并且在一个实施方案中可使用PRAME的所有509个氨基酸。已在PRAME上鉴定出数种细胞毒性T淋巴细胞(CTL)表位，例如：
VLDGLDVLL(PRA100-108；SEQ ID NO：13)；
SLYSFPEPEA(PRA142-151；SEQ ID NO：14)；
ALYVDSLFFL(PRA300-309；SEQ ID NO：15)；
LYVDSLFFL(PRA301-309；SEQ ID NO：16)和
SLLQHLIGL(PRA425-433；SEQ ID NO：17).
通常需要在抗原中包括尽可能多的这些表位以产生强免疫应答并确保抗原尽可能的具有免疫原性。即使如此，有可能通过应用具有有效免疫佐剂的制剂补偿给定构建体的较低免疫原性。下文中更详细地讨论强佐剂。
本发明的一方面提供融合蛋白的PRAME部分，其包括，由或基本由完整长度的蛋白质组成。
但是，本发明还延伸至具有保守替换的PRAME构建体。在一个实施方案中，可替换1、2、3、4、5、6、7、8、9或更多个氨基酸。如本文所述的PRAME构建体与野生型PRAME序列相比可附加地或可选择地包含氨基酸序列内部的缺失或插入。在一个实施方案中可插入或缺失1、2、3、4、5、6、7、8、9或更多个氨基酸。
保守替换是熟知的并在序列比对计算机程序中通常被设置成的缺省评分矩阵。这些程序包括PAM250(Dayhoft M.O.等，(1978)，″A modelof evolutionary changes in proteins″，″Atlas of Protein sequence andstructure″5(3)M.O.Dayhoft(编辑)，345-352)，National BiomedicalResearch Foundation，Washington，和Blosum 62(Steven Henikoft andJorja G.Henikoft(1992)，″Amino acid substitution matricies from proteinblocks″)，Proc.Natl.Acad.Sci.USA 89(Biochemistry)：10915-10919。
总体而言，下列组内替换为保守替换，但是组间替换为非保守的。这些组为：
i)天冬氨酸/天冬酰胺/谷氨酸/谷氨酰胺
ii)丝氨酸/苏氨酸
iii)赖氨酸/精氨酸
iv)苯丙氨酸/酪氨酸/色氨酸
v)亮氨酸/异亮氨酸/缬氨酸/甲硫氨酸
vi)甘氨酸/丙氨酸。
通常用于本发明融合蛋白的PRAME序列/氨基酸与天然PRAME的同一性大于80％，例如85、90、95并且更具体而言为99％。然而，本领域技术人员注意到克隆操作产生的氨基酸残基可保留在重组合成的蛋白质中。如果这些不会对产物的特性产生不利影响，可任选是否将它们移除。
一方面本发明提供如本文所述的融合蛋白，其包括、由或基本由全长PRAME蛋白组成。另一方面本发明融合蛋白的PRAME部分包括、由或基本由一个或更多个下列表位组成：
VLDGLDVLL(PRA100-108；SEQ ID NO：13)；
SLYSFPEPEA(PRA142-151；SEQ ID NO：14)；
ALYVDSLFFL(PRA300-309；SEQ ID NO：15)；
LYVDSLFFL(PRA301-309；SEQ ID NO：16)和
SLLQHLIGL(PRA425-433；SEQ ID NO：17).
融合蛋白
在本发明的另一个实施方案中，不是PRAME的或除PRAME之外的肿瘤抗原可用于本文所述的融合蛋白中。在一个实施方案中，提供一种融合蛋白，其包括如本文所述的融合伴侣蛋白和一个或更多个下列肿瘤抗原或肿瘤抗原衍生物或可引导(direct)对该抗原免疫应答的其免疫原性部分：MAGE抗原，例如MAGE-A抗原如MAGE 1、MAGE2、MAGE 3、MAGE 4、MAGE 5、MAGE 6、MAGE 7、MAGE8、MAGE 9、MAGE 10、MAGE 11、MAGE 12。这些抗原有时也称作MAGE A1、MAGE A2、MAGE A3、MAGE A4、MAGE A5、MAGE A6、MAGE A7、MAGE A8、MAGE A9、MAGE A10、MAGEA11和/或MAGE A12(MAGE A家族)。在一个实施方案中，可使用来自另外两个MAGE家族之一的抗原：MAGE B和MAGE C组。MAGE B家族包括MAGE B1(也称作MAGE Xp1和DAM 10)、MAGEB2(也称作MAGE Xp2和DAM 6)MAGE B3和MAGE B4-该Mage C家族目前包括MAGE C1和MAGE C2。
用于本发明的MAGE抗原可包括全长MAGE抗原。或者，该MAGE抗原可包括MAGE的免疫原性部分，其中可缺失或替换氨基酸序列中的1、2、3、4、5、6、7、8、9、10或更多个氨基酸。在本发明的一个实施方案中，可从MAGE的N-末端缺失2个氨基酸。在本发明的一个实施方案中(其中所述抗原为MAGE-A3或其免疫原性部分)，MAGE-A3的序列可来自MAGE-A3的氨基酸3至314。
在另一个实施方案中，用于本发明的肿瘤抗原或衍生物可为PRAME、BAGE、LAGE 1、LAGE 2(也称作NY-ESO-1)、SAGE、HAGE、XAGE、PSA、PAP、PSCA、P501S(也称作prostein)、HASH1、HASH2、Cripto、B726、NY-BR1.1、P510、MUC-1、Prostase、STEAP、酪氨酸酶、端粒酶、存活素、CASB616、P53和/或Her-2/neu或可引导对该抗原免疫应答的其免疫原性部分。
在本发明的另一个实施方案中，所述肿瘤抗原可包括或由下列抗原之一或可引导对该抗原免疫应答的其免疫原性部分组成：SSX-2；SSX-4；SSX-5；NA17；MELAN-A；P790；P835；B305D；B854；CASB618(如WO00/53748中描述)；CASB7439(如WO01/62778中描述)；C1491；C1584和C1585。
在一个实施方案中，用于本发明的抗原可包括或由P501S组成。P501S，也称作prostein(Xu等，Cancer Res.61，2001，1563-1568)，已知为WO98/37814的SEQ ID NO.113并且为553个氨基酸的蛋白质。如上文所引用专利申请公开的包括至少20个、优选50个，更优选100个连续氨基酸的其免疫原性片段或部分可用于本发明的融合蛋白中。优选的片段公开于WO 98/50567(PS 108抗原)并且是前列腺癌相关蛋白(WO 99/67384的SEQ ID NO：9)。其它优选片段为全长P501S蛋白的氨基酸51-553、34-553或55-553。
在一个实施方案中，所述抗原可包括或由以下组成：Wilm′s肿瘤基因表达的WT-1；或可引导对该抗原免疫应答的其免疫原性部分；或包括大约或大致WT-1氨基酸1-249的N-末端片段WT-IF。
在另一实施方案中，所述抗原可包括或由Her-2/neu基因表达的抗原，或可引导对该抗原免疫应答的其片段或其免疫原性部分组成。在一个实施方案中，Her-2/neu抗原可为描述于WO00/44899中的下列融合蛋白之一。
用于本发明的抗原可包括或由″HER-2/neu ECD-ICD融合蛋白″组成，所述融合蛋白也称作″ECD-ICD″或″ECD-ICD融合蛋白″，其指包括HER-2/neu蛋白的胞外结构域(或其片段)和胞内结构域(或其片段)的融合蛋白(或其片段)。在一个实施方案中，该ECD-ICD融合蛋白不包括大部分的HER-2/neu跨膜结构域或不包括任何HER-2/neu跨膜结构域。
在另一个实施方案中，该抗原包括或由″HER-2/neu ECD-PD融合蛋白″组成，所述融合蛋白也称作″ECD-PD″或″ECD-PD融合蛋白″或″HER-2/neu ECD-ΔPD融合蛋白″，也称作″ECD-ΔPD″或″ECD-ΔPD融合蛋白″，其指包括HER-2/neu蛋白的胞外结构域(或其片段)和磷酸化结构域(或其片段，例如ΔPD)的融合蛋白(或其片段)。在一个实施方案中，所述ECD-PD和ECD-ΔPD融合蛋白不包括大部分的HER-2/neu跨膜结构域或不包括任何HER-2/neu跨膜结构域。
如本文所述的PRAME抗原和蛋白质D融合伴侣蛋白的融合蛋白可化学结合，但是优选以重组融合蛋白表达，与不具有融合伴侣(例如蛋白质D或经修饰的蛋白质D蛋白)的单独PRAME相比其可使在表达系统中产生更高水平的PRAME蛋白。
附加地或可选地，本文描述的肿瘤抗原和本发明的融合伴侣蛋白可化学结合或以重组融合蛋白表达，与不具有融合伴侣(例如蛋白质D或经修饰的蛋白质D蛋白)的单独PRAME蛋白质或单独另一种肿瘤抗原相比其可使在表达系统中产生更高水平的PRAME蛋白或另一种肿瘤抗原。
如本文所述的本发明融合蛋白可在融合伴侣蛋白和肿瘤抗原或其免疫原性部分之间，或融合伴侣蛋白和组氨酸尾或其他亲和标签(如果存在的话)之间，或肿瘤抗原或其免疫原性部分和组氨酸尾或其他亲和标签(如果存在的话)之间附加地包括一个或更多个接头序列。接头序列中的氨基酸可与抗原和/或融合伴侣的序列无关。
如本文所述的本发明融合蛋白可在该融合蛋白序列的N末端附加地包括氨基酸Met-Asp-Pro。该Met氨基酸可来自原始蛋白质D序列或来自不相关序列。
融合伴侣可以高于天然重组蛋白的产率辅助表达该蛋白质(表达增强子)。由于其外源性，该融合伴侣蛋白D可尤其具有体内免疫原性并可通过提供T辅助表位(优选被CD4T-细胞识别的T辅助表位)辅助包括PRAME或另一种肿瘤抗原的融合蛋白。该类CD4-T细胞被认为有助于产生有利的免疫应答尤其是CD8细胞毒性T-细胞应答。
在一个实施方案中，该融合伴侣可作为表达增强伴侣和免疫原性融合伴侣二者。
一方面本发明提供融合蛋白，其中所述蛋白质D的N-末端部分(如上文和此处所述)与PRAME的N-末端或其免疫原性片段融合。更具体而言，实施蛋白质D片段和PRAME N-末端的融合这样PRAME替换了已被切除的蛋白质D的C-末端片段。因此蛋白质D的N-末端成为融合蛋白的N-末端。
在另一方面本发明提供融合蛋白，其中所述蛋白质D的N-末端部分(如本文所述)与肿瘤抗原或其免疫原性片段的N-末端或另一部分融合。更具体而言可实施蛋白质D片段和肿瘤抗原的N-末端或其他部分的融合这样如本文所述的PRAME或其他肿瘤抗原或其衍生物替换了已被切除的蛋白质D的C-末端片段。因此蛋白质D的N-末端成为融合蛋白的N-末端。
其它融合伴侣或其片段也可包括在本发明融合蛋白中或可取代本发明的蛋白质D元件，例如在包括如本文所述的PRAME抗原或其片段或部分的实施方案中。其它融合伴侣的实例包括：
·流感病毒的非结构蛋白NS1(红细胞凝集素)-通常利用N-末端的81个氨基酸，尽管可使用不同的片段只要它们包括T-辅助表位。
·来源自肺炎链球菌(Streptococcus pneumoniae)的LYTA，其合成N-乙酰基-L-丙氨酸酰胺酶，酰胺酶LYTA(由lytA基因编码{Gene，43(1986)第265-272页})，例如存在于C-末端(例如起始于残基178，如残基188-305)的Lyta分子的重复部分。
在氨基端包含C-LYTA片段的杂交蛋白的纯化已描述于{Biotechnology：10，(1992)第795-798页}。
本发明融合蛋白可包括亲和标签，例如包括5至9个例如6个组氨酸残基的组氨酸尾。这些残基例如可位于蛋白质D的末端部分(例如蛋白质D的N-末端)和/或与如本文所述的PRAME抗原或其衍生物，或肿瘤抗原或其衍生物的末端部分融合。然而组氨酸尾通常位于如本文所述的PRAME抗原或其衍生物，或肿瘤抗原或其衍生物的末端部分，例如如本文所述的PRAME抗原或其衍生物，或肿瘤抗原或其衍生物的C-末端。组氨酸尾有利于辅助纯化。
本发明也提供编码本发明蛋白质的核酸。该类序列可插入适当的表达载体并用于DNA/RNA疫苗接种或表达于适当的宿主中。表达核酸的微生物载体可用作疫苗。该类载体包括例如痘病毒、腺病毒、α病毒、李斯特菌和monophage。
编码本发明蛋白质的DNA序列可使用标准DNA合成技术合成，例如通过如D.M.Roberts等Biochemistry 1985，24，5090-5098中描述的酶连接，通过化学合成，通过体外酶聚合反应或通过使用例如热稳定聚合酶的PCR技术或通过这些技术的组合合成。
使用DNA聚合酶例如DNA聚合酶I(Klenow片段)在包含三磷酸核苷dATP、dCTP、dGTP和dTTP的适当缓冲液中按需要于10°-37℃的温度下通常在50μl或更小的体积中体外进行DNA的酶聚合反应。可使用DNA连接酶例如T4DNA连接酶在适当的缓冲液例如0.05MTris(pH 7.4)、0.01M MgCl2、0.01M二硫苏糖醇、1mM亚精胺、1mMATP和0.1mg/ml牛血清白蛋白于4℃至室温的条件下通常在50ml或更小的体积中进行DNA片段的酶连接。DNA聚合物或片段的化学合成可通过常规的磷酸三酯、亚磷酸酯或亚磷酰胺化学使用固相技术进行，例如下列文献中描述的那些：′Chemical and Enzymatic Synthesis ofGene Fragments-A Laboratory Manual′(H.G.Gassen和A.Lang编辑)，Verlag Chemie，Weinheim(1982)或其他科学出版物，例如MJ.Gait，H.W.D.Matthes，M.Singh，B.S.Sproat和R.C.Titmas，Nucleic AcidsResearch，1982，10，6243；B.S.Sproat和W.Bannwarth，TetrahedronLetters，1983，24，5771；M.D.Matteucci和M.H.Caruthers，TetrahedronLetters，1980，21，719；M.D.Matteucci和M.H.Caruthers，Journal oftheAmerican Chemical Society，1981，103，3185；S.P.Adams等，Journal ofthe American Chemical Society，1983，105，661；N.D.Sinha，J.Biernat，J.McMannus和H.Koester，Nucleic Acids Research，1984，12，4539；以及H.W.D.Matthes等，EMBO Journal，1984，3，801。
本发明方法可通过常规重组技术进行，例如Maniatis等，MolecularCloning-A Laboratory Manual；Cold Spring Harbor，1982-1989中描述的那些。
具体而言，该方法可包括下列步骤：
i)制备可在宿主细胞中表达包括编码所述蛋白或其免疫原性衍生物的核苷酸序列的DNA聚合物的可复制或整合表达载体；
ii)用所述载体转化宿主细胞；
iii)在允许所述DNA聚合物表达的条件下培养所述转化宿主细胞生产所述蛋白；和
iv)回收所述蛋白质。
本文使用的术语“转化”指将外源DNA引入宿主细胞。这可通过例如用适当的质粒或病毒载体使用例如Genetic EnGlneering；S.M.Kingsman和AJ.Kingsman编辑；Blackwell Scientific Publications；Oxford，England，1988中描述的常规技术转化、转染或感染而实现。术语“经转化的”或“转化子”在下文中应用于所得到的包含并表达所关注外源基因的宿主细胞。
这些表达载体是新的并为本发明一部分。
可根据本发明制备可复制的表达载体，其方式为通过切割与宿主细胞相容的载体提供具有完整复制子的线性DNA区段，并将所述线性区段与一种或更多种与所述线性区段一起编码期望产物的DNA分子例如编码本发明蛋白质的DNA聚合物或其衍生物在连接条件下组合。
因此，DNA聚合物可如所需在载体构建过程中预形成或形成。
载体的选择部分取决于宿主细胞(其可为原核的或真核的但通常为大肠杆菌(E.coli)或CHO细胞)。适当的载体可包括质粒例如TMCP14或pET21或pET26、pcDNA3、噬菌体、粘粒和重组病毒。
可使用适当的用于DNA限制性切割、聚合反应和连接的酶根据例如上文引用的Maniatis等描述的方法进行可复制表达载体的常规制备。
根据本发明可通过用本发明可复制表达载体在转化条件下转化宿主细胞来制备重组宿主细胞。适当的转化条件为常规的并描述于例如上文引用的Maniatis等或″DNA Cloning″第II卷，D.M.Glover编辑，IRL Press Ltd，1985。
转化条件的选择取决于宿主细胞。因此细菌宿主例如大肠杆菌可用CaCl2溶液(Cohen等，Proc.Nat.Acad.Sci.，1973，69，2110)或包括RbCl、MnCl2、醋酸钾和甘油的混合物溶液处理，接着用3-(N-吗啉代)-丙磺酸、RbCl和甘油处理。培养物中的哺乳动物细胞可通过载体DNA钙共沉淀至细胞上而被转化。本发明也涉及用本发明的可复制表达载体转化的宿主细胞。
可经标准技术进行DNA的密码子优化以进一步促进相关宿主的表达。在本发明的一个实施方案中提供了编码包括如本文所述的PRAME抗原或其部分或片段的融合蛋白的DNA，其中PRAME抗原或其部分或片段的核苷酸序列为经密码子优化的。在一个实施方案中，该蛋白质D的核苷酸序列未经密码子优化。
如例如上文引用的Maniatis等和″DNA Cloning″中所描述常规性地在允许DNA聚合物表达的条件下培养已转化的宿主细胞。因此，优选与营养成分一起提供细胞并在低于50℃的温度下培养。
本发明蛋白质可在原核细胞或真核细胞中表达，例如酵母，但通常在大肠杆菌中表达。可使用大肠杆菌的具体菌株例如AR58和BLRDE3。
通常并入例如卡那霉素抗性或氨苄西林抗性的选择标记以促进鉴定重组基因/构建体成功并入表达系统中。
根据宿主细胞并根据表达产物的定位(细胞内或分泌至培养基中或至细胞的周质)通过常规方法回收产物。在本发明的一个实施方案中，所述表达产物是细胞内的。在本发明的一个实施方案中，所述表达产物为不溶性蛋白。因此，当宿主细胞是细菌时，例如大肠杆菌，可物理、化学或酶裂解细胞并从所得溶菌产物中分离蛋白质产物。当宿主细胞为哺乳细胞时，可从营养培养基或无细胞提取物中分离产物。常规蛋白质分离技术包括选择性沉淀、吸附色谱法和包括单克隆抗体亲和柱的亲和色谱法。
在本发明的一个实施方案中提供了生产如本文所述的融合蛋白的方法，其包括在细胞中表达包括如本文所述融合伴侣蛋白的融合蛋白的步骤。所述细胞可为细菌。在所述细胞为细菌的实施方案中，该细菌可为大肠杆菌。本发明方法可包括在细胞中以不溶性蛋白表达如本文所述的融合蛋白的步骤。该方法可进一步包括裂解细胞并从已裂解细胞中纯化已表达融合蛋白的步骤。
在本发明的一个实施方案中提供了根据本文所述方法获得或可获得的融合蛋白。
本发明蛋白可以溶于液体的形式或冻干形式提供。
通常预计每个人用剂量包括1至1000μg蛋白质并优选30-300μg。
本发明也提供药用组合物例如包括本发明融合蛋白在药学可接受赋形剂中的疫苗。
该疫苗可任选包含一种或更多种其它肿瘤相关抗原或多肽，或优选与其他基于肿瘤相关抗原的癌症疫苗组合。举例而言，这些肿瘤相关抗原可为如本文所述的抗原和/或属于MAGE、LAGE和GAGE家族或WT-1的成员。在一个实施方案中所述肿瘤相关抗原可包括或由MAGE-A3抗原组成。
疫苗制备一般性描述于Vaccine Design(″The subunit and adjuvantapproach″(Powell M.F.&Newman MJ编辑).(1995)Plenum Press NewYork)。在脂质体内包囊描述于Fullerton，美国专利4235877。
本发明蛋白可优选在本发明的疫苗制剂中加入佐剂。适当的佐剂包括铝盐，例如氢氧化铝凝胶(明矾)或磷酸铝，但也可为钙、铁或锌盐，或可为不溶性的酰化酪氨酸混悬液，或酰化糖、阳离子或阴离子衍生多糖或聚磷腈。其它已知佐剂包括含CpG的寡核苷酸。该寡核苷酸的特点为该CpG二核苷酸是非甲基化的。该类寡核苷酸为熟知的并描述于例如WO 96/02555中。
在本发明制剂中需要该佐剂组合物可优先诱导TH1型免疫应答。在一个实施方案中提供了佐剂系统包括例如单磷酰类脂A优选3-O-脱酰单磷酰类脂A(3D-MPL)和铝盐的组合。该佐剂也可任选包括CpG寡核苷酸以优先诱导TH1应答。
可用于本发明的增强系统包括单磷酰类脂A和皂苷衍生物的组合，尤其是如WO 94/00153中公开的QS21和3D-MPL组合，或者例如反应原性较低的组合物，其中如WO 96/33739所公开，QS21被胆固醇淬灭。
可用于本发明制剂中一种制剂，其包括QS21 3D-MPL&生育酚，例如在水包油乳剂中，如WO 95/17210中描述。
可用于本发明制剂的另一种佐剂制剂为QS2、3D-MPL&CpG或其等同物，例如在水包油乳剂中或为脂质体制剂。
因此在本发明的一个实施方案中提供了包括如本文所述的融合蛋白或融合伴侣蛋白和佐剂的疫苗，例如上文所描述的。
PRAME和MAGE组合
本发明的一个实施方案中提供了一种组合物，其包括(a)包括本文所述PRAME抗原或融合蛋白的抗原组分和b)包括如本文所述的MAGE抗原或融合蛋白的抗原组分。在一个实施方案中该组合物可进一步包括如本文所述的佐剂。
用于该组合的MAGE抗原可包括全长MAGE抗原。或者，该MAGE抗原可包括MAGE的免疫原性部分，其中可缺失或替换氨基酸序列中的1、2、3、4、5、6、7、8、9、10或更多个氨基酸。在本发明的一个实施方案中，可从MAGE序列的N-末端缺失2个氨基酸。在本发明的一个实施方案中(其中所述抗原为MAGE-A3或其免疫原性部分)，MAGE-A3序列可来自MAGE-A3的氨基酸3至314。
对于上文所述的组合，PRAME和/或MAGE抗原之一或二者可为如本文描述的融合蛋白或蛋白质的一部分，或该抗原可能存在于其它融合蛋白中或以单独抗原提供。
在本发明的一个实施方案中提供了一种组合物，其包括包含如本文所述的PRAME抗原和融合伴侣蛋白的融合蛋白，以及包含如本文所述的MAGE-A3抗原和融合伴侣蛋白的融合蛋白。在可选择性的实施方案中，包括MAGE-A3抗原的融合蛋白包括或由MAGE-A3抗原和包括约蛋白质D起始109个氨基酸的融合伴侣蛋白组成，其中可任选替换蛋白质D的一个或两个或更多个氨基酸并且其中除了蛋白质D起始109个氨基酸之外还任选存在蛋白质D的信号序列。
本发明的融合蛋白可另外任选包括作为抗原序列和融合伴侣蛋白序列之间或抗原和组氨酸尾(如果存在的话)之间“接头”的一个或更多个氨基酸。这些氨基酸可与抗原和/或融合伴侣的序列不相关。
如本文所述，本发明的融合蛋白可在该融合蛋白序列的N-末端附加包括氨基酸Met-Asp-Pro。该Met氨基酸可来自原始蛋白质D序列或可来自不相关序列。
在一个实施方案中，用于本发明的包括MAGE-A3和蛋白质D的融合蛋白序列显示于图12和SEQ ID NO：43。
本发明也涉及制备所述疫苗/组合物的方法。
实施例
制备四种融合构建体并在本文中命名为实施例/构建体1、2、3和4。从实施例3制备密码子优化的构建体并在本文中命名为实施例3a。从实施例4制备密码子优化的构建体并在本文命名为实施例4a。
在实施例3a和4a中，分子中关于蛋白质D部分的序列是相同的。但是，PRAME区中的一些密码子是经修饰的以进一步提高表达并且在实施例3a中移除了PRAME和组氨酸尾之间的接头。
表A实施例/构建体1至4的融合蛋白质结构和质粒详情
N-末端---------------------------------------------------------------C-末端
上述实施例的融合蛋白质包含蛋白质D的氨基酸20-127。氨基酸Met、Asp和Pro包括于蛋白质D片段(即氨基酸MDP-20-127蛋白质D)的N-末端。人们认为这三个附加氨基酸有助于蛋白质的稳定性和/或提高其蛋白质表达水平。将蛋白质D的氨基酸127融合至全长PRAME的N-末端(即将蛋白质D的氨基酸127融合至PRAME的N-末端)。为辅助纯化，这六个蛋白质中的三个包括组氨酸标签尾。尾部的确切序列取决于所用质粒。
构建三种不同类型的质粒TCMP 14和pET21或pET26：每个质粒包括编码含有和不含组氨酸尾的融合蛋白质的DNA。
除非另外说明，在制备各实施例中采用下列一般性策略。
使用TCMP 14载体生成PD1/3-PRAME(含有或不含His标记)重组蛋
白的克隆策略：
质粒TCMP 14中所显示序列的扩增采用三步PCR策略完成。包含编码全部蛋白质D基因的DNA序列的载体pHIC348获得自Dr.A.Forsgren，Department of Medical Microbiology，University of Lund，
General Ho spital，
Sweden。蛋白质D的DNA序列由Janson等(1991)公开{Janson H，LO Heden，A Grubb，M Ruan，&AForsgren.1991.Infect Immun 59：119-125}。表达载体pMG81为pBR322衍生物，其中引入了用于转录和翻译外源插入基因的λ噬菌体衍生控制元件(Shatzman等，1983){Shatzman A，YS Ho，&M Rosenberg.1983.Experimental Manipulation of Gene Expression.Inouya(编辑)第1-14页.Academic NY}。此外，用卡那霉素抗性基因交换氨苄西林抗性基因。部分NS1蛋白质(氨基酸4至81)的编码序列替换了多克隆位点以获得pMG81MCS。用BamHI和NcoI限制性酶切位点将1/3蛋白质D(氨基酸20至127)的编码序列克隆至pMG81MCS以获得pMG81-1/3PD。首先，用pMG81-1/3PD载体作为模板以及正义寡核苷酸：
5’ATA TAA CAT ATG GAT CCA AGC AGC CAT TCA TCA AAT 3’(CAN008；SEQ ID NO：18)和反义寡核苷酸：
5’CCA CAA ACG CCT TCG TTC CAT GGT TTC AAA GTT TTC TGT C 3’(CAN037；SEQID NO：19).
完成对应于蛋白质D氨基酸20-127的部分的PCR扩增。将获自LudwigInstitute，Brussels，Belgium的PRAME cDNA插入pCDNA1载体(Invitrogen)的Bstx1-Not1位点以生成pCDNA-1-PRAME重组载体。用pcDNA-1-PRAME载体(GSKBio)作为模板以及正义寡核苷酸：
5’GAC AGA AAA CTT TGA AAC CAT GGA ACG AAG GCG TTT GTG G 3’(CAN036；SEQID NO：20)和反义寡核苷酸：
5’AGA GAG ACT AGT CTA GTT AGG CAT GAA ACA GGG GCA CAG 3’(CAN029；SEQ IDNO：21)或
5’GGA GGA ACT AGT GTT AGG CAT GAA ACA GGG GCA CAG 3’(CAN002；SEQ ID NO：22)
(取决于添加(CAN002)或未添加(CAN029)组氨酸尾)完成对应于PRAME蛋白质氨基酸的部分的PCR扩增。使用在初始步骤中生成的1/3PD和PRAME基因模板作为模板以及正义寡核苷酸：CAN008和反义寡核苷酸：CAN029或CAN002(取决于存在(CAN002)或不存在(CAN029)组氨酸尾)的PCR扩增后获得插入TCMP 14质粒的最终PRAME序列。在5’末端位点添加NdeI并在3’末端位点添加SpeI使该片段可克隆至TCMP 14载体。
构建载体设计以用pET21载体表达含有或不含His标签重组蛋白质的
重组蛋白质1/3PD-PRAME：
使用包含PRAME基因编码序列的称作pcDNA1-PRAME(如先前策略所述)的重组cDNA质粒和含蛋白质D编码序列N-末端部分的载体PMG81-1/3PD(如先前策略所述)。克隆策略包括以下步骤。
a)首先，用正义寡核苷酸：5’AGAGAGCATATGAGCAGCCATTCATCAAATATGGCG(CAN040；SEQ ID NO：22)，和反义寡核苷酸：
5’ACGTGGGCGGCCGCGGTTTCAAAGTTTTCTGTCATTTCTAA(CAN032；SEQ ID NO：23)；
从质粒PMG81-1/3PD PCR扩增不含分泌信号(分泌或信号序列)的1/3PD序列；
在5’末端位点添加Nde1并在3’末端位点添加Not1使该片段可克隆至pET21b(+)载体。
b)用正义寡核苷酸：
5’TTGTTGGCGGCCGCAATGGAACGAAGGCGTTTGTGGGGT(CAN033；SEQ ID NO：25)，和反义寡核苷酸：
5’GGAGGACTCGAGGTTAGGCATGAAACAGGGGCACAG(CAN034；SEQ ID NO：26)；
从质粒pcDNA1-PRAME PCR扩增PRAME序列；在5’末端位点添加Not1并在3’末端位点添加Xho1使该片段可克隆至pET21b载体。该扩增引起在pET21b(+)质粒中蛋白质的C-末端添加两个氨基酸Leu和Glu，其后为6个His。为生成不含His标签的蛋白质，用CAN033和CAN035(反义寡核苷酸：
5’GGAGGACTCGAGCTAGTTAGGCATGAAACAGGGGCACAG(CAN035；SEQ ID NO：XX)
而不是CAN033和CAN034在PRAME基因的3’末端添加终止密码子(TAG)。
c)将上述扩增片段克隆至pET21b(+)质粒(InVitrogen)。
d)使用QuikChange II位点定向诱变试剂盒(Stratagene)以及正义寡核苷酸：
5’CAGAAAACTTTGAAACCATGGAACGAAGGCG(CAN106；SEQ ID NO：XX)，和反义寡核苷酸：
5’cgccttcgttccatggtttcaaagttttctg(CAN107；SEQ ID NO：XX).
去除1/3PD和PRAME之间的Not1位点。
e)经诱变并使用正义寡核苷酸：
5’GGAGATATACATATGGATCCAAGCAGCCATTCATCAAATATGG(CAN104；SEQ ID NO：XX)和反义寡核苷酸：
5’CCATATTTGATGAATGGCTGCTTGGATCCATATGTATATCTCC(CAN105；SEQ ID NO：XX).
在蛋白质D 1/3N-末端1位的Met后添加两个氨基酸Asp和Pro。
构建载体设计用于在pET26载体中表达密码子优化(不含或含His标
签)的重组蛋白质1/3PD-PRAME：
将PRAME基因进行密码子优化并克隆至pGA4主链中，分别在已优化基因的5’末端和3’末端添加Not1和Xho1位点。
使用下列步骤用该质粒(名为0606420pGA4)克隆与pET26载体中PD 1/3融合的基因。
a)去除Not1/Xho1片段，其对应于在来自0606420pGA4质粒的基因3’末端具有终止密码子的已优化PRAME序列。
b)将经优化的PRAME片段克隆至pET26b(+)质粒，其包括之前用如上文所述的CAN040和CAN032寡核苷酸Nde1/Not1克隆的1/3PD并且其中用CAN104和CAN105寡核苷酸经诱变方法在N-末端添加了Asp和Pro氨基酸。
c)用下述寡核苷酸经诱变去除Not1位点：正义
5’GACAGAAAACTTTGAAACCATGGAACGTCGTCGTCTGTGG(CAN 123；SEQ ID NO：XX)和反义
5’CCACAGACGACGACGTTCCATGGTTTCAAAGTTTTCTGTC(CAN124；SEQ ID NO：XX).
这样得到不含组氨酸尾的1/3PD-PRAME密码子优化融合蛋白质。
d)接着将该质粒用作模板以生成含6His的1/3PD-PRAME密码子优化质粒。用正义寡核苷酸
5’GGAATTCCATATGGATCCAAGCAGCCATTC(CAN199；SEQ ID NO：XX)和反义寡核苷酸：
5’GGAGCTCTCGAGTCAGTGGTGGTGGTGGTGGTGGTTCGGCATAAAGCACGGGC(CAN198；SEQ IDNO：XX)；
完成融合蛋白的PCR扩增；为将该片段克隆至pET26b(+)载体，还添加了5’末端位点的Nde1、3’末端位点的Xho1及其后的6个His和终止密码子。
e)将扩增片段在Invitrogen的pET26b(+)质粒中克隆。
为生成融合蛋白质，将DNA构建体克隆至表达载体TCMP 14。该质粒利用来自λ噬菌体DNA的信号驱动插入的外源基因的转录和翻译。载体包含λPL启动子PL、操纵子OL和两个利用位点(NutL和NutR)以缓解提供N蛋白质时的转录极性效应(Gross等，1985.Mol.&Cell.Biol.5：1015)。
设计表达pD-PRAME融合蛋白质的质粒从而将PRAME氨基酸添加至不含它的信号序列(分泌或信号序列)(即残基20-127)的pD的108个氨基酸衍生物的C-末端。对于该构建体，在pD衍生物的N-末端添加三个不相关氨基酸(Met、Asp和脯氨酸)，并且对于一些构建体在PRAME氨基酸C-末端包括组氨酸尾(见上述表A)。如果认为N-末端Met来自pD序列，那么该构建体也可可选择地描述为包含pD的109个氨基酸衍生物。
宿主菌株和转化
用实施例/构建体1和2的质粒DNA转化来自大肠杆菌菌株AR58的宿主(Mott等，Proc.Natl.Acad.Sci.USA，第82卷，第88-92页，January1985，Biochemistry)。
用于制备实施例/构建体1和2的AR58溶源性大肠杆菌菌株为标准NIH大肠杆菌K12菌株N99(F-su-galK2，lacZ-thr-)的衍生物。它包含缺陷溶源性λ噬菌体(galE::TN10，1Kil-cI857 DH1)。Kil表型可防止宿主大分子合成的关闭。cI857突变使cI阻遏物具有温度敏感损害。DH1缺失可去除λ噬菌体的右侧操纵子和宿主bio、uvr3和chlA座位。通过用先前在SA500衍生物上生长的Pλ噬菌体原种(galE::TN10，1Kil-cI857 DH1)转导N99生成AR58菌株。依靠相邻galE基因中存在编码四环素抗性的TN10转座子用四环素筛选缺陷性溶源体引入N99。N99和SA500为获自National Institutes of Health的Dr.Martin Rosenberg′s laboratory的大肠杆菌K12菌株。
将含有PL启动子的载体引入大肠杆菌溶源性宿主以稳定质粒DNA。溶源性宿主菌株包含整合入基因组的复制缺陷λ噬菌体(Shatzman等，1983；In Experimental Manipulation of Gene Expression.Inouya(编辑)第1-14页.Academic Press NY)。λ噬菌体DNA引导cI阻遏物蛋白质的合成，其可结合载体OL阻遏物并防止RNA聚合酶与PL启动子结合并藉此防止插入基因的转录。表达菌株AR58的cI基因包含温度敏感性突变，这样PL引导的转录可经温度变化调控，即培养物温度升高使阻遏物失活并起始外源蛋白质的合成。该表达系统允许外源蛋白质尤其是那些可能具有细胞毒性的蛋白质的可控制合成(Shimataka&Rosenberg，1981.Nature 292：128)。
来自大肠杆菌菌株BLR(DE3)Novagen，WI，USA(目录编号：69053-4)BLR(DE3)Novagen，WI，USA(目录编号：69053-4)的宿主BLR为BL21的recA-衍生物，其可提高质粒单体产率并有助于稳定包含重复序列或其产物可导致DE3前噬菌体(1，2)损失的目标质粒，用实施例/构建体3和4的质粒DNA转化该宿主。
各转化均根据标准方法用经CaCl2处理的细胞进行(Hanahan D.《Plasmid transformation by Simanis.》In Glover，D.M.(编辑)，DNAcloning.IRL Press London.(1985)：第109-135页)。
细菌宿主菌株的培养和诱导
·培养
将细菌培养于20ml Luria-Bertani(LB)肉汤(BD)+1％(w/v)葡萄糖(Laboratoire MAT，目录编号：GR-0101)+抗生素(100μg/ml羧苄西林用于pET21b，40μg/ml卡那霉素用于TCMP14)。于33℃培养AR58细胞并于37℃培养BLR(DE3)细胞直至O.D.600nm达到约0.8。
·诱导
当O.D.600nm约0.8时，以1mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG；EMD Chemicals Inc.，目录编号：5815)诱导培养物BLR(DE3)并于37℃培养2小时或3小时，尽管使用更低的温度可能会增加溶解度。
当O.D.600nm约0.8时，于37℃通过热激活诱导培养物AR58并培养7小时。
细菌生长对于两个表达系统是足够的。
·蛋白质的提取和纯化
当多肽在培养物中表达时，通常经离心收获细胞，接着通过物理或化学方法(如果所表达多肽没有分泌至培养基中)破裂细胞并保留所得粗提取物以分离所关注的多肽。在供应商(Novagen)推荐的条件下使用BugBusterTM蛋白质提取试剂。
PDl/3-Prame-his蛋白质的纯化
将大肠杆菌细胞糊重悬于pH 8.5的20mM Tris缓冲液中，接着通过匀浆器系统(Niro Soavi S.p.A.的Panda-通过2次-750巴)。加入2mMMgCl2和Benzonase(50U/ml)之后，将匀浆于室温(RT)在温和搅拌下温育1小时，接着于15900g和室温离心30分钟。将所得沉淀重悬于包含1％十二烷基硫酸钠(SDS)和60mM谷胱甘肽的pH 8.5的20mM Tris缓冲液中并于室温在温和搅拌下温育30分钟，在15900g和室温离心30分钟后，丢弃沉淀。
将离心上清液在包含6.66M尿素、0.333M氯化钠(NaCl)和11.11mM咪唑的20mM Tris缓冲液中稀释10倍，接着在镍铁金属亲和柱(IMAC Sepharose 6FF-GE Healthcare)上进行色谱分离，该柱子经包含0.1％SDS、6.0M尿素，0.3M NaCl和10mM咪唑的pH 8.5的20mM Tris缓冲液平衡。用包含0.5％十二烷基肌氨酸钠、6.0M尿素、0.3M NaCl和10mM咪唑的pH 8.5的20mM Tris缓冲液洗涤柱子后，通过增加咪唑在相同洗涤缓冲液中的浓度直至40mM从柱上洗脱抗原。加入磷酸盐至50mM后，将阳性抗原洗脱液通过Macro-Prep II型陶瓷羟基磷灰石柱(Bio-Rad)，其已用包含50mM磷酸盐、0.5％十二烷基肌氨酸钠、6.0M尿素和0.3M NaCl的pH 8.5的20mM Tris缓冲液平衡。用包含3.15％蔗糖的pH 9.8的5mM硼酸盐缓冲液在Omega 30kDa膜(Pall)上渗滤包含抗原的羟基磷灰石流出液(flow-through)。用0.45/0.22μm醋酸纤维素膜(Sartorius)将超滤截留物过滤灭菌。经纯化的材料保存于-70℃。
也可使用另一种纯化方法，其与上述方法在下列步骤中存在差异：
-无benzonase处理
-IMAC柱不需从SDS至十二烷基肌氨酸钠的转变(从提取至HA步骤一直使用SDS)
-用于渗滤的缓冲液为pH 8.5的5mM Tris缓冲液-0.5M精氨酸。
这一可选择的纯化方法得到不完全的SDS清除，残留值约0.05和0.085％。
·纯化
根据树脂生产商的使用说明使用组氨酸结合金属螯合树脂(QIAgen，Chatsworth，CA)从离心经诱导的大肠杆菌获得的上清组分纯化已表达的重组蛋白。
该蛋白质的特点
SDS-Page：
凝胶：NuPAGE 4-12％Bis-Tris凝胶1.0mm 15或26孔(Invitrogen目录编号：NP0323BOX)
见下文中的图1和2，其分别显示了实施例3、4、3a和4a的SDSpage分析，其中包含或不含组氨酸标签的不同重组1/3pD-PRAME蛋白在凝胶上以～70kDa的表观分子量迁移。诱导后重组蛋白以包涵体存在于大肠杆菌细胞裂解物中。
在供应商(Invitrogen)推荐的条件下制备样品、缓冲液和迁移条件。上样10μl的所有制备物(诱导前(BI)和诱导后(AI))至对应于100μl等同培养物的孔中。
图1图解：IPTG诱导用重组pET21转化的大肠杆菌BLR DE3菌株后经考马斯蓝染色重组1/3PD-PRAME后的SDS page分析。将1mMIPTG于25、30或37℃在BLR DE3菌株中诱导2小时后的相同的100μl培养物上样至凝胶上。克隆#3(1/3PD-PRAME/pET21)和克隆#4(1/3PD-PRAME-His/pET21)诱导前(BI)和诱导后(AI)以可溶(上清)和不溶(沉淀)组分呈现在凝胶上。泳道1和10：标准宽范围预染色(Standard Broad Range prestain)(BioRad Cat#161-0318)，泳道2(克隆#3，BI，上清)，泳道3(克隆#3，BI，沉淀)，泳道4(克隆#3，AI，25℃，上清)，泳道5(克隆#3，AI，25℃，沉淀)，泳道6(克隆#3，AI，30℃，上清)，泳道7(克隆#3，AI，30℃，沉淀)，泳道8(克隆#3，AI，37℃，上清)，泳道9(克隆#3，AI，37℃，沉淀)，泳道11(克隆#4，BI，上清)，泳道12(克隆#4，BI，沉淀)，泳道13(克隆#4，AI，25℃，上清)，泳道14(克隆#4，AI，25℃，沉淀)，泳道15(克隆#4，AI，30℃，上清)，泳道16(克隆#4，AI，30℃，沉淀)，泳道17(克隆#4，AI，37℃，上清)，泳道18(克隆#4，AI，37℃，沉淀)。
图2图解：IPTG诱导用重组pET26转化的大肠杆菌BLR DE3菌株后经考马斯蓝染色重组1/3PD-PRAME后的SDS page分析。将1mMIPTG于25、30或37℃在BLR DE3菌株中诱导2小时后的相同的100μl培养物上样至凝胶上。克隆#3a(1/3PD-PRAME密码子优化/pET26)和克隆#4a(1/3PD-PRAME-His密码子优化/pET26)诱导前(BI)和诱导后(AI)以可溶(上清)和不溶(沉淀)组分呈现在凝胶上。泳道2和10：标准宽范围预染色(BioRad Cat#161-0318)，泳道1(克隆#3a，BI，上清)，泳道3(克隆#3a，BI，沉淀)，泳道4(克隆#3a，AI，25℃，上清)，泳道5(克隆#3a，AI，25℃，沉淀)，泳道6(克隆#3a，AI，30℃，上清)，泳道7(克隆#3a，AI，30℃，沉淀)，泳道8(克隆#3a，AI，37℃，上清)，泳道9(克隆#3a，AI，37℃，沉淀)，泳道11(克隆#4a，BI，上清)，泳道12(克隆#4a，BI，沉淀)，泳道13(克隆#4a，AI，25℃，上清)，泳道14(克隆#4a，AI，25℃，沉淀)，泳道15(克隆#4a，AI，30℃，上清)，泳道16(克隆#4a，AI，30℃，沉淀)，泳道17(克隆#4a，AI，37℃，上清)，泳道18(克隆#4a，AI，37℃，沉淀)。
蛋白质印迹法
用3％milk/PBS 1X新鲜溶液于37℃，60RPM将膜封闭30分钟。封闭温育后加入1∶5000稀释的一抗(兔抗-PRAME；GSK BiologicalsSA)或于37℃，60RPM在1小时内加入用3％milk/PBS 1X新鲜溶液1∶3000稀释的α-6X组氨酸标签(AbCam)。之后用0.02％Tween20/PBS1X于室温下将膜洗涤三次各5分钟。加入用3％milk/PBS 1X新鲜溶液以1∶20000稀释的二抗(perox驴抗-IgG(H+L)兔(Jackson laboratory)。37℃、60RPM温育膜1小时。之后，根据供应商的建议在膜暴露于过氧化物酶底物(KH2PO4，10mM；(NH4)2SO4，10mM；邻-联茴香胺，0.01％&过氧化氢0.045％)或碱性磷酸酶底物(Sigma Fast)之前用0.02％Tween20/PBS 1X于室温下将膜洗涤三次各5分钟。
分子分析
实施例/构建体1
| 分析 | 完整蛋白 |
| 长度 | 629氨基酸 |
| 分子量 | 71629.96m.w. |
| 1微克＝ | 13.961皮摩尔 |
| 摩尔消光系数 | 67860 |
| 1A[280]相当于 | 1.06mg/ml |
| 1mg/ml的A[280] | 0.94AU |
| 等电点 | 6.41 |
| pH7时的电荷 | -5.84 |
| 等电点 | 6.41 |
| pH7时的电荷 | -5.84 |
实施例/构建体2
| 分析 | 完整蛋白 |
| 长度 | 620氨基酸 |
| 分子量 | 70561.90m.w. |
| 1微克＝ | 14.172皮摩尔 |
| 摩尔消光系数 | 67680 |
| 1A[280]相当于 | 1.04mg/ml |
| 1mg/ml的A[280] | 0.96AU |
| 等电点 | 6.28 |
| pH7时的电荷 | -6.36 |
实施例/构建体3
| 分析 | 完整蛋白 |
| 长度 | 628氨基酸 |
| 分子量 | 71627.01m.w. |
| 1微克＝ | 13.961皮摩尔 |
| 摩尔消光系数 | 67680 |
| 1A[280]相当于 | 1.06mg/ml |
| 1mg/ml的A[280] | 0.94AU |
| 等电点 | 6.34 |
| pH7时的电荷 | -6.84 |
实施例/构建体4
| 分析 | 完整蛋白 |
| 长度 | 620氨基酸 |
| 分子量 | 70561.90m.w. |
| 1微克＝ | 14.172皮摩尔 |
| 摩尔消光系数 | 67680 |
| 1A[280]相当于 | 1.04mg/ml |
| 1mg/ml的A[280] | 0.96AU |
| 等电点 | 6.28 |
| pH7时的电荷 | -6.36 |
实施例5
评价融合蛋白中蛋白质D1/3的含有或不含分泌信号(分泌或信号序列)的蛋白质的生产
表B
蛋白质 表达水平
含有分泌信号的PD1/3-PRAME +
不含分泌信号的PD1/3-PRAME +++
图11：IPTG诱导用重组pET21转化的大肠杆菌BL21DE3菌株后经考马斯蓝染色含有或不含有分泌信号的重组1/3PD-PRAME后的SDS page分析。将1mM IPTG于37℃在BL21DE3菌株中诱导3小时后的相同的100μl培养物上样至凝胶上。这些构建体诱导前(BI)和诱导后(AI)以可溶(上清)和不溶(沉淀)组分呈现在胶上。泳道1：标准宽范围预染色(BioRad Cat#161-0318)，泳道2(pD1/3-PRAME+SS，BI，上清)，泳道3(pD1/3-PRAME+SS，BI，沉淀)，泳道4(pD1/3-PRAME+SS，AI，上清)，泳道5(pD1/3-PRAME+SS，AI，沉淀)，泳道6(pD1/3-PRAME+SS+His，BI，上清)，泳道7(pD1/3-PRAME+SS+His，BI，沉淀)，泳道8(pD1/3-PRAME+SS+His，AI，上清)，泳道9(pD1/3-PRAME+SS+His，AI，沉淀)，泳道10(pD1/3-PRAME w/o SS，BI，上清)，泳道11(pD1/3-PRAME w/o SS，BI，沉淀)，泳道12(pD1/3-PRAME w/o SS，AI，上清)，泳道13(pD1/3-PRAME w/o SS，AI，沉淀)，泳道14(pD1/3-PRAME w/o SS+His，BI，上清)，泳道15(pD1/3-PRAME w/o SS+His，BI，沉淀)，泳道16(pD1/3-PRAME w/o SS+His，AI，上清)，泳道17(pD1/3-PRAME w/o SS+His，AI，沉淀)。
实施例6：AS01B或AS15中配制的PD-PRAME-His的免疫原性：恒
定剂量佐剂中的抗原剂量范围
目标：选择用于临床前试验的最佳剂量的抗原剂量范围
方案：
在第0和第14天使6组12只CB6F1小鼠接受肌内(IM)注射：
1.PBS
2.AS01B或AS15中的PRAME(50*μg)
3.AS01B或AS15中的PRAME(10μg)
4.AS01B或AS15中的PRAME(2μg)
5.AS01B或AS15中的PRAME(0.4μg)
6.AS01B或AS15中的PRAME(0.08μg)
*实际上给予了44.7μg而不是50μg的预期剂量
AS01B为包括QS21和3D-MPL的脂质体佐剂制剂；AS 15为包括QS21、3D-MPL和CpG的脂质体佐剂制剂。
用于该实施例的构建体为实施例/构建体3a(含组氨酸尾的pET26)，其提供于5mM pH 8.5的Tris缓冲液-0.5M精氨酸中。也可使用提供于含有蔗糖的硼酸盐缓冲液中的蛋白质。
读出(Read-outs)：
●两次注射后14天以1μg/ml/肽(15-mer)的肽PRAME库在体外重新刺激脾细胞(每组3只小鼠，共4库)之后进行胞内细胞因子染色(ICS)
CD4应答(AS01B佐剂)
AS01B佐剂的CD4细胞因子的ICS结果显示于图3。在这一实验中可推断在这些条件下诱导AS01B中CD4应答的PRAME抗原最佳剂量为2μg。
CD8应答(AS01B佐剂)
AS01B佐剂的CD8细胞因子的ICS结果显示于图4。数据显示非常低的CD8应答和组内应答的不均一性。
CD4应答(AS15佐剂)
AS15佐剂的CD4细胞因子的ICS结果显示于图5。这些数据显示用AS15配制的44μg、10μg、2μg和0.4μg PRAME诱导相似的CD4应答；0.08μg PRAME诱导的应答降低。
CD8应答(AS15佐剂)
AS15佐剂的CD8细胞因子的ICS结果显示于图6。这些数据显示没有CD8应答(背景为PBS组)
实施例7
总之，对于本文所描述的发明，可使用下列概述描述至今已生成的PD1/3-PRAME的具体构建体：
用于PD1/3-PRAME的构建体
-不包括蛋白质D的信号序列(蛋白质D的氨基酸2至19)
-包括蛋白质D的甲硫氨酸(蛋白质D的氨基酸1)
-两个不相关氨基酸(Asp和Pro)替换了蛋白质D的氨基酸2-Lys和3-Leu
-包括蛋白质D信号序列后的蛋白质D的起始109个氨基酸(109个氨基酸包括N-末端第一个甲硫氨酸+蛋白质D的氨基酸20至127)
-包括PRAME的氨基酸1-509(全长原始PRAME序列)
-含有或不含组氨酸尾，其由以下之一组成：
●三个不相关的氨基酸(Thr、Ser和GIy)+6组氨酸残基用于在TCMP 14质粒中克隆；或
●两个不相关的氨基酸(Leu和GIu)+6组氨酸残基用于在pET21质粒中克隆；或
●6组氨酸残基用于在pET26质粒中克隆。
pD1/3-PRAME+/-组氨酸尾蛋白：
| PD1/3 20-127 | PRAME1-509 | ||
| N末端MDP | PD1/3 | PRAME | C末端 |
| N末端MDP | PD1/3 | PRAME | TSG 6×His(TCMP14中)C末端 |
| N末端MDP | PD1/3 | PRAME | LE 6×His(pET21中)C末端 |
| N末端MDP | PD1/3 | PRAME | 6×His(pET26中)C末端 |
本发明构建体实施例的标记氨基酸序列显示于图7中。
下列构建体的比对显示于图8、9和10。
LipoD-MAGE3-His和D1/3-PRAME-His之间的比对(图8)
流感嗜血杆菌的原始蛋白质D和LipoD-MAGE3-His的共享序列之间的比对(图9)
流感嗜血杆菌的原始蛋白质D、LipoD-MAGE3-His和pD1/3-PRAME-His的共享序列之间的比对(图10)
使用融合蛋白配制疫苗制剂
本发明融合蛋白可配置成含有或不含佐剂的疫苗。在一个实施方案中，所述制剂可包括3-O-脱酰单磷酰类脂A(3D-MPL)和QS21在油/水乳剂中的混合物作为佐剂。佐剂系统SBAS2之前已描述于WO95/17210。用于本发明的佐剂可选择性地包括水包油制剂或脂质体制剂中的3-O-脱酰单磷酰类脂A(3D-MPL)、QS21和CpG。
3D-MPL：为来源自革兰氏阴性细菌明尼苏达沙门氏菌(Salmonellaminnesota)脂多糖(LPS)的免疫刺激剂。MPL经脱酰化并缺失类脂A部分的磷酸基团。这一化学处理显著降低了毒性并同时保留了免疫刺激性质(Ribi，1986)。
据信与各种载体组合的3D-MPL可显著增强体液和TH1型细胞免疫。
QS21：为从南美皂皮树(Quillaja saponaria Molina)的树皮提取的天然皂苷分子。已研发的纯化技术可从树皮的粗提取物中分离单独的皂苷，使得可分离特异皂苷QS21，与母体组分相比其为显示更强佐剂活性和更低毒性的三萜糖苷。QS21已显示可活化对若干亚单位抗原的MHC-I类限制性CTL以及刺激抗原特异性淋巴细胞的增殖(Kensil，1992)。
据信MPL和QS21组合在诱导体液和TH1型细胞免疫应答中具有协同效应。
油/水乳剂包括由两种油(生育酚和角鲨烯)组成的有机相以及包含吐温80作为乳化剂的PBS水相。该乳剂包括5％角鲨烯、5％生育酚、0.4％吐温80并且平均粒径为180nm，也称作SB62(参见WO 95/17210)。所得油珠的尺寸应为约180nm。
用于本发明的佐剂可以MPL和QS21的组合配制在油/水乳剂或脂质体制剂中。这一制剂应提供于0.7ml的小瓶中与冻干抗原或融合蛋白混合(包含30至300μg抗原的小瓶)。
也可使用免疫刺激寡核苷酸。用于本发明佐剂或疫苗中的实例寡核苷酸包括含CpG的寡核苷酸，其通常包含被至少三个，更通常为至少六个或更多个核苷酸分隔的两个或更多个二核苷酸CpG基序。CpG基序为其后为鸟嘌呤核苷酸的胞嘧啶核苷酸。CpG寡核苷酸通常为脱氧核苷酸。在一个实施方案中所述寡核苷酸中的核苷酸中间物(internucleotide)为二硫代磷酸酯，或更优选为硫代磷酸酯键，尽管磷酸二酯和其他核苷酸间键合属于本发明范围。本发明范围也包括含有混合核苷酸间键的寡核苷酸。生产硫代磷酸寡核苷酸或二硫代磷酸酯的方法描述于美国专利5666153、5278302和WO 95/26204。
寡核苷酸的实例如下：
TCC ATG ACG TTC CTG ACG TT(CpG 1826；SEQ ID NO：36)
TCT CCC AGC GTG CGC CAT(CpG 1758；SEQ ID NO：37)
ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG TCG TCG TTT TGT CGT TTT GTC GTT(CpG2006；SEQ ID NO：38)
TCC ATG ACG TTC CTG ATG CT(CpG 1668；SEQ ID NO：39)
TCG ACG TTT TCG GCG CGC GCC G(CpG 5456；SEQ ID NO：40)，
这些序列可包含经硫代磷酸酯修饰的核苷酸间键。
可选择的CpG寡核苷酸可包括一个或更多个上述序列因为它们具有不连续缺失或添加。
可经本领域任何已知方法合成CpG寡核苷酸(例如参见EP468520)。可使用自动合成仪方便地合成这些寡核苷酸。
在本发明的一个实施方案中用于本发明的佐剂组合包括一个或更多个下列组分：3D-MPL和QS21(EP 0 671 948B1)；包括3D-MPL和QS21的水包油乳剂(WO 95/17210，WO 98/56414)；或与其他载体配制的3D-MPL(EP 0 689 454B1)。可用于本发明的其他佐剂系统包括如US6558670和US6544518中描述的3D-MPL、QS21和CpG寡核苷酸的组合。
将冻干制剂重构后可得到最终的疫苗。
参考文献：
1.A.Roca(U.of Wisconsin)，personal communication.
2.Studier，F.W.(1991)J.Mol.Biol.219，37-44.
3.Jan H.Kesslera等The Journal of Experimental Medicine，Volume 193，Number1，January 1，2001 73-88，
4.Ikeda H等Immunity，Feb；6(2)：1997，199-208
SEQ ID NO：1
实施例1的DNA序列
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgaaggcgtttgtggggttccattcagagccgatacatcagcatgagtgtgtggacaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctggccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcctttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgcctccctctgggagtgctgatgaagggacaacatcttcacctggagaccttcaaagctgtgcttgatggacttgatgtgctccttgcccaggaggttcgccccaggaggtggaaacttcaagtgctggatttacggaagaactctcatcaggacttctggactgtatggtctggaaacagggccagtctgtactcatttccagagccagaagcagctcagcccatgacaaagaagcgaaaagtagatggtttgagcacagaggcagagcagcccttcattccagtagaggtgctcgtagacctgttcctcaaggaaggtgcctgtgatgaattgttctcctacctcattgagaaagtgaagcgaaagaaaaatgtactacgcctgtgctgtaagaagctgaagatttttgcaatgcccatgcaggatatcaagatgatcctgaaaatggtgcagctggactctattgaagatttggaagtgacttgtacctggaagctacccaccttggcgaaattttctccttacctgggccagatgattaatctgcgtagactcctcctctcccacatccatgcatcttcctacatttccccggagaaggaagagcagtatatcgcccagttcacctctcagttcctcagtctgcagtgcctgcaggctctctatgtggactctttatttttccttagaggccgcctggatcagttgctcaggcacgtgatgaaccccttggaaaccctctcaataactaactgccggctttcggaaggggatgtgatgcatctgtcccagagtcccagcgtcagtcagctaagtgtcctgagtctaagtggggtcatgctgaccgatgtaagtcccgagcccctccaagctctgctggagagagcctctgccaccctccaggacctggtctttgatgagtgtgggatcacggatgatcagctccttgccctcctgccttccctgagccactgctcccagcttacaaccttaagcttctacgggaattccatctccatatctgccttgcagagtctcctgcagcacctcatcgggctgagcaatctgacccacgtgctgtatcctgtccccctggagagttatgaggacatccatggtaccctccacctggagaggcttgcctatctgcatgccaggctcagggagttgctgtgtgagttggggcggcccagcatggtctggcttagtgccaacccctgtcctcactgtggggacagaaccttctadtgacccggagcccatcctgtgcccctgtttcatgcctaacactagtggccaccatcaccatcaccat
SEQ ID NO：2
实施例1的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNTSGHHHHHH
SEQ ID NO：3
实施例2的DNA序列
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgaaggcgtttgtggggttccattcagagccgatacatcagcatgagtgtgtggacaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctggccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcctttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgcctccctctgggagtgctgatgaagggacaacatcttcacctggagaccttcaaagctgtgcttgatggacttgatgtgctccttgcccaggaggttcgccccaggaggtggaaacttcaagtgctggatttacggaagaactctcatcaggacttctggactgtatggtctggaaacagggccagtctgtactcatttccagagccagaagcagctcagcccatgacaaagaagcgaaaagtagatggtttgagcacagaggcagagcagcccttcattccagtagaggtgctcgtagacctgttcctcaaggaaggtgcctgtgatgaattgttctcctacctcattgagaaagtgaagcgaaagaaaaatgtactacgcctgtgctgtaagaagctgaagatttttgcaatgcccatgcaggatatcaagatgatcctgaaaatggtgcagctggactctattgaagatttggaagtgacttgtacctggaagctacccaccttggcgaaattttctccttacctgggccagatgattaatctgcgtagactcctcctctcccacatccatgcatcttcctacatttccccggagaaggaagagcagtatatcgcccagttcacctctcagttcctcagtctgcagtgcctgcaggctctctatgtggactctttatttttccttagaggccgcctggatcagttgctcaggcacgtgatgaaccccttggaaaccctctcaataactaactgccggctttcggaaggggatgtgatgcatctgtcccagagtcccagcgtcagtcagctaagtgtcctgagtctaagtggggtcatgctgaccgatgtaagtcccgagcccctccaagctctgctggagagagcctctgccaccctccaggacctggtctttgatgagtgtgggatcacggatgatcagctccttgccctcctgccttccctgagccactgctcccagcttacaaccttaagcttctacgggaattccatctccatatctgccttgcagagtctcctgcagcacctcatcgggctgagcaatctgacccacgtgctgtatcctgtccccctggagagttatgaggacatccatggtaccctccacctggagaggcttgcctatctgcatgccaggctcagggagttgctgtgtgagttggggcggcccagcatggtctggcttagtgccaacccctgtcctcactgtggggacagaaccttctatgacccggagcccatcctgtgcccctgtttcatgcctaac
SEQ ID NO：4
实施例2的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN
SEQ ID NO：5
实施例3的DNA序列
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgaaggcgtttgtggggttccattcagagccgatacatcagcatgagtgtgtggacaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctggccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcctttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgcctccctctgggagtgctgatgaagggacaacatcttcacctggagaccttcaaagctgtgcttgatggacttgatgtgctccttgcccaggaggttcgccccaggaggtggaaacttcaagtgctggatttacggaagaactctcatcaggacttctggactgtatggtctggaaacagggccagtctgtactcatttccagagccagaagcagctcagcccatgacaaagaagcgaaaagtagatggtttgagcacagaggcagagcagcccttcattccagtagaggtgctcgtagacctgttcctcaaggaaggtgcctgtgatgaattgttctcctacctcattgagaaagtgaagcgaaagaaaaatgtactacgcctgtgctgtaagaagctgaagatttttgcaatgcccatgcaggatatcaagatgatcctgaaaatggtgcagctggactctattgaagatttggaagtgacttgtacctggaagctacccaccttggcgaaattttctccttacctgggccagatgattaatctgcgtagactcctcctctcccacatccatgcatcttcctacatttccccggagaaggaagagcagtatatcgcccagttcacctctcagttcctcagtctgcagtgcctgcaggctctctatgtggactctttatttttccttagaggccgcctggatcagttgctcaggcacgtgatgaaccccttggaaaccctctcaataactaactgccggctttcggaaggggatgtgatgcatctgtcccagagtcccagcgtcagtcagctaagtgtcctgagtctaagtggggtcatgctgaccgatgtaagtcccgagcccctccaagctctgctggagagagcctctgccaccctccaggacctggtctttgatgagtgtgggatcacggatgatcagctccttgccctcctgccttccctgagccactgctcccagcttacaaccttaagcttctacgggaattccatctccatatctgccttgcagagtctcctgcagcacctcatcgggctgagcaatctgacccacgtgctgtatcctgtccccctggagagttatgaggacatccatggtaccctccacctggagaggcttgcctatctgcatgccaggctcagggagttgctgtgtgagttggggcggcccagcatggtctggcttagtgccaacccctgtcctcactgtggggacagaaccttctatgacccggagcccatcctgtgcccctgtttcatgcctaacctcgagcaccaccaccaccaccac
SEQ ID NO：6
实施例3的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNLEHHHHHH
SEQ ID NO：7
实施例4的DNA序列：
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgaaggcgtttgtggggttccattcagagccgatacatcagcatgagtgtgtggacaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctggccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcctttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgcctccctctgggagtgctgatgaagggacaacatcttcacctggagaccttcaaagctgtgcttgatggacttgatgtgctccttgcccaggaggttcgccccaggaggtggaaacttcaagtgctggatttacggaagaactctcatcaggacttctggactgtatggtctggaaacagggccagtctgtactcatttccagagccagaagcagctcagcccatgacaaagaagcgaaaagtagatggtttgagcacagaggcagagcagcccttcattccagtagaggtgctcgtagacctgttcctcaaggaaggtgcctgtgatgaattgttctcctacctcattgagaaagtgaagcgaaagaaaaatgtactacgcctgtgctgtaagaagctgaagatttttgcaatgcccatgcaggatatcaagatgatcctgaaaatggtgcagctggactctattgaagatttggaagtgacttgtacctggaagctacccaccttggcgaaattttctccttacctgggccagatgattaatctgcgtagactcctcctctcccacatccatgcatcttcctacatttccccggagaaggaagagcagtatatcgcccagttcacctctcagttcctcagtctgcagtgcctgcaggctctctatgtggactctttatttttccttagaggccgcctggatcagttgctcaggcacgtgatgaaccccttggaaaccctctcaataactaactgccggctttcggaaggggatgtgatgcatctgtcccagagtcccagcgtcagtcagctaagtgtcctgagtctaagtggggtcatgctgaccgatgtaagtcccgagcccctccaagctctgctggagagagcctctgccaccctccaggacctggtctttgatgagtgtgggatcacggatgatcagctccttgccctcctgccttccctgagccactgctcccagcttacaaccttaagcttctacgggaattccatctccatatctgccttgcagagtctcctgcagcacctcatcgggctgagcaatctgacccacgtgctgtatcctgtccccctggagagttatgaggacatccatggtaccctccacctggagaggcttgcctatctgcatgccaggctcagggagttgctgtgtgagttggggcggcccagcatggtctggcttagtgccaacccctgtcctcactgtggggacagaaccttctatgacccggagcccatcctgtgcccctgtttcatgcctaac
SEQ ED NO：8
实施例4的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN
SEQ ID NO：9
实施例3a的密码子优化DNA序列
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgtcgtcgtctgtggggcagcattcagagccgttatattagcatgagcgtgtggaccagcccgcgtcgtctggttgagctggccggccagagcctgctgaaagatgaagcgctggccattgcggcgctggagctgctgccgcgtgagctgtttccgccgctgtttatggcggcgtttgatggccgtcatagccagaccctgaaagcgatggtgcaggcgtggccgtttacctgtctgccgctgggcgtgctgatgaaaggccagcatctgcatctggaaacctttaaagcggtgctggatggcctggatgtgctgctggcccaggaagttcgtccgcgtcgttggaaactgcaagtgctggatctgcgtaaaaacagccatcaggatttttggaccgtgtggagcggcaatcgtgcgagcctgtatagctttccggaaccggaagcggcgcagccgatgaccaaaaaacgtaaagtggatggcctgagcaccgaagcggaacagccgtttattccggtggaagtgctggttgacctgtttctgaaagaaggcgcctgcgacgagctgtttagctatctgatcgaaaaagtgaaacgcaaaaaaaacgtgctgcgtctgtgctgcaaaaaactgaaaatcttcgcgatgccgatgcaggatattaaaatgatcctgaaaatggtgcagctggatagcattgaggacctggaagtgacctgcacctggaaactgccgaccctggccaaatttagcccgtatctgggccagatgattaacctgcgtcgtctgctgctgtctcatattcatgcgagcagctatattagcccggaaaaagaagaacagtatatcgcgcagtttaccagccagtttctgagcctgcaatgcctgcaagcgctgtatgtggatagcctgttttttctgcgtggccgtctggatcagctgctgcgtcatgtgatgaatccgctggaaaccctgagcattaccaactgccgtctgagcgaaggcgatgtgatgcatctgagccagagcccgagcgttagccagctgtctgttctgagcctgagcggcgtgatgctgaccgatgtgagcccggaaccgctgcaagccctgctggaacgtgcgagcgcgaccctgcaagacctggtgtttgatgaatgcggcattaccgatgatcagctgctggccctgctgccgagcctgagccattgcagccagctgaccaccctgagcttttatggcaacagcattagcattagcgcgctgcaaagcctgctgcaacatctgattggcctgagcaacctgacccatgtgctgtatccggtgccgctggaaagctatgaagatattcatggcaccctgcatctggaacgtctggcctatctgcacgcgcgtctgcgtgagctgctgtgcgagctgggccgtccgagcatggtttggctgtctgcgaatccgtgcccgcattgcggcgatcgtaccttttatgatccggaaccgattctgtgcccgtgctttatgccgaaccaccaccaccaccaccac
SEQ ID NO：10
实施例3a的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNHHHHHH
SEQID NO：11
实施例4a的密码子优化DNA序列
atggatccaagcagccattcatcaaatatggcgaatacccaaatgaaatcagacaaaatcattattgctcaccgtggtgctagcggttatttaccagagcatacgttagaatctaaagcacttgcgtttgcacaacaggctgattatttagagcaagatttagcaatgactaaggatggtcgtttagtggttattcacgatcactttttagatggcttgactgatgttgcgaaaaaattcccacatcgtcatcgtaaagatggccgttactatgtcatcgactttaccttaaaagaaattcaaagtttagaaatgacagaaaactttgaaaccatggaacgtcgtcgtctgtggggcagcattcagagccgttatattagcatgagcgtgtggaccagcccgcgtcgtctggttgagctggccggccagagcctgctgaaagatgaagcgctggccattgcggcgctggagctgctgccgcgtgagctgtttccgccgctgtttatggcggcgtttgatggccgtcatagccagaccctgaaagcgatggtgcaggcgtggccgtttacctgtctgccgctgggcgtgctgatgaaaggccagcatctgcatctggaaacctttaaagcggtgctggatggcctggatgtgctgctggcccaggaagttcgtccgcgtcgttggaaactgcaagtgctggatctgcgtaaaaacagccatcaggatttttggaccgtgtggagcggcaatcgtgcgagcctgtatagctttccggaaccggaagcggcgcagccgatgaccaaaaaacgtaaagtggatggcctgagcaccgaagcggaacagccgtttattccggtggaagtgctggttgacctgtttctgaaagaaggcgcctgcgacgagctgtttagctatctgatcgaaaaagtgaaacgcaaaaaaaacgtgctgcgtctgtgctgcaaaaaactgaaaatcttcgcgatgccgatgcaggatattaaaatgatcctgaaaatggtgcagctggatagcattgaggacctggaagtgacctgcacctggaaactgccgaccctggccaaatttagcccgtatctgggccagatgattaacctgcgtcgtctgctgctgtctcatattcatgcgagcagctatattagcccggaaaaagaagaacagtatatcgcgcagtttaccagccagtttctgagcctgcaatgcctgcaagcgctgtatgtggatagcctgttttttctgcgtggccgtctggatcagctgctgcgtcatgtgatgaatccgctggaaaccctgagcattaccaactgccgtctgagcgaaggcgatgtgatgcatctgagccagagcccgagcgttagccagctgtctgttctgagcctgagcggcgtgatgctgaccgatgtgagcccggaaccgctgcaagccctgctggaacgtgcgagcgcgaccctgcaagacctggtgtttgatgaatgcggcattaccgatgatcagctgctggccctgctgccgagcctgagccattgcagccagctgaccaccctgagcttttatggcaacagcattagcattagcgcgctgcaaagcctgctgcaacatctgattggcctgagcaacctgacccatgtgctgtatccggtgccgctggaaagctatgaagatattcatggcaccctgcatctggaacgtctggcctatctgcacgcgcgtctgcgtgagctgctgtgcgagctgggccgtccgagcatggtttggctgtctgcgaatccgtgcccgcattgcggcgatcgtaccttttatgatccggaaccgattctgtgcccgtgctttatgccgaac
SEQ ID NO：12
实施例4a的氨基酸序列
MDPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN
序列表
<110>GlaxoSmithKline Biologicals SA
<120>疫苗
<130>VB62293
<150>0700760.2
<151>2007-01-15
<150>0701262.8
<151>2007-01-23
<160>45
<170>FastSEQ for Windows Version 4.0
<210>1
<211>1887
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-TSGHHHHHH
(质粒TCMP14)
<400>1
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
actagtggcc accatcacca tcaccat 1887
<210>2
<211>629
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-TSGHHHHHH
(质粒TCMP14)
<400>2
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn Thr Ser Gly His
610 615 620
His His His His His
625
<210>3
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒TCMP14)
<400>3
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
<210>4
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒TCMP14)
<400>4
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>5
<211>1884
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-LEHHHHHH
(质粒pET21)
<400>5
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
ctcgagcacc accaccacca ccac 1884
<210>6
<211>628
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-LEHHHHHH
(质粒pET21)
<400>6
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn Leu Glu His His
610 615 620
His His His His
625
<210>7
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET21)
<400>7
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
<210>8
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET21)
<400>8
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>9
<211>1878
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-HHHHHH(质粒
pET26)
<400>9
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gtcgtcgtct gtggggcagc 360
attcagagcc gttatattag catgagcgtg tggaccagcc cgcgtcgtct ggttgagctg 420
gccggccaga gcctgctgaa agatgaagcg ctggccattg cggcgctgga gctgctgccg 480
cgtgagctgt ttccgccgct gtttatggcg gcgtttgatg gccgtcatag ccagaccctg 540
aaagcgatgg tgcaggcgtg gccgtttacc tgtctgccgc tgggcgtgct gatgaaaggc 600
cagcatctgc atctggaaac ctttaaagcg gtgctggatg gcctggatgt gctgctggcc 660
caggaagttc gtccgcgtcg ttggaaactg caagtgctgg atctgcgtaa aaacagccat 720
caggattttt ggaccgtgtg gagcggcaat cgtgcgagcc tgtatagctt tccggaaccg 780
gaagcggcgc agccgatgac caaaaaacgt aaagtggatg gcctgagcac cgaagcggaa 840
cagccgttta ttccggtgga agtgctggtt gacctgtttc tgaaagaagg cgcctgcgac 900
gagctgttta gctatctgat cgaaaaagtg aaacgcaaaa aaaacgtgct gcgtctgtgc 960
tgcaaaaaac tgaaaatctt cgcgatgccg atgcaggata ttaaaatgat cctgaaaatg 1020
gtgcagctgg atagcattga ggacctggaa gtgacctgca cctggaaact gccgaccctg 1080
gccaaattta gcccgtatct gggccagatg attaacctgc gtcgtctgct gctgtctcat 1140
attcatgcga gcagctatat tagcccggaa aaagaagaac agtatatcgc gcagtttacc 1200
agccagtttc tgagcctgca atgcctgcaa gcgctgtatg tggatagcct gttttttctg 1260
cgtggccgtc tggatcagct gctgcgtcat gtgatgaatc cgctggaaac cctgagcatt 1320
accaactgcc gtctgagcga aggcgatgtg atgcatctga gccagagccc gagcgttagc 1380
cagctgtctg ttctgagcct gagcggcgtg atgctgaccg atgtgagccc ggaaccgctg 1440
caagccctgc tggaacgtgc gagcgcgacc ctgcaagacc tggtgtttga tgaatgcggc 1500
attaccgatg atcagctgct ggccctgctg ccgagcctga gccattgcag ccagctgacc 1560
accctgagct tttatggcaa cagcattagc attagcgcgc tgcaaagcct gctgcaacat 1620
ctgattggcc tgagcaacct gacccatgtg ctgtatccgg tgccgctgga aagctatgaa 1680
gatattcatg gcaccctgca tctggaacgt ctggcctatc tgcacgcgcg tctgcgtgag 1740
ctgctgtgcg agctgggccg tccgagcatg gtttggctgt ctgcgaatcc gtgcccgcat 1800
tgcggcgatc gtacctttta tgatccggaa ccgattctgt gcccgtgctt tatgccgaac 1860
caccaccacc accaccac 1878
<210>10
<211>626
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-HHHHHH(质粒
pET26)
<400>10
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn His His His His
610 615 620
His His
625
<210>11
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET26)
<400>11
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gtcgtcgtct gtggggcagc 360
attcagagcc gttatattag catgagcgtg tggaccagcc cgcgtcgtct ggttgagctg 420
gccggccaga gcctgctgaa agatgaagcg ctggccattg cggcgctgga gctgctgccg 480
cgtgagctgt ttccgccgct gtttatggcg gcgtttgatg gccgtcatag ccagaccctg 540
aaagcgatgg tgcaggcgtg gccgtttacc tgtctgccgc tgggcgtgct gatgaaaggc 600
cagcatctgc atctggaaac ctttaaagcg gtgctggatg gcctggatgt gctgctggcc 660
caggaagttc gtccgcgtcg ttggaaactg caagtgctgg atctgcgtaa aaacagccat 720
caggattttt ggaccgtgtg gagcggcaat cgtgcgagcc tgtatagctt tccggaaccg 780
gaagcggcgc agccgatgac caaaaaacgt aaagtggatg gcctgagcac cgaagcggaa 840
cagccgttta ttccggtgga agtgctggtt gacctgtttc tgaaagaagg cgcctgcgac 900
gagctgttta gctatctgat cgaaaaagtg aaacgcaaaa aaaacgtgct gcgtctgtgc 960
tgcaaaaaac tgaaaatctt cgcgatgccg atgcaggata ttaaaatgat cctgaaaatg 1020
gtgcagctgg atagcattga ggacctggaa gtgacctgca cctggaaact gccgaccctg 1080
gccaaattta gcccgtatct gggccagatg attaacctgc gtcgtctgct gctgtctcat 1140
attcatgcga gcagctatat tagcccggaa aaagaagaac agtatatcgc gcagtttacc 1200
agccagtttc tgagcctgca atgcctgcaa gcgctgtatg tggatagcct gttttttctg 1260
cgtggccgtc tggatcagct gctgcgtcat gtgatgaatc cgctggaaac cctgagcatt 1320
accaactgcc gtctgagcga aggcgatgtg atgcatctga gccagagccc gagcgttagc 1380
cagctgtctg ttctgagcct gagcggcgtg atgctgaccg atgtgagccc ggaaccgctg 1440
caagccctgc tggaacgtgc gagcgcgacc ctgcaagacc tggtgtttga tgaatgcggc 1500
attaccgatg atcagctgct ggccctgctg ccgagcctga gccattgcag ccagctgacc 1560
accctgagct tttatggcaa cagcattagc attagcgcgc tgcaaagcct gctgcaacat 1620
ctgattggcc tgagcaacct gacccatgtg ctgtatccgg tgccgctgga aagctatgaa 1680
gatattcatg gcaccctgca tctggaacgt ctggcctatc tgcacgcgcg tctgcgtgag 1740
ctgctgtgcg agctgggccg tccgagcatg gtttggctgt ctgcgaatcc gtgcccgcat 1800
tgcggcgatc gtacctttta tgatccggaa ccgattctgt gcccgtgctt tatgccgaac 1860
<210>12
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET26)
<400>12
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>13
<211>9
<212>PRT
<213>智人
<400>13
Val Leu Asp Gly Leu Asp Val Leu Leu
1 5
<210>14
<211>10
<212>PRT
<213>智人
<400>14
Ser Leu Tyr Ser Phe Pro Glu Pro Glu Ala
1 5 10
<210>15
<211>10
<212>PRT
<213>智人
<400>15
Ala Leu Tyr Val Asp Ser Leu Phe Phe Leu
1 5 10
<210>16
<211>9
<212>PRT
<213>智人
<400>16
Leu Tyr Val Asp Ser Leu Phe Phe Leu
1 5
<210>17
<211>9
<212>PRT
<213>智人
<400>17
Ser Leu Leu Gln His Leu Ile Gly Leu
1 5
<210>18
<211>36
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN008
<400>18
atataacata tggatccaag cagccattca tcaaat 36
<210>19
<211>40
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN037
<400>19
ccacaaacgc cttcgttcca tggtttcaaa gttttctgtc 40
<210>20
<211>40
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN036
<400>20
gacagaaaac tttgaaacca tggaacgaag gcgtttgtgg 40
<210>21
<211>39
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN029
<400>21
agagagacta gtctagttag gcatgaaaca ggggcacag 39
<210>22
<211>36
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN002
<400>22
ggaggaacta gtgttaggca tgaaacaggg gcacag 36
<210>23
<211>36
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN040
<400>23
agagagcata tgagcagcca ttcatcaaat atggcg 36
<210>24
<211>41
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN032
<400>24
acgtgggcgg ccgcggtttc aaagttttct gtcatttcta a 41
<210>25
<211>39
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN033
<400>25
ttgttggcgg ccgcaatgga acgaaggcgt ttgtggggt 39
<210>26
<211>36
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN034
<400>26
ggaggactcg aggttaggca tgaaacaggg gcacag 36
<210>27
<211>39
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN035
<400>27
ggaggactcg agctagttag gcatgaaaca ggggcacag 39
<210>28
<211>31
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN106
<400>28
cagaaaactt tgaaaccatg gaacgaaggc g 31
<210>29
<211>31
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN107
<400>29
cgccttcgtt ccatggtttc aaagttttct g 31
<210>30
<211>43
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN104
<400>30
ggagatatac atatggatcc aagcagccat tcatcaaata tgg 43
<210>31
<211>43
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN105
<400>31
ccatatttga tgaatggctg cttggatcca tatgtatatc tcc 43
<210>32
<211>40
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN123
<400>32
gacagaaaac tttgaaacca tggaacgtcg tcgtctgtgg 40
<210>33
<211>40
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN124
<400>33
ccacagacga cgacgttcca tggtttcaaa gttttctgtc 40
<210>34
<211>30
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN199
<400>34
ggaattccat atggatccaa gcagccattc 30
<210>35
<211>53
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN19
<400>35
ggagctctcg agtcagtggt ggtggtggtg gtggttcggc ataaagcacg ggc 53
<210>36
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG 1826
<400>36
tccatgacgt tcctgacgtt 20
<210>37
<211>18
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG 1758
<400>37
tctcccagcg tgcgccat 18
<210>38
<211>54
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG2006
<400>38
accgatgacg tcgccggtga cggcaccacg tcgtcgtttt gtcgttttgt cgtt 54
<210>39
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG1668
<400>39
tccatgacgt tcctgatgct 20
<210>40
<211>22
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG5456
<400>40
tcgacgtttt cggcgcgcgc cg 22
<210>41
<211>127
<212>PRT
<213>流感嗜血杆菌b
<400>41
Met Lys Leu Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
115 120 125
<210>42
<211>111
<212>PRT
<213>人工序列
<220>
<223>1-Met；2-Asp；3-Pro；后面是蛋白质D的氨基酸20到127
<400>42
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala PheAla Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
100 105 110
<210>43
<211>450
<212>PRT
<213>人工序列
<220>
<223>蛋白质D-MAGE-A3-His
<400>43
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
115 120 125
Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
130 135 140
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
145 150 155 160
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
165 170 175
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
180 185 190
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
195 200 205
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
210 215 220
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
225 230 235 240
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
245 250 255
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
260 265 270
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
275 280 285
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
290 295 300
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
305 310 315 320
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
325 330 335
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
340 345 350
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
355 360 365
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
370 375 380
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
385 390 395 400
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
405 410 415
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
420 425 430
Glu Trp Val Leu Arg Glu Gly Glu Glu Gly Gly His His His His His
435 440 445
His His
450
<210>44
<211>109
<212>PRT
<213>人工序列
<220>
<223>用于pD1/3-PRAME-His序列中的流感嗜血杆菌蛋白质D的氨基酸
<400>44
Met Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp
1 5 10 15
Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30
Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45
Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His
50 55 60
Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His
65 70 75 80
Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95
Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
100 105
<210>45
<211>127
<212>PRT
<213>人工序列
<220>
<223>用于pD1/3-MAGE-His序列中的流感嗜血杆菌蛋白质D的氨基酸
<400>45
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Mal Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
115 120 125
序列表
<110>GlaxoSmithKline Biologicals SA
<120>疫苗
<130>VB62293
<150>0700760.2
<151>2007-01-15
<150>0701262.8
<151>2007-01-23
<160>45
<170>FastSEQ for Windows Version 4.0
<210>1
<211>1887
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-TSGHHHHHH
(质粒TCMP14)
<400>1
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
actagtggcc accatcacca tcaccat 1887
<210>2
<211>629
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-TSGHHHHHH
(质粒TCMP14)
<400>2
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn Thr Ser Gly His
610 615 620
His His His His His
625
<210>3
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒TCMP14)
<400>3
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
<210>4
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒TCMP14)
<400>4
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>5
<211>1884
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-LEHHHHHH
(质粒pET21)
<400>5
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
ctcgagcacc accaccacca ccac 1884
<210>6
<211>628
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-LEHHHHHH
(质粒pET21)
<400>6
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn Leu Glu His His
610 615 620
His His His His
625
<210>7
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET21)
<400>7
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gaaggcgttt gtggggttcc 360
attcagagcc gatacatcag catgagtgtg tggacaagcc cacggagact tgtggagctg 420
gcagggcaga gcctgctgaa ggatgaggcc ctggccattg ccgccctgga gttgctgccc 480
agggagctct tcccgccact cttcatggca gcctttgacg ggagacacag ccagaccctg 540
aaggcaatgg tgcaggcctg gcccttcacc tgcctccctc tgggagtgct gatgaaggga 600
caacatcttc acctggagac cttcaaagct gtgcttgatg gacttgatgt gctccttgcc 660
caggaggttc gccccaggag gtggaaactt caagtgctgg atttacggaa gaactctcat 720
caggacttct ggactgtatg gtctggaaac agggccagtc tgtactcatt tccagagcca 780
gaagcagctc agcccatgac aaagaagcga aaagtagatg gtttgagcac agaggcagag 840
cagcccttca ttccagtaga ggtgctcgta gacctgttcc tcaaggaagg tgcctgtgat 900
gaattgttct cctacctcat tgagaaagtg aagcgaaaga aaaatgtact acgcctgtgc 960
tgtaagaagc tgaagatttt tgcaatgccc atgcaggata tcaagatgat cctgaaaatg 1020
gtgcagctgg actctattga agatttggaa gtgacttgta cctggaagct acccaccttg 1080
gcgaaatttt ctccttacct gggccagatg attaatctgc gtagactcct cctctcccac 1140
atccatgcat cttcctacat ttccccggag aaggaagagc agtatatcgc ccagttcacc 1200
tctcagttcc tcagtctgca gtgcctgcag gctctctatg tggactcttt atttttcctt 1260
agaggccgcc tggatcagtt gctcaggcac gtgatgaacc ccttggaaac cctctcaata 1320
actaactgcc ggctttcgga aggggatgtg atgcatctgt cccagagtcc cagcgtcagt 1380
cagctaagtg tcctgagtct aagtggggtc atgctgaccg atgtaagtcc cgagcccctc 1440
caagctctgc tggagagagc ctctgccacc ctccaggacc tggtctttga tgagtgtggg 1500
atcacggatg atcagctcct tgccctcctg ccttccctga gccactgctc ccagcttaca 1560
accttaagct tctacgggaa ttccatctcc atatctgcct tgcagagtct cctgcagcac 1620
ctcatcgggc tgagcaatct gacccacgtg ctgtatcctg tccccctgga gagttatgag 1680
gacatccatg gtaccctcca cctggagagg cttgcctatc tgcatgccag gctcagggag 1740
ttgctgtgtg agttggggcg gcccagcatg gtctggctta gtgccaaccc ctgtcctcac 1800
tgtggggaca gaaccttcta tgacccggag cccatcctgt gcccctgttt catgcctaac 1860
<210>8
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET21)
<400>8
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>9
<211>1878
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-HHHHHH(质粒
pET26)
<400>9
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gtcgtcgtct gtggggcagc 360
attcagagcc gttatattag catgagcgtg tggaccagcc cgcgtcgtct ggttgagctg 420
gccggccaga gcctgctgaa agatgaagcg ctggccattg cggcgctgga gctgctgccg 480
cgtgagctgt ttccgccgct gtttatggcg gcgtttgatg gccgtcatag ccagaccctg 540
aaagcgatgg tgcaggcgtg gccgtttacc tgtctgccgc tgggcgtgct gatgaaaggc 600
cagcatctgc atctggaaac ctttaaagcg gtgctggatg gcctggatgt gctgctggcc 660
caggaagttc gtccgcgtcg ttggaaactg caagtgctgg atctgcgtaa aaacagccat 720
caggattttt ggaccgtgtg gagcggcaat cgtgcgagcc tgtatagctt tccggaaccg 780
gaagcggcgc agccgatgac caaaaaacgt aaagtggatg gcctgagcac cgaagcggaa 840
cagccgttta ttccggtgga agtgctggtt gacctgtttc tgaaagaagg cgcctgcgac 900
gagctgttta gctatctgat cgaaaaagtg aaacgcaaaa aaaacgtgct gcgtctgtgc 960
tgcaaaaaac tgaaaatctt cgcgatgccg atgcaggata ttaaaatgat cctgaaaatg 1020
gtgcagctgg atagcattga ggacctggaa gtgacctgca cctggaaact gccgaccctg 1080
gccaaattta gcccgtatct gggccagatg attaacctgc gtcgtctgct gctgtctcat 1140
attcatgcga gcagctatat tagcccggaa aaagaagaac agtatatcgc gcagtttacc 1200
agccagtttc tgagcctgca atgcctgcaa gcgctgtatg tggatagcct gttttttctg 1260
cgtggccgtc tggatcagct gctgcgtcat gtgatgaatc cgctggaaac cctgagcatt 1320
accaactgcc gtctgagcga aggcgatgtg atgcatctga gccagagccc gagcgttagc 1380
cagctgtctg ttctgagcct gagcggcgtg atgctgaccg atgtgagccc ggaaccgctg 1440
caagccctgc tggaacgtgc gagcgcgacc ctgcaagacc tggtgtttga tgaatgcggc 1500
attaccgatg atcagctgct ggccctgctg ccgagcctga gccattgcag ccagctgacc 1560
accctgagct tttatggcaa cagcattagc attagcgcgc tgcaaagcct gctgcaacat 1620
ctgattggcc tgagcaacct gacccatgtg ctgtatccgg tgccgctgga aagctatgaa 1680
gatattcatg gcaccctgca tctggaacgt ctggcctatc tgcacgcgcg tctgcgtgag 1740
ctgctgtgcg agctgggccg tccgagcatg gtttggctgt ctgcgaatcc gtgcccgcat 1800
tgcggcgatc gtacctttta tgatccggaa ccgattctgt gcccgtgctt tatgccgaac 1860
caccaccacc accaccac 1878
<210>10
<211>626
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-HHHHHH(质粒
pET26)
<400>10
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn His His His His
610 615 620
His His
625
<210>11
<211>1860
<212>DNA
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET26)
<400>11
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60
attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300
caaagtttag aaatgacaga aaactttgaa accatggaac gtcgtcgtct gtggggcagc 360
attcagagcc gttatattag catgagcgtg tggaccagcc cgcgtcgtct ggttgagctg 420
gccggccaga gcctgctgaa agatgaagcg ctggccattg cggcgctgga gctgctgccg 480
cgtgagctgt ttccgccgct gtttatggcg gcgtttgatg gccgtcatag ccagaccctg 540
aaagcgatgg tgcaggcgtg gccgtttacc tgtctgccgc tgggcgtgct gatgaaaggc 600
cagcatctgc atctggaaac ctttaaagcg gtgctggatg gcctggatgt gctgctggcc 660
caggaagttc gtccgcgtcg ttggaaactg caagtgctgg atctgcgtaa aaacagccat 720
caggattttt ggaccgtgtg gagcggcaat cgtgcgagcc tgtatagctt tccggaaccg 780
gaagcggcgc agccgatgac caaaaaacgt aaagtggatg gcctgagcac cgaagcggaa 840
cagccgttta ttccggtgga agtgctggtt gacctgtttc tgaaagaagg cgcctgcgac 900
gagctgttta gctatctgat cgaaaaagtg aaacgcaaaa aaaacgtgct gcgtctgtgc 960
tgcaaaaaac tgaaaatctt cgcgatgccg atgcaggata ttaaaatgat cctgaaaatg 1020
gtgcagctgg atagcattga ggacctggaa gtgacctgca cctggaaact gccgaccctg 1080
gccaaattta gcccgtatct gggccagatg attaacctgc gtcgtctgct gctgtctcat 1140
attcatgcga gcagctatat tagcccggaa aaagaagaac agtatatcgc gcagtttacc 1200
agccagtttc tgagcctgca atgcctgcaa gcgctgtatg tggatagcct gttttttctg 1260
cgtggccgtc tggatcagct gctgcgtcat gtgatgaatc cgctggaaac cctgagcatt 1320
accaactgcc gtctgagcga aggcgatgtg atgcatctga gccagagccc gagcgttagc 1380
cagctgtctg ttctgagcct gagcggcgtg atgctgaccg atgtgagccc ggaaccgctg 1440
caagccctgc tggaacgtgc gagcgcgacc ctgcaagacc tggtgtttga tgaatgcggc 1500
attaccgatg atcagctgct ggccctgctg ccgagcctga gccattgcag ccagctgacc 1560
accctgagct tttatggcaa cagcattagc attagcgcgc tgcaaagcct gctgcaacat 1620
ctgattggcc tgagcaacct gacccatgtg ctgtatccgg tgccgctgga aagctatgaa 1680
gatattcatg gcaccctgca tctggaacgt ctggcctatc tgcacgcgcg tctgcgtgag 1740
ctgctgtgcg agctgggccg tccgagcatg gtttggctgt ctgcgaatcc gtgcccgcat 1800
tgcggcgatc gtacctttta tgatccggaa ccgattctgt gcccgtgctt tatgccgaac 1860
<210>12
<211>620
<212>PRT
<213>人工序列
<220>
<223>MDP-20-127-蛋白质D-PRAME-无His尾
(质粒pET26)
<400>12
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110
Glu Arg Arg Arg Leu Trp Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met
115 120 125
Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser
130 135 140
Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro
145 150 155 160
Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His
165 170 175
Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu
180 185 190
Pro Leu Gly Val Leu Met Lys Gly Gln His Leu His Leu Glu Thr Phe
195 200 205
Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg
210 215 220
Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His
225 230 235 240
Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser
245 250 255
Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val
260 265 270
Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val
275 280 285
Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser
290 295 300
Tyr Leu Ile Glu Lys Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys
305 310 315 320
Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met Gln Asp Ile Lys Met
325 330 335
Ile Leu Lys Met Val Gln Leu Asp Ser Ile Glu Asp Leu Glu Val Thr
340 345 350
Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly
355 360 365
Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser
370 375 380
Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gln Tyr Ile Ala Gln Phe Thr
385 390 395 400
Ser Gln Phe Leu Ser Leu Gln Cys Leu Gln Ala Leu Tyr Val Asp Ser
405 410 415
Leu Phe Phe Leu Arg Gly Arg Leu Asp Gln Leu Leu Arg His Val Met
420 425 430
Asn Pro Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly
435 440 445
Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val
450 455 460
Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu
465 470 475 480
Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe
485 490 495
Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu Ala Leu Leu Pro Ser
500 505 510
Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser
515 520 525
Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu
530 535 540
Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu
545 550 555 560
Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala
565 570 575
Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp
580 585 590
Leu Ser Ala Asn Pro Cys Pro His Cys Gly Asp Arg Thr Phe Tyr Asp
595 600 605
Pro Glu Pro Ile Leu Cys Pro Cys Phe Met Pro Asn
610 615 620
<210>13
<211>9
<212>PRT
<213>智人
<400>13
Val Leu Asp Gly Leu Asp Val Leu Leu
1 5
<210>14
<211>10
<212>PRT
<213>智人
<400>14
Ser Leu Tyr Ser Phe Pro Glu Pro Glu Ala
1 5 10
<210>15
<211>10
<212>PRT
<213>智人
<400>15
Ala Leu Tyr Val Asp Ser Leu Phe Phe Leu
1 5 10
<210>16
<211>9
<212>PRT
<213>智人
<400>16
Leu Tyr Val Asp Ser Leu Phe Phe Leu
1 5
<210>17
<211>9
<212>PRT
<213>智人
<400>17
Ser Leu Leu Gln His Leu Ile Gly Leu
1 5
<210>18
<211>36
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN008
<400>18
atataacata tggatccaag cagccattca tcaaat 36
<210>19
<211>40
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN037
<400>19
ccacaaacgc cttcgttcca tggtttcaaa gttttctgtc 40
<210>20
<211>40
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN036
<400>20
gacagaaaac tttgaaacca tggaacgaag gcgtttgtgg 40
<210>21
<211>39
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN029
<400>21
agagagacta gtctagttag gcatgaaaca ggggcacag 39
<210>22
<211>36
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN002
<400>22
ggaggaacta gtgttaggca tgaaacaggg gcacag 36
<210>23
<211>36
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN040
<400>23
agagagcata tgagcagcca ttcatcaaat atggcg 36
<210>24
<211>41
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN032
<400>24
acgtgggcgg ccgcggtttc aaagttttct gtcatttcta a 41
<210>25
<211>39
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN033
<400>25
ttgttggcgg ccgcaatgga acgaaggcgt ttgtggggt 39
<210>26
<211>36
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN034
<400>26
ggaggactcg aggttaggca tgaaacaggg gcacag 36
<210>27
<211>39
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN035
<400>27
ggaggactcg agctagttag gcatgaaaca ggggcacag 39
<210>28
<211>31
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN106
<400>28
cagaaaactt tgaaaccatg gaacgaaggc g 31
<210>29
<211>31
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN107
<400>29
cgccttcgtt ccatggtttc aaagttttct g 31
<210>30
<211>43
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN104
<400>30
ggagatatac atatggatcc aagcagccat tcatcaaata tgg 43
<210>31
<211>43
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN105
<400>31
ccatatttga tgaatggctg cttggatcca tatgtatatc tcc 43
<210>32
<211>40
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN123
<400>32
gacagaaaac tttgaaacca tggaacgtcg tcgtctgtgg 40
<210>33
<211>40
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN124
<400>33
ccacagacga cgacgttcca tggtttcaaa gttttctgtc 40
<210>34
<211>30
<212>DNA
<213>人工序列
<220>
<223>正义寡核苷酸CAN199
<400>34
ggaattccat atggatccaa gcagccattc 30
<210>35
<211>53
<212>DNA
<213>人工序列
<220>
<223>反义寡核苷酸CAN19
<400>35
ggagctctcg agtcagtggt ggtggtggtg gtggttcggc ataaagcacg ggc 53
<210>36
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG 1826
<400>36
tccatgacgt tcctgacgtt 20
<210>37
<211>18
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG 1758
<400>37
tctcccagcg tgcgccat 18
<210>38
<211>54
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG2006
<400>38
accgatgacg tcgccggtga cggcaccacg tcgtcgtttt gtcgttttgt cgtt 54
<210>39
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG1668
<400>39
tccatgacgt tcctgatgct 20
<210>40
<211>22
<212>DNA
<213>人工序列
<220>
<223>免疫刺激寡核苷酸CpG5456
<400>40
tcgacgtttt cggcgcgcgc cg 22
<210>41
<211>127
<212>PRT
<213>流感嗜血杆菌b
<400>41
Met Lys Leu Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
115 120 125
<210>42
<211>111
<212>PRT
<213>人工序列
<220>
<223>1-Met；2-Asp；3-Pro；后面是蛋白质D的氨基酸20到127
<400>42
Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
1 5 10 15
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
100 105 110
<210>43
<211>450
<212>PRT
<213>人工序列
<220>
<223>蛋白质D-MAGE-A3-His
<400>43
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
115 120 125
Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
130 135 140
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
145 150 155 160
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
165 170 175
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
180 185 190
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
195 200 205
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
210 215 220
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
225 230 235 240
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
245 250 255
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
260 265 270
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
275 280 285
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
290 295 300
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
305 310 315 320
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
325 330 335
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
340 345 350
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
355 360 365
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
370 375 380
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
385 390 395 400
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
405 410 415
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
420 425 430
Glu Trp Val Leu Arg Glu Gly Glu Glu Gly Gly His His His His His
435 440 445
His His
450
<210>44
<211>109
<212>PRT
<213>人工序列
<220>
<223>用于pD1/3-PRAME-His序列中的流感嗜血杆菌蛋白质D的氨基酸
<400>44
Met Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp
1 5 10 15
Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30
Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45
Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His
50 55 60
Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His
65 70 75 80
Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95
Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
100 105
<210>45
<211>127
<212>PRT
<213>人工序列
<220>
<223>用于pD1/3-MAGE-His序列中的流感嗜血杆菌蛋白质D的氨基酸
<400>45
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu
1 5 10 15
Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30
Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
35 40 45
Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60
Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
100 105 110
Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
115 120 125
Claims (35)
1.一种融合蛋白，其包括
(a)PRAME或其免疫原性片段，和
(b)来源自蛋白质D的异源融合伴侣蛋白，
其中所述融合伴侣蛋白不包括蛋白质D的分泌序列或信号序列。
2.来源自蛋白质D的融合伴侣蛋白，其中所述融合伴侣蛋白在融合蛋白序列的N-末端处或其中包括氨基酸Met-Asp-Pro并且其中所述融合伴侣蛋白不包括蛋白质D的分泌序列或信号序列。
3.根据权利要求2的融合伴侣蛋白，其中所述蛋白质D序列大约或确切地包括或由蛋白质D的氨基酸17至127、18至127、19至127或20至127组成。
4.任一前述权利要求的融合伴侣蛋白，其中缺失或通过替换而取代了蛋白质D融合伴侣蛋白的一个或更多个氨基酸。
5.根据权利要求4的融合伴侣蛋白，其中所述氨基酸被利用保守替换而替换。
6.根据权利要求4或5的融合伴侣蛋白，其中1、2、3、4、5、6、7、8、9或更多个氨基酸被替换。
7.根据任一前述权利要求的融合伴侣蛋白，其中蛋白质D的分泌序列或信号序列指大约天然蛋白的氨基酸1至16、17、18或19。
8.根据任一前述权利要求的融合伴侣蛋白，其中蛋白质D的分泌序列或信号序列为蛋白质D的N-末端19个氨基酸。
9.一种融合蛋白，其包括权利要求2至8中任一项的融合伴侣蛋白。
10.一种融合蛋白，其包括权利要求2至8中任一项的融合伴侣蛋白和一个或更多个肿瘤抗原或其免疫原性部分。
11.根据权利要求9或10的融合蛋白，其包括肿瘤抗原PRAME或其免疫原性部分。
12.根据权利要求1或11的融合蛋白，其中所述PRAME的免疫原性片段或部分包括或由一个或更多个下列表位组成：
VLDGLDVLL(PRA100-108；SEQ ID NO：XX)；
SLYSFPEPEA(PRA142-151；SEQ ID NO：XX)；
ALYVDSLFFL(PRA300-309；SEQ ID NO：XX)；
LYVDSLFFL(PRA301-309；SEQ ID NO：XX)和
SLLQHLIGL(PRA425-433；SEQ ID NO：XX).
13.根据权利要求9或10的融合蛋白，其包括一个或更多个下列肿瘤抗原或肿瘤抗原衍生物或其免疫原性部分：MAGE 1、MAGE 2、MAGE 3、MAGE 4、MAGE 5、MAGE 6、MAGE 7、MAGE 8、MAGE9、MAGE 10、MAGE 11、MAGE 12、MAGE B1、MAGE B2、MAGEB3、MAGE B4、MAGE C1、MAGE C2。
14.根据权利要求13的融合蛋白，其中MAGE抗原的氨基酸序列中可缺失或替换1、2、3、4、5、6、7、8、9、10或更多个氨基酸。
15.根据权利要求14的融合蛋白，其中从MAGE序列的N-末端缺失了2个氨基酸。
16.根据权利要求13、14或15的融合蛋白，其中所述抗原为MAGE-A3或其免疫原性部分，其中所述MAGE-A3抗原包括或由MAGE-A3的氨基酸3至314组成。
17.根据权利要求10的融合蛋白，其中所述肿瘤抗原或其衍生物选自下列抗原之一或可引导对该抗原的免疫应答的其免疫原性部分：WT-1、WT-1F、BAGE、LAGE 1、LAGE 2(也称作NY-ESO-1)、SAGE、HAGE、XAGE、PSA、PAP、PSCA、P501S(也称作prostein)、HASH1、HASH2、Cripto、B726、NY-BR1.1、P510、MUC-1、Prostase、STEAP、酪氨酸酶、端粒酶、存活素、CASB616、P53和/或Her-2/neu、SSX-2；SSX-4；SSX-5；NA17；MELAN-A；P790；P835；B305D；B854；CASB618(如WO00/53748中所述)；CASB7439(如WO01/62778中所述)；C1491；C1584和C1585。
18.根据权利要求1或9至17中任一项的融合蛋白，其进一步包括亲和标签。
19.根据权利要求1或10至18中任一项的融合蛋白，其还包括融合伴侣蛋白和肿瘤抗原或其免疫原性部分之间，和/或融合伴侣蛋白和组氨酸尾或其他亲和标签之间，和/或肿瘤抗原或其免疫原性部分和组氨酸尾或其他亲和标签之间的一个或更多个接头序列。
20.一种核酸序列，其编码如权利要求1至19中任一项所要求的融合蛋白或融合伴侣蛋白。
21.一种载体，其包括权利要求20的核酸序列。
22.一种宿主细胞，其被权利要求21的载体转化。
23.一种疫苗，其包含权利要求1至19中任一项所要求的融合蛋白或融合伴侣蛋白或权利要求20中要求的核酸或权利要求21中要求的载体。
24.权利要求23中要求的疫苗，其还包括佐剂和/或免疫刺激细胞因子或趋化因子。
25.权利要求24中要求的疫苗，其中所述佐剂包括3D-MPL，QS21和/或CpG寡核苷酸。
26.用于药物的权利要求23至25中任一项中所要求的疫苗。
27.本文要求的蛋白质或核酸或载体在制备用于免疫疗法治疗癌症患者的疫苗中的用途。
28.生产融合蛋白的方法，其包括在细胞中表达包括根据权利要求1至8中任一项的融合伴侣蛋白的融合蛋白的步骤。
29.根据权利要求28的方法，其中所述细胞为细菌。
30.根据权利要求29的方法，其中所述细菌为大肠杆菌。
31.根据权利要求28或30中任一项的方法，其中所述融合蛋白以不溶性蛋白表达于细胞中。
32.根据权利要求31的方法，其进一步包括裂解细胞并从已裂解细胞中纯化已表达融合蛋白的步骤。
33.通过权利要求28至32中任一项的方法获得的或可获得的融合蛋白。
34.治疗癌症患者的方法，其包括给予本文要求的蛋白质、核酸、载体或疫苗的步骤。
35.权利要求27的用途或权利要求34的方法，其中所述癌症选自黑色素瘤、乳腺癌、膀胱癌、肺癌例如NSCLC、肉瘤、卵巢癌、头颈部癌、肾癌、结肠直肠癌、多发性骨髓瘤、包括急性白血病的白血病和食道癌。
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| CN103717613A (zh) * | 2011-07-22 | 2014-04-09 | 葛兰素史密丝克莱恩生物有限公司 | Prame纯化 |
| CN105254767A (zh) * | 2015-11-17 | 2016-01-20 | 重庆科润生物医药研发有限公司 | 一种Protein D与HER2融合蛋白及其制备方法和应用 |
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| CN103269719A (zh) * | 2010-10-27 | 2013-08-28 | 葛兰素史密丝克莱恩生物有限公司 | 治疗神经病症的免疫原性组合物和方法 |
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| CN108264550B (zh) * | 2017-01-04 | 2021-04-23 | 香雪生命科学技术（广东）有限公司 | 一种识别源自于prame抗原短肽的tcr |
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| CN109400697A (zh) * | 2017-08-17 | 2019-03-01 | 广东香雪精准医疗技术有限公司 | 一种识别prame抗原短肽的tcr及其相关组合物 |
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