CN1675237A - 免疫原性组合物 - Google Patents
免疫原性组合物 Download PDFInfo
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- CN1675237A CN1675237A CNA038194872A CN03819487A CN1675237A CN 1675237 A CN1675237 A CN 1675237A CN A038194872 A CNA038194872 A CN A038194872A CN 03819487 A CN03819487 A CN 03819487A CN 1675237 A CN1675237 A CN 1675237A
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Abstract
本发明涉及作为免疫融合伴侣、表达增强子的融合伴侣,优选涉及具有双重功能的融合伴侣。特别的,所述融合伴侣含有所谓的胆碱结合域,例如所述的融合物包含来源于肺炎链球菌属的LytA,或肺炎球菌噬菌体CP1溶菌酶(CPL1),其中所述的胆碱结合域被修饰包括异源的T-辅助表位,并且与抗原融合,特别是那些免疫原性较弱的抗原,如自身抗原、如肿瘤特异性或组织特异性抗原。本发明还涉及含有融合伴侣的融合蛋白,及其制造方法,和其在免疫原性组合物和疫苗中的应用和其在制药中的应用。
Description
本发明涉及作为免疫融合伴侣表达增强子的融合伴侣,优选涉及具有双重功能的融合伴侣。本发明还涉及含有融合伴侣的融合蛋白,及其制造方法,和其在疫苗中的应用和在制药中的应用。特别的,提供了含有所谓的胆碱结合域的融合伴侣,例如包括来源于肺炎链球菌属的LytA,或肺炎球菌噬菌体CP1溶菌酶(CPL1)的融合物,其中所述的胆碱结合域被修饰包括异源的T-辅助表位。这类融合伴侣被显示能提高其附着的异源蛋白的表达水平,并且被发现当与免疫原性较弱的蛋白或肽融合时将特别有效,可作为疫苗抗原。更特别的,这类融合伴侣在包含自身抗原,如肿瘤特异性或组织特异性抗原的构建体中非常有效。
发明背景
肺炎链球菌合成了N乙酰-L-丙氨酸酰胺酶,LytA,一种特异性降解细胞壁肽聚糖骨架并最终导致细胞溶解的自溶素。其多肽链具有两个域。其N-末端域具有催化活性,而LytA的C-末端具有吸附胆碱并锚定至细胞壁的作用。已知其C-末端域可与胆碱或胆碱类似物结合,并且也可与叔胺,如常用于层析中的DEAE(二乙基氨基乙基)相结合。
LytA是长318个氨基酸的蛋白,其C-末端部分包括由六个20或21氨基酸的不完全重复序列组成的串联排列结构和一短COOH-末尾结构。所述的重复序列位于如下位置:
R1:177-191
R2:192-212
R3:213-234
R4:235-254
R5:255-275
R6:276-298
据预测,这类重复序列呈β-转角构象。C-末端负责结合胆碱。同样的,CPL1的C-末端负责结合亲合性,重复结构中的芳香基团有助于这种结合。这类蛋白可被用作亲和标记从而允许快速纯化(SanchezPuelles,Eur J Biochem.1992,203,153-9)。
肺炎链球菌属中的具有胆碱结合域的其他蛋白也被进行了研究。
其中的一种PspA(或肺炎球菌表面蛋白A)是一种毒力因子(Yother J & Briles(1992)J Bacteriol 174(2)p 601)。该蛋白具有抗原和免疫原性。它具有由10个与LytA重复序列同源的20个氨基酸的重复序列组成的C-末端域。
CbpA(或胆碱结合蛋白A)涉及肺炎球菌对人细胞的吸附(Rosenow等(1997)Mol Microbiol 25(5)p 819)。显示在C-末端结合域中的10个20氨基酸的重复序列几乎与PspA中的完全一致。
LytB和LytC与上述蛋白具有不同的模块组织,其分别由15个和11个重复结构组成的胆碱结合域位于N-末端,而不是C-末端(GarciaP Mol Microbiol(1999)31(4)p1275和Garcia P等(1999)Mol Microbiol33(1)p128)。序列比较显示LytB具有氨基葡萄糖苷酶活性。LytC显示在体外具有溶菌酶活性。此外,1995年克隆得到了被命名为PepA,PepB和PepC的三个基因。虽然其功能未知,这些基因同样也具有数目不等的与LytA中的重复结构同源的重复序列。
在其感染周期中,噬菌体合成了胞壁质水解酶,这有助于其进入细菌中。这些水解酶具有胆碱结合域。
噬菌体Cp-1的CPL1溶菌酶已进行了详细的研究。研究表明C-末端的6个20氨基酸的重复序列涉及胆碱的特异性识别(Garica J.L.J.Virol 61(8)p2573-80;(1987)和Garcia E Prol Natl Acad Sci(1988)p914)。对于LytA和CPL1重复序列的比较表明这些重复序列大致相同。
噬菌体Dp-1的胞壁质水解酶(Garcia P等(1983)J Gen Microbiol129(2)p489,Cpl-9(Garcia P等(1989)Biochem Biophys Res Commun158(1)p251,HB-3 Romero等1990 J Bacteriol 172(9)p5064-5070)和EJ-1 Diaz(1992)J Bacteriol 174(17)p5516)同样显示出胆碱结合域的特性。
由肺炎球菌噬菌体CP-1编码的溶菌酶同样也具有这种特性。WO99/10375中记载了人***瘤病毒蛋白E6,或E7与His标记和LytA的C-末端部分(即C-LytA)结合,和通过不同的亲和色谱对蛋白的纯化。WO 99/40188中记载了包含MAGE抗原和His尾及分子N-末端的C-LytA部分的融合蛋白。
令人惊奇的发现,本发明中的融合伴侣在与异源蛋白融合时,能增强其附着的异源蛋白的免疫原性。同时还发现可提高其附着的异源蛋白的表达水平。相应的在一优选实施例中,本发明还提供了一种能作为表达增强子的改进的免疫融合伴侣。
发明简述
相应的,本发明包括一融合伴侣分子,其包含胆碱结合域、或其片段、或其类似物,和异源通用(promiscuous)T辅助表位,优选通用MHC II类T-表位。所述的融合伴侣显示出可作为免疫融合伴侣,或作为表达增强子的能力,并且优选的具有作为免疫伴侣和表达增强子的双重能力。通用T-辅助表位是与多于一种的MHC II类等位基因,优选的与多于3种的MHC II类等位基因结合的表位。优选地,这种表位可在大量表达不同MHC单倍型的个体中引发辅助T细胞应答。可选的,所述融合蛋白可保留其结合胆碱的能力。
在一优选实施方案中,所述的胆碱结合部分来源于LytA的C末端。优选的,所述C-LytA或其衍生物包括至少4个如图1所示的R1至R6重复序列(SEQ ID NO:1-6)中的任意重复序列。在一最优选实施方案中,C-LytA包括177-298氨基酸,其包括第一重复序列的一部分和其他的完整的5个重复序列。
在本发明的进一步的方面,提供如本文所定义的进一步包括异源蛋白的融合伴侣。所述的异源蛋白既可以是化学偶联,也可以是与融合伴侣融合。优选的所述异源蛋白是肿瘤相关抗原或其免疫片段。
本发明的进一步的方面,提供了一种编码如本文所定义的蛋白的核酸序列。还提供了包括所述核酸的表达载体,和采用所述核酸或载体转化的宿主细胞。
在本发明的进一步的方面,提供了一种免疫原性组合物,其包括如本文所述的蛋白或核酸序列、和药学上可接受的赋形剂、稀释剂或载体。优选地所述免疫原性组合物进一步包括Th-1诱导佐剂。
在进一步的实施例中,本发明提供了免疫原性组合物或蛋白和核酸用于医药应用。特别的,提供了一种本发明所述的蛋白或核酸,用于制备在病人中引发免疫应答的、或用于治疗或预防传染病或癌症的药物。
本发明进一步提供了一种通过施用安全和有效量的本文所述的组合物或核酸,从而来治疗患有传染病或癌症,尤其是乳腺癌、肺癌(特别是非小细胞肺癌)、结肠直肠癌、卵巢癌、***癌、胃癌和其他GI(胃肠的)癌的病人的方法。
另外,本发明的进一步的实施例提供了一种通过将本发明所述的核酸或蛋白与药学上可接受的赋形剂、稀释剂或载体混合,从而来生产如本文所述的免疫原性组合物的方法。
发明详述
如本文所述,在本发明的一实施例中,修饰的胆碱结合域(融合伴侣)具有作为表达增强子的能力,所得到的融合蛋白与未融合蛋白相比,在宿主细胞中具有更高的表达量,优选的具有高出约100%(2倍以上)或150%或更多的表达量,所述表达量通过SDS-PAGE,随后经考马斯亮蓝染色或银染,可选的经凝胶扫描来测定。本发明所述的修饰的胆碱结合域还具有免疫伴侣的能力,其与异源蛋白融合的融合蛋白与未融合的异源蛋白相比在宿主中具有更大的免疫原性。
在本发明的另一实施例中,修饰的胆碱结合域具有免疫融合伴侣的能力,与单独的异源蛋白相比,融合蛋白能获得增强的免疫应答。
在一优选实施例中,修饰的胆碱结合域具有双重功能,具有作为免疫融合伴侣和作为表达增强子的能力。
在一优选实施例中,胆碱结合部分来源于LytA的C末端。优选C-LytA或其衍生物包含至少两个重复序列,优选至少四个重复序列。在本文上下文中,C-LytA衍生物是指本发明所述的C-LytA的变异体,也就是说是指那些保留了既作为免疫伴侣又作为表达增强子的能力的变异体。优选变异体包括,例如,包含与如图1所示的R1至R6(SEQID NO:1至6)中的任意重复序列具有至少85%同一性,优选至少90%同一性,更优选至少95%同一性,最优选至少97-99%同一性的氨基酸序列的肽,或包含如图1(SEQ ID NO:1至8)所示的氨基酸序列中的至少15,20,30,40,50或100个连续氨基酸的肽。
因此,本发明一方面提供了包含修饰的胆碱结合域和异源通用T辅助表位的融合伴侣蛋白,其中所述的胆碱结合域选自包含如下的组:
a) 如SEQ ID NO:7所示的LytA的C-末端域;
b) SEQ ID NO:8所示的序列;
c) 包含与SEQ ID NO:1至6中任意序列具有至少85%同一性,优选至少90%同一性,更优选至少95%同一性,最优选至少97-99%同一性的氨基酸序列的肽序列;
d) 包括SEQ ID NO:7或SEQ ID NO:8的氨基酸序列中的至少15,20,30,40,50或100个连续氨基酸的氨基酸序列的肽序列。
在一最优选实施方案中,C-LytA包括177-298氨基酸,其含有第一重复序列的一部分和另外五个完整的重复序列,如图1所示。
融合伴侣的第二组分,异源T-细胞表位,优选地选自这样一组表位,所述表位能与在人体中表达多于一种MHC II类分子的许多个体结合。例如,特别考虑的表位为来源于破伤风类毒素的P2和P30表位,Panina-Bordignon Eur.J.Immunol 19(12),2237(1989)。在一优选实施例中异源T-细胞表位为来源于破伤风毒素的P2或P30。
P2表位具有QYIKANSKFIGITE序列,并且相应于破伤风毒素830-843位的氨基酸。P30表位(破伤风毒素的947-967位残基)具有FNNFTVSFWLRVPKVSASHLE序列。可选的,可缺失FNNFTV序列。其他通用T表位可来源于恶性疟原虫的环子孢子蛋白,特别是具有DIEKKIAKMEKASSVFNVVNS序列的378-398区域(Alexander J,(1994)Immunity 1(9),p751-761)。另一表位来源于麻疹病毒融合蛋白的具有LSEIKGVIVHRLEGV序列的288-302位残基(PartidosCD,1990,J.Gen.Virol 71(9)2099-2105)。另一表位来源于肝炎B病毒表面抗原,特别是具有FFLLTRILTIPQSLD序列的氨基酸。另一组表位来源于白喉毒素。这些肽图谱(氨基酸271-290,321-340,331-350,351-370)中的4个位于毒素片段B的T域中,并且剩下的2个图谱位于R域(411-430,431-450):
PVFAGANYAAWAVNVAQVI
VHHNTEEIVAQSIALSSLMV
QSIALSSLMVAQAIPLVGEL
VDIGFAAYNFVESIINLFQV
QGESGHDIKITAENTPLPIA
GVLLPTIPGKLDVNKSKTHI
(Raju R.,Navaneetham D.,Okita D.,Diethelm-Okita B.,McCormick D.,Conti-Fine B.M.(1995)Eur.J.Immunol.25:3207-14.)
异源T-表位优选的与含有至少4个重复序列,优选包含2-5个重复序列的C-LytA融合。一个或多个随后的重复序列可选的与T-表位的C-末端融合。可选的,异源T-表位优选的***含有总共至少4个重复序列的C-LytA的两个连续重复序列之间,或***含有总共至少4个重复序列的C-LytA的1个重复序列中。更优选,C-LytA含有6个重复序列,并且异源表位***C-LytA的第六个重复序列之中或者其起始位置。
在另外的方面,本发明进一步提供了一种包含上述多肽中的至少一种的融合蛋白,以及编码这种融合蛋白的多核苷酸,典型的以药物组合物的形式,如,包含生理学上可接受的载体和/或免疫刺激剂的疫苗组合物。
因此,自身蛋白或其他免疫原性较弱的蛋白可与获得的融合伴侣的N-末端或C-末端融合。可选的,自身蛋白或免疫原性较弱的蛋白可被***融合伴侣中。在另一优选实施例中,组氨酸标记或至少4个,优选多于6个的组氨酸残基可与免疫原性较弱蛋白的任意末端融合。这样可允许所述蛋白经亲和层析步骤来纯化,因为典型的包括至少4个、优选6个或更多残基的组氨酸尾结构可与金属离子结合,并且因此适合于金属固相化金属离子亲和层析法(IMAC)。
从而典型的构建体包括:
-免疫原性较弱蛋白-C-LytA重复序列1-4-P2表位(***或替换C-LytA重复序列5)-C-LytA重复序列6
-C-LytA重复序列1-4-P2表位(***或替换C-LytA重复序列5)-C-LytA重复序列6-免疫原性较弱蛋白
-免疫原性较弱蛋白-C-LytA重复序列2-5-P2表位(***C-LytA重复序列6)
-C-LytA重复序列2-5-P2表位(***C-LytA重复序列6)-免疫原性较弱蛋白
-免疫原性较弱蛋白C-LytA重复序列1-5-P2表位-***C-LytA重复序列6
-C-LytA重复序列1-5-P2表位-***C-LytA重复序列6-免疫原性较弱蛋白
-免疫原性较弱蛋白-P2表位***C-LytA重复序列1-C-LytA重复序列2-5
-P2表位***C-LytA重复序列1-C-LytA重复序列2-5-免疫原性较弱蛋白
-免疫原性较弱蛋白-P2表位***C-LytA重复序列1-C-LytA重复序列2-6
-P2表位***C-LytA重复序列1-C-LytA重复序列2-6-免疫原性较弱蛋白
-免疫原性较弱蛋白-C-LytA重复序列1-P2表位***C-LytA重复序列2-C-LytA重复序列3-6
-C-LytA重复序列1-P2表位***C-LytA重复序列2-C-LytA重复序列3-6-免疫原性较弱蛋白
其中“***”是指位于所述重复中的任意位置,例如在残基1和2之间,或位于2和3之间等。
通用T辅助表位可被***重复区域中,如C-LytA重复序列2-5-C-LytA重复序列6a-P2表位-C-LytA重复序列6b,其中P2表位被***第六个重复序列中(见图2)。
在其他优选实施例中,CPL1的C-末端(C-CPL1)可被用作C-LytA的一种替换。
可选的,上述构建体中的P2表位可被其他通用T表位替换,如P30。在本发明一具体实施例中,两个或更多的通用表位为融合构建体的一部分。不过,优选的使融合伴侣尽可能的小,从而限制了潜在的干涉CD8+和B表位的数目。因此,融合伴侣优选的不大于100-140个氨基酸,进一步优选不大于120个氨基酸,典型的为约100个氨基酸。
与融合伴侣融合的抗原可来源于细菌、病毒、原生动物、真菌或哺乳动物,包括人来源。
本发明的融合伴侣优选的与自身抗原,如那些***癌、胸腺癌、结肠直肠癌、肺癌、胰腺癌、卵巢癌、肾脏癌、黑素瘤等的肿瘤相关或组织特异性抗原融合。特意考虑将所述自身或肿瘤抗原的片段与本发明的融合伴侣融合。典型的,所述片段含有全长序列的至少20,优选50,更优选100个连续的氨基酸。典型的这类片段将缺乏一个或多个穿膜结构域或具有约3,5,8,10,15,20,28,33,50,54个氨基酸的N-末端或C-末端缺失。当以适当的方式呈递时,这类片段将能产生识别全长蛋白的免疫应答。本发明特别阐释的多肽包括与融合伴侣融合的肿瘤相关或组织特异性蛋白的至少10个,优选20,更优选30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180个相连的氨基酸。
本发明的多肽具有免疫原性,也就是,它们在免疫测定中(如ELISA或T-细胞刺激检测),以可检测的量与来源于患有cripto表达癌症的病人的抗血清和/或T-细胞反应。可采用本领域熟练技术人员已知的技术来进行免疫活性筛选。例如,可采用那些在Harlow & Lane,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,1988中记载的方法来进行筛选。在一示例实施例中,多肽可固定于固相支持物上并与病人血清接触,从而允许血清中的抗体与固定的多肽相结合。随后可移去未固定的血清,如采用125I-标记的蛋白A来检测结合的抗体。正如本领域熟练技术人员可理解的那样,肿瘤相关或肿瘤特异性抗原的免疫原性部分也包含在本发明的范围内。本文所采用的“免疫原性部分”,是指一片段,其自身可与B-细胞和/或识别多肽的T-细胞表面抗原受体发生免疫反应(也就是,特异性结合)。通常采用已知的技术,如那些在Paul,Fundamental Immunology,3rd ed.,243-247(Raven Press,1993)中概述的技术来鉴别免疫原性部分,该文献在此引入参考。这类技术包括筛选那些能与抗原特异性抗体、抗血清和/或T-细胞系或克隆反应的多肽。如本文所采用的,如果它们能特异性结合抗原(也就是在ELISA或其他免疫测定中与所述蛋白反应,并且不与无关蛋白可检测量的发生反应),则抗血清和抗体具有“抗原特异性”。这类抗血清和抗体可采用常规技术如本文所述进行制备。在一优选实施例中,多肽的免疫原性部分是指那些以基本上不低于全长多肽的反应水平的水平与抗血清和/或T-细胞反应的部分(如,在ELISA和/或T-细胞反应检测中)。优选,免疫原性部分的免疫原性水平至少约为全长多肽免疫性的约50%,优选至少约为70%,最优选大于约90%。在一些实施例中,优选鉴别那些免疫活性水平高于相应的全长多肽的部分,例如,具有高于约100%或150%或更高的免疫活性。
在某些其他实施例中,示例性的免疫原性部分可包括其中N-末端前导肽序列和/或穿膜结构域缺失的肽。其他示范性的免疫原性部分可包含相对于成熟蛋白的小的N-和/或C-末端缺失(如,约1-50个氨基酸,优选约1-30个氨基酸,更优选约5-15氨基酸)。
示范性的抗原或其片段可包括MAGE 1,Mage 3和MAGE 4或其他如WO 99/40188中所公开的MAGE抗原,PRAME(WO 96/10577),BAGE,RAGE,LAGE 1(WO 98/32855),LAGE 2(也称为NY-ESO-1,WO 98/14464),XAGE(Liu等,Cancer Res,2000,60:4752-4755;WO 02/18584),SAGE,和HAGE(WO 99/53061)或GAGE(Robbins&Kawakami,1996,Current Opinions in Immunology 8,pps 628-636;Van den Eynde等,International Journal of Clinical & LaboratoryResearch(submitted 1997);Correale等(1997),Journal of the NationalCancer Institute 89,p293。事实上,这类抗原在广泛的肿瘤类型范围内表达,如黑素瘤、肺癌、肉瘤和膀胱癌中。
在一优选实施例中采用了***抗原,如***特异抗原(PSA),PAP,PSCA(PNAS 95(4)1735-1740 1998),PSMA或已知作为***酶的抗原。
在一特定优选实施例中,***抗原为P501S或其片段。P501S,也被命名为prostein(Xu etal.,Cancer Res.61,2001,1563-1568),为W098/37814中的SEQ ID NO.113,并且为一553个氨基酸的蛋白。免疫原性片段或其部分包括至少20,优选50,更优选100个连续的如上文所述参考专利申请并经本发明特别指出的氨基酸。优选片段为WO98/50567(PS108抗原)中公开的片段和***癌相关蛋白(WO99/67384中的SEQ ID NO:9)。其他优选片段为全长P501S蛋白的51-553,34-553或55-553位氨基酸。特别是,特意考虑设计了构建体1,2和3(参见图2,SEQ ID NOs.27-32),其可在酵母***中表达,例如编码这类多肽的DNA序列可在酵母***中表达。
***酶是一种***特异性丝氨酸蛋白酶(胰蛋白酶类似物),长254个氨基酸,具有保守丝氨酸蛋白酶催化三联体H-D-S和具有潜在分泌功能的氨基末端前前肽序列(P.Nelson,Lu Gan,C.Ferguson,P.Moss,R.linas,L.Hood & K.Wand,″Molecular cloning andcharacterisation of prostase,an androgen-regulated serine protease withprostate restricted expression”,In Proc.Natl.Acad.Sci.USA(1999)96,3114-3119),并描述了一假设的糖基化位点。其预测的结构与其他已知的丝氨酸蛋白酶非常类似,显示出成熟多肽折叠成为单结构域。成熟蛋白长224个氨基酸,并具有一显示出经天然加工的A2表位。***酶核苷酸序列和推导的多肽序列和同源序列在Ferguson,等(Proc.Natl.Acad.Sci.USA 1999,96,3114-3119)中,和国际专利申请No.WO98/12302(和其相应的授权专利US 5,955,306),WO 98/20117(和其相应的授权专利US 5,840,871和US 5,786,148)(***酶特异性激肽释放酶)和WO 00/04149(P703P)中有公开。
其他***特异性抗原见于W098/37418,和WO/004149。另外一种为STEAP(PNAS96 14523 14528 7-12 1999)。
本发明上下文中所采用的其他肿瘤相关抗原包括:Plu-1 JBiol.Chem 274(22)15633-15645,1999,HASH-1,HASH-2(Alders,M.等,Hum.Mol.Genet.1997,6,859-867),Cripto(Salomon等Bioessays199,2161-70,US专利5654140),CASB616(WO 00/53216),Criptin(US 5,981,215)。另外,用于癌症治疗中的疫苗特定相关的抗原还包括酪氨酸酶、端粒酶、P53、NY-Br1.1(WO 01/47959)及其片段,如在WO 00/43420中所公开的,B726(WO00/60076,SEQ ID nos 469和463;WO 01/79286,SEQ ID nos 474和475),P510(WO 01/34802SEQ ID nos 537和538)以及存活素。
当与乳腺癌抗原,如Her-2/neu,乳腺珠蛋白(US专利5,668,267),B305D(WO 00/61753 SEQ ID nos299,304,305和315),和那些WO00/52165,WO 99/33869,WO 99/19479,WO 98/45328中记载的抗原联用时,本发明也非常有效。Her-2/neu抗原在US专利5,801,005中公开。优选Her-2/neu包括完整的胞外域(包括约1-645个氨基酸)或其片段,和至少其免疫原性部分或约C末端580个氨基酸的完整胞外域。特别是,细胞内部分应当包括磷酸化域或其片段。WO 00/44899公开了这类构建体。一已知的特别优选的构建体为ECD-PhD,第二个为ECD δ PhD(参见WO 00/44899)。本文所采用的Her-2/neu来源于大鼠、小鼠或人。
某些肿瘤抗原为小肽抗原(即小于约50个氨基酸)。这类抗原可经化学方法偶联至本发明的修饰的胆碱结合蛋白上。
示范性肽包括粘蛋白(Mucin)衍生的肽如,MUC-1(参见US5,744,144;US 5,827,666;WO 88/05054,US 4,963,484)。特别设计的为MUC-1来源的肽,其包括MUC-1肽的至少一个重复单元,优选至少两个能被SM3抗体(US 6,054,438)识别的重复序列。其他粘蛋白来源的肽包括来源于MUC-5的肽。
可选的,所述抗原为白介素,如优选IL13和IL14。或者所述抗原可以是一种自身肽激素,例如全长***释放激素(GnRH,WO95/20600),短的10个氨基酸长的肽,其可用于治疗多种癌症,或用于免疫去势。
适合于与本发明中的修饰的胆碱结合蛋白结合的其他肿瘤特异性抗原包括,但不限于肿瘤特异性神经节苷脂,如GM2,和GM3。
可通过本领域常用方法来使所述肽与修饰的胆碱结合蛋白共价结合。因此,例如,为了直接共价结合,利用常用的商业化可得的异双功能接头,如CDAP和SPDP(按照制造商的说明),采用碳二亚胺、戊二醛或(N-[γ-马来酰亚胺氧化丁酰]琥珀酰亚胺酯来进行。在联接反应后,通过透析方法、凝胶过滤方法、分馏方法等途径可很容易的分离和纯化免疫原。
抗原还可来源于对人类致病的来源,如人类免疫缺陷病毒HIV-1(如tat,nef,反转录酶,gag,gp120和gp160),人单疱疹病毒,如gD或其衍生物,或者立即早期蛋白,如来源于HSV1或HSV2的ICP27,巨细胞病毒(尤其是人类的)(如gB或其衍生物),轮状病毒(包括活减毒病毒),EB病毒(如gp350或其衍生物),水痘带状疱疹病毒(如gpI,II和IE63),或者来源于肝炎病毒,如肝炎B病毒(如肝炎B表面抗原或其衍生物),肝炎A病毒,肝炎C病毒,和肝炎E病毒,或来源于其他病毒病原体,如副粘病毒:呼吸道合胞体病毒(如F和G蛋白或其衍生物),副流感病毒,麻疹病毒,流行性腮腺炎病毒,人***瘤病毒(如HPV6,11,16,18,…),黄病毒(如,黄热病毒,登革热病毒,蜱传播的脑炎病毒,日本脑炎病毒),或流感病毒(全活或失活病毒,培养于卵黄或MDCK细胞中的裂解的流行性感冒病毒,或完整的流感病毒颗粒(如R.Gluck,Vaccine,1992,10,915-920所述),或其纯化或重组蛋白,如HA,NP,NA,或M蛋白或其组合物),或者来源于细菌病原体,如奈瑟氏菌属(Neisseria spp),其包括淋病奈瑟氏菌(N.gonorrhoea)和脑膜炎奈瑟氏菌(N.meningitidis)(例如囊膜多糖和其偶联物,转铁蛋白结合蛋白,乳铁蛋白结合蛋白,PiIC,粘结素);化脓链球菌(S.pyogenes)(如M蛋白或其片段,C5A蛋白酶,脂磷壁酸),无乳链球菌(S.agalactiae),变异链球菌(S.mutans);杜克雷嗜血杆菌(H.ducreyi);莫拉氏菌(Moraxella spp),包括粘膜炎莫拉氏菌(M catarrhalis),其也被称为粘膜炎布兰汉氏球菌(Branhamella catarrhalis)(例如高和低分子量的粘结素和侵袭素);博代氏杆菌(Bordetella spp),其包括百日咳博代氏杆菌(B.pertussis)(例如百日咳杆菌粘着素,百日咳毒素或其衍生物,丝状凝血素,腺苷酸环化酶,菌毛),副百日咳博德特菌(B.parapertussis)和支气管炎博德特菌(B.bronchiseptica);分枝杆菌,包括结核分枝杆菌(例如ESAT6,抗原85A,-B或-C),牛分枝杆菌,麻风分枝杆菌,鸟分枝杆菌,副结核分枝杆菌,耻垢分枝杆菌;军团菌,包括侵肺军团菌;埃希氏菌,包括肠毒素大肠埃希菌(例如,克隆化因子,热不稳定毒素或其衍生物,热稳定毒素或其衍生物),肠出血大肠埃希菌,肠致病性大肠埃希菌(例如志贺氏毒素类毒素或其衍生物);弧菌,包括霍乱弧菌(例如霍乱毒素或其衍生物);志贺氏菌,包括索氏志贺氏菌,痢疾志贺氏菌,弗氏志贺氏菌;耶尔森氏菌,包括小肠结肠炎耶尔森氏菌(例如Yop蛋白),鼠疫耶尔森氏菌,假结核耶尔森氏菌;弯曲杆菌,包括空肠弯曲杆菌(例如毒素,粘结素和侵袭素)和大肠弯曲杆菌;沙门氏菌,包括伤寒沙门氏菌,副伤寒沙门氏菌,猪霍乱沙门氏菌,肠炎沙门氏菌;李斯特氏菌,包括单核细胞增生李斯特氏菌;螺杆菌,包括幽门螺杆菌(例如脲酶,过氧化氢酶,空泡毒素);假单胞菌,包括铜绿假单胞菌;葡萄球菌,包括金黄葡萄球菌,表皮葡萄球菌;肠球菌,包括粪肠球菌,屎肠球菌;梭菌,包括破伤风梭菌(例如破伤风毒素和其衍生物),肉毒梭菌(例如肉毒杆菌毒素和其衍生物),艰难梭菌(例如梭菌毒素A或B及其衍生物);芽孢杆菌,包括炭疽芽孢杆菌(例如肉毒杆菌毒素及其衍生物);棒杆菌,包括白喉棒杆菌(例如白喉毒素及其衍生物);疏螺旋体,包括布氏疏螺旋体(例如OspA,OspC,DbpA,DbpB),嘎氏疏螺旋体(例如OspA,OspC,DbpA,DbpB),阿氏疏螺旋体(例如OspA,OspC,DbpA,DbpB),B.andersonii(例如OspA,OspC,DbpA,DbpB),赫氏蜱疏螺旋体;埃里希氏体,包括马埃里希氏体和人粒细胞埃立克体病的病原;立克次氏体,包括立氏立克次氏体;衣原体,包括砂眼衣原体(例如MOMP,肝素结合蛋白),肺炎衣原体(例如MOMP,肝素结合蛋白),鹦鹉热衣原体;钩端螺旋体,包括问号钩端螺旋体;密螺旋体,包括苍白密螺旋体(例如稀有外膜蛋白),齿垢密螺旋体,猪痢疾密螺旋体;或者来源于寄生虫,如疟原虫,包括恶性疟原虫;弓形虫,包括刚地弓形虫(例如SAG2,SAG3,Tg34);阿米巴,包括溶组织内阿米巴;巴贝斯虫属,包括巴贝斯原虫;冈比亚锥虫,包括枯氏锥虫;贾第鞭毛虫,包括蓝氏贾第鞭毛虫;利什曼原虫,包括硕大利什曼原虫;肺孢子虫,包括卡氏肺孢子虫;毛滴虫,包括***毛滴虫;血吸虫,包括曼氏血吸虫,或来源于酵母,例如念珠菌,包括白色念珠菌;隐球菌,包括新生隐球菌。
结核分枝杆菌的其他优选特异性抗原为,如Tb Ra 12,Tb H9,Tb Ra35,Tb38-1,Erd 14,DPV,MTI,MSL,mTTC2和hTCC1(WO99/41748)。结核分枝杆菌的蛋白还包括融合蛋白及其突变体,其中至少两个,优选三个结核分枝杆菌的多肽与一较大蛋白融合。优选的融合体包括Ra12-TbH9-Ra35,Erd14-DPV-MTI,DPV-MTI-MSL,Erd14-DPV-MTI-MSL-mTCC2,Erd14-DPV-MTI-MSL,DPV-MTI-MSL-mTCC2,TbH9-DPV-MTI(WO99/51748)。
衣原体最优选的抗原包括,例如,高分子量蛋白(HWMP)(WO99/17741),ORF3(EP 366 412),和假定膜蛋白(Pmps)。疫苗制剂的其他衣原体抗原可选自WO 99/28475中所记载的抗原组。
优选的细菌抗原来源于链球菌,包括肺炎链球菌(例如囊膜多糖和其偶联物PsaA,PspA,链球菌融血素,胆碱结合蛋白)和蛋白抗原肺炎球菌自融酶(Biochem Biophys Acta,1989,67,1007;Rubins等,Microbial Pathogenesis,25,337-342),及其去毒突变体衍生物(WO90/06951;WO 99/03884)。其他优选的细菌抗原来源于嗜血杆菌,包括B流感嗜血杆菌(例如PRP及其偶联物),非可分型流感嗜血杆菌,例如OMP26,高分子量粘合素,P5,P6,蛋白D和脂蛋白D,和丝束蛋白和丝束蛋白衍生肽(US 5,843,464)或多拷贝变异体或其融合蛋白。
本领域已知肝炎B表面抗原的各种衍生物,其包括,那些在欧洲专利申请EP-A-414 374;EP-A-0304578,和EP198-474中所记载的PreS1,PreS2 S抗原。一优选方面,所述HBV抗原为HBV聚合酶(JiHoon Jeong等,1996,BBRC 223,264-271;Lee H.J.等,Biotechnol.Lett.15,821-826)。在另一优选方面,融合体中的抗原为HIV-1抗原,gp120,特别是那些在CHO细胞中表达的。在一进一步的实施例中,抗原包括如本文上文中所定义的gD2t。
在本发明的优选实施例中,所述融合体包括来源于人***瘤病毒的抗原(HPV 6a,6b,11,16,18,31,33,35,39,45,51,52,56,58,59和68),尤其是那些被认为与生殖疣相关的HPV血清型(HPV 6或HPV 11和其他),以及那些被认为与***相关的HPV病毒(HPV16,HPV18和其他)。
合适的HPV抗原为E1,E2,E4,E5,E6,E7,L1和L2。生殖疣预防、或治疗融合体的特别优选形式包括L1颗粒或衣壳体,并且融合蛋白包括一个或多个选自HPV 6和HPV 11蛋白E6,E7,L1和L2的抗原。
融合蛋白最优选的形式为:如WO 96/26277中所述的L2E7,和如GB 9717953.5(PCT/EP98/05285)中所述的蛋白D(1/3)-E7。
优选的HPV宫颈感染或癌症,预防或治疗的疫苗组合物可包括HPV 16或18抗原。例如,L1或L2抗原单体,或L1或L2抗原以病毒样颗粒(VLP)的形式一起存在,或单一的L1蛋白单独的在VLP或衣壳体结构中存在。这种抗原、病毒样颗粒和衣壳体其本身是已知的。例如,参见WO94/00152,WO94/20137,WO94/05792和WO93/02184。
另外的早期蛋白可包括单一或融合蛋白,例如E7,E2或优选E5。其特别优选的实施例包括包含L1E7融合蛋白的VLP(WO 96/11272)。特别优选HPV 16抗原包含早期蛋白E6或E7,其与蛋白D载体融合,从而形成来源于HPV16的蛋白D-E6或E7融合体,或其组合物;或者E6或E7与L2的组合物(WO 96/26277)。
可选的,HPV 16或18早期蛋白E6和E7,可以单一分子的形式存在,优选的为蛋白D-E6/E7融合体。其他融合体可选的含有HPV18中的E6和E7中的任意一种或全部,优选的呈蛋白D-E6或蛋白D-E7融合蛋白,或者蛋白D E6/E7融合蛋白的形式。融合体中可包含来源于其他HPV株的抗原,优选地来源于HPV 31或33株。
本发明的融合体包括来源于导致疟疾的寄生虫的抗原。例如,优选来源于恶性疟原虫的抗原,包括RTS,S和TRAP。RTS是杂合蛋白,其包括恶性疟原虫环子孢子(CS)蛋白的基本上全部C-末端部分,该蛋白通过肝炎B表面抗原的preS2部分的四个氨基酸与肝炎B病毒表面(S)抗原连接。公开号为WO 93/10152并要求UK专利申请No.9124390.7的优先权的国际专利申请No.PCT/EP92/02591公开了其完整结构。当在酵母中表达时,RTS以脂蛋白颗粒的形式产生,并且当其与来源于HBV的S抗原共表达时,其产生了一种RTS,S的混合颗粒。公开号为WO 90/01496的国际专利申请No.PCT/GB89/00895中记载了TRAP抗原。本发明优选实施例为一种融合体,其中抗原制剂包括RTS,S和TRAP的组合物。可作为融合体组分的可能候选的其他疟原虫抗原为恶性疟原虫(P.Faciparum)MSP1,AMA1,MSP3,EBA,GLURP,RAP1,RAP2,钳合蛋白(Sequestrin),PfEMP1,Pf332,LSA1,LSA3,STARP,SALSA,PfEXP1,Pfs25,Pfs28,PFS27/25,Pfs16,Pfs48/45,Pfs230和其在疟原虫属的类似物。
本发明还提供了编码本发明融合伴侣的多核苷酸。本发明进一步涉及与本文图1(SEQ ID NO:9至16)所示的多核苷酸序列杂交的多核苷酸。在这一点上,本发明特别涉及在严谨条件下与本文所述的多核苷酸杂交的多核苷酸。如本文所应用的,术语“严谨条件”和“严谨杂交条件”是指仅在序列间存在至少95%,优选至少97%的同一性的情况下,杂交才会发生。严谨杂交条件的特定实施例为,在如下溶液中42℃培养过夜:50%甲酰胺,5×SSC(150mMNaCl,15mM柠檬酸三钠),50mM磷酸钠(pH7.6),5×Denhardt′s溶液,10%硫酸右旋糖酐,和20微克/毫升的变性、剪切的鲑鱼精DNA,随后在65℃在0.1×SSC中漂洗杂交载体。杂交和漂洗的条件是本领域已知的,并且在Sambrook等,Molecular Cloning:A Laboratory Manual,SecondEdition,Cold Spring Harbor,N.Y.,(1989),特别是Chapter 11中进行了举例说明。杂交溶液也可与本发明所述的多核苷酸一起使用。
本发明还提供了编码包括与肿瘤相关抗原或其片段融合的、本发明所述的融合伴侣的多肽的多核苷酸。特别是,本发明提供编码融合伴侣蛋白的多核苷酸序列,其中所述蛋白包括胆碱结合域和异源通用T辅助表位,优选其中胆碱结合域来源于LytA的C末端。在一更优选实施例中,本发明所述的多核苷酸的C-LytA部分包括SEQ ID NO.9-14中任一序列的至少四个重复序列,更优选包括SEQ ID NO.15序列,更进一步优选SEQ ID NO.16序列。在其他相关实施例中,本发明提供了与本文SEQ ID NOs:9-16所公开的序列具有基本上同一性的多核苷酸变异体,例如所述的序列包括与本发明所述的多核苷酸序列的至少70%序列同一性,优选至少75%,80%,85%,90%,95%,96%,97%,98%,或99%或更高,序列同一性比较采用常规方法,例如采用标准参数的BLAST分析。在更进一步的实施例中,如权利要求所要求包含的多核苷酸进一步还包括异源蛋白。
这种多核苷酸序列可被***适当的表达载体中,并在适当的宿主中表达。可采用这样的载体,其可编码本发明所述的修饰的胆碱结合蛋白,并且其含有合适的限制酶切位点,从而编码含量极少的免疫蛋白的DNA可被***以产生融合蛋白。在本发明的另外实施例中,编码本发明所述的多肽融合体的多核苷酸序列或其片段可在重组DNA分子中应用,从而在适当的宿主细胞中引导所述多肽的表达。由于遗传密码子的固有简并性,可采用编码实质上相同或功能等同的氨基酸序列的其他DNA序列,并且这类序列可被用于克隆和表达特定的多肽。
如本领域熟练技术人员能理解的那样,在某些情况中,产生使用非自然发生的密码子的多肽编码核酸序列是十分有利的。DNA编码采用4字母(A,T,C,和G),并且利用其拼出物种基因编码得到的蛋白质中的氨基酸的三字母的“密码子”。沿着DNA分子的密码子的线性序列被转录成由这些基因编码的蛋白质中的氨基酸的线性序列。编码具有高度的简并性,61个密码子用于编码20种天然氨基酸,同时3个密码子表示“终止”信号。因此,绝大多数氨基酸由多于一个的密码子来编码,事实上许多是由4种或更多的不同密码子来编码的。
当多于一个的密码子用于编码一种特定的氨基酸时,可以观察到物种中的密码子使用模式具有高度的随机性。不同的物种在其密码子选择方面具有不同的偏好性,此外,在单一物种中的高和低水平表达的基因之间的密码子选用可显著不同。在病毒、植物、细菌和哺乳动物细胞中这种偏好不同,并且一些物种比其他的物种显示出更强的随机密码子选择偏好性。例如,人和其他哺乳动物比细菌或病毒表现出更少一些的偏好性。基于这种原因,很有可能在E.Coli中表达的哺乳动物基因或在哺乳动物细胞中表达的病毒基因将具有对于高效表达不合适的密码子分布。可以认为当在其表达发生的宿主中很少观察到的密码子簇的异源DNA序列存在时,可以预计宿主中的低异源表达水平。
其结果是,特定的原核(例如E.Coli或酵母)或真核宿主优选的密码子可被优化,其被选择用于提高蛋白的表达率,从而产生具有理想特性的重组RNA转录产物,例如,比天然产生的序列获得的转录产物的半衰期更长的半衰期,或者用于优化人体内的免疫应答。密码子优化的过程可包括任何序列,可通过手工或计算机软件来产生,其中天然序列的一些或全部密码子被修饰。已经公开了许多方法(Nakamura等,Nucleic Acids Research 1996,24:214-215;W098/34640)。本发明的优选方法为Syngene法,一种Calcgene法的改进方法(R.S.Hale和G Thompson(Protein Expression andPurification Vol.12 pp.185-188(1998))。
相应的在一优选实施例中,蛋白质的DNA序列具有至少0.65的RSCU(相对同义密码子使用值(也称作密码子指数CI)),并且与相对的野生型区域相比具有小于85%的同一性。
这种密码子优化的处理和获得的构建体十分有用,其可能具有如下的一些或全部的益处:1)通过采用更常用的密码子来替换稀有或不常用的密码子可提高基因产物的表达;2)除去或包含限制性酶切位点从而有助于随后的克隆和3)降低DNA载体中的***序列和基因组序列间的同源重组可能和4)通过增加抗目的抗原的细胞和/或抗体应答(优选两种应答)来提高免疫应答。本发明的序列非常有效的降低了重组的可能性,但是其表达至少与野生型序列在同一水平上。由于用于产生密码子优化序列的SynGene程序中应用的算法的特性,可能会产生具有类似功能的、非常多数量的不同的密码子优化序列。简单的说,密码子采用统计方法来赋值,从而产生一种具有更接近于在高效表达的E.coli和人基因中发现的天然密码子频率的密码子频率的合成基因。简单的说,密码子采用统计方法来赋值,从而产生一种具有更接近于在高效表达的人基因,如P-Actin中发现的天然密码子频率的密码子频率的合成基因。非限定性的,举例来说,合适的密码子优化序列如SEQ ID NOs:19-22和SEQ ID NOs:24-26所示。
在本发明的多核苷酸中,密码子的使用模式从典型的目的抗原转变为更接近于表现在目的有机体中高效表达的基因,如人β-肌动蛋白的密码子偏好性。“密码子使用系数”是特定多核苷酸序列密码子模式如何接近于表现目的种类的密码子模式的测量。密码子频率可来源于许多物种的高效表达基因的文献资料(参见,如Nakamura等NucleicAcids Research 1996,24:214-215)。61种密码子的每一种的密码子频率(表现为每1000个选择的基因类型的密码子的事件发生的数目)在20种天然氨基酸的每一种中被标准化,从而每种氨基酸的最常用的密码子的值被设定为1,较少常用的密码子的频率被度量为0到1之间。从而为了在目的物种中高效表达基因,61种密码子中的每一种被赋值为1或更低。为了计算特定多核苷酸的密码子使用系数,相对于该物种中的高效表达基因来说,记录特定多核苷酸的每一密码子的度量值,并且取全部这些值的几何平均值(通过采用这些值的自然对数和来除以密码子的总数并取反对数来获得)。系数具有介于0和1之间的值,并且系数越高,则多核苷酸中的密码子为越常用的密码子。如果多核苷酸序列具有值为1的密码子使用系数,则所有的密码子均为“最常用”的密码子用于在目的物种中高效表达基因。
根据本发明,多核苷酸的密码子使用模式优选的排除表示用于特定氨基酸的<10%的密码子的密码子。相对同义密码子使用值为观察到的密码子的数目除以假设所有的氨基酸密码子使用频率相等得到的预期数目。本发明的多核苷酸优选的除去那些在目的有机体的高效表达基因中RSCU值小于0.2的密码子。本发明的多核苷酸通常具有大于0.6的高效表达人基因的密码子使用系数,优选大于0.65,最优选大于0.7。也可在Genbank中查阅密码子使用表。
经比较,高效表达的β-肌动蛋白基因具有0.747的RSCU值。
用于智人属(homo sapiens)的密码子使用表(表1)如下:
表1.人(高效表达的)基因1/24/91的密码子使用(human_high.cod)
氨基酸 密码子 数目 /1000 分数
Gly GGG 905.00 18.76 0.24
Gly GGA 525.00 10.88 0.14
Gly GGT 441.00 9.14 0.12
Gly GGC 1867.00 38.70 0.50
Glu GAG 2420.00 50.16 0.75
Glu GAA 792.00 16.42 0.25
Asp GAT 592.00 12.27 0.25
Asp GAC 1821.00 37.75 0.75
Val GTG 1866.00 38.68 0.64
Val GTA 134.00 2.78 0.05
Val GTT 198.00 4.10 0.07
Val GTC 728.00 15.09 0.25
Ala GCG 652.00 13.51 0.17
Ala GCA 488.00 10.12 0.13
Ala GCT 654.00 13.56 0.17
Ala GCC 2057.00 42.64 0.53
Arg AGG 512.00 10.61 0.18
Arg AGA 298.00 6.18 0.10
Ser AGT 354.00 7.34 0.10
Ser AGC 1171.00 24.27 0.34
Lys AAG 2117.00 43.88 0.82
Lys AAA 471.00 9.76 0.18
Asn AAT 314.00 6.51 0.22
Asn AAC 1120.00 23.22 0.78
Met ATG 1077.00 22.32 1.00
Ile ATA 88.00 1.82 0.05
Ile ATT 315.00 6.53 0.18
Ile ATC 1369.00 28.38 0.77
Thr ACG 405.00 8.40 0.15
Thr ACA 373.00 7.73 0.14
Thr ACT 358.00 7.42 0.14
Thr ACC 1502.00 31.13 0.57
Trp TGG 652.00 13.51 1.00
End TGA 109.00 2.26 0.55
Cys TGT 325.00 6.74 0.32
Cys TGC 706.00 14.63 0.68
End TAG 42.00 0.87 0.21
End TAA 46.00 0.95 0.23
Tyr TAT 360.00 7.46 0.26
Tyr TAC 1042.00 21.60 0.74
Leu TTG 313.00 6.49 0.06
Leu TTA 76.00 1.58 0.02
Phe TTT 336.00 6.96 0.20
Phe TTC 1377.00 28.54 0.80
Ser TCG 325.00 6.74 0.09
Ser TCA 165.00 3.42 0.05
Ser TCT 450.00 9.33 0.13
Ser TCC 958.00 19.86 0.28
Arg CGG 611.00 12.67 0.21
Arg CGA 183.00 3.79 0.06
Arg CGT 210.00 4.35 0.07
Arg CGC 1086.00 22.51 0.37
Gln CAG 2020.00 41.87 0.88
Gln CAA 283.00 5.87 0.12
His CAT 234.00 4.85 0.21
His CAC 870.00 18.03 0.79
Leu CTG 2884.00 59.78 0.58
Leu CTA 166.00 3.44 0.03
Leu CTT 238.00 4.93 0.05
Leu CTC 1276.00 26.45 0.26
Pro CCG 482.00 9.99 0.17
Pro CCA 456.00 9.45 0.16
Pro CCT 568.00 11.77 0.19
Pro CCC 1410.00 29.23 0.48
编码本发明的融合蛋白或修饰的胆碱结合蛋白的DNA序列可采用标准的DNA合成技术来合成,如通过如D.M.Roberts等,在Biochemistry 1985,24,5090-5098中所述的酶联反应,或通过,如利用热稳定聚合酶的PCR技术,或通过这类技术的组合。
利用DNA聚合酶,如DNA聚合酶I(Klenow片段)或Taq聚合酶,在含有必须的核苷三磷酸dATP,dCTP,dGTP和dTTP的适当缓冲液,在10-37℃下,通常以50μl或更少的体积,可在体外进行DNA酶聚合反应。DNA片段的酶连接反应可采用DNA连接酶,如T4 DNA连接酶在适当的缓冲液,如0.05M Tris(pH 7.4),0.01M MgCl2,0.01M二巯基苏糖醇,1mM亚精胺,1mM ATP和0.1mg/ml牛血清白蛋白,在4℃孵育,一般以50μl或更少的体积进行。DNA聚合体或片段的化学合成可通过传统的磷酸三酯、磷酸酯或亚磷酰胺化学方法,利用如“Chemical and Enzymatic Synthesis of Gene Fragments-A LaboratoryManual”(ed.H.G.Gassen和A.Lang),Verlag Chemie,Weinheim(1982),或其他的科技出版物,如M.J.Gait,H.W.D.Matthes,M.Singh,B.S.Sproat,和R.C.Titmas,Nucleic Acids Research,1982,10,6243;B.S.Sproat,和W.Bannwarth,Tetrahedron Letters,1983,24,5771;M.D.Matteucci和M.H.Caruthers,TetrahedronLetters,1980,21,719;M.D.Matteucci和M.H.Caruthers,Journal ofthe Anerican Chemical Society,1981,103,3185;S.P.Adams等,Journalof the American Chemical Society,1983,105,661;N.D.Sinha,J.Biernat,J.McMannus,和H.Koester,Nucleic Acids Research,1984,12,4539;和H.W.D.Matthes等,EMBO Journal,1984,3,801中所述的固相技术来合成。
本发明的过程可按如Maniatis等,Molecular Cloning-ALaboratory Manual;Cold Spring Harbor,1982-1989中所述的传统重组技术来进行。
特别是,所述过程可包括如下步骤:
i)制备可复制或整合的表达载体,所述载体能在宿主细胞中表达包含编码所述蛋白或其衍生物的核酸序列的DNA聚合体
ii)采用所述载体转化宿主细胞
iii)在允许所述的DNA聚合体表达产生所述蛋白质的条件下培养所述转化的宿主细胞;和
iv)回收所述蛋白质
本文所采用的术语“转化”是指向宿主细胞中导入外源DNA。这可利用如Genetic Engineering;Eds.S.M.Kingsman和A.J.Kingsman;Blackwell Scientific Publications;Oxford,England,1988中所述的传统技术,通过采用适当的质粒或病毒载体,进行转化、转染或感染来实现。术语“被转化的”或“转化子”将随后用于得到的含有和表达感兴趣的外源基因的宿主细胞。
所述的表达载体也是新的,同时也形成了本发明的一部分。
可按本发明所述来制备可复制的表达载体:通过酶切与宿主细胞相容的载体获得含有完整复制子的线性DNA片段,和在连接条件下将所述线性片段与一个或多个DNA分子结合,其中所述DNA分子与所述线性片段一起编码目的产物,如编码本发明的蛋白质或其衍生物的DNA聚合体。
因此,可根据需要,在构建载体的过程中获得或形成DNA聚合体。
载体的选择部分地由宿主细胞来决定,所述的宿主细胞可以是原核或真核的,但是优选E.Coli、酵母或CHO细胞。合适的载体包括质粒、噬菌体、粘粒和重组病毒。表达和克隆载体优选含有选择性标记,从而只有表达了标记的宿主细胞才能在选择性条件下存活。选择性基因包括,但不局限于,具有抗氨苄青霉素、四环素或卡那霉素抗性的编码蛋白质。表达载体还可含有与指定宿主相容的调控序列。例如,用于E.Coli,和更通用的用于原核生物的表达调控序列包括启动子和核糖体结合位点。启动子序列可天然存在,如β-内酰胺酶(青霉素酶)(Weissman 1981,In Interferon 3(ed.L.Gresser),乳糖(lac)(Chang等Nature,1977,198:1056),和色氨酸(trp)(Goeddel等Nucl.AcidsRes.1980,8,4057)和λ-来源的PL启动子***。此外,非天然存在的合成启动子同时也具有细菌启动子的功能。这种情况存在,如来源于trp和lac启动子序列的tac合成杂交启动子(De Boer等,Proc.NatlAcad Sci.USA 1983,80,21-26)。该***特别适合E.Coli。
酵母相容载体也可携带允许选择成功转化子的标记,所述标记可使营养缺陷型突变体变为原养型,或具有针对对野生型有害的重金属的抗性。酵母载体的表达调控序列包括糖酵解的启动子(Hess等,J.Adv.Enzyme Reg.1968,7,149),编码酸性磷酸酶的PHO5基因,CUP1基因,ARG3基因,GAL基因启动子和合成启动子序列。在酵母表达***中有用的其他调控元件为终止子和mRNA前导序列。由于其典型的编码由疏水氨基酸组成的引导蛋白从细胞中分泌的信号肽,因此5’端编码序列特别有用。合适的信号序列可由分泌型酵母蛋白的基因来编码,如酵母转化酶基因和a-因子基因,酸性磷酸酶,杀手毒素,α-配对因子基因,以及最近来源于克鲁维酵母(Kluyveromycesmarxianus)INU1A基因的异源菊粉酶信号序列。已经开发出了用于在毕赤酵母(Pichia pastoris)和酿酒酵母(Saccharomyces cerevisiae)中表达的合适的载体。
可根据各种可诱导的或基本启动子来获得酵母表达载体的变体(Cereghino和Cregg,FEMS Microbiol.Rev.2000,24:45-66)。为了生产细胞内和分泌性蛋白,最常用的毕赤酵母(P.pastoris)含有非常强和紧密调控的乙醇氧化酶(AOX1)启动子。该载体还含有毕赤酵母组氨醇脱氢酶(HIS4)基因用于在his4宿主中进行选择。外源蛋白的分泌需要信号序列的存在,并且酿酒酵母前原α-配对因子已在毕赤酵母表达***中广泛和成功的应用了。表达载体被整合至毕赤酵母基因组从而使表达菌株的稳定性最大化。与酿酒酵母中一样,在宿主基因组剪切序列内部的毕赤酵母表达载体的酶切片段(AOX1或HIS4)刺激了同源重组发生,从而有效的使载体靶向整合至基因组部位。一般来说,含有多个表达盒整合拷贝的重组菌株与单拷贝菌株相比可产生更多的异源蛋白。获得高拷贝数目转化子的最有效的方法需要通过球形原生质体技术(Cregg等1985,Mol.Cell.Biol.5:3376-3385)来转化毕赤酵母受体菌株。
一般可采用用于DNA酶切、聚合和连接的合适的酶,采用如上文所引用的Maniatis等中记载的方法来制备可复制的表达载体。
根据本发明,采用在转化条件下得到的本发明的可复制的表达载体来转化宿主细胞,从而制备重组宿主细胞。适当的转化条件是常规的,并记载于上文引用的Maniatis等中,或″DNA Cloning′Vol.II,D.M.Glover ed.,IRL Press Ltd,1985中。
根据待转化的宿主细胞的选择来选择转化条件。例如,体内转化是采用活的病毒载体作为本发明所述的多核苷酸的转化介质。通过经CaCl2溶液处理宿主(Cohen等,Proc.Nat.Acad.Sci.,1973,69,2110)或采用包含氯化铷(RbCl)、MnCl2、醋酸钾和甘油混合物的溶液处理,然后采用3-[N-吗啉]-丙烷-磺酸,RbCl和甘油处理后直接吸收多核苷酸(可以是含有目的序列的表达载体),或经电转化,可实现宿主,如E.coli的细菌转化。可采用,如Hinnen等(Proc.Natl.Acad.Sci.1978,75:1929-1933)的方法来实现低等真核生物如酵母细胞在培养基中通过直接吸收而发生的转化。可采用磷酸钙与载体DNA共沉淀至细胞的方法来转化培养基中的哺乳动物细胞(Graham & Van der Eb,Virology 1978,52,546)。向哺乳动物细胞导入多核苷酸的其他方法包括:右旋糖酐介导的转染、多聚季铵盐介导的转染、原生质融合、电穿孔、多核苷酸在脂质体中的包被、和向细胞核中直接进行多核苷酸显微注射。
本发明还包括采用编码本发明的蛋白的核酸或本发明的可复制的表达载体转化的宿主细胞。
依照如上文所引用的Maniatis等和“DNA Cloning”中所述的方法,在允许DNA聚合物表达的条件下常规的培养转化的宿主细胞。因此,优选细胞在添加营养素的情况下在低于50℃的温度下,优选介于25℃至42℃之间,更优选介于25℃和35℃之间,最优选30℃下培养。所述的培养时间将根据经SDS-PAGE或Western blot测定的多肽在细菌细胞中的比例,从几分钟到几小时不等。
可根据宿主细胞和根据表达产物的定位(细胞内或分泌至培养介质或分泌至细胞周质),通过常规方法回收产物。因此,当宿主细胞为细菌,如E.coli时,可经物理的、化学的或酶学方法来溶解,并从获得的溶解产物中分离蛋白产物。当宿主细胞为哺乳动物时,产物通常可从营养培养基或细胞游离提取物中分离。当宿主为酵母,如酿酒酵母或毕赤酵母时,产物通常可从溶解的细胞或从培养基中分离,并随后采用常规技术纯化。可经western blot或通过利用直接抗感兴趣的多肽的抗体的ELISA方法来测定表达***的特异性。
常规的蛋白分离技术包括:选择性沉淀、吸附层析、和包括单克隆抗体亲和柱的亲和层析。当本发明的蛋白质与组氨酸尾(His标记)共表达时,其可以很容易的采用利用离子金属亲和层析柱(IMAC)的亲和层析来进行纯化。所述的金属离子可以是任何合适的离子,如锌、镍、铁、镁或铜,但是优选锌或镍。优选IMAC缓冲液含有去垢剂、优选阴离子去垢剂,如SDS,更优选非离子去垢剂,如Tween 80,或两性离子去垢剂如Empigen BB,这将使终产物内仅存在较低水平的内毒素。
进一步的层析步骤包括,如Q-Sepharose步骤,其可在IMAC柱层析步骤之前或之后进行。优选的pH在7.5到10的范围内,更优选7.5到9.5,最佳的介于8和9之间。
本发明的蛋白随后可根据如下方案来纯化。裂解细胞后,将含有蛋白的细胞提取物溶解在含有尿素(例如8.0M),和SDS(例如从0.5%至1%)的pH 8.5的Tris缓冲液中。离心后,得到的上清液随后被加样至经pH 8.5 Tris缓冲液平衡过的IMAC(镍)Sepharose FF柱中。随后采用含有缓冲液的高盐溶液漂洗(例如,0.75-1.5mNaCl,15mM pH 8.5 Tris缓冲液)。随后,可选的采用无盐的磷酸缓冲液漂洗层析柱。采用含咪唑的缓冲溶液从柱中洗脱本发明的蛋白。随后,可将该蛋白经另一层析步骤处理,如阴离子交换层析柱(例如QSepharose)。
本发明的蛋白既可以溶于液体的形式,又可以优选的冷冻干燥的形式保存。一般希望每一人体剂量包括1至1000μg的蛋白,优选30-300μg。所述的纯化过程还可包括羧基酰胺化步骤,其中所述蛋白首先在谷胱甘肽存在的情况下被还原,并随后在碘代乙酰胺存在的条件下被羧甲基化。该步骤有利于控制分子之间或与宿主细胞蛋白污染物间通过二硫键共价桥接形成氧化聚集。
本发明还提供包含在药学上可接受的赋形剂中的本发明的蛋白的药物和免疫原性组合物。优选的疫苗组合物包含至少一种本发明的蛋白。所述的蛋白,优选的,具有封闭的硫醇基团并且经高度纯化,例如,具有少于5%的宿主细胞污染物。这种疫苗可优选的含有一种或多种其他的肿瘤相关抗原和衍生物。例如,合适的其他相关抗原包括,***酶、PAP-1、PSA(***特异抗原)、PSMA(***特异膜抗原)、PSCA(***干细胞抗原)、STEAP。
在另一实施例中,示例性的免疫原性组合物,如本发明的疫苗组合物,包含编码一个或多个上文所述的融合多肽,如原位产生的融合多肽的DNA。如上文所指出的那样,所述的多核苷酸可采用本领域熟练技术人员已知的各种传递***中的任意一种进行施用。实际上,本领域已知多种基因传递技术,如那些在Rolland,Crit.Rev.Therap.Drug Carrier Systems 15:143-198,1998中所记载的,在此作为参考文献引用。毫无疑问,适当的多核苷酸表达***包含在病人中表达必须的调控DNA调节子序列(如适当的启动子和终止信号)。可选的,细菌传递***涉及施用细菌(如卡介苗),其可在其细胞表面表达多肽的免疫原性部分或分泌这种表位。
因此,在特定实施例中,编码本文所述的免疫原性多肽的多核苷酸可通过已知的众多以病毒基础的***中的任意一种被导入适当的哺乳动物宿主细胞中进行表达。在示例性实施例中,逆转录病毒为基因传递***提供了方便和有效的平台。选择的编码本发明的多核苷酸的核苷酸序列可经本发明公知的方法,被***载体中并被包装成逆转录病毒颗粒。随后,分离重组病毒并递送给受试者。目前已公开了多个示例性的逆转录病毒***(例如,U.S.Pat.No.5219740;Miller和Rosman(1989)BioTechniques 7:980-990;Miller,A.D.(1990)HumanGene Therapy 1:5-14;Scarpa等(1991)Virology 180:849-852;Burnsetal.(1993)Proc.Natl.Acad.Sci.USA 90:8033-8037;和Boris-Lawrie& Temin(1993)Cur.Opin.Genet.Develop.3:102-109)。
此外,还记载了许多示例性的基于腺病毒的***。与整合至宿主基因组的逆转录病毒不同,腺病毒保持独立于染色体之外,从而最小化了***突变造成的风险(Haj-Ahmad & Graham(1986)J.Virol.57:267-274;Bett等(1993)J.Virol.67:5911-5921;Mittereder等(1994)Human Genc Therapy 5:717-729;Seth等(1994)J.Virol.68:933-940;Barr等(1994)Gene Therapy 1:51-58;Berkner,K.L.(1988)BioTechniques 6:616-629;和Rich等(1993)Human Gene Therapy4:461-476)。由于人常常受到常见的人腺病毒血清型,如AdHu5的感染,因此很大比例的人群产生抗腺病毒的中和抗体应答,其容易对重组疫苗***中的异源抗原产生免疫应答。非人类的灵长目腺病毒载体,如黑猩猩腺病毒68(AdC68,Fitzgerald等(2003)J.Immunol 170(3):1416-22)可作为一种可选择的没有之前存在的中和抗体应答缺陷的腺病毒***。
各种腺相关病毒(AAV)载体***也被开发用于多核苷酸传递。AAV载体可采用本领域已知的技术很容易的构建得到。参见,例如,U.S.Pat.Nos.5,173,414和5,139,941;国际公布Nos.WO 92/01070和WO 93/03769;Lebkowski等(1988)Molec.Cell.Biol.8:3988-3996;Vincent等(1990)Vaccines 90(Cold Spring Harbor Laboratory Press);Carter,B.J.(1992)Current Opinionin Biotechnology 3:533-539;Muzyczka,N(1992)Current Topics in Microbiol.and Immunol.158:97-129;Kotin,R.M.(1994)Human Gene Therapy 5:793-801;Shelling和Smith(1994)Gene Therapy 1:165-169;和Zhou等(1994)J.Exp.Med.179:1867-1875。
通过基因传递,用于传递编码本发明的多肽的核酸分子的另外的病毒载体包括那些来源于痘病毒科的病毒,如痘苗病毒和禽痘病毒。举例来说,表达新分子的痘苗病毒重组体按照如下方法构建。首先将编码多肽的DNA***适当的载体中,从而它与痘苗启动子相邻,并与痘苗DNA序列侧接,例如编码胸腺激酶(TK)的序列。随后采用该载体转染细胞,并同时用痘苗感染该细胞。同源重组发生,痘苗启动子和编码感兴趣多肽的基因被***病毒基因组中。通过在5-溴脱氧尿嘧啶核苷存在下培养细胞并挑选病毒抗性空斑来筛选TK.sup.(-)重组体。
基于牛痘的感染/转染***可方便地用于在有机体的宿主细胞中发生本发明所述的一个或多个多核苷酸的可诱导的瞬时表达或共表达。在该特定***中,首先采用编码噬菌体T7RNA聚合酶的痘苗病毒重组体在体外感染细胞。该聚合酶显示出精确的特异性,仅转录带有T7启动子的模板。感染后,采用在T7启动子调控下的感兴趣的多核苷酸来转染细胞。牛痘病毒重组体将转染的DNA转录成为RNA,并随后通过宿主翻译机制翻译成为多肽,从而聚合酶在细胞质中表达。该方法提供了大量RNA和其翻译产物的高水平、瞬时的、细胞质生产方法。参见,例如,Elroy-Stein和Moss,Proc.Natl.Acad.Sci.USA(1990)87:6743-6747;Fuerst等Proc.Natl.Acad.Sci.USA(1986)83:8122-8126。
可选的,禽痘病毒,如禽痘病毒和金丝雀痘病毒,也可被用于传递感兴趣的编码序列。已知当给予非禽类物种时,表达哺乳动物病原体免疫原的重组鸟痘病毒可产生保护性免疫反应。在人类和其他哺乳动物物种中使用禽痘病毒载体是非常理想的,因为禽痘病毒属的成员仅能在易感染的鸟类物种中产生复制,从而在哺乳动物细胞中不具有传染性。生产重组鸟痘病毒的方法是本领域已知的方法,并且如上文所述关于牛痘病毒的生产可采用遗传重组方法。参见,例如,WO91/12882;WO 89/03429;和WO 92/03545。
众多α病毒载体中的任意一种也可被用于传递本发明的多核苷酸组合物,如那些在美国专利Nos.5,843,723;6,015,686;6,008,035和6,015,694中所述的载体。某些基于委内瑞拉马脑炎病毒(VEE)的载体也可被使用,其具体的例子可参见美国专利Nos.5,505,947和5,643,576。
可通过许多途径,如肌内的、皮下的、腹膜内的或静脉内的方式来传递本发明的组合物。
本发明的另一实施例中,多核苷酸以“裸露”DNA的方式施用/传递,例如,如Ulmer等,Science 259:1745-1749,1993和Cohen,Science259:1691-1692,1993中所述。通过将DNA包裹至有效的转移至细胞内的生物可降解珠粒表面可增加裸露DNA的吸收。特别是,所述组合物通过基因枪(特别是粒子轰击)施用技术的途径进行传递,所述技术涉及将载体包裹至珠粒(例如金)表面,并在高压下施用至表皮中;例如,如Haynes等J Biotechnology 44:37-42(1996)中所述。
在示例性实施例中,气体驱动的颗粒的加速可采用由PowderjectPharmaceutical PLC(Oxford,UK)和Powderject Vaccines Inc.(Madison,WI)公司制造的装置来实现,所述装置的例子可参见美国专利Nos.5,846,796;6,010,478;5,865,796;5,584,807;和欧洲专利No.0500 799的记载。该方法提供了一种无需针的传递方式,其中显微颗粒,如多核苷酸,的干粉制剂,可在由手持装置产生的氦气喷射流中被加速至很高速度,氦气推动颗粒至感兴趣的靶组织,典型的为皮肤中。所述的颗粒优选为0.4-4.0μm的金粒,更优选0.6-2.0μm的直径,并且将DNA偶联物包裹至颗粒表面,并随后将其装入药筒或药盒中用于置于“基因枪”内。
在相关实施例中,其他可用于本发明组合物的无针气体驱动注射的装置和方法包括由Bioject,Inc.(Portland,OR)所提供的装置和方法,其具体记载于美国专利Nos.4,790,824;5,064,413;5,312,335;5,383,851;5,399,163;5,520,639和5,993,412中。
包含编码抗原肽的核苷酸序列的免疫原组分可通过一次性的或重复的进行施用,例如介于1到7次之间,优选的介于1到4次之间,其施用间隔介于1天至约18个月。不过,这种治疗方案将根据病人的体积、待治疗/预防的疾病、施用的核苷酸序列的量、施用途径、和对熟练医师来说非常明显的其他因素的不同而显著不同。
因此,本发明的另一方面涉及本发明所述的编码所述蛋白的DNA或蛋白在制备用于引起病人中免疫应答的免疫原性组合物中的应用。优选的免疫应答通过如下施用顺序引发i)所述蛋白,接着是所述DNA序列;或ii)所述DNA序列,接着是所述蛋白。更优选,所述DNA序列被包裹至生物可降解珠粒表面,或通过粒子轰击途径传递。更进一步优选所述蛋白添加佐剂,优选的添加TH-1诱导剂,更优选基于CpG/QS21的佐剂。
包含编码抗原肽的核苷酸序列的载体以预防或治疗有效的剂量进行施用。施用的剂量一般在1皮克至16毫克之间,对于颗粒介导的传递优选每剂量1皮克至10微克,对于核酸的其他施用途径优选每剂量10微克至16毫克之间。确切的量将根据待免疫的病人的体重和施用方式的变化而大不相同。
用于向病人中导入裸露的多核苷酸或载体的合适的技术还包括通过适当的载体进行的局部施用。所述核酸可局部施用至皮肤,或粘膜表面,例如通过鼻内的、口腔的、***内的或直肠内施用。所述的裸露的多核苷酸或载体可与药学上可接受的赋形剂,如磷酸缓冲盐溶液(PBS)共同施用。还可通过使用促进剂,如布比卡因,以与DNA制剂分开或包括在其内部的方式来进一步促进DNA的吸收。对受体直接施用所述核酸的其他方法包括超声波、电刺激、电穿孔和记载于US5,697,901中的显微植入法。
核酸构建体的摄取可通过许多已知转染技术来增强,例如那些包括使用转染剂的方法。这些试剂的例子包括阳离子剂,例如,磷酸钙和DEAE-右旋糖酐及脂质转染剂,例如lipofectam和transfectam。施用的核酸的剂量是可变的。
本发明中的融合蛋白和编码的多肽可配制成药用/免疫原性组合物的形式,例如,作为疫苗。因此,本发明还提供了包含在药学上可接受的赋形剂中的本发明的融合蛋白的药用/免疫原性组合物。因此,还提供了一种制备本发明的免疫原性组合物的方法,其包括将本发明的融合蛋白或编码的多核苷酸与合适的佐剂,稀释剂或其他的药学上可接受的载体混合。
如SDS PAGE结果所显示的那样,本发明的融合蛋白优选地呈至少80%的纯度,更优选90%的纯度。优选地在SDS PAGE中,所述的蛋白呈单一条带。
疫苗的制备方法常规的记载于Vaccine Design(″The subunit andadjuvant approach″(eds.Powell M.F.& Newman M.J).(1995)PlenumPress New Vork)中。采用脂质体包裹的方法由Fullerton在美国专利4,235,877进行了记载。
在本发明的疫苗制剂中,本发明的融合蛋白和编码的多核苷酸优选的添加了佐剂。特定的佐剂是商业化可购得的,例如,Freund′s不完全佐剂和完全佐剂(Difco Laboratories,Detroit,MI);Merck佐剂65(Merck & Company,Inc.,Rahway,NJ);AS-2(SmithKlineBeecham,Philadelphia,PA);铝盐,如氢氧化铝凝胶(明矾)或磷酸铝;钙盐,铁盐或锌盐;酰化酪氨酸的不溶悬浮颗粒;酰化糖;阳离子或阴离子的衍生多糖;含磷氮链聚合物;生物可降解的微球体;单磷酰脂A和quil A。细胞因子,如GM-CSF,白介素-2,-7,-12,和其他类似的生长因子也可用作佐剂。
在本发明的特定实施例中,佐剂组合物优选的为主要能引起Th1型免疫应答的物质。高水平的Th1-型细胞因子(例如,IFN-γ,TNFα,IL-2和IL-12)有助于诱导产生针对施用抗原的细胞介导的免疫应答。与之相对,高水平的Th2-型细胞因子(例如,IL-4,IL-5,IL-6和IL-10)有助于诱导产生体液免疫应答。施用了如本文所述的疫苗后,病人将经历包括Th1-和Th2-型应答的免疫应答。在免疫应答主要为Th1-型的优选实施例中,Th1-型细胞因子水平将增至远超过Th2-型细胞因子的水平。采用标准的检测方法可以很容易的检测这类细胞因子的水平。对于细胞因子家族的相关内容,参见Mosmann & Coffman,Ann.Rev.Immunol.7:145-173,1989。
优选TH-1诱导佐剂选自如下的一组佐剂:3D-MPL,QS21,QS21和胆固醇的混合物,和CpG寡核苷酸或两种或更多所述佐剂的混合物。主要引起Th1-型应答的特别优选的佐剂包括,例如,单磷酰脂质A,优选3-脱-O-酰化单磷酰脂质A与铝盐的组合物。MPL佐剂购于Corixa Corporation(Seattle,WA;参见,如美国专利Nos.4,436,727;4,877,611;4,866,034和4,912,094)。含CpG-寡核苷酸(其中CpG二核苷酸未甲基化)也能主要诱导Th1应答。这种寡核苷酸是已知的,并且记载于,如WO 96/02555,WO 99/33488和美国专利Nos.6,008,200和5,856,462。免疫刺激DNA序列还记载于,例如,Sato等,Science273:352,1996。其他优选的佐剂包括皂甙,如Quil A,或其衍生物,包括QS21和QS7(Aquila Biopharmaceuticals Inc.,Framingham,MA);七叶树溶血皂角素;洋地黄皂甙;或满天星(Gypsophila)或美洲土荆芥(Chenopodium quinoa)皂甙。其他优选的制剂包括在本发明的佐剂组合物中存在一种以上的皂甙,例如,如下组中的至少两种物种的组合:QS21,QS7,Quil A,β-七叶树溶血皂角素,或洋地黄皂甙。
可选的,所述的皂甙制剂可与其他疫苗载体联合使用,所述载体包括:壳聚糖或其他多聚阳离子聚合物,聚交酯和聚交酯共乙交酯颗粒,几丁质(poly-N-acetyl glucosamine-based)聚合物基质,由多糖或化学修饰的多糖组成的颗粒,脂质体和脂类颗粒,由甘油单酯组成的颗粒等。还可在胆固醇存在的条件下配制皂甙从而形成微粒结构,如脂质体或ISCOMs。此外,皂甙还可与聚氧化乙烯醚或酯一起配制而成,呈一种非微粒溶液或悬浮液的形式,或呈微粒结构的形式,如paucilamelar脂质体或ISCOM。皂甙还可与赋形剂,如CarbopolR一起配制来增加粘性,或与粉末赋形剂如乳糖一起配制成干粉形式。
在一优选实施例中,所述的佐剂***包括单磷酰脂质A和皂甙衍生物的组合物,如记载于WO 94/00153中的QS21与3D-MPL佐剂的组合物,或一更少反应原性组合物,如WO 96/33739中所述,其中QS21的作用被胆固醇猝灭。其他优选的制剂包括水包油乳剂和生育酚。另外特别优选的佐剂制剂采用了呈水包油乳剂的QS21,3D-MPL佐剂和生育酚,其记载于WO 95/17210。
另一增强的佐剂***涉及含CpG-寡核苷酸和皂甙衍生物的组合物,尤其是如WO 00/09159和WO 00/62800中所述的CpG和QS21的组合物。优选所述制剂另外还包含水包油乳剂和生育酚。
在进一步的实施例中,本发明提供了免疫原性组合物,所述组合物包含本发明的融合蛋白,并进一步包含D3-MPL,一种皂甙,优选QS21和CpG寡核苷酸,可选的配制成水包油乳剂的形式。
用于本发明的药物组合物的另外的示例性的佐剂包括MontanideISA 720(Seppic,France),SAF(Chiron,California,United States),ISCOMS(CSL),MF-59(Chiron),SBAS系列佐剂(如SBAS-2或SBAS-4,购于SmithKline Beecham,Rixensart,Belgium),Detox(Enhanzyn)(Corixa,Hamilton,MT),RC-529(Corixa,Hamilton,MT)和其他的氨基烷基氨基葡萄糖苷4-磷酸酯(AGPs),如在本文中整体作为参考的美国待决专利申请Nos.08/853,826和09/074,720中所述的,以及如WO99/52549 A1中所述的聚氧化乙烯醚。
其他优选的佐剂包括具有如下通式(I)的佐剂分子:
HO(CH2CH2O)n-A-R,其中,n为1-50,A为结合键或-C(O)-,R为C1-50烷基或苯基C1-50烷基。本发明的一实施例包括由具有通式(I)的聚氧化乙烯醚构成的疫苗制剂,其中n介于1和50之间,优选4-24,最优选9;R组分为C1-50,优选C4-C20烷基,最优选C12烷基,同时A为结合键。聚氧化乙烯醚的浓度应当在0.1-20%,优选在0.1-10%之间,最优选在0.1-1%的范围内。优选聚氧化乙烯醚选自如下组:聚氧化乙烯-9-月桂醚,聚氧化乙烯-9-硬酯醇(steoryl)醚,聚氧化乙烯-8-高醚,聚氧化乙烯-4-月桂醚,聚氧化乙烯-35-月桂醚,和聚氧化乙烯-23-月桂醚。聚氧化乙烯醚,如聚氧化乙烯月桂醚记载于Merck索引(12th版:目录7717)。这些佐剂分子记载于WO 99/52549中。如上文所述的具有通式(I)的聚氧化乙烯醚,理想的与其他佐剂联合使用。例如,优选的佐剂组合物,更可取的与如待决英国专利申请GB9820956.2中所述的CpG联合使用。
在本发明一实施例中,本发明的包括核酸载体的所述抗原与免疫刺激剂一起使用。优选的所述免疫刺激剂与本发明的抗原一起施用,并且在一优选实施例中,二者被配成制剂施用。在本发明的另一实施例中,所述的抗原和免疫刺激剂(或反之亦然)按先后顺序施用至相同或邻近的位点,其相隔0-100小时之间的间隔。这类免疫刺激剂包括,但不限于:合成的咪唑喹啉,如咪喹莫特(imiquimod)[S-26308,R-837],(Harrison等,Vaccine 19:1820-1826,2001);和瑞西喹漠(resiquimod)[S-28463,R-848](Vasilakos等,Cellular immunology204:64-74,2000);在抗原呈递细胞和T-细胞表面按组成表达的羰基席夫碱和胺,如妥卡雷琐(tucaresol)(Rhodes,J.等,Nature 377:71-75,1995),细胞因子,化学活性因子和可为蛋白或肽的共刺激分子,其包括前炎细胞因子,如干扰素,GM-CSF,IL-1α,IL-1β,TGF-α,FGF-β,Th1诱导剂,如干扰素γ,IL-2,IL-12,IL-15,IL-18和IL-21,Th2诱导剂如,IL-4,IL-5,IL-6,IL-10和IL-13,以及其他的化学活性因子和共刺激基因,如MCP-1,MIP-1α,MIP-1β,RANTES,TCA-3,CD80,CD86和CD40L,其他免疫刺激靶配体,如CTLA-4和L-selectin,凋亡刺激蛋白和肽,如Fas,(49),合成脂质佐剂,如vaxfectin(Reyes等,Vaccine 19:3778-3786,2001),角鲨烯,α-生育酚,聚山梨醇酐脂肪酸酯80,DOPC和胆固醇,内毒素,[LPS],(Beutler,B.,Current Opinion in Microbiology 3:23-30,2000);CpG寡核苷酸和二核苷酸(Sato,Y.等,Science 273(5273):352-354,1996;Hemmi,H.etal.,Nature 408:740-745,2000)和其他引发Toll受体产生Th1细胞因子的可能的配体,如合成的分枝杆菌脂蛋白,分枝杆菌蛋白p19,肽聚糖,磷壁酸和脂质A。
其他适当的佐剂包括CT(霍乱毒素,亚单位A和B)和LT(来源于E.coli的热不稳定肠毒素亚单位A和B),热休克蛋白家族(H SPs),和LLO(listeriolysin O;WO 01/72329)。
当免疫刺激剂为蛋白质时,该免疫刺激剂既可以蛋白形式,又可以编码蛋白的多核苷酸的形式施用。
其他合适的传递***包括微球体,其中抗原材料掺入或结合至生物可降解的聚合体/微球体上,从而抗原材料可与适当的药物载体混合并且作为疫苗使用。术语“微球体”一般用于描述大体上呈球状、并且具有10nm至2mm直径的胶质颗粒。由各种天然和合成聚合体制备的微球体被发现广泛的应用于生物医药应用领域。该传递***特别适合那些在体内具有很短半衰期从而需要多次治疗以达到效果,或者那些在生物液体中不稳定,或者那些由于其相对较高的分子量从而经肠胃途径不能完全吸收的蛋白。许多聚合物被记载作为蛋白释放的基质。合适的聚合物包括:明胶,胶原,藻酸盐,右旋糖酐。优选的传递***包括生物可降解的聚合(DL-乳酸)(PLA),聚乳酸-甘醇酸共聚物(PLG),聚(甘醇酸)(PGA),聚(ε-己内酯)(PCL),和共聚物聚(DL-乳糖-共聚-甘醇酸)(PLGA)。其他优选的***包括异源水凝胶,如聚(醚酯)多嵌段共聚物,其含有重复的嵌段:亲水的聚乙烯基乙二醇(PEG)和疏水的聚对苯二甲酸丁二醇酯(PBT),或聚乙烯基乙二醇-对苯二酸酯/聚对苯二甲酸丁二醇酯(PEGT/PBT)(Sohier等Eur.J.Pharm and Biopharm,2003,55,221-228)。优选那些能持续释放1至3个月的***,如PLGA,PLA和PEGT/PBT。
免疫原性或疫苗组合物可一次性施用,或优选的按所需的次数进行重复施用,例如,1至7次,优选的1至4次,其间隔介于约1天至约18个月之间,优选1个月。可选的,可按照在病人的剩余生命阶段在介于1到12个月的固定间隔内按剂量施用。在一优选实施例中,病人通过“基础加强接种”方案接受不同形式的抗原。因此,例如,所述抗原,融合蛋白首先以蛋白佐剂制剂的形式施用,并随后以DNA疫苗的形式施用。这种施用模式是优选的。优选的佐剂是含CpG寡核苷酸和皂甙衍生物的组合物,特别是如WO 00/09159和WO 00/62800中所述的CpG和QS21的组合物。通过将DNA包裹至能有效的转移至细胞内的生物可降解珠粒表面,可增加裸露DNA的摄取。可选的,所述DNA可经粒子轰击途径来传递,例如,利用由如本文所述的PowderiectPharmaceutical PLC(Oxford,UK)和Powderject Vaccines Inc.(Madison,WI)公司生产的装置,进行的气体驱动的颗粒加速。该途径提供了一种无需针的传递途径,其中显微颗粒的干粉制剂,如多核苷酸或多肽颗粒在手持装置所产生的氦气体喷射流中被加速至高速,其中氦气推动颗粒到达感兴趣的靶组织。
在另一优选实施例中,首先施用DNA疫苗,然后施用蛋白佐剂制剂。另一实施例涉及DNA构建体通过特定的传递载体的传递,优选的通过病毒***的途径,最优选通过腺病毒***的途径。其他DNA传递的合适的病毒***包括:逆转录病毒,慢病毒,腺相关病毒,疱疹病毒和痘苗病毒***。
在另一优选实施例中,蛋白佐剂制剂和DNA疫苗可在邻近或重叠的位点共同施用。根据DNA疫苗制剂的特性,这可通过在施用前混合DNA和蛋白佐剂制剂,或通过按顺序施用DNA和蛋白佐剂制剂来实现。
根据涉及的病人的体积和种类、核酸疫苗的量和/或施用的蛋白组合物、施用途径、所采用的任意佐剂化合物的效能和剂量、以及对于熟练的药剂师来说非常显而易见的其他因素的不同,所述的治疗方法也将显著变化。
进一步的方面,本发明提供了一种刺激病人产生免疫应答,优选在人类病人中产生T细胞免疫应答的方法,其包括施用如本文所述的药物组合物。所述病人可能患有肺癌、结肠癌或结肠直肠癌或乳腺癌,在这种情况下,该方法提供了对疾病,或者对被认为具有患这类可进行预防性治疗的疾病的风险的病人的治疗。
进一步的方面,本发明提供抑制病人中的癌症发展的方法,其包括对病人施用如上文所述的药物组合物。所述病人可能患有,如肉瘤、***癌、卵巢癌、膀胱癌、肺癌、结肠癌、结肠直肠癌或乳腺癌,在这种情况下,该方法提供了对疾病,或者对被认为具有患这类可进行预防性治疗的疾病的风险的病人的治疗。
另外的方面,本发明进一步提供了从生物样品中除去肿瘤细胞的方法,其包括将生物样品同与本发明的多肽发生特异性反应的T细胞接触,其中所述的接触步骤在允许将表达所述蛋白的细胞从样品中除去的条件下和足够的时间下进行。
在相关的方面,本发明提供了抑制病人的癌症发展的方法,其包括对病人施用如上文所述进行处理的生物样品。
在其他方面,本发明进一步提供了刺激和/或扩增本发明多肽特异性T细胞的方法,其包括在允许刺激和/或扩增T细胞的条件和足够的时间下,将T细胞与一种或多种如下物质接触:(i)如上文所述的多肽;(ii)编码所述多肽的多核苷酸;和/或(iii)表达这种多肽的抗原呈递细胞。同时,本发明还提供了按照如上文所述方法制备的包括T细胞的T细胞类群。
在进一步的方面,本发明提供了抑制病人癌症发展的方法,其包括对病人施用有效量的如上文所述的T细胞类群。
本发明进一步提供了抑制病人癌症发展的方法,其包括如下步骤:(a)将从病人中分离的CD4+和/或CD8+T细胞与一种或多种如下物质共培养:(i)如本文所述的多肽;(ii)编码所述多肽的多核苷酸;和(iii)表达这种多肽的抗原呈递细胞;和(b)给病人施用有效量的增殖的T细胞,从而抑制了病人中的癌症发展。在给病人施用前,增殖的细胞可以,但不是必须进行克隆。
根据本发明的另一实施例,本文所述的免疫原性组合物通过抗原呈递细胞(APCs),如树突细胞、巨噬细胞、B细胞、单核细胞和可被工程改造成为有效的APCs的其他细胞,传递至宿主中。这类细胞可以,但不是必须的,经遗传修饰以增加呈递抗原的能力,从而提高和/或保持T细胞应答的活性,从而具有自身抗肿瘤效果和/或与受体具有免疫相容性(即匹配的HLA单体型)。APCs一般可从任何的各类生物液体和器官中分离,包括肿瘤和肿瘤周围组织,并且可以是自体固有的、同种异体的、同源的或异源的细胞。
本发明的特定的优选实施例采用树突细胞或其前体作为抗原呈递细胞。树突细胞是非常有效的APCs(Banchereau & Steinman,Nature392:245-251,1998),其显示出是一种非常有效的生理佐剂用于引起预防性或治疗性的抗肿瘤免疫(参见Timmerman & Levy,Ann.Rev.Med.50:507-529,1999)。一般而言,可根据其典型形状(通过体外标记细胞质方法(树突)在原位呈星状),其高效摄取,处理和呈递抗原的能力,和其活化未经抗原刺激的T细胞应答的能力来鉴定树突细胞。毫无疑问,树突细胞可经工程改造用于表达那些通常不存在于体内或体外树突细胞中的特定细胞表面受体或配体,并且这种修饰的树突细胞也由本发明构思得到。作为树突细胞的一种选择,可在疫苗中使用携带分泌泡抗原的树突细胞(称为外泌小体)(参见Zitvogel等,NatureMed.4:594-600,1998)。
树突细胞和前体可从外周血、骨髓、肿瘤浸润细胞、肿瘤周围组织浸润细胞、***、脾脏、皮肤、脐带血和任何其他合适的组织或体液中分离得到。例如,树突细胞可通过向从外周血中收获的单核细胞培养物中添加细胞因子的混合物,如GM-CSF,IL-4,IL-13和/或TNFα来分化得到。可选的,从外周血、脐带血或骨髓中收获的CD34阳性细胞可通过向培养物培养基中添加GM-CSF,IL-3,TNFα,CD40配体,LPS,flt3配体和/或其他诱导树突细胞分化、成熟和增殖的化合物来分化成为树突细胞。
树突细胞可分为“不成熟”和“成熟”细胞,这允许通过一种简单的方式来区分两种特征明确的表型。不过,这种命名法则不应当被理解为排除了全部可能的分化中间状态。免疫树突细胞具有能很好的摄取和加工抗原的APC的特征,这与Fcγ受体和甘露糖受体的高水平表达相关。成熟类型典型地具有这些标记的较低水平表达,但引起T细胞活化的细胞表面分子,如MHC I和II类分子,粘附分子(例如,CD54和CD11)和协同刺激分子(例如,CD40,CD80和4-1BB)则高表达。
一般可采用本发明的多核苷酸(或其部分或其他变体)来转染APCs,从而其编码的多肽,或其免疫原性部分在细胞表面表达。这种转染可在体外发生,并且包括这种转染的细胞的药物组合物可随后用于治疗目的,如本文所述的。可选的,靶向树突或其他抗原呈递细胞的基因传递载体可被施用至病人,导致在体内发生转染。树突细胞的体内和体外转染,例如,一般可采用本发明已知的任何方法来进行,如那些记载于WO 97/24447中的方法,或者采用由Mahvi等,Imnunology and cell Biology 75:456-460,1997中记载的基因枪途径。通过将树突细胞或其前体细胞与肿瘤多肽、DNA(裸露的或存在质粒载体中的)或RNA;或与抗原表达重组细菌或病毒(如痘苗病毒、禽痘病毒、腺病毒或慢病毒载体)一起培养,可使树突细胞携带抗原。在携带前,所述的多肽可与提供T细胞辅助(例如,载体分子)的免疫伴侣共价结合。可选的,树突细胞可与非结合的免疫伴侣,单独的或在多肽存在的情况下进行脉冲电击。
定义
本发明还提供了用于分析特征序列或序列串,尤其是基因序列或编码的蛋白序列的方法。优选的序列分析方法包括,例如,序列同一性分析方法,如同一性和类似性分析,DNA、RNA和蛋白结构分析,序列组合,支序分析,序列基元分析,开放阅读框确定,核酸碱基确定,密码子使用分析,核酸碱基微调,和序列色谱峰分析。
提供了一种基于计算机的方法来进行同一性确定。该方法包括如下步骤:以计算机可读形式提供包括本发明的多核苷酸序列的第一多核苷酸序列;和将所述的第一多核苷酸序列与至少一条第二多核苷酸或多肽序列来比较以确定同一性。还提供了一种基于计算机的方法来进行同一性确定,所述方法包括如下步骤:以计算机可读形式提供包括本发明的多核苷酸序列的第一多核苷酸;和将所述的第一多核苷酸序列与至少一种第二多核苷酸和多肽序列来比较以确定同一性。
“同一性”,如本领域所知的那样,视情况而定,为通过序列比较确定的,两个或多个多肽序列或两个或多个多核苷酸序列之间的关系。在本领域中,“同一性”视情况而定,还表示通过所述序列串间比较来确定的多肽或多核苷酸序列间的序列相关程度。“同一性”可很容易的通过已知方法来计算得到,所述方法包括,但不仅限于那些在Computational Molecular Biology,Lesk,A.M.,ed.,OxfordUniversity Press,New York,1988;Biocomputing:Informatics andGenome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,& Griffin,H.G.,eds.,Humana Press,New Jersey,1994;SequenceAnalvsis in Molecular Biology,von Heine,G.,Academic Press,1987;和Sequence Analysis Primer,Gribskov,M.& Devereux,J.,eds.,M Stockton Press,New York,1991;和Carillo,H.,and Lipman,D.,SIAMJ.Applied Math.,48:1073(1988)中所述的方法。确定同一性的方法被设计在测试的序列间产生最大程度的配对。此外,在公众可获得的计算机程序中编写确定同一性的方法。用于确定两序列间同一性的计算机程序方法包括,但不限于GCG程序软件包中的GAP程序(Devereux,J.,等,Nucleic Acids Research12(1):387(1984)),BLASTP,BLASTN(Altschul,S.F.等,J.Molec.Biol.215:403-410(1990),和FASTA(Pearson & Lipman Proc.Natl.Acad.Sci.USA 85;2444-2448(1988)。公众可从NCBI和其他来源获得BLAST类程序(BLAST Manual,Altschul,S.,等,NCBI NLM NIH Bethesda,MD 20894;Altschul,S.,等,J.Mol.Biol.215:403-410(1990))。著名的Smith Waterman算法也可被用于确定同一性。
多肽序列比较的参数包括如下:
算法:Needleman & Wunsch,J.Mol Biol.48:443-453(1970)
比较矩阵:来源于Henikoff和Henikoff的BLOSSUM62,Proc.Natl.Acad.Sci.USA.89:10915-10919(1992)
空位罚分:8
空位长度罚分:2
采用这类参数的程序为一种公众可获得的、来源于GeneticsComputer Group,Madison WI的“gap”程序。上述参数为肽序列比较的缺省参数(同时末端空位不罚分)。
多核苷酸比较参数包括如下:
算法:Needleman & Wunsch,J.Mol Biol.48:443-453(1970)
比较矩阵:配对=+10,错配=0
空位罚分:50
空位长度罚分:3
可获得于来源于Genetics Computer Group,MadisonWl的“gap”程序。这些为核酸比较的缺省参数。
多核苷酸和多肽的“同一性”的优选含义,视情况而定,如下文(1)和(2)所述。
(1)多核苷酸实施方案进一步包括分离的多核苷酸,所述的多核苷酸包含的多核苷酸序列与SEQ ID NO:9至SEQ ID NO:16参考序列中的任意一种具有至少50,60,70,80,85,90,95,97或100%的同一性,其中所述多核苷酸序列可与SEQ ID NO:9至SEQ ID NO:16参考序列中的任意一种完全相同,或与参考序列相比可包括一定整数目的核酸变更,其中所述的变更选自如下组:至少一个核苷酸的缺失、取代,包括转换和颠换,或***,并且所述的变更可发生在参考核酸序列的5’或3’末端,或在这些末端位置中的任意位置,其既可单独分布在参考序列中的核苷酸之间,又可在参考序列中的一个或多个连续的基团之间分布,并且所述的核苷酸变更的数目由SEQ ID NO:9至SEQ ID NO:16任意序列中的总核苷酸数目乘以同一性百分比中的整数值并除以100,并从所述SEQ ID NO:9至SEQ ID NO:16任意序列核酸总数目中减去该乘积,或:
nn≤xn-(xn·y)
其中nn为核苷酸变更的数目,xn为SEQ ID NO:9至SEQ ID NO:16任意序列中核苷酸的总数,y为表示50%的0.50,表示60%的0.60,表示70%的0.70,表示80%的0.80,表示85%的0.85,表示90%的0.90,表示95%的0.95,表示97%的0.97,或表示100%的1.00,并且·表示乘号,并且在被从xn中减去之前,xn和y的非整数乘积被下舍入最接近的整数。编码SEQ ID NO:1至SEQ ID NO:8的任意多肽的多核苷酸序列变更可能在编码序列中产生无义、错义或读框移码突变,从而改变由如下的变更多核苷酸编码的多肽。
举例来说,本发明的多核苷酸序列可与SEQ ID NO:9至SEQ IDNO:16中的任意参考序列一致,即可以是100%的一致,或其可能包括与参考序列相比,一定整数目的核酸变更,从而同一性百分比小于100%。所述的变更选自如下组:至少一个核苷酸的缺失、取代,包括转换和颠换,或***,并且所述的变更可发生在参考多核苷酸序列的5’或3’末端,或在这些末端位置之间的任意位置,其既可单独分布在参考序列中的核苷酸之间,又可在参考序列中的一个或多个连续的基团之中分布。特定百分比同一性的核酸变更的数目由SEQ ID NO:9至SEQ ID NO:16任意序列中的总核酸数目乘以同一性百分比中的整数值并除以100,并从所述SEQ ID NO:9至SEQ ID NO:16任意序列核酸总数中减去该乘积,或:
nn≤xn-(xn·y)
其中nn为核苷酸变更的数目,xn为SEQ ID NO:9至SEQ ID NO:16任意序列中核苷酸的总数,例如,y为表示70%的0.70,表示80%的0.80,表示85%的0.85等,并且·表示乘号,并且在被从xn中减去之前,xn和y的非整数乘积被下舍入最接近的整数。
(2)多肽实施例进一步包括分离的多肽,所述分离的多肽包括与SEQ ID NO:1至SEQ ID NO:8任意参考序列的多肽具有至少50,60,70,80,85,90,95,97或100%同一性的多肽,其中所述多肽序列可与SEQ ID NO:1至SEQ ID NO:8参考序列中的任意一种完全相同,或与参考序列相比可包括一定整数目的氨基酸变更,其中所述的变更选自如下组:至少一个氨基酸的缺失、取代,包括保守和非保守的取代,或***,并且所述的变更可发生在参考多肽序列的氨基或羧基末端的位置,或在这些末端位置之间的任意位置,其既可单独分布在参考序列中的多肽之间,又可在参考序列中的一个或多个连续的基团之中分布,并且所述的氨基酸变更的数目由SEQ ID NO:1至SEQ IDNO:8任意序列中的总氨基酸数目乘以同一性百分比中的整数值并除以100,并从所述SEQ ID NO:1至SEQ ID NO:8任意序列氨基酸总数目中减去该乘积,或:
na≤xa-(xa·y)
其中na为氨基酸变更的数目,xa为SEQ ID NO:2中氨基酸总数,y为表示50%的0.50,表示60%的0.60,表示70%的0.70,表示80%的0.80,表示85%的0.85,表示90%的0.90,表示95%的0.95,表示97%的0.97,或表示100%的1.00,并且·表示乘号,并且在被从xa中减去之前,xa和y的任何非整数乘积被下舍入最接近的整数。
举例来说,本发明的多肽序列可与SEQ ID NO:1至SEQ ID NO:8中的任意参考序列一致,即可以是100%的一致,或其可能包括与参考序列相比,一定整数目的氨基酸变更,从而同一性百分比小于100%。所述的变更选自如下组:至少一个氨基酸的缺失、取代,包括保守和非保守的取代,或***,并且所述的变更可发生在参考多肽序列的氨基或羧基末端,或在这些末端位置之间的任意位置,其既可单独的在参考序列中的氨基酸之间,又可在参考序列中的一个或多个连续的基团之中分布。一特定%同一性的氨基酸变更的数目由SEQ ID NO:1至SEQ ID NO:8任意序列中的总氨基酸数目乘以同一性百分比中的整数值并除以100,并从所述SEQ ID NO:1至SEQ ID NO:8任意序列氨基酸总数中减去该乘积,或:
na≤xa-(xa·y)
其中na为氨基酸变更的数目,xa为SEQ ID NO:1至SEQ ID NO:8任意序列中氨基酸的总数,例如,y为表示70%的0.70,表示80%的0.80,表示85%的0.85等,并且·表示乘号,并且在被从xa中减去之前,xa和y的非整数乘积被下舍入最接近的整数。
附图简述
图1:C-LytA的序列信息。采用如下序列对比程序,在基于多个序列对比和第二结构预测的基础上可定义每一重复序列:1)MatchBox(Depiereux E等(1992)Comput Applic Biosci 8:501-9);2)ClustalW(Thompson JD等(1994)Nucl Acid Res 22:4673-80);3)Block-Maker(Henikoff S等(1995)Gene 163:gc17-26)
图2:CPC和天然构建体(SEQ ID NOs.27-36)
图3:在酿酒酵母(S.cerevisiae)中表达的CPC-p501 His融合蛋白的图解结构
图4:CPC-P501 His融合蛋白(SEQ ID NO.41)的一级结构
图5:CPC P501 His(pRIT15201)(SEQ ID NO.42)的核苷酸序列
图6:质粒pRIT 15201的生产的克隆策略
图7:pRIT15201的质粒图谱
图8:CPC P501和P501在酿酒酵母(S.cerevisiae)菌株DC5中的比较表达
图9:CPC-P501S HIS(Y1796)的小规模生产。图9A表示了通过SDS-PAGE及银染估计的抗原生产率;图9B表示了通过western blot估计的抗原产率。
图10:Y1796产生的CPC-P501-His的纯化方案。
图11:CPC P501 His纯化蛋白的电泳图谱(4-12%的Novex Nu-Page聚丙烯酰胺预制凝胶)。
图12:天然全长P501S序列(SEQ ID NO:17)。
图13:JNW735的CPC-P501S表达盒序列(SEQ ID NO:18)。
图14:两个密码子优化的P501S序列(SEQID NO:19-20)。
图15:再工程化的密码子优化序列19(SEQ ID NO:21)。
图16:再工程化的密码子优化序列20(SEQ ID NO:22)。
图17:CPC优化的起始序列(SEQ ID NO:23)。
图18:代表密码子优化的CPC序列(SEQ ID NO:24-25)。
图19:改造的CPC密码子优化序列(SEQ ID NO:26)。
图20:P501S CPC融合候选构建体和序列(SEQ ID NOs.37-40&45-48)。
图21:当采用P501S(JNW680),CPC-P501S(JNW735),对照空载体瞬时转染后,进行的CHO细胞Western blot分析。
图22:当在第0,21,和42天采用pVAC-P501S(JNW680,小鼠B1-9)或空载体(pVAC,小鼠A1-6)进行免疫后,抗P501S抗体的应答。在一1天进行预先放血。随后在第28天和第49天(鼠A1-3,B1-3)和第56天(鼠A4-6,B4-9)进行放血。所有的血清在1/100稀释后进行检测。将pVAC免疫小鼠的结果进行平均。个体pVAC-P501S免疫小鼠的结果列于图表中。作为阳性对照,来源于腺-P501S免疫小鼠的血清(Corixa Corp,稀释1/100)也包括在内。
图23:采用在第0,21,42,和70天通过pVAC-P501S(JNW680)免疫的C57BL/6小鼠进行肽文库筛选。所有的肽均在50μg/ml的终浓度下使用。购于Corixa的肽1-50重叠15-20链节。从Mimotopes(UK)定购的肽51-70预测8-9链节Kb和Db表位。样品71-72和73-78分别为DMSO对照和无肽对照。图A表示IFN-γ应答,而图B表示IL-2应答。被选择用于随后的免疫分析的肽以黑色表示。
图24:在第0,21,42,和70天采用pVAC-P501S(JNW680,B6-9)和pVAC空(A4-6)进行PMID免疫后在第77天通过ELISPOT测定的细胞应答。肽18,22,和48以50μg/ml的浓度使用。CPC-P501S蛋白在20μg/ml的浓度下使用。图A表示IFN-γ应答,而图B表示IL-2应答。
图25:P501S和CPC-P501S的比较。采用肽22(10μg/ml)在第28天通过IL-2 ELISPOT来检测细胞应答。通过PMID在第0和21天,采用pVAC空载体(对照),pVAC-P501S(JNW680)和CPC-P501S(JNW735)来免疫小鼠。
图26:当采用CPC-P501S进行蛋白免疫后的免疫应答(脾细胞淋巴增殖)。
图27:对不同CPC-P501S构建体的免疫应答评价。通过IL-2ELISPOT在第28天检测细胞应答。通过PMID在第0,和21天采用p7313-即空载体(对照),JNW735和CPC-P501S构建体(JNW770,771和773)来免疫小鼠。
图28:MUC-1 CPC序列(SEQ ID NOs.49&50)
图29:ss-CPC-MUC-1序列(SEQ ID NOs.51&52)
通过参考如下实施例,将进一步详述本发明:
实施例1:表达含有C-LytA-P2-C-LYtA(CPC)作为融合伴侣的P501融合蛋白的重组酵母菌株Y1796的制备
1.-蛋白设计
图3中描述了在酿酒酵母中表达的融合蛋白C-P2-C-p501(还可称为CPC-P501)的结构。该融合蛋白含有基因LytA(残基187至306)的C-末端区域,其中破伤风毒素的P2片段(残基830-843)被***。P2片段被置于C-Lyt-A的277和278残基之间。含有P2***的C-lytA片段之后为P501(氨基酸残基51至553)和His尾结构。
获得的融合蛋白的一级结构具有如图4所述的序列,并且相应于上述蛋白设计的编码序列如图5所示。
2.-生产表达CPC-P501(51-553)-His融合蛋白的酵母质粒的克隆策略
●起始材料为酵母载体pRIT15068(UK专利申请0015619.0)。
●该载体含有酵母Cup1启动子,酵母α前原信号编码序列和相应于P501S的55至553残基的编码序列之后为His尾结构。
●图6所概述的克隆策略包括如下步骤:
a)第一步为P2序列(已进行酵母表达的密码子优化)按阅读框***C-lytA编码序列内部。质粒pRIT 14662(PCT/EP99/00660)携带C-lytA编码序列。用由两个命名为P21和P22的互补寡核苷酸形成的适配子***预先采用Ncol酶切切开的质粒pRIT 14662。
P21和P22的序列为:
P21 5′catgcaatacatcaaggctaactctaagttcattggtatcactgaaggcgt 3′
P22 3′gttatgtagttccgattgagattcaagtaaccatagtgacttccgcagtac 5′
在连接和E.coli转化和转化子鉴定后,即获得了命名为pRIT15199的质粒。
b)第二步为通过PCR扩增来制备C-lytA-P2-C-lytA DNA片段。采用pRIT15199作为模板进行扩增,并且所述寡核苷酸命名为C-LytANOTATG和C-LytA-aa55。两个寡核苷酸的序列为:
C-LytANOTATG
=5′aaaaccatggcggccgcttacgtacattccgacggctcttatccaaaagacaag 3′
C-LytA-aa55=5′aaacatgtacatgaacttttctggcctgtctgccagtgttc 3′
采用限制性内切酶Ncol和AflIII来处理扩增的片段并分别产生粘性末端。
c)下一步为将上述片段与载体pRIT15068(经Ncol处理后的最大片段)连接,从而产生完全融合蛋白的编码序列。在连接和E.coli转化后,获得了命名为pRIT15200的质粒。在该质粒中,剩下的唯一Ncol位点含有ATG用作起始密码子。
d)在下一步骤中,从质粒PRIT 15202中制备含有CUP1启动子的Ncol片段和2μ质粒序列的一部分。质粒pRIT 15202为一含有在ATG处有Ncol位点(ATG序列:AAACC ATG)的CUP1启动子的酵母2μ衍生物。
e)将从pRIT 15202中分离的Ncol片段按照正确的方向与预先采用Ncol酶切切开的pRIT15200连接,在这种方式中pCUP1启动子在编码序列的5’端。这导致了命名为pRIT15201的最终表达载体的产生(参见图7)。
3.-重组酵母菌株Y1796(RIX4440)的制备
质粒pRIT 15201用于转化酿酒酵母(S.cerevisiae)菌株DC5(ATCC 20820)。在含有质粒pRIT 15201的酵母转化子筛选和鉴定后,获得了命名为Y1796的表达CPC-P501-His融合蛋白的重组酵母菌株。通过亲和层析(IMAC)和随后的阴离子交换层析(Q SepharoseFF),可分离和纯化得到经还原和羧基酰胺化的蛋白。
实施例II
利用其中描述的相应的DNA序列,可表达如图2所示的类似的蛋白构建体。特别是,酵母菌株SC333(构建体2)相应于Y1796菌株,但是表达缺乏CPC融合伴侣的P50155-553。酵母菌株Y1800(构建体3)相应于Y1796菌株,但是还包括P501S(aa1-aa34)的天然序列信号,而酵母菌株Y1802(构建体4)包括α前信号序列上游CPC-P501S序列。酵母菌株Y1790(构建体5)表达缺乏CPC并具有α前原信号序列的P501S构建体。
实施例III纯化CPC-P501的制备
1.-CPC-P501S HIS(Y1796)的小规模生产
对于Y1796,在添加有组氨酸的基本培养基中,可通过在对数期添加100至500μM范围的CuSO4,并在30℃保持培养的条件下诱导表达。在诱导后8或24小时后收获细胞。仅在使用前加入铜,并且其不能事先与培养基混合。
为了进行SDS PAGE分析,在柠檬酸磷酸盐缓冲液pH4.0+130mM NaCl中进行酵母细胞提取。采用玻璃珠进行小细胞量的提取,采用高压细胞均质机(French press)进行较大细胞量的提取,并随后与样品缓冲液混合并进行SDS-PAGE分析。图8中描述了不同构建体的SDS PAGE比较分析结果,并将其总结于下表2中。
如下表表1所示,与表达相应的不含CPC伴侣P501S-His的亲本菌株SC333的表达水平相比,Y1796菌株的培养物的表达水平更高。同样的,信号序列(α前体)的存在也不影响上述讨论的结果:与表达相应的不含CPC伴侣P501S-His的菌株Y1790的表达水平相比,Y1802菌株的培养物的表达水平更高。
表2
重组菌株 | 质粒 | 启动子 | 信号序列 | 融合伴侣 | P501 aa序列 | 表达水平 |
SC333 | Ma333 | CUP 1 | - | - | 55-553-His | ND |
Y1796 | pRIT 15201 | CUP 1 | - | CPC | 51-553-His | +++ |
Y1802 | pRIT 15219 | CUP 1 | α前体 | CPC | 51-553-His | ++++ |
Y1790 | pRIT 15068 | CUP 1 | α前原 | - | 55-553-His | + |
CPC=clyta P2 clyta
ND=即使采用western blot也无法检测到
+=采用western blot可检测到
+++/++++=采用western blot可检测到,并且在银染色凝胶中肉眼可见
2.-Y1796(RIX4440)的大规模发酵
将100μl的工作种子接种于固体培养基上,并在30℃下培养约24小时。该固体预培养物随后被用于接种置于摇动烧瓶中的液体预培养基中。
该液体预培养物在30℃下培养20小时,然后转移至20L的发酵罐中。连续分批式发酵包括约44小时的生长期和约22小时的诱导期。
通过连续添加的方式将碳源(葡萄糖)补充至培养物中。为了使发酵产生的乙醇的量最小化,使残留的葡萄糖浓度保持较低水平(≤50mg/L)。这可通过限制葡萄糖添加速率从而限制微生物的增殖来实现。
为了生产抗原,在生长期的末尾,通过添加CuSO4来诱导CUP1启动子。
通过将106细胞接种至添加有制霉菌素的标准TSB和THI小瓶中,并分别在20-25℃和30-35℃下培养14天来检测污染物的去除。如所预计的那样,没有观察到细菌生长。
3.-抗原鉴定和生产
利用高压细胞均质机(French press)处理在诱导期的不同时间收获的发酵样品来制备细胞匀浆,并通过SDS-PAGE和Western Blot来进行分析。结果显示,感兴趣的蛋白的主要部分定位于来源于离心后的细胞匀浆的不溶部分。采用来源于这些细胞匀浆离心后的沉淀来进行如下图中显示的SDS-PAGE和Western Blot分析。
图8A和B显示了培养物PRO127在诱导期的抗原产生的动力学曲线。其显示在生长期,没有抗原表达发生。特异性抗原的产生大致在诱导期开始至第6小时持续增加,并随后保持稳定直至诱导期结束。但是,由于在该相同时间段观察到生物量积累,其体积产量以1.5到2的系数增加。通过比较纯化的参考抗原和原始提取物在采用银染(图9A)的SDS-PAGE中的结果和利用抗-P501S抗体(一种在1/1000倍稀释下使用的,直接抗P501S aa439-aa459的小鼠腹水)进行的WB分析结果,可估计抗原产生率约为500mg每升发酵培养基。
实施例IV由Y1796产生的CPC-P501(51-553)-His融合蛋白的纯化
当细胞破裂后,所述蛋白与颗粒部分结合。在该过程中,为了解决分子之间或分子与宿主细胞蛋白污染物通过二硫键共价桥接发生氧化聚集的问题,可在处理中引入分子脲基甲基化作用。同时,为了保持该蛋白的疏水特性(预测存在12个穿膜结构域),还需要使用去垢剂。
用于1L培养物OD(光密度)120规模的纯化方案记载于图10中。所有的操作均在室温下进行(RT)。
根据DOC TCA BCA蛋白检测,普遍的纯化产量为30-70mg纯化的抗原/L培养物OD 120。该产量与培养物表达水平相关,同时,当与表达未融合的P501S-His的亲本菌株的纯化产量相比时,其产量更高。
按如下步骤进行蛋白分析:首先采用TCA(三氯乙酸)在DOC(脱氧胆酸)存在的条件下沉淀蛋白,然后在SDS存在的条件下将其溶于碱性介质中。然后蛋白与BCA(双金鸡纳酸)(Pierce)反应,从而形成一可溶的紫色络合物,其在562nm下有高吸收值,并且所述络合物与样品中存在的蛋白的量成比例。
在还原和非还原条件下,3份纯化的SDS-PAGE分析(图11)显示没有区别(将2,3,4道与5,6,7相比较)。电泳图谱由位于70kDa的主带,模糊的更高MW的微弱的降解带组成。所有的带均通过特异性抗P501 S的单克隆抗体来检测。
实施例V采用CPC-P501S His蛋白来制备疫苗
实施例3或4中的蛋白可被配制成呈水油乳剂形式的含QS21和3D-MPL的疫苗。
1.-疫苗制备
按照实施例1-3的方法制备抗原C-LytA-P2-P501S His。作为佐剂,所述制剂包括呈油/水乳剂形式的3去氧酰化单磷酰脂A(3D-MPL)和QS21。在WO 95/17210中记载了佐剂***SBAS2。
3D-MPL:是一种来源于革兰氏阴性细菌明尼苏达沙门氏菌(Salmonella minnesota)脂多糖(LPS)的免疫刺激剂。MPL被去酰化,并在脂质A部分缺失磷酸基团。该化学处理显著降低了毒性并保持了其免疫刺激性(Ribi,1986)。Ribi lmmunochemistry生产并且向SB-Biologicals提供MPL。在Smith Kline Beecham Biologicals进行的实验显示与各种赋形剂结合的3D-MPL显著的增强了体液和TH1型的细胞免疫。
QS21:是一种从南美洲树木Quillaja saponaria Molina皂树树皮中提取的皂甙分子。纯化技术被发展用于从树皮粗提物中分离皂甙单体,该技术允许分离特定的皂甙,QS21,这是一种与亲本化合物相比显示出较强佐剂活性和较低毒性的三萜糖苷。QS21显示出对一些亚单位抗原的MHC I类限制性CTL的活化,以及刺激Ag特异性淋巴细胞增殖(Kensil,1992)。Aquila(正式名称为Cambridge BiotechCorporation)生产和向SB-Biologicals提供QS21。在Smith KlineBeecham Biologicals进行的实验显示在体液和TH1型细胞免疫应答的诱导中MPL和QS21的联合使用具有明确的协同效应。
水/油乳剂包括由2种油(生育酚和角鲨烯)制成的有机相,和含Tween 80作为乳化剂的PBS的水相。所述的乳剂含有5%角鲨烯、5%生育酚、0.4%Tween 80,并且还包括平均颗粒大小为180nm并被称为SB62的物质(参见WO 95/17210)。
在SmithKline Beecham Biologicals进行的实验证实这种O/W乳剂向3D-MPL/QS21(SBAS2)中的添加可进一步增加后者抗各种亚单位抗原的免疫刺激性。
2.-乳剂SB62的制备(2倍浓缩物)
将Tween 80溶于磷酸缓冲盐溶液(PBS)中从而获得在PBS中的2%的溶液。为了制备100ml的2倍浓缩液,5g的DLα生育酚和5ml的角鲨烯被加入,并振荡从而彻底混合。将得到的乳剂通过一注射器,并最终采用M110S微流体机使其微流体化。获得的油滴具有约180nm的大小。
3.-制剂:
一种呈油/水乳剂形式,并含有3D-MPL和QS21的典型制剂采用如下步骤制备:在连续的加入SB62(50μl),MPL(20μg),QS21(20μg)后,将20μg-25μg C-LytA P2-P501S稀释于10倍浓缩的PBS pH6.8和水中,可选的包括CpG寡核苷酸(100μg)和1μg/ml作为防腐剂的柳硫汞。每一组分的量可根据需要改变。所有的诱导在室温下搅拌进行。
实施例VI.密码子优化的P501S序列
1.-对照重组质粒的生产:
将全长P501S序列克隆至pVAC(Thomsen,Immunology,1998;95:510P105),产生表达质粒JNW680。SEQ ID NO:17表示质粒JNW680中的人P501S表达盒,其结构示于图12中。SEQ ID NO:17的蛋白序列以单字母的形式表示,其起始和终止密码子以粗体显示。Kozak序列以混杂符号表示。人P501S序列(SEQ ID NO:17)的密码子使用指数为0.618,其通过SynGene程序来计算。
SynGene程序
基本上,密码子通过统计方法来赋值,从而使合成基因具有接近于在高效表达的E.coli中和人基因中天然发现的密码子频率。
SynGene是被称作Calcgene的Visual Basic的更新版,其由R.S.Hale和G Thompson编写(Protein Expression and Purification Vol.12pp.185-188(1998)。对于原始序列中的每一氨基酸残基,根据其出现在高效表达的E.coli基因中的频率来赋值密码子。从作者处可获得在Microsoft Windows 3.1环境下运行的Calcgene程序的详细信息。由于该程序采用统计方法来赋值合成基因的密码子,因此不是所有的获得的密码子都是在目标有机体中最常用的。而是,通过在适当的比例中赋值密码子来在合成序列中反映靶有机体的常用和不常用密码子。不过,由于在将一特定密码子赋值于序列中的特定位置方面没有严格而牢固的规则,因此每次运行该程序时将产生一种不同的合成基因,虽然每次均具有相同的密码子使用模式,并且每次都编码相同的氨基酸序列。如果针对特定的氨基酸序列和特定的靶有机体来多次运行该程序,将产生许多不同的核酸序列,这些序列在数目、类型和限制性内切酶位点的位置、内含子结合信号等方面将不同,其中某些序列是不理想的。本领域的熟练技术人员在这些特征的基础上能选择适当的序列用于表达多肽。
此外,由于密码子在统计基础上随机赋值,因此可能出现(虽然也许不太可能)两个或多个在靶有机体中相对较少使用的密码子聚集在相近的位置上。可以认为这种聚集将可能妨碍转录机制,并特别导致较低的表达率,从而为了避免稀有密码子偶然的选择发生聚集,在优化基因中选择密码子的算法将排除那些对于高效表达基因来说RSCU值小于0.2的任何密码子。然后,根据高效表达的E.coli的频率来分配剩下的密码子的分布,从而在典型的这类基因(系数=0.85)和高效表达的人基因(系数=0.50)的合成基因中整体分布。
Syngene(Peter Ertl,未发布),Calcgene程序的更新版,允许排除优选的稀有密码子,并且也根据高效表达的人基因的密码子频率模式来用于分配密码子。
从载体pRIT15201(参见图7)中将CPC-P501S表达盒序列克隆至pVAC中,从而产生质粒JNW735,其序列示于SEQ ID NO:18,且如图13所示。除了除去了His尾结构和添加Kozak序列(GCCACC)和适当的限制性内切酶位点外,该序列与pRIT15201序列完全一致。SEQ ID NO:18的氨基酸序列以单字母形式表示,起始和终止密码子以粗体表示。方框标明的残基为破伤风毒素的P2辅助表位。下划线的残基为Clyta纯化标签。Kozak序列采用混杂符号标明。
2.-具有P501S密码子优化序列的重组质粒的产生:
虽然P501S的天然序列的密码子系数指数(CI)已经很高(0.618),但还可进一步增加CI值。这将具有两种潜在的优点:提高抗原表达和/或免疫原性并且减少P501S载体和基因组序列的重组可能性。
采用Syngene程序,获得了密码子优化的序列(SEQ ID NO:19至SEQ ID NO:20)(图14)。下表3显示了起始P501S序列与两个典型的密码子优化序列的密码子系数指数的比较结果,所述的优化序列是基于合适的限制性内切酶位点图谱和好的CI指数的基础上筛选得到的。
表3-两个密码子优化P501S基因的密码子系数指数比较
序列 | 密码子系数指数(CI) |
P501SSEQ ID NO:19SEQ ID NO:20 | 0.6180.7250.755 |
3.密码子优化序列的进一步评价
序列SEQ ID NO:19
虽然SEQ ID NO:19具有好的CI指数(0.725),但是在氨基酸位置202和203上其含有成对的稀有密码子。通过将DNA序列从TTGTTG改为CTGCTG,这些密码子被人工替换为更常用的密码子。为了有助于克隆和表达,添加了限制性酶切位点和Kozak序列。最终改造的序列(SEQ ID NO:21)示于图15中。Syngene程序用于将该序列片段化成为具有最小重叠为19-20碱基的寡核苷酸。因此,图15显示了重新工程改造的P501S密码子优化序列SEQ ID NO.19。用下划线标明了限制性内切酶酶切位点,用粗体表示Kozak序列,除去稀有密码子成对物的重新工程改造的DNA序列被用方框标出。
采用两步PCR方法,利用PCR合成方法(如下详述),首先采用由Syngene程序产生的重叠引物进行合成。合成反应产生各种不同的片段。采用PCR回收方法和末端引物来回收/扩增正确的全长片段。从琼脂糖凝胶中切下得到的PCR片段,进行纯化,采用Nhel和Xhol进行限制性酶切,并克隆至pVAC中。采用限制性内切酶分析鉴定阳性克隆子,并通过双链测序来验证。这将产生质粒JNW766,该质粒由于PCR过程的出错倾向的性质,含有一单个沉默突变(在SEQ IDNO:21的360位置上C突变为T)。
1.合成反应-PCR条件,通用方法
反应混合物(总体积=50μl):
-1×反应缓冲液(Pfx或Proofstart)
-1μl寡核苷酸(所有重叠的寡核苷酸等量混合)
-0.5mM dNTPs
-DNA聚合酶(Pfx或Proofstart,2.5-5U)
-+/-1mM MgSO4
-+/-1×增强溶液(Pfx增强剂或Proofstart缓冲液Q)
1. 94℃,120秒(仅Proofstart应用)
2. 94℃,30秒
3. 40℃,120秒
4. 72℃,10秒
5. 94℃,15秒
6. 40℃,30秒
7. 72℃,20秒+3秒/循环
8.循环至步骤5,25次
9.在4℃保持
2.回收反应-PCR条件(通用方法)
反应混合物(总体积=50μl):
-1×反应缓冲液(Pfx或Proofstart)
-5-10μl合成反应混合物
-0.3-0.75mM dNTPs
-50pmol引物(5’末端引物,有义方向)
-50pmol引物(3’末端引物,反义方向)
-DNA聚合酶(Pfx或Proofstart,2.5-5U)
-+/-1mM MgSO4
-+/-1×增强溶液(Pfx增强剂或Proofstart缓冲液Q)
1. 94℃,120秒(仅Proofstart应用)
2. 94℃,45秒
3. 60℃,30秒
4. 72℃,120秒
5.循环至步骤2,25次
6. 72℃,240秒
7.在4℃保持
序列SEQ ID NO:20
虽然SEQ ID NO:20具有非常好的CI指数(0.755),但是可注意到在氨基酸位置131和132上其含有成对的稀有密码子。通过将DNA序列从TTGTTG改为CTGCTG,这些密码子被人工替换为更常用的密码子。为了有助于克隆,通过突变G至C,可将一内部的BamHI位点除去(参见图16中的双下划线核苷酸)。为了有助于克隆和表达,添加了限制性酶切位点和Kozak序列。最终改造的序列(SEQ IDNO:22)示于图16中。Syngene程序用于将该序列片段化成为具有最小重叠为19-20碱基的寡核苷酸。
因此,图16显示了重新工程改造的P501S密码子优化序列20(SEQID NO.22)。用下划线标明了限制性内切酶酶切位点,用粗体表示Kozak序列,除去稀有密码子成对物的重新工程改造的DNA序列被用方框标出,并用双下划线标明了除去BamHI位点的沉默点突变的位置。
采用与上文所述的类似两步PCR方法,可扩增全长P501S片段并克隆至pVAC。采用限制性内切酶分析鉴定阳性克隆,并通过双链测序来验证。这将产生质粒JNW764,编码表达盒的P501S的序列示于图16中(SEQ ID NO:22)。
DNA序列相似性
通过ClustalV(加权的)方法进行的成对距离随后序列比较(Pairdistances following alignment)示于下表3中。下表4显示了起始人P501S序列和选择的用于进一步研究的两条密码子优化序列SEQ IDNO:21和22之间的百分比相似性。该数据证实密码子优化的DNA序列与原始P501S序列具有约80%的相似性。
表4
SEQ ID NO: | 与起始P501 S序列的%相似性 |
2122 | 79.679.4 |
实施例VII.密码子优化CPC序列
1.-获得途径
由于原始CPC序列最初设计用于优化在酵母中的表达,本部分记载了用于在人中表达的密码子优化过程。
2.-序列设计
用于优化CPC的起始序列示于图17中(SEQ ID NO:23)。其完全来源于pRIT15201,其含有CPC的完整编码序列加上P501 S的四个氨基酸从而有助于下游克隆。采用Syngene程序得到了一种密码子优化序列的选择,其中典型序列示于图18中(SEQ ID NO:24-25)。下表5显示了起始CPC序列和两条典型密码子优化序列的密码子系数指数的比较结果。
表5.两条CPC优化序列的密码子系数指数
序列 | 密码子系数指数(CI) |
原始 CPC=SEQ ID NO:23SEQ ID NO:24SEQ ID NO:25 | 0.5060.8090.800 |
除了密码子优化外,还筛选了所有序列的限制性酶切克隆位点。在最高CI值和最佳限制性酶切位点图谱的基础上,筛选得到SEQ IDNO:24用于构建。为了有助于克隆和表达,添加了5’和3’克隆位点,并在初始ATG起始密码子的5’端***Kozak序列(GCCACC)。这种工程改造的序列如图19所示(SEQ ID NO:26)。该序列包括P501S的4个氨基酸(用方框标明),限制性酶切克隆位点(Nhel和Xhol,下划线标明),Kozak序列(粗体),终止密码子(斜体表示)和4bp的侧接无关DNA从而有助于克隆。
Syngene程序用于将该序列片段化成为具有18-20碱基的最小重叠的50-60链节的寡核苷酸。
采用与上文所述类似的两步PCR方法,将正确的片段回收/扩增和克隆至pVAC。采用限制性内切酶分析和序列验证来鉴别阳性克隆,并产生载体JNW759。
3.-DNA序列相似性
成对距离随后序列比较ClustalV(加权的)(Pair distances followingalignment ClustalV(Weighted))示于下表6中。该表显示CPC起始序列和密码子优化序列间在DNA水平上的百分比相似性,并且证实了密码子优化序列与原始CPC序列具有约80%的相似性。
表6
序列SEQ ID NO: | 与起始CPC序列的%相似性 |
2425 | 80.281.6 |
实施例VIII.P501S融合候选物的构建
下图所示的所有候选物均进行了密码子优化,并通过重叠PCR方法,以质粒JNW764和JNW759作为模板(分别为SEQ ID NO:22和SEQ ID NO:26),并克隆至表达载体,即p7313。
下图所示的4个候选物均以CPC-P501S为基础。密码子优化的CPC-P501S为构建体A。候选物B、C、D还包括编码P501S N末端50个氨基酸的序列,其可以位于CPC-P501S(构建体D)的N末端,CPC-P501S(构建体C)的C末端,或位于CPC和P501S之间(构建体B)。表示构建体的图表示于图20中。
四个构建体的每一种的核酸和蛋白序列示于:SEQ ID NO:37-40表示核酸序列,和SEQ ID NO.45-48表示相应的多肽序列。在构建体A、C和D中,下划线标明的密码子优选的编码酪氨酸(TAC或TAT),但是可改变核酸序列使其编码苏氨酸(ACA,ACC,ACG或ACT)。在构建体B中,下划线标明的密码子优选的编码苏氨酸(ACA,ACC,ACG或ACT),但也可改变核酸序列使其编码酪氨酸(TAC或TAT)。在所有的构建体中,编码序列两侧均有适当的限制性内切酶克隆位点(在本实施例中为NotI和BamHI),并且在起始ATG的直接上游存在一Kozak序列。下表7表示上述构建体的质粒鉴定:
表7
构建体 | 下划线密码子表示氨基酸 | 密码子序列 | 质粒ID |
A | 酪氨酸 | TAC | JNW771 |
B | 苏氨酸 | ACA | JNW773 |
B | 酪氨酸 | TAC | JNW770 |
C | 酪氨酸 | TAC | JNW777 |
D | 酪氨酸 | TAC | JNW769 |
在第0天通过PMID进行初次免疫,和分别在第21,42和70天进行3次加强免疫后,通过ELISPOT来检测经p7313-即(空载体),pVAC-P501 S(JNW735),JNW770,JNW771和JNW773免疫的细胞应答。在加强免疫后的7天进行检测。图27显示,在用JNW770,JNW771和JNW773免疫的小鼠中可检测到良好的IL-2ELISPOT应答。
实施例IX.采用颗粒介导的皮内递送(PMID)进行的免疫实验研究
当全长P501S通过颗粒介导的皮内传递***(PMID)来进行传递时,将产生好的抗体和细胞应答。这些数据标明PMID是一种非常有效的传递途径。此外,通过肽ELISPOT进行P501 S和CPC-P501 S的比较证实CPC-P501 S诱导更强的免疫应答。
1.-材料和方法
1.1 皮肤基因枪免疫
采用氯化钙和亚精胺将质粒DNA沉淀至2μm直径的金粒上。将装载了DNA的珠粒按文献所述(Eisenbraum等,1993;Pertmer et al,1996)的方法覆盖至Tefzel管上。采用Accell基因传递***(PCT WO95/19799)进行粒子轰击。对于每一种质粒,雌性C57BL/6鼠分别在第0,21,42和70天进行免疫。每次施用由两次DNA/金轰击组成,并且总剂量为约4-5μg质粒。
1.2对P501S基因产物的T细胞应答的ELISPOT分析
a)脾细胞的制备
脾来源于加强免疫后的7-14天的免疫动物。通过在玻片间进行研磨来处理脾组织从而产生细胞悬浮液。通过氯化铵处理来裂解红细胞,同时除去细胞碎片从而获得好的脾细胞悬浮液。细胞以8×106/ml的浓度重悬于RPMI不完全培养基中用于ELISPOT检测。
b)肽文库筛选
覆盖了大部分的P501S序列的肽文库来源于Corixa Corp公司。该文库中含有50条重叠4-11氨基酸的15-20链节长的肽。这些肽被编号为1-50。此外,一预测程序(H-G.Rammensee等:Immunogenetics,1999,50:213-219)(http://svfpeithi.bmi-heidelberg.com/)被用于预测P501S序列的Kb和Db表位。从Mimotopes(UK)定购了10个最好的Kb和Db表位,并包括在本文库中(肽51-70)。为了进行肽文库筛选,按照如下所述的方法,所述肽在IFNγ和IL-2 ELISPOTS中以50μg/ml(约25-50μM)的终浓度使用。对于IFNγ ELISPOTS,IL-2以10ng/ml的浓度加入检测中。用于筛选的脾细胞是在第84天,从分别在第0,21,42和70天免疫的C57BL/6鼠中分离得到的。从文库筛选中确定了3条肽-肽18(HCRQAYSVYAFMISLGGCLG),22(GLSAPSLSPHCCPCRARLAF)和48(VCLAAGITYVPPLLLEVGV)。这些肽随后用于ELISPOT检测。
c)ELISPOT检测
采用15μg/ml(溶于PBS)的大鼠抗小鼠IFNγ或大鼠抗小鼠IL-2(Pharmingen)来包被检测板。检测板在+4℃下包被过夜。在使用前,采用PBS洗板三次。脾细胞以4×105细胞/孔的浓度加入到检测板中。重新向Genemed Synthesis定购在文库筛选中确定的肽,并在50μg/ml的终浓度下使用。CPC-P501S蛋白(GSKBio)在检测中以20μg/ml的浓度使用。在存在IL-2(10ng/ml),IL-7(10ng/ml),或不存在细胞因子的情况下进行ELISPOT检测。每孔中的总体积为200μl。含有肽刺激的细胞的检测板在潮湿的37℃培养箱中培养16个小时。
e)ELISPOT检测板的建立
通过用水漂洗一次(浸泡10分钟从而保证细胞裂解)和采用PBS漂洗三次,从检测板中除去细胞。与生物素结合的大鼠抗小鼠IFNg或IL-2(Phamingen)溶于PBS在1μg/ml的浓度下添加。检测板在室温下震荡温育2小时。在添加1/1000倍稀释的链霉菌抗生物素蛋白碱性磷酸酶(Caltag)之前,采用PBS洗板三次。在采用PBS洗涤三次后,通过采用BCICP(Biorad)底物温育15-45分钟来显现结合点。用水洗去底物,并让检测板晾干。
采用Brian Hayes,Asthma Cell Biology unit,GSK设计的图象分析***来对结合点计数。
1.3.P501 S基因产物的抗体的ELISA分析
通过静脉穿刺术,在第-1,28,49和56天从动物中获取血清样品,并进行抗P501S抗体存在的检测分析。在4℃下,采用溶于碳酸钠缓冲液中的0.5μg/ml的CPC-P501S蛋白(GSKBio)过夜包被Nunc Maxisorp检测板,利用上述检测板进行ELISA检测。经TBS-Tween(Tris-缓冲盐,含有0.05%的Tween 20,pH 7.4)漂洗后,采用封闭缓冲液(3%BSA溶于TBS-Tween缓冲液)在室温下将检测板封闭2小时。所有的血清以1∶100的稀释度稀释于封闭缓冲液中,并在室温下温育1小时。采用以1∶2000稀释度在封闭缓冲液中稀释的HRP-结合的兔抗鼠免疫球蛋白(#P0260,Dako)来检测抗体的结合。再次洗板,并采用快速OPD颜色试剂(Sigma,UK)来检测结合。通过添加3M硫酸来终止反应,并通过检测490nm处的光吸收来定量OPD产物。
1.4.瞬时转染检测
通过质粒向CHO(中国仓鼠卵巢细胞)细胞瞬时转染,以及随后的针对总细胞蛋白的Western blotting分析,可分析各种DNA构建体中的人P501 S的表达。采用转染剂(Promega),按照制造商的说明可进行瞬时转染。简单的说,将细胞以5×104 CHO细胞每孔的量接种于含有1 ml DMEM完全培养基(DMEM,10%FCS,2mM L-谷氨酸盐,青霉素1001U/ml,链霉素100μg/ml)的24孔组织培养板中,并在37℃下培养16小时。将0.5μg DNA加入25μl的0.3M的NaCl(每孔足量的)中,并将2μl的转染剂加入25μl的Milli-Q中。将DNA和转染剂溶液轻微混合并在室温下孵育15分钟。在该孵育步骤中,细胞采用PBS漂洗一次,并采用150μl的不含血清的培养基(DMEM,2mML-谷氨酸盐)来覆盖。DNA转染剂溶液逐滴加入细胞中,将培养板轻轻摇动并在37℃下培养4-6小时。加入500μl的DMEM完全培养基,并在37℃下进一步培养48-72小时。
2.采用P501S质粒瞬时转染的CHO细胞的Western blot分析
瞬时转染的CHO细胞采用PBS来漂洗,并采用Versene(1∶5000)/0.025%胰蛋白酶溶液来处理从而将细胞转移至悬浮液中。经胰蛋白酶消化后,沉淀CHO细胞并重悬于50μl的PBS中。加入等体积的2×NP40裂解缓冲液并在冰上温育细胞30分钟。加入100μl的含50mMDTT的2×TRIS-甘氨酸SDS样品缓冲液(Invitrogen),并将该溶液在95℃下加热5分钟。将1-20μl的样品加样至4-20%TRIS-甘氨酸凝胶1.5mm(Invitrogen)中,并在1×TRIS-甘氨酸缓冲液(Invitrogen)中在稳定电压下(125V)电泳90分钟。预染的宽范围的分子量标记(NewEngland Biolabs,#P7708S)用于确定样品分子的大小。在凝胶电泳后,采用XcellIII Blot Module(Invitrogen),和含有20%甲醇的1×转移缓冲液(Invitrogen),在25V稳定电压下电泳90分钟,从而将样品转移至预先采用甲醇浸湿的Immobilon-P PVDF膜(Millipore)上。将膜在4℃下,置于含3%的脱脂奶粉(Marvel)的TBS-Tween(Tris-缓冲盐,含0.05%的Tween 20,pH 7.4)中封闭过夜。以1∶1000的比例稀释一抗(10E3),并在室温下与膜孵育1小时。在用TBS-Tween漂洗后,将二抗(HRP结合的兔抗小鼠免疫球蛋白(#P0260,Dako))以1∶2000的比例稀释于含3%脱脂奶粉的TBS-Tween中,并与膜在室温下一起温育1小时。漂洗后,膜与Supersignal West Pico化学发光底物(Pierce)一起温育5分钟。除去多余的液体,并将膜密封于两张粘性膜之间,暴露于Hyperfilm ECL膜(Amersham-PharmaciaBiotech)1-30分钟。
3.全长人P501S表达盒的产生
P501S表达盒构建的起始点为质粒pcDNA3.1-P501S(CorixaCorp),其以pcDNA3.1为主链(Invitrogen),并含有克隆于EcoRI和NotI位点之间的全长人P501S cDNA表达盒。该载体同样被命名为JNW673。通过荧光测序证实了P501S的存在。cDNA表达盒的序列来源于NCBI/Genbank序列(登录号AY033593)。从JNW673模板DNA经PCR扩增获得人P501 S,并采用XbaI和SalI进行限制性酶切,并将其克隆至pVAC的NheI/XhoI位点,从而产生载体JNW680。通过PCR和DNA序列测定来确定片段相对于CMV启动子的正确方向。表达盒的序列示于图12中(SEQ ID NO:17)。
为了构建CPC-P501 S表达盒,从载体pRIT15201(参见7)中经PCR扩增获得CPC-P501 S,并通过XbaI和SalI的限制性酶切克隆至pVAC的NheI和XhoI位点,从而产生质粒JNW735。通过PCR和DNA序列测定来确定正确的***方向。CPC-P501 S表达盒的序列示于图13中(SEQ ID NO:18)。
4.质粒JNW680和JNW735的人P501S的表达
P501S表达质粒被瞬时转染至CHO细胞中,并按上文所述的方法制备完全细胞裂解产物。完全细胞裂解产物的Western blot分析从分别用JNW680和JNW735转染的样品中鉴定出约55kDa和62kDa的单条带(图21)。这与预测的P501S和CPC-P501S的分子量59.3kDa和63.3kDa相一致。CPC标签的添加没有对P501S的表达产生不利影响。
5.结果
5.1.PMID免疫后对人P501S的抗体应答
在经PMID在第0天进行初次免疫,和在第21天和第42天和第70天进行3次加强免疫后,通过ELISA方法检测了经pVAC(空载体)和pVAC-P501S(JNW680)免疫后的抗体应答。图22显示了分别从第-1天,第28天和第49天(小鼠A1-3,B1-3)和第56天(小鼠A4-6,B4-9)取样的血清中检测到的抗体应答。虽然对pVAC空载体存在非特异性应答,在9只小鼠中有5只观察到对P501 S构建体的特异性应答。
5.2.通过筛选P501S肽文库从C57BL/6小鼠的人P501S中鉴定新的T细胞表位
用JNW680(pVAC-P501S)经PMID在第0天免疫及在第21天和第42天和第70天进行3次加强免疫,在第84天进行ELISPOT检测。来源于P501 S文库的肽以50μg/ml的终浓度进行检测。从该初步筛选中,发现3种肽能刺激IFNγ和/或IL-2分泌。为肽18,22和48(图23)。这些肽用于随后的细胞分析。
5.3.PMID免疫后的对pVAC-P501S(JNW680)的细胞应答
经PMID在第0天免疫,和在第21,42和70天进行三次加强免疫后,通过ELISPOT检测用pVAC(空载体)和pVAC-P501 S进行免疫后的细胞应答。在加强免疫后的第7天进行检测。采用了两种不同的检测条件:1)从肽文库筛选中鉴定得到的肽18,22和48在50μg/ml的终浓度下使用;和2)CPC-P501S蛋白在20μg/ml的终浓度下使用。图24A显示,虽然对于空载体(A4-6)不产生P501 S特异性应答,但pVAC-P501 S构建体在所有小鼠(B6-9)中均诱导针对肽18和22的特异性IFN-γ应答,不过还有一只鼠(B7)中还显示出针对肽48的IFN-γ应答。图24B表明,所有的小鼠均显示出针对肽18,22和48的特异性IL-2应答。此外,pVAC-P501S免疫小鼠(B6-9)还显示出针对CPC-P501S的中度的IL-2应答,其中采用空载体免疫的小鼠(A4-6)不产生应答。
5.4.经PMID免疫后的针对P501S和CPC-P501S的细胞应答比较
在经PMID在第0天初次免疫及在第21和第42天加强免疫后,通过ELISPOT可检测经pVAC(空载体)、pVAC-P501S(JNW680)和CPC-P501S(JNW735)免疫后的细胞应答。所述检测在加强免疫后的第7天开始进行。采用了两种不同的检测条件:1)从肽文库筛选中鉴定得到的肽18,22和48在50μg/ml的终浓度下使用;和2)CPC-P501S蛋白在20μg/ml的终浓度下使用。图25显示,在第28天,CPC-P501S诱导针对10μg/ml的肽22的良好的IL-2应答,然而针对空载体或pVAC-P501S均不存在P501S-特异应答。在采用CPC-P501 S蛋白再次免疫脾细胞时还可观察到这种结果。在第49天(在第二次加强免疫后),由P501S和CPC-P501S诱导的应答是相等的。这些数据表明,CPC标签的添加提高了针对P501 S应答的动力学和/或数量。
实施例IX.在小鼠中采用P501S蛋白+佐剂进行免疫原性实验的研究
1.实验设计和佐剂的配制
用与佐剂一起配制的重组纯化的CPC-P501S蛋白进行疫苗接种而诱导的免疫应答通过用小鼠进行实验来表征。采用10μg的与不同佐剂***一起配制的CPC-P501S蛋白以2星期的时间间隔进行肌肉内注射2-6次,从而对5到10组,8周大的C57BL6鼠进行免疫。其施用量相当于人剂量的1/10(50μl)。
采用如Gerard,c.等,2001,Vaccine 19,2583-2589中所述的标准方法,对最后一次免疫后6-14天的脾细胞进行了血清学(总Ig应答)和细胞应答的分析。
代表性实验的数据如下文所示。其包括5组每组8只分别在第0,14,28和42天接受4次肌肉内CPC P501(10μg)+佐剂(A,B,C)注射的C57BI/6小鼠。实施例5提供了如何配制制剂的方法。简单的,所示的佐剂制剂按如下方法配制(定量为100μl每剂量):
-佐剂A:QS21(10μg),MPL(10μg)和CPG7909(100μg)按照WO 00/62800中所公开的方法制备;
-佐剂B:QS21(20μg),MPL(20μg),CPG7909(100μg)和50μl SB62水包油乳剂(WO 95/17210)配成的制剂;
-佐剂C:QS21(10μg),MPL(10μg),CpG7909(100μg)和10μl SB62水包油乳剂(WO 99/12565)配成的制剂。
2.血清学
采用CPC-P501或RA12-P501(C末端来源于如Skeiky等,Infectionand Immun.(1999)67:3998-4007所述的MTB32A抗原,所述的C末端是P501蛋白的截短形式,其相应于在其N末端与TB衍生蛋白RA12-Ra12融合的蛋白的C末端),经ELISA方法来检测接种诱导的总Ig应答。
在免疫后,添加了佐剂的CPC-P501 S蛋白产生了良好的抗体应答。
3.细胞应答
3.1.淋巴细胞增殖
最后一次免疫后7天,在个别脾细胞中出现了淋巴细胞增殖现象。将2.10e5脾细胞以一式四份的方式加入含1%正常鼠血清的RPMI培养基的96孔微孔板中。在用不同浓度的免疫原(CPC-P501)或截短蛋白(RA12 P501)重新刺激后72小时,加入1μCi 3H胸腺嘧啶(Amersham5Ci/ml)。16小时后,将细胞收集于过滤板上。采用β计数器来计量合并的放射性。结果通过CPM或刺激指数*来表现(在含有抗原的培养物中的几何平均数CPM/在不含抗原的培养物中的几何平均数CPM)。
作为阳性对照的采用ConA(2μg/ml)进行重新刺激也包括在内作为阳性对照。
如图26所示,在采用由另外的表达***(E coli)制备的免疫原或其他的P501蛋白在体外重新刺激后,所有接受了佐剂配制的蛋白的小鼠脾脏中均观察到P501特异性淋巴细胞增殖现象,这表明通过疫苗接种在体内诱导了T细胞。
3.2.通过脾细胞细胞内染色检测IFNg生产
从在存在GMCSF的条件下培养的小鼠PBL的7天培养物中获取骨髓树突细胞(BMDC)。
在最后一次疫苗接种后7天,收集脾或PBL并制备细胞悬浮物。将10e6细胞(每组混合在一起)与10e5 BMDC一起培养+/-18小时,并在10μg/ml的CPCp501蛋白或RA12的刺激下过夜。
在经2.4.G.2抗体处理后,采用荧光抗CD4和CD8抗体(抗CD4-APC和抗CD8PerCP)染色脾细胞。在渗透和固定步骤后,采用荧光抗IFNg-FITC抗体染色细胞。
在经与不同佐剂配合使用的CPC P501疫苗接种的小鼠中,CD4和CD8 T细胞均显示出产生针对采用在E coli中制备的免疫原或C-末端p501刺激的DC而产生的IFNg(如脾和PBLs的细胞内染色所示)。与单独的蛋白相比,在接受了添加佐剂的CPC-P501S的组中,在产生这种细胞因子的细胞%方面存在4-10×的增加,同时0.1-10%的CD4或CD8 T细胞显示出产生IFNg。
总的来说,从这些数据中能得出这样的结论:添加佐剂的CPC-P501蛋白在小鼠中具有免疫原性。
在采用添加佐剂的CPC P501进行数次肌肉内疫苗接种后,包括由CD4和CD8 T细胞产生的IFNg的P501特异性体液和细胞应答均可检测到。
实施例X.CPC-MUC-1构建体和其序列
CPC序列来源于核苷酸SEQ ID NO.28。
MUC1序列可从Genbank数据库获得(登录号NM_002456)
1.MUC1-CPC构建体
由于转录后将被切除的信号序列存在于MUC1中,CPC基元序列被置于C末端。获得的MUC1-CPC DNA序列如SEQ ID NO.xx(图28A)所示,并且相应的MUC1-CPC蛋白序列如SEQ ID NO.yy(图28B)所示。
2.ss-CPC-MUC1构建体
由于翻译后将被切除的信号序列存在于MUC1中,MUC1信号序列被异源前导肽序列(来源于人免疫球蛋白的重链)所取代,并且CPC基元序列被***异源前导肽序列和MUC1序列之间,从而产生如图29所示的被命名为ss-CPC-MUC1的序列。
序列表
<110>GlaxosmithKline Biologicals sa
Glaxo Group Ltd
<120>免疫原性组合物
<130>B45311
<160>52
<170>Windows版本4.0 FastSEQ
<210>1
<211>15
<212>PRT
<213>肺炎链球菌
<400>1
Gly Trp Gln Lys Asn Asp Thr Gly Tyr Trp Tyr Val His Ser Asp
1 5 10 15
<210>2
<211>21
<212>PRT
<213>肺炎链球菌
<400>2
Gly Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr
1 5 10 15
Tyr Phe Asp Ser Ser
20
<210>3
<211>22
<212>PRT
<213>肺炎链球菌
<400>3
Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp
1 5 10 15
Tyr Trp Phe Asp Asn Ser
20
<210>4
<211>20
<212>PRT
<213>肺炎链球菌
<400>4
Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp Lys Trp Tyr Tyr
1 5 10 15
Phe Asn Glu Glu
20
<210>5
<211>21
<212>PRT
<213>肺炎链球菌
<400>5
Gly Ala Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr
1 5 10 15
Leu Asp Ala Lys Glu
20
<210>6
<211>23
<212>PRT
<213>肺炎链球菌
<400>6
Gly Ala Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly
1 5 10 15
Trp Tyr Tyr Leu Lys Pro Asp
20
<210>7
<211>142
<212>PRT
<213>肺炎链球菌
<400>7
Gly Trp Gln Lys Asn Asp Thr Gly Tyr Trp Tyr Val His Ser Asp Gly
1 5 10 15
Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr
20 25 30
Phe Asp Ser Ser Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr
35 40 45
Asp Gly Asn Trp Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly
50 55 60
Trp Lys Lys Ile Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala
65 70 75 80
Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp
85 90 95
Ala Lys Glu Gly Ala Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp
100 105 110
Gly Thr Gly Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg
115 120 125
Pro Glu Phe Thr Val Glu Pro Asp Gly Leu Ile Thr Val Lys
130 135 140
<210>8
<211>112
<212>PRT
<213>肺炎链球菌
<400>8
Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile
1 5 10 15
Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr Met Leu Ala Asp
20 25 30
Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp Phe Asp Asn Ser
35 40 45
Gly G1u Met Ala Thr Gly Trp Lys Lys Ile Ala Asp Lys Trp Tyr Tyr
50 55 60
Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val Lys Tyr Lys Asp
65 70 75 80
Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met Val Ser Asn Ala
85 90 95
Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr Leu Lys Pro Asp
100 105 110
<210>9
<211>45
<212>DNA
<213>肺炎链球菌
<400>9
ggctggcaga agaatgacac tggctactgg tacgtacatt cagac 45
<210>10
<211>63
<212>DNA
<213>肺炎链球菌
<400>10
ggctcttatc caaaagacaa gtttgagaaa atcaatggca cttggtacta ctttgacagt 60
tca 63
<210>11
<211>66
<212>DNA
<213>肺炎链球菌
<400>11
ggctatatgc ttgcagaccg ctggaggaag cacacagacg gcaactggta ctggttcgac 60
aactca 66
<210>12
<211>60
<212>DNA
<213>肺炎链球菌
<400>12
ggcgaaatgg ctacaggctg gaagaaaatc gctgataagt ggtactattt caacgaagaa 60
<210>13
<211>63
<212>DNA
<213>肺炎链球菌
<400>13
ggtgccatga agacaggctg ggtcaagtac aaggacactt ggtactactt agacgctaaa 60
gaa 63
<210>14
<211>69
<212>DNA
<213>肺炎链球菌
<400>14
ggcgccatgg tatcaaatgc ctttatccag tcagcggacg gaacaggctg gtactacctc 60
aaaccagac 69
<210>15
<211>429
<212>DNA
<213>肺炎链球菌
<400>15
ggctggcaga agaatgacac tggctactgg tacgtacatt cagacggctc ttatccaaaa 60
gacaagtttg agaaaatcaa tggcacttgg tactactttg acagttcagg ctatatgctt 120
gcagaccgct ggaggaagca cacagacggc aactggtact ggttcgacaa ctcaggcgaa 180
atggctacag gctggaagaa aatcgctgat aagtggtact atttcaacga agaaggtgcc 240
atgaagacag gctgggtcaa gtacaaggac acttggtact acttagacgc taaagaaggc 300
gccatggtat caaatgcctt tatccagtca gcggacggaa caggctggta ctacctcaaa 360
ccagacggaa cactggcaga caggccagaa ttcacagtag agccagatgg cttgattaca 420
gtaaaataa 429
<210>16
<211>336
<212>DNA
<213>肺炎链球菌
<400>16
tacgtacatt ccgacggctc ttatccaaaa gacaagtttg agaaaatcaa tggcacttgg 60
tactactttg acagttcagg ctatatgctt gcagaccgct ggaggaagca cacagacggc 120
aactggtact ggttcgacaa ctcaggcgaa atggctacag gctggaagaa aatcgctgat 180
aagtggtact atttcaacga agaaggtgcc atgaagacag gctgggtcaa gtacaaggac 240
acttggtact acttagacgc taaagaaggc gccatggtat caaatgcctt tatccagtca 300
gcggacggaa caggctggta ctacctcaaa ccagac 336
<210>17
<211>1674
<212>DNA
<213>人类
<400>17
gccaccatgg tccagaggct gtgggtgagc cgcctgctgc ggcaccggaa agcccagctc 60
ttgctggtca acctgctaac ctttggcctg gaggtgtgtt tggccgcagg catcacctat 120
gtgccgcctc tgctgctgga agtgggggta gaggagaagt tcatgaccat ggtgctgggc 180
attggtccag tgctgggcct ggtctgtgtc ccgctcctag gctcagccag tgaccactgg 240
cgtggacgct atggccgccg ccggcccttc atctgggcac tgtccttggg catcctgctg 300
agcctctttc tcatcccaag ggccggctgg ctagcagggc tgctgtgccc ggatcccagg 360
cccctggagc tggcactgct catcctgggc gtggggctgc tggacttctg tggccaggtg 420
tgcttcactc cactggaggc cctgctctct gacctcttcc gggacccgga ccactgtcgc 480
caggcctact ctgtctatgc cttcatgatc agtcttgggg gctgcctggg ctacctcctg 540
cctgccattg actgggacac cagtgccctg gccccctacc tgggcaccca ggaggagtgc 600
ctctttggcc tgctcaccct catcttcctc acctgcgtag cagccacact gctggtggct 660
gaggaggcag cgctgggccc caccgagcca gcagaagggc tgtcggcccc ctccttgtcg 720
ccccactgct gtccatgccg ggcccgcttg gctttccgga acctgggcgc cctgcttccc 780
cggctgcacc agctgtgctg ccgcatgccc cgcaccctgc gccggctctt cgtggctgag 840
ctgtgcagct ggatggcact catgaccttc acgctgtttt acacggattt cgtgggcgag 900
gggctgtacc agggcgtgcc cagagctgag ccgggcaccg aggcccggag acactatgat 960
gaaggcgttc ggatgggcag cctggggctg ttcctgcagt gcgccatctc cctggtcttc 1020
tctctggtca tggaccggct ggtgcagcga ttcggcactc gagcagtcta tttggccagt 1080
gtggcagctt tccctgtggc tgccggtgcc acatgcctgt cccacagtgt ggccgtggtg 1140
acagcttcag ccgccctcac cgggttcacc ttctcagccc tgcagatcct gccctacaca 1200
ctggcctccc tctaccaccg ggagaagcag gtgttcctgc ccaaataccg aggggacact 1260
ggaggtgcta gcagtgagga cagcctgatg accagcttcc tgccaggccc taagcctgga 1320
gctcccttcc ctaatggaca cgtgggtgct ggaggcagtg gcctgctccc acctccaccc 1380
gcgctctgcg gggcctctgc ctgtgatgtc tccgtacgtg tggtggtggg tgagcccacc 1440
gaggccaggg tggttccggg ccggggcatc tgcctggacc tcgccatcct ggatagtgcc 1500
ttcctgctgt cccaggtggc cccatccctg tttatgggct ccattgtcca gctcagccag 1560
tctgtcactg cctatatggt gtctgccgca ggcctgggtc tggtcgccat ttactttgct 1620
acacaggtag tatttgacaa gagcgacttg gccaaatact cagcgtaggt cgag 1674
<210>18
<211>1947
<212>DNA
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S之间的杂交基因
<400>18
gccaccatgg cggccgctta cgtacattcc gacggctctt atccaaaaga caagtttgag 60
aaaatcaatg gcacttggta ctactttgac agttcaggct atatgcttgc agaccgctgg 120
aggaagcaca cagacggcaa ctggtactgg ttcgacaact caggcgaaat ggctacaggc 180
tggaagaaaa tcgctgataa gtggtactat ttcaacgaag aaggtgccat gaagacaggc 240
tgggtcaagt acaaggacac ttggtactac ttagacgcta aagaaggcgc catgcaatac 300
atcaaggcta actctaagtt cattggtatc actgaaggcg tcatggtatc aaatgccttt 360
atccagtcag cggacggaac aggctggtac tacctcaaac cagacggaac actggcagac 420
aggccagaaa agttcatgta catggtgctg ggcattggtc cagtgctggg cctggtctgt 480
gtcccgctcc taggctcagc cagtgaccac tggcgtggac gctatggccg ccgccggccc 540
ttcatctggg cactgtcctt gggcatcctg ctgagcctct ttctcatccc aagggccggc 600
tggctagcag ggctgctgtg cccggatccc aggcccctgg agctggcact gctcatcctg 660
ggcgtggggc tgctggactt ctgtggccag gtgtgcttca ctccactgga ggccctgctc 720
tctgacctct tccgggaccc ggaccactgt cgccaggcct actctgtcta tgccttcatg 780
atcagtcttg ggggctgcct gggctacctc ctgcctgcca ttgactggga caccagtgcc 840
ctggccccct acctgggcac ccaggaggag tgcctctttg gcctgctcac cctcatcttc 900
ctcacctgcg tagcagccac actgctggtg gctgaggagg cagcgctggg ccccaccgag 960
ccagcagaag ggctgtcggc cccctccttg tcgccccact gctgtccatg ccgggcccgc 1020
ttggctttcc ggaacctggg cgccctgctt ccccggctgc accagctgtg ctgccgcatg 1080
ccccgcaccc tgcgccggct cttcgtggct gagctgtgca gctggatggc actcatgacc 1140
ttcacgctgt tttacacgga tttcgtgggc gaggggctgt accagggcgt gcccagagct 1200
gagccgggca ccgaggcccg gagacactat gatgaaggcg ttcggatggg cagcctgggg 1260
ctgttcctgc agtgcgccat ctccctggtc ttctctctgg tcatggaccg gctggtgcag 1320
cgattcggca ctcgagcagt ctatttggcc agtgtggcag ctttccctgt ggctgccggt 1380
gccacatgcc tgtcccacag tgtggccgtg gtgacagctt cagccgccct caccgggttc 1440
accttctcag ccctgcagat cctgccctac acactggcct ccctctacca ccgggagaag 1500
caggtgttcc tgcccaaata ccgaggggac actggaggtg ctagcagtga ggacagcctg 1560
atgaccagct tcctgccagg ccctaagcct ggagctccct tccctaatgg acacgtgggt 1620
gctggaggca gtggcctgct cccacctcca cccgcgctct gcggggcctc tgcctgtgat 1680
gtctccgtac gtgtggtggt gggtgagccc accgaggcca gggtggttcc gggccggggc 1740
atctgcctgg acctcgccat cctggatagt gccttcctgc tgtcccaggt ggccccatcc 1800
ctgtttatgg gctccattgt ccagctcagc cagtctgtca ctgcctatat ggtgtctgcc 1860
gcaggcctgg gtctggtcgc catttacttt gctacacagg tagtatttga caagagcgac 1920
ttggccaaat actcagcgta ggtcgag 1947
<210>19
<211>1662
<212>DNA
<213>人工序列
<220>
<223>密码子优化的人P501S
<400>19
atggtgcagc ggctctgggt gagccgcctc ctgcggcatc gcaaggccca gctcctgctg 60
gtgaatctgc tcacattcgg cctggaggtg tgcctggccg ccggcatcac ctacgtgccc 120
cccctcctgc tggaggtggg agtcgaggag aagttcatga ccatggtgct gggcattggg 180
cccgtcctgg gcctcgtgtg cgtgcctctc ctcggcagcg cttccgacca ttggcgcggc 240
cggtatggcc gcaggagacc cttcatctgg gctctgagtc tcggcatcct gctgagcctg 300
ttcctgatcc ctcgggccgg ctggctggcc gggctgctgt gccccgatcc tcggcccctg 360
gagctggccc tgctgatcct cggcgtgggc ctgctggact tctgcggcca ggtgtgcttc 420
acgcccctgg aggcactgct gagcgacctg ttccgggacc ccgaccattg ccgccaggcg 480
tacagcgtgt acgccttcat gatctccctg ggaggctgcc tgggctacct gctccccgcc 540
atcgattggg acaccagcgc actcgccccc tatctcggaa cacaggagga atgcctgttc 600
ggattgttga cgctcatctt cctcacgtgc gtcgcggcca ccctgttggt ggccgaggag 660
gccgccctgg ggcccaccga gccggccgag ggactgagcg ccccgagcct gagtccacac 720
tgctgccctt gccgggcccg cctggccttc cgtaatctgg gcgccctcct gcctcggctc 780
catcagctgt gttgcagaat gcctaggacg ctgcggcgcc tgttcgtcgc tgagttgtgc 840
tcctggatgg ctctcatgac cttcaccctg ttttatacgg acttcgtcgg ggagggcctg 900
taccaggggg tgccgcgcgc cgagcccggg acagaggcgc gccgccacta cgacgaggga 960
gtgcgtatgg gctccctggg cctcttcttg cagtgcgcca tcagtctggt tttctctctg 1020
gtcatggaca ggctggtgca gcgcttcgga acccgggcgg tgtacctggc gagcgtggcc 1080
gccttccccg tggctgccgg cgccacctgc ctctctcact cggtggccgt ggtcaccgcc 1140
agcgccgccc tgaccgggtt caccttctct gccctgcaga ttctgcctta caccctggcc 1200
agcctgtacc atcgcgagaa acaggtgttt ctccccaagt acagaggcga caccgggggc 1260
gcctccagcg aggacagcct catgacctcc ttcctgcctg gccccaagcc cggcgcccct 1320
ttccccaacg ggcacgtggg cgccggcggg agtgggctcc tgcccccccc tcctgcgctg 1380
tgcggggcca gcgcctgcga cgtgagcgtg cgcgtggtgg tgggcgagcc caccgaggcc 1440
cgcgtggtgc cgggcagagg catttgtctg gacctggcca tcctcgactc cgccttcctc 1500
ctcagccagg tggccccgtc cctcttcatg ggctctatcg tccagctgtc tcagagcgtc 1560
accgcttaca tggtgtccgc tgctggactg ggcttggtgg ctatttattt cgccacccag 1620
gtggtgttcg acaagagcga cctggccaaa tactccgcct ga 1662
<210>20
<211>1662
<212>DNA
<213>人工序列
<220>
<223>密码子优化的人P501S
<400>20
atggtgcagc ggctgtgggt gtcccggctg ctgcgccata gaaaggccca gttgctgctg 60
gtgaacctgc tgactttcgg actggaggtg tgcctggctg ccgggatcac gtacgtgccc 120
cccctgctgc tggaggtggg cgtggaggag aagttcatga caatggtgct gggcatcggc 180
cccgtcctgg gcctcgtgtg tgtgcccctc ctcgggagtg cgtccgatca ttggcggggc 240
cgctacggcc gccgcagacc gttcatctgg gccctgagcc tggggatcct gctctctctc 300
ttcctgatcc cccgggccgg ctggctggcc ggcctgctgt gtcccgaccc ccgccctctg 360
gagctggccc tcctgatcct gggcgtgggc ttgttggact tctgcggcca ggtgtgtttc 420
actcccctgg aggctctgct ctccgacctc ttccgcgacc ccgaccactg taggcaggct 480
tacagcgtgt acgccttcat gatcagtctg gggggatgcc tgggctatct gctgcccgct 540
atcgactggg acaccagcgc cctggccccc tacctgggga ctcaggagga gtgcctgttc 600
ggcctgctca ccttgatctt cctgacgtgc gtcgccgcca ccctgctggt ggccgaggag 660
gcggccctgg ggcccaccga gcccgccgag ggcctgagcg ctcccagcct gagcccccat 720
tgctgcccgt gcagggctag gctcgccttc aggaatctgg gcgctttgct gccccgcctg 780
catcagctgt gctgtcgcat gcctcgcacc ctgcgccgcc tgttcgtcgc tgagctctgt 840
tcctggatgg ccctgatgac gttcaccctc ttctacaccg acttcgtggg ggagggcctg 900
taccagggcg tgcccagggc cgagcccggc accgaggcta ggcgccatta cgacgagggc 960
gtcaggatgg gctctctggg cctcttcctg cagtgcgcca tcagtctggt gttctctctg 1020
gtgatggacc ggctggtgca gcgcttcggc acccgggccg tgtacctcgc ctctgtggcg 1080
gctttccccg tcgccgccgg cgcgacctgc ctgtctcatt ctgtcgccgt ggtgaccgcc 1140
agcgccgccc tgaccggctt caccttcagt gcgctccaga ttctgcccta caccctggcg 1200
tctctgtacc atcgcgagaa gcaggtgttc ctgcccaagt accgcgggga cacaggggga 1260
gcttcctctg aggacagcct gatgaccagc ttcttgcccg gccccaagcc gggggcccct 1320
ttccccaacg gccatgtcgg ggcgggcggc agcggcctgc tccctccccc ccccgccctg 1380
tgcggcgcta gtgcctgcga cgtgagcgtg cgggtggtgg tgggggagcc caccgaggct 1440
agggtcgtgc ctggccgggg gatctgcctg gacctggcca tcctcgactc cgccttcctg 1500
ctctcccagg tggcgcccag cctgttcatg ggcagtatcg tgcagctgag ccagagcgtg 1560
accgcctaca cggtgagcgc cgccggcctg gggttggtgg ccatctactt tgccacccag 1620
gtcgtgttcg acaagagcga tctcgccaagtatagcgcct ga 1662
<210>21
<211>1688
<212>DNA
<213>人工序列
<220>
<223>密码子优化的人P501S
<400>21
gacggctagc gccaccatgg tgcagcggct ctgggtgagc cgcctcctgc ggcatcgcaa 60
ggcccagctc ctgctggtga atctgctcac attcggcctg gaggtgtgcc tggccgccgg 120
catcacctac gtgccccccc tcctgctgga ggtgggagtc gaggagaagt tcatgaccat 180
ggtgctgggc attgggcccg tcctgggcct cgtgtgcgtg cctctcctcg gcagcgcttc 240
cgaccattgg cgcggccggt atggccgcag gagacccttc atctgggctc tgagtctcgg 300
catcctgctg agcctgttcc tgatccctcg ggccggctgg ctggccgggc tgctgtgccc 360
cgatcctcgg cccctggagc tggccctgct gatcctcggc gtgggcctgc tggacttctg 420
cggccaggtg tgcttcacgc ccctggaggc actgctgagc gacctgttcc gggaccccga 480
ccattgccgc caggcgtaca gcgtgtacgc cttcatgatc tccctgggag gctgcctggg 540
ctacctgctc cccgccatcg attgggacac cagcgcactc gccccctatc tcggaacaca 600
ggaggaatgc ctgttcggac tgctgacgct catcttcctc acgtgcgtcg cggccaccct 660
gttggtggcc gaggaggccg ccctggggcc caccgagccg gccgagggac tgagcgcccc 720
gagcctgagt ccacactgct gcccttgccg ggcccgcctg gccttccgta atctgggcgc 780
cctcctgcct cggctccatc agctgtgttg cagaatgcct aggacgctgc ggcgcctgtt 840
cgtcgctgag ttgtgctcct ggatggctct catgaccttc accctgtttt atacggactt 900
cgtcggggag ggcctgtacc agggggtgcc gcgcgccgag cccgggacag aggcgcgccg 960
ccactacgac gagggagtgc gtatgggctc cctgggcctc ttcttgcagt gcgccatcag 1020
tctggttttc tctctggtca tggacaggct ggtgcagcgc ttcggaaccc gggcggtgta 1080
cctggcgagc gtggccgcct tccccgtggc tgccggcgcc acctgcctct ctcactcggt 1140
ggccgtggtc accgccagcg ccgccctgac cgggttcacc ttctctgccc tgcagattct 1200
gccttacacc ctggccagcc tgtaccatcg cgagaaacag gtgtttctcc ccaagtacag 1260
aggcgacacc gggggcgcct ccagcgagga cagcctcatg acctccttcc tgcctggccc 1320
caagcccggc gcccctttcc ccaacgggca cgtgggcgcc ggcgggagtg ggctcctgcc 1380
cccccctcct gcgctgtgcg gggccagcgc ctgcgacgtg agcgtgcgcg tggtggtggg 1440
cgagcccacc gaggcccgcg tggtgccggg cagaggcatt tgtctggacc tggccatcct 1500
cgactccgcc ttcctcctca gccaggtggc cccgtccctc ttcatgggct ctatcgtcca 1560
gctgtctcag agcgtcaccg cttacatggt gtccgctgct ggactgggct tggtggctat 1620
ttatttcgcc acccaggtgg tgttcgacaa gagcgacctg gccaaatact ccgcctgact 1680
cgaggcag 1688
<210>22
<211>1688
<212>DNA
<213>人工序列
<220>
<223>密码子优化的人P501S
<400>22
gacggctagc gccaccatgg tgcagcggct gtgggtgtcc cggctgctgc gccatagaaa 60
ggcccagttg ctgctggtga acctgctgac tttcggactg gaggtgtgcc tggctgccgg 120
gatcacgtac gtgccccccc tgctgctgga ggtgggcgtg gaggagaagt tcatgacaat 180
ggtgctgggc atcggccccg tcctgggcct cgtgtgtgtg cccctcctcg ggagtgcgtc 240
cgatcattgg cggggccgct acggccgccg cagaccgttc atctgggccc tgagcctggg 300
catcctgctc tctctcttcc tgatcccccg ggccggctgg ctggccggcc tgctgtgtcc 360
cgacccccgc cctctggagc tggccctcct gatcctgggc gtgggcctgc tggacttctg 420
cggccaggtg tgtttcactc ccctggaggc tctgctctcc gacctcttcc gcgaccccga 480
ccactgtagg caggcttaca gcgtgtacgc cttcatgatc agtctggggg gatgcctggg 540
ctatctgctg cccgctatcg actgggacac cagcgccctg gccccctacc tggggactca 600
ggaggagtgc ctgttcggcc tgctcacctt gatcttcctg acgtgcgtcg ccgccaccct 660
gctggtggcc gaggaggcgg ccctggggcc caccgagccc gccgagggcc tgagcgctcc 720
cagcctgagc ccccattgct gcccgtgcag ggctaggctc gccttcagga atctgggcgc 780
tttgctgccc cgcctgcatc agctgtgctg tcgcatgcct cgcaccctgc gccgcctgtt 840
cgtcgctgag ctctgttcct ggatggccct gatgacgttc accctcttct acaccgactt 900
cgtgggggag ggcctgtacc agggcgtgcc cagggccgag cccggcaccg aggctaggcg 960
ccattacgac gagggcgtca ggatgggctc tctgggcctc ttcctgcagt gcgccatcag 1020
tctggtgttc tctctggtga tggaccggct ggtgcagcgc ttcggcaccc gggccgtgta 1080
cctcgcctct gtggcggctt tccccgtcgc cgccggcgcg acctgcctgt ctcattctgt 1140
cgccgtggtg accgccagcg ccgccctgac cggcttcacc ttcagtgcgc tccagattct 1200
gccctacacc ctggcgtctc tgtaccatcg cgagaagcag gtgttcctgc ccaagtaccg 1260
cggggacaca gggggagctt cctctgagga cagcctgatg accagcttct tgcccggccc 1320
caagccgggg gcccctttcc ccaacggcca tgtcggggcg ggcggcagcg gcctgctccc 1380
tccccccccc gccctgtgcg gcgctagtgc ctgcgacgtg agcgtgcggg tggtggtggg 1440
ggagcccacc gaggctaggg tcgtgcctgg ccgggggatc tgcctggacc tggccatcct 1500
cgactccgcc ttcctgctct cccaggtggc gcccagcctg ttcatgggca gtatcgtgca 1560
gctgagccag agcgtgaccg cctacatggt gagcgccgcc ggcctggggt tggtggccat 1620
ctactttgcc acccaggtcg tgttcgacaa gagcgatctc gccaagtata gcgcctgact 1680
cgaggcag 1688
<210>23
<211>435
<212>DNA
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S的5′末端的
一小部分之间的杂交基因
<400>23
atggcggccg cttacgtaca ttccgacggc tcttatccaa aagacaagtt tgagaaaatc 60
aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120
cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180
aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240
aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatgca atacatcaag 300
gctaactcta agttcattgg tatcactgaa ggcgtcatgg tatcaaatgc ctttatccag 360
tcagcggacg gaacaggctg gtactacctc aaaccagacg gaacactggc agacaggcca 420
gaaaagttca tgtac 435
<210>24
<211>435
<212>DNA
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S的5′末端的
一小部分之间的杂交基因-优化密码子
<400>24
atggccgccg cctacgtgca tagcgacggg agctacccca aggacaagtt cgagaagatc 60
aacgggacat ggtactactt cgactcctcc ggctacatgc tcgccgaccg ctggcggaag 120
cacaccgacg gcaactggta ctggttcgat aactcgggag agatggccac cggctggaag 180
aagatcgcgg acaagtggta ctatttcaac gaggagggcg ccatgaagac cggctgggtg 240
aagtataagg acacctggta ctacctcgac gccaaggagg gcgccatgca gtatatcaag 300
gccaacagca agttcatcgg catcaccgag ggagtgatgg tcagcaacgc ctttatccag 360
agcgccgacg gcaccggatg gtactacttg aagccggacg gcaccctcgc ggatcggccc 420
gagaagttca tgtac 435
<210>25
<211>435
<212>DNA
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S的5′末端的
一小部分之间的杂交基因-优化密码子
<400>25
atggccgccg cctacgtgca cagcgacggg tcctacccaa aggacaagtt cgagaagatc 60
aacggcacgt ggtactattt cgacagcagc ggctacatgc tcgccgatcg ctggcgcaag 120
cacaccgacg ggaactggta ctggttcgac aactctggcg agatggctac ggggtggaag 180
aagatcgccg acaagtggta ctacttcaac gaggagggcg ccatgaagac cgggtgggtg 240
aagtacaagg acacctggta ctacctggac gctaaggagg gcgccatgca gtacatcaag 300
gccaactcga agttcatcgg gatcaccgag ggcgtgatgg tcagtaacgc tttcatccag 360
agcgcggacg gcacaggctg gtattacctg aagcccgatg gcaccctggc ggacagacct 420
gagaaattca tgtac 435
<210>26
<211>464
<212>DNA
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S的5′末端的
一小部分之间的杂交基因-优化密码子
<400>26
gacggctagc gccaccatgg ccgccgccta cgtgcatagc gacgggagct accccaagga 60
caagttcgag aagatcaacg ggacatggta ctacttcgac tcctccggct acatgctcgc 120
cgaccgctgg cggaagcaca ccgacggcaa ctggtactgg ttcgataact cgggagagat 180
ggccaccggc tggaagaaga tcgcggacaa gtggtactat ttcaacgagg agggcgccat 240
gaagaccggc tgggtgaagt ataaggacac ctggtactac ctcgacgcca aggagggcgc 300
catgcagtat atcaaggcca acagcaagtt catcggcatc accgagggag tgatggtcag 360
caacgccttt atccagagcg ccgacggcac cggatggtac tacttgaagc cggacggcac 420
cctcgcggat cggcccgaga agttcatgta ctgactcgag gcag 464
<210>27
<211>652
<212>PRT
<213>人工序列
<220>
<223>肺炎链球菌C-LytA,P2 T辅助表位和人P501S的51-553
氨基酸之间的杂交蛋白-优化密码子
<400>27
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe Met
130 135 140
Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro
145 150 155 160
Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg
165 170 175
Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe
180 185 190
Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro
195 200 205
Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp
210 215 220
Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp
225 230 235 240
Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala
245 250 255
Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile
260 265 270
Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu
275 280 285
Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala
290 295 300
Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala
305 310 315 320
Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg
325 330 335
Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His
340 345 350
Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala
355 360 365
Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr
370 375 380
Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro
385 390 395 400
Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser
405 410 415
Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val
420 425 430
Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala
435 440 445
Ser Val Ala Ala Phe pro Val Ala Ala Gly Ala Thr Cys Leu Ser His
450 455 460
Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe
465 470 475 480
Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg
485 490 495
Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala
500 505 510
Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro
515 520 525
Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu
530 535 540
Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser
545 550 555 560
Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly
565 570 575
Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu
580 585 590
Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser
595 600 605
Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val
610 615 620
Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala
625 630 635 640
Lys Tyr Ser Ala Gly Gly His His His His His His
645 650
<210>28
<211>1959
<212>DNA
<213>人工序列
<220>
<223>编码肺炎链球菌C-LytA,P2 T辅助表位和人P501S的51-553
氨基酸之间的杂交蛋白的DNA
<400>28
atggcggccg cttacgtaca ttccgacggc tcttatccaa aagacaagtt tgagaaaatc 60
aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120
cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180
aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240
aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatgca atacatcaag 300
gctaactcra agttcattgg tatcactgaa ggcgtcatgg tatcaaatgc ctttatccag 360
tcagcggacg gaacaggctg gtactacctc aaaccagacg gaacactggc agacaggcca 420
gaaaagttca tgtacatggt gctgggcatt ggtccagtgc tgggcctggt ctgtgtcccg 480
ctcctaggct cagccagtga ccactggcgt ggacgctatg gccgccgccg gcccttcatc 540
tgggcactgt ccttgggcat cctgctgagc ctctttctca tcccaagggc cggctggcta 600
gcagggctgc tgtgcccgga tcccaggccc ctggagctgg cactgctcat cctgggcgtg 660
gggctgctgg acttctgtgg ccaggtgtgc ttcactccac tggaggccct gctctctgac 720
ctcttccggg acccggacca ctgtcgccag gcctactctg tctatgcctt catgatcagt 780
cttgggggct gcctgggcta cctcctgcct gccattgact gggacaccag tgccctggcc 840
ccctacctgg gcacccagga ggagtgcctc tttggcctgc tcaccctcat cttcctcacc 900
tgcgtagcag ccacactgct ggtggctgag gaggcagcgc tgggccccac cgagccagca 960
gaagggctgt cggccccctc cttgtcgccc cactgctgtc catgccgggc ccgcttggct 1020
ttccggaacc tgggcgccct gcttccccgg ctgcaccagc tgtgctgccg catgccccgc 1080
accctgcgcc ggctcttcgt ggctgagctg tgcagctgga tggcactcat gaccttcacg 1140
ctgttttaca cggatttcgt gggcgagggg ctgtaccagg gcgtgcccag agctgagccg 1200
ggcaccgagg cccggagaca ctatgatgaa ggcgttcgga tgggcagcct ggggctgttc 1260
ctgcagtgcg ccatctccct ggtcttctct ctggtcatgg accggctggt gcagcgattc 1320
ggcactcgag cagtctattt ggccagtgtg gcagctttcc ctgtggctgc cggtgccaca 1380
tgcctgtccc acagtgtggc cgtggtgaca gcttcagccg ccctcaccgg gttcaccttc 1440
tcagccctgc agatcctgcc ctacacactg gcctccctct accaccggga gaagcaggtg 1500
ttcctgccca aataccgagg ggacactgga ggtgctagca gtgaggacag cctgatgacc 1560
agcttcctgc caggccctaa gcctggagct cccttcccta atggacacgt gggtgctgga 1620
ggcagtggcc tgctcccacc tccacccgcg ctctgcgggg cctctgcctg tgatgtctcc 1680
gtacgtgtgg tggtgggtga gcccaccgag gccagggtgg ttccgggccg gggcatctgc 1740
ctggacctcg ccatcctgga tagtgccttc ctgctgtccc aggtggcccc atccctgttt 1800
atgggctcca ttgtccagct cagccagtct gtcactgcct atatggtgtc tgccgcaggc 1860
ctgggtctgg tcgccattta ctttgctaca caggtagtat ttgacaagag cgacttggcc 1920
aaatactcag cgggtggaca ccatcaccat caccattaa 1959
<210>29
<211>507
<212>PRT
<213>人工序列
<220>
<223>与6个组氨酸残基融合的人P501S(氨基酸55-553)
<400>29
Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro Leu
1 5 10 15
Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg Arg
20 25 30
Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe Leu
35 40 45
Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro Arg
50 55 60
Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp Phe
65 70 75 80
Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp Leu
85 90 95
Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala Phe
100 105 110
Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile Asp
115 120 125
Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu Cys
130 135 140
Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala Thr
145 150 155 160
Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala Glu
165 170 175
Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg Ala
180 185 190
Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His Gln
195 200 205
Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala Glu
210 215 220
Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr Asp
225 230 235 240
Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro Gly
245 250 255
Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser Leu
260 265 270
Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val Met
275 280 285
Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala Ser
290 295 300
Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His Ser
305 310 315 320
Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe Ser
325 330 335
Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg Glu
340 345 350
Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala Ser
355 360 365
Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro Gly
370 375 380
Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu Leu
385 390 395 400
Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser Val
405 410 415
Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly Arg
420 425 430
Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu Ser
435 440 445
Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser Gln
450 455 460
Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val Ala
465 470 475 480
Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala Lys
485 490 495
Tyr Ser Ala Gly Gly His His His His His His
500 505
<210>30
<211>1524
<212>DNA
<213>人工序列
<220>
<223>与6个组氨酸残基融合的人P501S(氨基酸55-553)
<400>30
atggtgctgg gcattggtcc agtgctgggc ctggtctgtg tcccgctcct aggctcagcc 60
agtgaccact ggcgtggacg ctatggccgc cgccggccct tcatctgggc actgtccttg 120
ggcatcctgc tgagcctctt tctcatccca agggccggct ggctagcagg gctgctgtgc 180
ccggatccca ggcccctgga gctggcactg ctcatcctgg gcgtggggct gctggacttc 240
tgtggccagg tgtgcttcac tccactggag gccctgctct ctgacctctt ccgggacccg 300
gaccactgtc gccaggccta ctctgtctat gccttcatga tcagtcttgg gggctgcctg 360
ggctacctcc tgcctgccat tgactgggac accagtgccc tggcccccta cctgggcacc 420
caggaggagt gcctctttgg cctgctcacc ctcatcttcc tcacctgcgt agcagccaca 480
ctgctggtgg ctgaggaggc agcgctgggc cccaccgagc cagcagaagg gctgtcggcc 540
ccctccttgt cgccccactg ctgtccatgc cgggcccgct tggctttccg gaacctgggc 600
gccctgcttc cccggctgca ccagctgtgc tgccgcatgc cccgcaccct gcgccggctc 660
ttcgtggctg agctgtgcag ctggatggca ctcatgacct tcacgctgtt ttacacggat 720
ttcgtgggcg aggggctgta ccagggcgtg cccagagctg agccgggcac cgaggcccgg 780
agacactatg atgaaggcgt tcggatgggc agcctggggc tgttcctgca gtgcgccatc 840
tccctggtct tctctctggt catggaccgg ctggtgcagc gattcggcac tcgagcagtc 900
tatttggcca gtgtggcagc tttccctgtg gctgccggtg ccacatgcct gtcccacagt 960
gtggccgtgg tgacagcttc agccgccctc accgggttca ccttctcagc cctgcagatc 1020
ctgccctaca cactggcctc cctctaccac cgggagaagc aggtgttcct gcccaaatac 1080
cgaggggaca ctggaggtgc tagcagtgag gacagcctga tgaccagctt cctgccaggc 1140
cctaagcctg gagctccctt ccctaatgga cacgtgggtg ctggaggcag tggcctgctc 1200
ccacctccac ccgcgctctg cggggcctct gcctgtgatg tctccgtacg tgtggtggtg 1260
ggtgagccca ccgaggccag ggtggttccg ggccggggca tctgcctgga cctcgccatc 1320
ctggatagtg ccttcctgct gtcccaggtg gccccatccc tgtttatggg ctccattgtc 1380
cagctcagcc agtctgtcac tgcctatatg gtgtctgccg caggcctggg tctggtcgcc 1440
atttactttg ctacacaggt agtatttgac aagagcgact tggccaaata ctcagcgggt 1500
ggacaccatc accatcacca ttaa 1524
<210>31
<211>685
<212>PRT
<213>人工序列
<220>
<223>与6个组氨酸残基融合的人P5 01S(氨基酸1-34与氨基酸
55-553融合)
<400>31
Met Ala Ala Val Gln Arg Leu Trp Val Ser Arg Leu Leu Arg His Arg
1 5 10 15
Lys Ala Gln Leu Leu Leu Val Asn Leu Leu Thr Phe Gly Leu Glu Val
20 25 30
Cys Leu Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp
35 40 45
Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly
50 55 60
Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr
65 70 75 80
Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala
85 90 95
Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp
100 105 110
Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala
115 120 125
Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly
130 135 140
Val Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp
145 150 155 160
Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe
165 170 175
Met Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val
180 185 190
Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg
195 200 205
Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu
210 215 220
Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp
225 230 235 240
Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu
245 250 255
Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser
260 265 270
Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr
275 280 285
Ala Phe Met lle Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala
290 295 300
Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu
305 310 315 320
Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala
325 330 335
Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro
340 345 350
Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys
355 360 365
Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu
370 375 380
His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val
385 390 395 400
Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr
405 410 415
Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu
420 425 430
Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly
435 440 445
Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu
450 455 460
Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu
465 470 475 480
Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser
485 490 495
His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr
500 505 510
Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His
515 520 525
Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly
530 535 540
Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys
545 550 555 560
Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly
565 570 575
Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val
580 585 590
Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro
595 600 605
Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu
610 615 620
Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu
625 630 635 640
Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu
645 650 655
Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu
660 665 670
Ala Lys Tyr Ser Ala Gly Gly His His His His His His
675 680 685
<210>32
<211>2058
<212>DNA
<213>人工序列
<220>
<223>编码与6个组氨酸残基融合的人P501S(氨基酸1-34与氨基酸
55-553融合)的DNA
<400>32
atggcggccg tgcagaggct atgggtatcg agactgctaa gacaccgcaa agctcagttg 60
ttgttggtta acttgttgac cttcgggctg gaagtctgtt tggcggccgc ttacgtacat 120
tccgacggct cttatccaaa agacaagttt gagaaaatca atggcacttg gtactacttt 180
gacagttcag gctatatgct tgcagaccgc tggaggaagc acacagacgg caactggtac 240
tggttcgaca actcaggcga aatggctaca ggctggaaga aaatcgctga taagtggtac 300
tatttcaacg aagaaggtgc catgaagaca ggctgggtca agtacaagga cacttggtac 360
tacttagacg ctaaagaagg cgccatgcaa tacatcaagg ctaactctaa gttcattggt 420
atcactgaag gcgtcatggt atcaaatgcc tttatccagt cagcggacgg aacaggctgg 480
tactacctca aaccagacgg aacactggca gacaggccag aaaagttcat gtacatggtg 540
ctgggcattg gtccagtgct gggcctggtc tgtgtcccgc tcctaggctc agccagtgac 600
cactggcgtg gacgctatgg ccgccgccgg cccttcatct gggcactgtc cttgggcatc 660
ctgctgagcc tctttctcat cccaagggcc ggctggctag cagggctgct gtgcccggat 720
cccaggcccc tggagctggc actgctcatc ctgggcgtgg ggctgctgga cttctgtggc 780
caggtgtgct tcactccact ggaggccctg ctctctgacc tcttccggga cccggaccac 840
tgtcgccagg cctactctgt ctatgccttc atgatcagtc ttgggggctg cctgggctac 900
ctcctgcctg ccattgactg ggacaccagt gccctggccc cctacctggg cacccaggag 960
gagtgcctct ttggcctgct caccctcatc ttcctcacct gcgtagcagc cacactgctg 1020
gtggctgagg aggcagcgct gggccccacc gagccagcag aagggctgtc ggccccctcc 1080
ttgtcgcccc actgctgtcc atgccgggcc cgcttggctt tccggaacct gggcgccctg 1140
cttccccggc tgcaccagct gtgctgccgc atgccccgca ccctgcgccg gctcttcgtg 1200
gctgagctgt gcagctggat ggcactcatg accttcacgc tgttttacac ggatttcgtg 1260
ggcgaggggc tgtaccaggg cgtgcccaga gctgagccgg gcaccgaggc ccggagacac 1320
tatgatgaag gcgttcggat gggcagcctg gggctgttcc tgcagtgcgc catctccctg 1380
gtcttctctc tggtcatgga ccggctggtg cagcgattcg gcactcgagc agtctatttg 1440
gccagtgtgg cagctttccc tgtggctgcc ggtgccacat gcctgtccca cagtgtggcc 1500
gtggtgacag cttcagccgc cctcaccggg ttcaccttct cagccctgca gatcctgccc 1560
tacacactgg cctccctcta ccaccgggag aagcaggtgt tcctgcccaa ataccgaggg 1620
gacactggag gtgctagcag tgaggacagc ctgatgacca gcttcctgcc aggccctaag 1680
cctggagctc ccttccctaa tggacacgtg ggtgctggag gcagtggcct gctcccacct 1740
ccacccgcgc tctgcggggc ctctgcctgt gatgtctccg tacgtgtggt ggtgggtgag 1800
cccaccgagg ccagggtggt tccgggccgg ggcatctgcc tggacctcgc catcctggat 1860
agtgccttcc tgctgtccca ggtggcccca tccctgttta tgggctccat tgtccagctc 1920
agccagtctg tcactgccta tatggtgtct gccgcaggcc tgggtctggt cgccatttac 1980
tttgctacac aggtagtatt tgacaagagc gacttggcca aatactcagc gggtggacac 2040
catcaccatc accattaa 2058
<210>33
<211>671
<212>PRT
<213>人工序列
<220>
<223>与酵母α前原信号序列下游的6个组氨酸残基融合的人P501S
(氨基酸55-553)融合的P2 T辅助表位融合的肺炎链球菌
C-LytA部分
<400>33
Met Ala Ala Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala
1 5 10 15
Ser Ser Ala Leu Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro
20 25 30
Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser
35 40 45
Ser Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn
50 55 60
Trp Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys
65 70 75 80
Ile Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr
85 90 95
Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu
100 105 110
Gly Ala Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr
115 120 125
Glu Gly Val Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr
130 135 140
Gly Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu
145 150 155 160
Lys Phe Met Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val
165 170 175
Cys Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr
180 185 190
Gly Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu
195 200 205
Ser Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys
210 215 220
Pro Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly
225 230 235 240
Leu Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu
245 250 255
Leu Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser
260 265 270
Val Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu
275 280 285
Pro Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr
290 295 300
Gln Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys
305 310 315 320
Val Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr
325 330 335
Glu Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys
340 345 350
Pro Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro
355 360 365
Arg Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu
370 375 380
Phe Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu
385 390 395 400
Phe Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg
405 410 415
Ala Glu Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg
420 425 430
Met Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe
435 440 445
Ser Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val
450 455 460
Tyr Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys
465 470 475 480
Leu Ser His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly
485 490 495
Phe Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu
500 505 510
Tyr His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr
515 520 525
Gly Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly
530 535 540
Pro Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly
545 550 555 560
Ser Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys
565 570 575
Asp Val Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val
580 585 590
Val Pro Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala
595 600 605
Phe Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val
610 615 620
Gln Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu
625 630 635 640
Gly Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser
645 650 655
Asp Leu Ala Lys Tyr Ser Ala Gly Gly His His His His His His
660 665 670
<210>34
<211>2477
<212>DNA
<213>人工序列
<220>
<223>编码与酵母α前原信号序列下游的6个组氨酸残基融合的人
P501S(氨基酸55-553)融合的P2 T辅助表位融合的肺炎链
球菌C-LytA部分的DNA
<400>34
tacgtacatt ccgacggctc ttatccaaaa gacaagtttg agaaaatcaa tggcacttgg 60
tactactttg acagttcagg ctatatgctt gcagaccgct ggaggaagca cacagacggc 120
aactggtact ggttcgacaa ctcaggcgaa atggctacag gctggaagaa aatcgctgat 180
aagtggtact atttcaacga agaaggtgcc atgaagacag gctgggtcaa gtacaaggac 240
acttggtact acttagacgc taaagaaggc gccatgcaat acatcaaggc taactctaag 300
ttcattggta tcactgaagg cgtcatggta tcaaatgcct ttatccagtc agcggacgga 360
acaggctggt actacctcaa accagacgga acactggcag acaggccaga aatggcggcc 420
agatttcctt caatttttac tgcagtttta ttcgcagcat cctccgcatt agcggccgct 480
tacgtacatt ccgacggctc ttatccaaaa gacaagtttg agaaaatcaa tggcacttgg 540
tactactttg acagttcagg ctatatgctt gcagaccgct ggaggaagca cacagacggc 600
aactggtact ggttcgacaa ctcaggcgaa atggctacag gctggaagaa aatcgctgat 660
aagtggtact atttcaacga agaaggtgcc atgaagacag gctgggtcaa gtacaaggac 720
acttggtact acttagacgc taaagaaggc gccatgcaat acatcaaggc taactctaag 780
ttcattggta tcactgaagg cgtcatggta tcaaatgcct ttatccagtc agcggacgga 840
acaggctggt actacctcaa accagacgga acactggcag acaggccaga agctggtatt 900
acttacgttc caccattgtt gttggaagtt ggtgttgaag aaaagttcat gtacatggtg 960
ctgggcattg gtccagtgct gggcctggtc tgtgtcccgc tcctaggctc agccagtgac 1020
cactggcgtg gacgctatgg ccgccgccgg cccttcatct gggcactgtc cttgggcatc 1080
ctgctgagcc tctttctcat cccaagggcc ggctggctag cagggctgct gtgcccggat 1140
cccaggcccc tggagctggc actgctcatc ctgggcgtgg ggctgctgga cttctgtggc 1200
caggtgtgct tcactccact ggaggccctg ctctctgacc tcttccggga cccggaccac 1260
tgtcgccagg cctactctgt ctatgcttca tgatcagtct tgggggctgc ctgggctacc 1320
tcctgcctgc cattgactgg gacaccagtg ccctggcccc ctacctgggc acccaggagg 1380
agtgcctctt tggcctgctc accctcatct tcctcacctg cgtagcagcc acactgctgg 1440
tggctgagga ggcagcgctg ggccccaccg agccagcaga agggctgtcg gccccctcct 1500
tgtcgcccca ctgctgtcca tgccgggccc gcttggcttt ccggaacctg ggcgccctgc 1560
ttccccggct gcaccagctg tgctgccgca tgccccgcac cctgcgccgg ctcttcgtgg 1620
ctgagctgtg cagctggatg gcactcatga ccttcacgct gttttacacg gatttcgtgg 1680
gcgaggggct gtaccagggc gtgcccagag ctgagccggg caccgaggcc cggagacact 1740
atgatgaagg cgttcggatg ggcagcctgg ggctgttcct gcagtgcgcc atctccctgg 1800
tcttctctct ggtcatggac cggctggtgc agcgattcgg cactcgagca gtctatttgg 1860
ccagtgtggc agctttccct gtggctgccg gtgccacatg cctgtcccac agtgtggccg 1920
tggtgacagc ttcagccgcc ctcaccgggt tcaccttctc agccctgcag atcctgccct 1980
acacactggc ctccctctac caccgggaga agcaggtgtt cctgcccaaa taccgagggg 2040
acactggagg tgctagcagt gaggacagcc tgatgaccag cttcctgcca ggccctaagc 2100
ctggagctcc cttccctaat ggacacgtgg gtgctggagg cagtggcctg ctcccacctc 2160
cacccgcgct ctgcggggcc tctgcctgtg atgtctccgt acgtgtggtg gtgggtgagc 2220
ccaccgaggc cagggtggtt ccgggccggg gcatctgcct ggacctcgcc atcctggata 2280
gtgccttcct gctgtcccag gtggccccat ccctgtttat gggctccatt gtccagctca 2340
gccagtctgt cactgcctat atggtgtctg ccgcaggcct gggtctggtc gccatttact 2400
ttgctacaca ggtagtattt gacaagagcg acttggccaa atactcagcg ggtggacacc 2460
atcaccatca ccattaa 2477
<210>35
<211>595
<212>PRT
<213>人工序列
<220>
<223>与酵母α前原信号序列下游的6个组氨酸残基融合的人P501S
(氨基酸55-553)
<400>35
Met Ser Phe Leu Asn Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala
1 5 10 15
Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile
20 25 30
Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Lsu Glu Gly Asp Phe Asp
35 40 45
Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe
50 55 60
Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser
65 70 75 80
Leu Glu Lys Arg Glu Ala Glu Ala Met Val Leu Gly Ile Gly Pro Val
85 90 95
Leu Gly Leu Val Cys Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp
100 105 110
Arg Gly Arg Tyr Gly Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu
115 120 125
Gly Ile Leu Leu Ser Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala
130 135 140
Gly Leu Leu Cys Pro Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile
145 150 155 160
Leu Gly Val Gly Leu Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro
165 170 175
Leu Glu Ala Leu Leu Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg
180 185 190
Gln Ala Tyr Ser Val Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu
195 200 205
Gly Tyr Leu Leu Pro Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro
210 215 220
Tyr Leu Gly Thr Gln Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile
225 230 235 240
Phe Leu Thr Cys Val Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala
245 250 255
Leu Gly Pro Thr Glu Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser
260 265 270
Pro His Cys Cys Pro Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly
275 280 285
Ala Leu Leu Pro Arg Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr
290 295 300
Leu Arg Arg Leu Phe Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met
305 310 315 320
Thr Phe Thr Leu Phe Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln
325 330 335
Gly Val Pro Arg Ala Glu Pro Gly Thr Glu Ala Arg Arg His Tyr Asp
340 345 350
Glu Gly Val Arg Met Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile
355 360 365
Ser Leu Val Phe Ser Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly
370 375 380
Thr Arg Ala Val Tyr Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala
385 390 395 400
Gly Ala Thr Cys Leu Ser His Ser Val Ala Val Val Thr Ala Ser Ala
405 410 415
Ala Leu Thr Gly Phe Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr
420 425 430
Leu Ala Ser Leu Tyr His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr
435 440 445
Arg Gly Asp Thr Gly Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser
450 455 460
Phe Leu Pro Gly Pro Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val
465 470 475 480
Gly Ala Gly Gly Ser Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly
485 490 495
Ala Ser Ala Cys Asp Val Ser Val Arg Val Val Val Gly Glu Pro Thr
500 505 510
Glu Ala Arg Val Val Pro Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile
515 520 525
Leu Asp Ser Ala Phe Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met
530 535 540
Gly Ser Ile Val Gln Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser
545 550 555 560
Ala Ala Gly Leu Gly Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val
565 570 575
Phe Asp Lys Ser Asp Leu Ala Lys Tyr Ser Ala Gly Gly His His His
580 585 590
His His His
595
<210>36
<211>1788
<212>DNA
<213>人工序列
<220>
<223>编码与酵母α前原信号序列下游的6个组氨酸残基融合的人
P501S(氨基酸55-553)的DNA
<400>36
atgagtttcc tcaattttac tgcagtttta ttcgcagcat cctccgcatt agctgctcca 60
gtcaacacta caacagaaga tgaaacggca caaattccgg ctgaagctgt catcggttac 120
tcagatttag aaggggattt cgatgttgct gttttgccat tttccaacag cacaaataac 180
gggttattgt ttataaatac tactattgcc agcattgctg ctaaagaaga aggggtatct 240
ctcgagaaaa gagaggctga agccatggtg ctgggcattg gtccagtgct gggcctggtc 300
tgtgtcccgc tcctaggctc agccagtgac cactggcgtg gacgctatgg ccgccgccgg 360
cccttcatct gggcactgtc cttgggcatc ctgctgagcc tctttctcat cccaagggcc 420
ggctggctag cagggctgct gtgcccggat cccaggcccc tggagctggc actgctcatc 480
ctgggcgtgg ggctgctgga cttctgtggc caggtgtgct tcactccact ggaggccctg 540
ctctctgacc tcttccggga cccggaccac tgtcgccagg cctactctgt ctatgccttc 600
atgatcagtc ttgggggctg cctgggctac ctcctgcctg ccattgactg ggacaccagt 660
gccctggccc cctacctggg cacccaggag gagtgcctct ttggcctgct caccctcatc 720
ttcctcacct gcgtagcagc cacactgctg gtggctgagg aggcagcgct gggccccacc 780
gagccagcag aagggctgtc ggccccctcc ttgtcgcccc actgctgtcc atgccgggcc 840
cgcttggctt tccggaacct gggcgccctg cttccccggc tgcaccagct gtgctgccgc 900
atgccccgca ccctgcgccg gctcttcgtg gctgagctgt gcagctggat ggcactcatg 960
accttcacgc tgttttacac ggatttcgtg ggcgaggggc tgtaccaggg cgtgcccaga 1020
gctgagccgg gcaccgaggc ccggagacac tatgatgaag gcgttcggat gggcagcctg 1080
gggctgttcc tgcagtgcgc catctccctg gtcttctctc tggtcatgga ccggctggtg 1140
cagcgattcg gcactcgagc agtctatttg gccagtgtgg cagctttccc tgtggctgcc 1200
ggtgccacat gcctgtccca cagtgtggcc gtggtgacag cttcagccgc cctcaccggg 1260
ttcaccttct cagccctgca gatcctgccc tacacactgg cctccctcta ccaccgggag 1320
aagcaggtgt tcctgcccaa ataccgaggg gacactggag gtgctagcag tgaggacagc 1380
ctgatgacca gcttcctgcc aggccctaag cctggagctc ccttccctaa tggacacgtg 1440
ggtgctggag gcagtggcct gctcccacct ccacccgcgc tctgcggggc ctctgcctgt 1500
gatgtctccg tacgtgtggt ggtgggtgag cccaccgagg ccagggtggt tccgggccgg 1560
ggcatctgcc tggacctcgc catcctggat agtgccttcc tgctgtccca ggtggcccca 1620
tccctgttta tgggctccat tgtccagctc agccagtctg tcactgccta tatggtgtct 1680
gccgcaggcc tgggtctggt cgccatttac tttgctacac aggtagtatt tgacaagagc 1740
gacttggcca aatactcagc gggtggacac catcaccatc accattaa 1788
<210>37
<211>1955
<212>DNA
<213>人工序列
<220>
<223>编码与肺炎链球菌C-LytA P2辅助表位C-Lyta融合的密码子
优化的人P501S(氨基酸51-553)的DNA
<400>37
gcggccgcgc caccatggcc gccgcctacg tgcatagcga cgggagctac cccaaggaca 60
agttcgagaa gatcaacggg acatggtact acttcgactc ctccggctac atgctcgccg 120
accgctggcg gaagcacacc gacggcaact ggtactggtt cgataactcg ggagagatgg 180
ccaccggctg gaagaagatc gcggacaagt ggtactattt caacgaggag ggcgccatga 240
agaccggctg ggtgaagtat aaggacacct ggtactacct cgacgccaag gagggcgcca 300
tgcagtatat caaggccaac agcaagttca tcggcatcac cgagggagtg atggtcagca 360
acgcctttat ccagagcgcc gacggcaccg gatggtacta cttgaagccg gacggcaccc 420
tcgcggatcg gcccgagaag ttcatgtaca tggtgctggg catcggcccc gtcctgggcc 480
tcgtgtgtgt gcccctcctc gggagtgcgt ccgatcattg gcggggccgc tacggccgcc 540
gcagaccgtt catctgggcc ctgagcctgg gcatcctgct ctctctcttc ctgatccccc 600
gggccggctg gctggccggc ctgctgtgtc ccgacccccg ccctctggag ctggccctcc 660
tgatcctggg cgtgggcctg ctggacttct gcggccaggt gtgtttcact cccctggagg 720
ctctgctctc cgacctcttc cgcgaccccg accactgtag gcaggcttac agcgtgtacg 780
ccttcatgat cagtctgggg ggatgcctgg gctatctgct gcccgctatc gactgggaca 840
ccagcgccct ggccccctac ctggggactc aggaggagtg cctgttcggc ctgctcacct 900
tgatcttcct gacgtgcgtc gccgccaccc tgctggtggc cgaggaggcg gccctggggc 960
ccaccgagcc cgccgagggc ctgagcgctc ccagcctgag cccccattgc tgcccgtgca 1020
gggctaggct cgccttcagg aatctgggcg ctttgctgcc ccgcctgcat cagctgtgct 1080
gtcgcatgcc tcgcaccctg cgccgcctgt tcgtcgctga gctctgttcc tggatggccc 1140
tgatgacgtt caccctcttc tacaccgact tcgtggggga gggcctgtac cagggcgtgc 1200
ccagggccga gcccggcacc gaggctaggc gccattacga cgagggcgtc aggatgggct 1260
ctctgggcct cttcctgcag tgcgccatca gtctggtgtt ctctctggtg atggaccggc 1320
tggtgcagcg cttcggcacc cgggccgtgt acctcgcctc tgtggcggct ttccccgtcg 1380
ccgccggcgc gacctgcctg tctcattctg tcgccgtggt gaccgccagc gccgccctga 1440
ccggcttcac cttcagtgcg ctccagattc tgccctacac cctggcgtct ctgtaccatc 1500
gcgagaagca ggtgttcctg cccaagtacc gcggggacac agggggagct tcctctgagg 1560
acagcctgat gaccagcttc ttgcccggcc ccaagccggg ggcccctttc cccaacggcc 1620
atgtcggggc gggcggcagc ggcctgctcc ctcccccccc cgccctgtgc ggcgctagtg 1680
cctgcgacgt gagcgtgcgg gtggtggtgg gggagcccac cgaggctagg gtcgtgcctg 1740
gccgggggat ctgcctggac ctggccatcc tcgactccgc cttcctgctc tcccaggtgg 1800
cgcccagcct gttcatgggc agtatcgtgc agctgagcca gagcgtgacc gcctacatgg 1860
tgagcgccgc cggcctgggg ttggtggcca tctactttgc cacccaggtc gtgttcgaca 1920
agagcgatct cgccaagtat agcgcctgag gatcc 1955
<210>38
<211>2045
<212>DNA
<213>人工序列
<220>
<223>编码与肺炎链球菌C-LytA P2辅助表位C-Lyta融合的密码子
优化的人P501S(氨基酸1-553)的DNA
<400>38
gcggccgcgc caccatggcc gccgcctacg tgcatagcga cgggagctac cccaaggaca 60
agttcgagaa gatcaacggg acatggtact acttcgactc ctccggctac atgctcgccg 120
accgctggcg gaagcacacc gacggcaact ggtactggtt cgataactcg ggagagatgg 180
ccaccggctg gaagaagatc gcggacaagt ggtactattt caacgaggag ggcgccatga 240
agaccggctg ggtgaagtat aaggacacct ggtactacct cgacgccaag gagggcgcca 300
tgcagtatat caaggccaac agcaagttca tcggcatcac cgagggagtg atggtcagca 360
acgcctttat ccagagcgcc gacggcaccg gatggtacta cttgaagccg gacggcaccc 420
tcgcggatcg gcccgagatg gtgcagcggc tgtgggtgtc ccggctgctg cgccatagaa 480
aggcccagtt gctgctggtg aacctgctga ctttcggact ggaggtgtgc ctggctgccg 540
tggtgctggg catcggcccc gtcctgggcc tcgtgtgtgt gcccctcctc gggagtgcgt 600
ccgatcattg gcggggccgc tacggccgcc gcagaccgtt catctgggcc ctgagcctgg 660
gcatcctgct ctctctcttc ctgatccccc gggccggctg gctggccggc ctgctgtgtc 720
ccgacccccg ccctctggag ctggccctcc tgatcctggg cgtgggcctg ctggacttct 780
gcggccaggt gtgtttcact cccctggagg ctctgctctc cgacctcttc cgcgaccccg 840
accactgtag gcaggcttac agcgtgtacg ccttcatgat cagtctgggg ggatgcctgg 900
gctatctgct gcccgctatc gactgggaca ccagcgccct ggccccctac ctggggactc 960
aggaggagtg cctgttcggc ctgctcacct tgatcttcct gacgtgcgtc gccgccaccc 1020
tgctggtggc cgaggaggcg gccctggggc ccaccgagcc cgccgagggc ctgagcgctc 1080
ccagcctgag cccccattgc tgcccgtgca gggctaggct cgccttcagg aatctgggcg 1140
ctttgctgcc ccgcctgcat cagctgtgct gtcgcatgcc tcgcaccctg cgccgcctgt 1200
tcgtcgctga gctctgttcc tggatggccc tgatgacgtt caccctcttc tacaccgact 1260
tcgtggggga gggcctgtac cagggcgtgc ccagggccga gcccggcacc gaggctaggc 1320
gccattacga cgagggcgtc aggatgggct ctctgggcct cttcctgcag tgcgccatca 1380
gtctggtgtt ctctctggtg atggaccggc tggtgcagcg cttcggcacc cgggccgtgt 1440
acctcgcctc tgtggcggct ttccccgtcg ccgccggcgc gacctgcctg tctcattctg 1500
tcgccgtggt gaccgccagc gccgccctga ccggcttcac cttcagtgcg ctccagattc 1560
tgccctacac cctggcgtct ctgtaccatc gcgagaagca ggtgttcctg cccaagtacc 1620
gcggggacac agggggagct tcctctgagg acagcctgat gaccagcttc ttgcccggcc 1680
ccaagccggg ggcccctttc cccaacggcc atgtcggggc gggcggcagc ggcctgctcc 1740
ctcccccccc cgccctgtgc ggcgctagtg cctgcgacgt gagcgtgcgg gtggtggtgg 1800
gggagcccac cgaggctagg gtcgtgcctg gccgggggat ctgcctggac ctggccatcc 1860
tcgactccgc cttcctgctc tcccaggtgg cgcccagcct gttcatgggc agtatcgtgc 1920
agctgagcca gagcgtgacc gcctacatgg tgagcgccgc cggcctgggg ttggtggcca 1980
tctactttgc cacccaggtc gtgttcgaca agagcgatct cgccaagtat agcgcctgag 2040
gatcc 2045
<210>39
<211>2105
<212>DNA
<213>人工序列
<220>
<223>编码与人P501S(氨基酸1-50)融合的人P501S(氨基酸51-
553)融合的肺炎链球菌C-LytA P2辅助表位C-Lyta的DNA-
优化的密码子
<400>39
gcggccgcgc caccatggcc gccgcctacg tgcatagcga cgggagctac cccaaggaca 60
agttcgagaa gatcaacggg acatggtact acttcgactc ctccggctac atgctcgccg 120
accgctggcg gaagcacacc gacggcaact ggtactggtt cgataactcg ggagagatgg 180
ccaccggctg gaagaagatc gcggacaagt ggtactattt caacgaggag ggcgccatga 240
agaccggctg ggtgaagtat aaggacacct ggtactacct cgacgccaag gagggcgcca 300
tgcagtatat caaggccaac agcaagttca tcggcatcac cgagggagtg atggtcagca 360
acgcctttat ccagagcgcc gacggcaccg gatggtacta cttgaagccg gacggcaccc 420
tcgcggatcg gcccgagaag ttcatgtaca tggtgctggg catcggcccc gtcctgggcc 480
tcgtgtgtgt gcccctcctc gggagtgcgt ccgatcattg gcggggccgc tacggccgcc 540
gcagaccgtt catctgggcc ctgagcctgg gcatcctgct ctctctcttc ctgatccccc 600
gggccggctg gctggccggc ctgctgtgtc ccgacccccg ccctctggag ctggccctcc 660
tgatcctggg cgtgggcctg ctggacttct gcggccaggt gtgtttcact cccctggagg 720
ctctgctctc cgacctcttc cgcgaccccg accactgtag gcaggcttac agcgtgtacg 780
ccttcatgat cagtctgggg ggatgcctgg gctatctgct gcccgctatc gactgggaca 840
ccagcgccct ggccccctac ctggggactc aggaggagtg cctgttcggc ctgctcacct 900
tgatcttcct gacgtgcgtc gccgccaccc tgctggtggc cgaggaggcg gccctggggc 960
ccaccgagcc cgccgagggc ctgagcgctc ccagcctgag cccccattgc tgcccgtgca 1020
gggctaggct cgccttcagg aatctgggcg ctttgctgcc ccgcctgcat cagctgtgct 1080
gtcgcatgcc tcgcaccctg cgccgcctgt tcgtcgctga gctctgttcc tggatggccc 1140
tgatgacgtt caccctcttc tacaccgact tcgtggggga gggcctgtac cagggcgtgc 1200
ccagggccga gcccggcacc gaggctaggc gccattacga cgagggcgtc aggatgggct 1260
ctctgggcct cttcctgcag tgcgccatca gtctggtgtt ctctctggtg atggaccggc 1320
tggtgcagcg cttcggcacc cgggccgtgt acctcgcctc tgtggcggct ttccccgtcg 1380
ccgccggcgc gacctgcctg tctcattctg tcgccgtggt gaccgccagc gccgccctga 1440
ccggcttcac cttcagtgcg ctccagattc tgccctacac cctggcgtct ctgtaccatc 1500
gcgagaagca ggtgttcctg cccaagtacc gcggggacac agggggagct tcctctgagg 1560
acagcctgat gaccagcttc ttgcccggcc ccaagccggg ggcccctttc cccaacggcc 1620
atgtcggggc gggcggcagc ggcctgctcc ctcccccccc cgccctgtgc ggcgctagtg 1680
cctgcgacgt gagcgtgcgg gtggtggtgg gggagcccac cgaggctagg gtcgtgcctg 1740
gccgggggat ctgcctggac ctggccatcc tcgactccgc cttcctgctc tcccaggtgg 1800
cgcccagcct gttcatgggc agtatcgtgc agctgagcca gagcgtgacc gcctacatgg 1860
tgagcgccgc cggcctgggg ttggtggcca tctactttgc cacccaggtc gtgttcgaca 1920
agagcgatct cgccaagtat agcgccatgg tgcagcggct gtgggtgtcc cggctgctgc 1980
gccatagaaa ggcccagttg ctgctggtga acctgctgac tttcggactg gaggtgtgcc 2040
tggctgccgg gatcacgtac gtgccccccc tgctgctgga ggtgggcgtg gaggagtgag 2100
gatcc 2105
<210>40
<211>2105
<212>DNA
<213>人工序列
<220>
<223>编码与人P501S(氨基酸51-553)融合的肺炎链球菌C-LytA
P2辅助表位C-Lyta融合的人P501S(氨基酸1-50)的DNA-
优化的密码子
<400>40
gcggccgcgc caccatggtg cagcggctgt gggtgtcccg gctgctgcgc catagaaagg 60
cccagttgct gctggtgaac ctgctgactt tcggactgga ggtgtgcctg gctgccggga 120
tcacgtacgt gccccccctg ctgctggagg tgggcgtgga ggagatggcc gccgcctacg 180
tgcatagcga cgggagctac cccaaggaca agttcgagaa gatcaacggg acatggtact 240
acttcgactc ctccggctac atgctcgccg accgctggcg gaagcacacc gacggcaact 300
ggtactggtt cgataactcg ggagagatgg ccaccggctg gaagaagatc gcggacaagt 360
ggtactattt caacgaggag ggcgccatga agaccggctg ggtgaagtat aaggacacct 420
ggtactacct cgacgccaag gagggcgcca tgcagtatat caaggccaac agcaagttca 480
tcggcatcac cgagggagtg atggtcagca acgcctttat ccagagcgcc gacggcaccg 540
gatggtacta cttgaagccg gacggcaccc tcgcggatcg gcccgagaag ttcatgtaca 600
tggtgctggg catcggcccc gtcctgggcc tcgtgtgtgt gcccctcctc gggagtgcgt 660
ccgatcattg gcggggccgc tacggccgcc gcagaccgtt catctgggcc ctgagcctgg 720
gcatcctgct ctctctcttc ctgatccccc gggccggctg gctggccggc ctgctgtgtc 780
ccgacccccg ccctctggag ctggccctcc tgatcctggg cgtgggcctg ctggacttct 840
gcggccaggt gtgtttcact cccctggagg ctctgctctc cgacctcttc cgcgaccccg 900
accactgtag gcaggcttac agcgtgtacg ccttcatgat cagtctgggg ggatgcctgg 960
gctatctgct gcccgctatc gactgggaca ccagcgccct ggccccctac ctggggactc 1020
aggaggagtg cctgttcggc ctgctcacct tgatcttcct gacgtgcgtc gccgccaccc 1080
tgctggtggc cgaggaggcg gccctggggc ccaccgagcc cgccgagggc ctgagcgctc 1140
ccagcctgag cccccattgc tgcccgtgca gggctaggct cgccttcagg aatctgggcg 1200
ctttgctgcc ccgcctgcat cagctgtgct gtcgcatgcc tcgcaccctg cgccgcctgt 1260
tcgtcgctga gctctgttcc tggatggccc tgatgacgtt caccctcttc tacaccgact 1320
tcgtggggga gggcctgtac cagggcgtgc ccagggccga gcccggcacc gaggctaggc 1380
gccattacga cgagggcgtc aggatgggct ctctgggcct cttcctgcag tgcgccatca 1440
gtctggtgtt ctctctggtg atggaccggc tggtgcagcg cttcggcacc cgggccgtgt 1500
acctcgcctc tgtggcggct ttccccgtcg ccgccggcgc gacctgcctg tctcattctg 1560
tcgccgtggt gaccgccagc gccgccctga ccggcttcac cttcagtgcg ctccagattc 1620
tgccctacac cctggcgtct ctgtaccatc gcgagaagca ggtgttcctg cccaagtacc 1680
gcggggacac agggggagct tcctctgagg acagcctgat gaccagcttc ttgcccggcc 1740
ccaagccggg ggcccctttc cccaacggcc atgtcggggc gggcggcagc ggcctgctcc 1800
ctcccccccc cgccctgtgc ggcgctagtg cctgcgacgt gagcgtgcgg gtggtggtgg 1860
gggagcccac cgaggctagg gtcgtgcctg gccgggggat ctgcctggac ctggccatcc 1920
tcgactccgc cttcctgctc tcccaggtgg cgcccagcct gttcatgggc agtatcgtgc 1980
agctgagcca gagcgtgacc gcctacatgg tgagcgccgc cggcctgggg ttggtggcca 2040
tctactttgc cacccaggtc gtgttcgaca agagcgatct cgccaagtat agcgcctgag 2100
gatcc 2105
<210>41
<211>652
<212>PRT
<213>人工序列
<220>
<223>与人P501S融合的肺炎链球菌C-LytA P2辅助表位C-Lyta
<400>41
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe Met
130 135 140
Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro
145 150 155 160
Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg
165 170 175
Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe
180 185 190
Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro
195 200 205
Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp
210 215 220
Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp
225 230 235 240
Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala
245 250 255
Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile
260 265 270
Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu
275 280 285
Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala
290 295 300
Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala
305 310 315 320
Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg
325 330 335
Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His
340 345 350
Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala
355 360 365
Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr
370 375 380
Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro
385 390 395 400
Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser
405 410 415
Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val
420 425 430
Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala
435 440 445
Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His
450 455 460
Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe
465 470 475 480
Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg
485 490 495
Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala
500 505 510
Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro
515 520 525
Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu
530 535 540
Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser
545 550 555 560
Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly
565 570 575
Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu
580 585 590
Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser
595 600 605
Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val
610 615 620
Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala
625 630 635 640
Lys Tyr Ser Ala Gly Gly His His His His His His
645 650
<210>42
<211>1959
<212>DNA
<213>人工序列
<220>
<223>编码与人P501S融合的肺炎链球菌C-LytA P2辅助表位C-Lyta
的DNA(加his标签)
<400>42
atggcggccg cttacgtaca ttccgacggc tcttatccaa aagacaagtt tgagaaaatc 60
aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120
cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180
aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240
aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatgca atacatcaag 300
gctaactcta agttcattgg tatcactgaa ggcgtcatgg tatcaaatgc ctttatccag 360
tcagcggacg gaacaggctg gtactacctc aaaccagacg gaacactggc agacaggcca 420
gaaaagttca tgtacatggt gctgggcatt ggtccagtgc tgggcctggt ctgtgtcccg 480
ctcctaggct cagccagtga ccactggcgt ggacgctatg gccgccgccg gcccttcatc 540
tgggcactgt ccttgggcat cctgctgagc ctctttctca tcccaagggc cggctggcta 600
gcagggctgc tgtgcccgga tcccaggccc ctggagctgg cactgctcat cctgggcgtg 660
gggctgctgg acttctgtgg ccaggtgtgc ttcactccac tggaggccct gctctctgac 720
ctcttccggg acccggacca ctgtcgccag gcctactctg tctatgcctt catgatcagt 780
cttgggggct gcctgggcta cctcctgcct gccattgact gggacaccag tgccctggcc 840
ccctacctgg gcacccagga ggagtgcctc tttggcctgc tcaccctcat cttcctcacc 900
tgcgtagcag ccacactgct ggtggctgag gaggcagcgc tgggccccac cgagccagca 960
gaagggctgt cggccccctc cttgtcgccc cactgctgtc catgccgggc ccgcttggct 1020
ttccggaacc tgggcgccct gcttccccgg ctgcaccagc tgtgctgccg catgccccgc 1080
accctgcgcc ggctcttcgt ggctgagctg tgcagctgga tggcactcat gaccttcacg 1140
ctgttttaca cggatttcgt gggcgagggg ctgtaccagg gcgtgcccag agctgagccg 1200
ggcaccgagg cccggagaca ctatgatgaa ggcgttcgga tgggcagcct ggggctgttc 1260
ctgcagtgcg ccatctccct ggtcttctct ctggtcatgg accggctggt gcagcgattc 1320
ggcactcgag cagtctattt ggccagtgtg gcagctttcc ctgtggctgc cggtgccaca 1380
tgcctgtccc acagtgtggc cgtggtgaca gcttcagccg ccctcaccgg gttcaccttc 1440
tcagccctgc agatcctgcc ctacacactg gcctccctct accaccggga gaagcaggtg 1500
ttcctgccca aataccgagg ggacactgga ggtgctagca gtgaggacag cctgatgacc 1560
agcttcctgc caggccctaa gcctggagct cccttcccta atggacacgt gggtgctgga 1620
ggcagtggcc tgctcccacc tccacccgcg ctctgcgggg cctctgcctg tgatgtctcc 1680
gtacgtgtgg tggtgggtga gcccaccgag gccagggtgg ttccgggccg gggcatctgc 1740
ctggacctcg ccatcctgga tagtgccttc ctgctgtccc aggtggcccc atccctgttt 1800
atgggctcca ttgtccagct cagccagtct gtcactgcct atatggtgtc tgccgcaggc 1860
ctgggtctgg tcgccattta ctttgctaca caggtagtat ttgacaagag cgacttggcc 1920
aaatactcag cgggtggaca ccatcaccat caccattaa 1959
<210>43
<211>553
<212>PRT
<213>人类
<400>43
Met Val Gln Arg Leu Trp Val Ser Arg Leu Leu Arg His Arg Lys Ala
1 5 10 15
Gln Leu Leu Leu Val Asn Leu Leu Thr Phe Gly Leu Glu Val Cys Leu
20 25 30
Ala Ala Gly Ile Thr Tyr Val Pro Pro Leu Leu Leu Glu Val Gly Val
35 40 45
Glu Glu Lys Phe Met Thr Met Val Leu Gly Ile Gly Pro Val Leu Gly
50 55 60
Leu Val Cys Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly
65 70 75 80
Arg Tyr Gly Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile
85 90 95
Leu Leu Ser Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu
100 105 110
Leu Cys Pro Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly
115 120 125
Val Gly Leu Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu
130 135 140
Ala Leu Leu Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala
145 150 155 160
Tyr Ser Val Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr
165 170 175
Leu Leu Pro Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu
180 185 190
Gly Thr Gln Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu
195 200 205
Thr Cys Val Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly
210 215 220
Pro Thr Glu Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His
225 230 235 240
Cys Cys Pro Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu
245 250 255
Leu Pro Arg Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg
260 265 270
Arg Leu Phe Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe
275 280 285
Thr Leu Phe Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val
290 295 300
Pro Arg Ala Glu Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly
305 310 315 320
Val Arg Met Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu
325 330 335
Val Phe Ser Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg
340 345 350
Ala Val Tyr Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala
355 360 365
Thr Cys Leu Ser His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu
370 375 380
Thr Gly Phe Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala
385 390 395 400
Ser Leu Tyr His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly
405 410 415
Asp Thr Gly Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu
420 425 430
Pro Gly Pro Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala
435 440 445
Gly Gly Ser Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser
450 455 460
Ala Cys Asp Val Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala
465 470 475 480
Arg Val Val Pro Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp
485 490 495
Ser Ala Phe Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser
500 505 510
Ile Val Gln Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala
515 520 525
Gly Leu Gly Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp
530 535 540
Lys Ser Asp Leu Ala Lys Tyr Ser Ala
545 550
<210>44
<211>644
<212>PRT
<213>人工序列
<220>
<223>与人P501S融合的肺炎链球菌C-LytA P2辅助表位C-Lyta
<400>44
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe Met
130 135 140
Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro
145 150 155 160
Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg
165 170 175
Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe
180 185 190
Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro
195 200 205
Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp
210 215 220
Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp
225 230 235 240
Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala
245 250 255
Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile
260 265 270
Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu
275 280 285
Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala
290 295 300
Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala
305 310 315 320
Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg
325 330 335
Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His
340 345 350
Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala
355 360 365
Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr
370 375 380
Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro
385 390 395 400
Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser
405 410 415
Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val
420 425 430
Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala
435 440 445
Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His
450 455 460
Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe
465 470 475 480
Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg
485 490 495
Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala
500 505 510
Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro
515 520 525
Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Hly Leu
530 535 540
Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser
545 550 555 560
Val Arg Val Val Val Gly Glu Pro Thr Glu Als Arg Val Val Pro Gly
565 570 575
Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu
580 585 590
Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser
595 600 605
Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val
610 615 620
Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala
625 630 635 640
Lys Tyr Ser Ala
<210>45
<211>644
<212>PRT
<213>人工序列
<220>
<223>融合于人P501S(氨基酸51-553)的肺炎链球菌C-LytA P2
辅助表位C-Lyta的密码子优化的杂交蛋白
<400>45
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe Met
130 135 140
Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro
145 150 155 160
Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg
165 170 175
Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe
180 185 190
Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro
195 200 205
Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp
210 215 220
Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp
225 230 235 240
Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala
245 250 255
Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile
260 265 270
Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu
275 280 285
Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala
290 295 300
Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala
305 310 315 320
Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg
325 330 335
Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His
340 345 350
Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala
355 360 365
Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr
370 375 380
Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro
385 390 395 400
Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser
405 410 415
Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val
420 425 430
Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala
435 440 445
Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His
450 455 460
Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe
465 470 475 480
Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg
485 490 495
Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala
500 505 510
Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro
515 520 525
Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu
530 535 540
Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser
545 550 555 560
Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly
565 570 575
Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu
580 585 590
Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser
595 600 605
Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val
610 615 620
Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala
625 630 635 640
Lys Tyr Ser Ala
<210>46
<211>694
<212>PRT
<213>人工序列
<220>
<223>与人P501S(氨基酸1-553)融合的肺炎链球菌C-LytA P2
辅助表位C-Lyta-优化的密码子
<400>46
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Met Val Gln
130 135 140
Arg Leu Trp Val Ser Arg Leu Leu Arg His Arg Lys Ala Gln Leu Leu
145 150 155 160
Leu Val Asn Leu Leu Thr Phe Gly Leu Glu Val Cys Leu Ala Ala Gly
165 170 175
Ile Thr Tyr Val Pro Pro Leu Leu Leu Glu Val Gly Val Glu Glu Lys
180 185 190
Phe Met Thr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys
195 200 205
Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly
210 215 220
Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser
225 230 235 240
Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro
245 250 255
Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu
260 265 270
Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu
275 280 285
Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val
290 295 300
Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro
305 310 315 320
Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln
325 330 335
Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val
340 345 350
Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu
355 360 365
Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro
370 375 380
Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg
385 390 395 400
Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe
405 410 415
Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe
420 425 430
Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala
435 440 445
Glu Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met
450 455 460
Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser
465 470 475 480
Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr
485 490 495
Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu
500 505 510
Ser His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe
515 520 525
Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr
530 535 540
His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly
545 550 555 560
Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro
565 570 575
Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser
580 585 590
Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp
595 600 605
Val Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val
610 615 620
Pro Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe
625 630 635 640
Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln
645 650 655
Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly
660 665 670
Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lya Ser Asp
675 680 685
Leu Ala Lys Tyr Ser Ala
690
<210>47
<211>694
<212>PRT
<213>人工序列
<220>
<223>与人P50lS(氨基酸1-50)融合的人P50lS(氨基酸51
-553)融合的肺炎链球菌C-LytA P2辅助表位C-Lyta-
优化的密码子
<400>47
Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr
20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
35 40 45
Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp
50 55 60
Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met
85 90 95
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Gly Val
100 105 110
Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr
115 120 125
Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys Phe Met
130 135 140
Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro
145 150 155 160
Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg
165 170 175
Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe
180 185 190
Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro
195 200 205
Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp
210 215 220
Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp
225 230 235 240
Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala
245 250 255
Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile
260 265 270
Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu
275 280 285
Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala
290 295 300
Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala
305 310 315 320
Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg
325 330 335
Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His
340 345 350
Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala
355 360 365
Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr
370 375 380
Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala Glu Pro
385 390 395 400
Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser
405 410 415
Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val
420 425 430
Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala
435 440 445
Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His
450 455 460
Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe
465 470 475 480
Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg
485 490 495
Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala
500 505 510
Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro
515 520 525
Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu
530 535 540
Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser
545 550 555 560
Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly
565 570 575
Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu
580 585 590
Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser
595 600 605
Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val
610 615 620
Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala
625 630 635 640
Lys Tyr Ser Ala Met Val Gln Arg Leu Trp Val Ser Arg Leu Leu Arg
645 650 655
His Arg Lys Ala Gln Leu Leu Leu Val Asn Leu Leu Thr Phe Gly Leu
660 665 670
Glu Val Cys Leu Ala Ala Gly Ile Thr Tyr Val Pro Pro Leu Leu Leu
675 680 685
Glu Val Gly Val Glu Glu
690
<210>48
<211>694
<212>PRT
<213>人工序列
<220>
<223>与人P501S(氨基酸51-553)融合的肺炎链球菌C-LytA P2
辅助表位C-Lyta融合的人P501S(氨基酸1-50)-优化的密码子
<400>48
Met Val Gln Arg Leu Trp Val Ser Arg Leu Leu Arg His Arg Lys Ala
1 5 10 15
Gln Leu Leu Leu Val Asn Leu Leu Thr Phe Gly Leu Glu Val Cys Leu
20 25 30
Ala Ala Gly Ile Thr Tyr Val Pro Pro Leu Leu Leu Glu Val Gly Val
35 40 45
Glu Glu Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro Lys
50 55 60
Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser
65 70 75 80
Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp
85 90 95
Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile
100 105 110
Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly
115 120 125
Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly
130 135 140
Ala Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu
145 150 155 160
Gly Val Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly
165 170 175
Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Lys
180 185 190
Phe Met Tyr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys
195 200 205
Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly
210 215 220
Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser
225 230 235 240
Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro
245 250 255
Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu
260 265 270
Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu
275 280 285
Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val
290 295 300
Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro
305 310 315 320
Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln
325 330 335
Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val
340 345 350
Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu
355 360 365
Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro
370 375 380
Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg
385 390 395 400
Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe
405 410 415
Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe
420 425 430
Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala
435 440 445
Glu Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met
450 455 460
Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser
465 470 475 480
Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr
485 490 495
Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu
500 505 510
Ser His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe
515 520 525
Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr
530 535 540
His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly
545 550 555 560
Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro
565 570 575
Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser
580 585 590
Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp
595 600 605
Val Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala Arg Val Val
610 615 620
Pro Gly Arg Gly Ile Cys Leu Asp Leu Ala Ile Leu Asp Ser Ala Phe
625 630 635 640
Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln
645 650 655
Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly
660 665 670
Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp
675 680 685
Leu Ala Lys Tyr Ser Ala
690
<210>49
<211>1971
<212>DNA
<213>人工序列
<220>
<223>编码与肺炎链球菌C-LytA P2辅助表位C-Lyta融合的人
MUC-1的DNA
<400>49
atgacaccgg gcacccagtc tcctttcttc ctgctgctgc tcctcacagt gcttacagtt 60
gttacaggtt ctggtcatgc aagctctacc ccaggtggag aaaaggagac ttcggctacc 120
cagagaagtt cagtgcccag ctctactgag aagaatgctg tgagtatgac cagcagcgta 180
ctctccagcc acagccccgg ttcaggctcc tccaccactc agggacagga tgtcactctg 240
gccccggcca cggaaccagc ttcaggttca gctgccacct ggggacagga tgtcacctcg 300
gtcccagtca ccaggccagc cctgggctcc accaccccgc cagcccacga tgtcacctca 360
gccccggaca acaagccagc cccgggctcc accgcccccc cagcccacgg tgtcacctcg 420
gccccggaca ccaggccgcc cccgggctcc accgcccccc cagcccacgg tgtcacctcg 480
gccccggaca ccaggccgcc cccgggctcc accgcgcccg cagcccacgg tgtcacctcg 540
gccccggaca ccaggccggc cccgggctcc accgcccccc cagcccatgg tgtcacctcg 600
gccccggaca acaggcccgc cttggcgtcc accgcccctc cagtccacaa tgtcacctcg 660
gcctcaggct ctgcatcagg ctcagcttct actctggtgc acaacggcac ctctgccagg 720
gctaccacaa ccccagccag caagagcact ccattctcaa ttcccagcca ccactctgat 780
actcctacca cccttgccag ccatagcacc aagactgatg ccagtagcac tcaccatagc 840
acggtacctc ctctcacctc ctccaatcac agcacttctc cccagttgtc tactggggtc 900
tctttctttt tcctgtcttt tcacatttca aacctccagt ttaattcctc tctggaagat 960
cccagcaccg actactacca agagctgcag agagacattt ctgaaatgtt tttgcagatt 1020
tataaecaag ggggttttct gggcctctcc aatattaagt tcaggccagg atctgtggtg 1080
gtacaattga ctctggcctt ccgagaaggt accatcaatg tccacgacgt ggagacacag 1140
ttcaatcagt ataaaacgga agcagcctct cgatataacc tgacgatctc agacgtcagc 1200
gtgagtgatg tgccatttcc tttctctgcc cagtctgggg ctggggtgcc aggctggggc 1260
atcgcgctgc tggtgctggt ctgtgttctg gttgcgctgg ccattgtcta tctcattgcc 1320
ttggctgtct gtcagtgccg ccgaaagaac tacgggcagc tggacatctt tccagcccgg 1380
gatacctacc atcctatgag cgagtacccc acctaccaca cccatgggcg ctatgtgccc 1440
cctagcagta ccgatcgtag cccctatgag aaggtttctg caggtaatgg tggcagcagc 1500
ctctcttaca caaacccagc agtggcagcc acttctgcca acttgatggc ggccgcttac 1560
gtacattccg acggctctta tccaaaagac aagtttgaga aaatcaatgg cacttggtac 1620
tactttgaca gttcaggcta tatgcttgca gaccgctgga ggaagcacac agacggcaac 1680
tggtactggt tcgacaactc aggcgaaatg gctacaggct ggaagaaaat cgctgataag 1740
tggtactatt tcaacgaaga aggtgccatg aagacaggct gggtcaagta caaggacact 1800
tggtactact tagacgctaa agaaggcgcc atgcaataca tcaaggctaa ctctaagttc 1860
attggtatca ctgaaggcgt catggtatca aatgccttta tccagtcagc ggacggaaca 1920
ggctggtact acctcaaacc agacggaaca ctggcagaca ggccagaatg a 1971
<210>50
<211>656
<212>PRT
<213>人工序列
<220>
<223>与肺炎链球菌C-LytA P2辅助表位C-Lyta融合的人MUC-1
<400>50
Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr
1 5 10 15
Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly
20 25 30
Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser
35 40 45
Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His
50 55 60
Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu
65 70 75 80
Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln
85 90 95
Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr
100 105 110
Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro
115 120 125
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
130 135 140
Arg Pro Pro Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
145 150 155 160
Ala Pro Asp Thr Arg Pro Pro Pro Gly Ser Thr Ala Pro Ala Ala His
165 170 175
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
180 185 190
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu
195 200 205
Ala Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala Ser Gly Ser
210 215 220
Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr Ser Ala Arg
225 230 235 240
Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser Ile Pro Ser
245 250 255
His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser Thr Lys Thr
260 265 270
Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro Leu Thr Ser Ser
275 280 285
Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser Phe Phe Phe
290 295 300
Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp
305 310 315 320
Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met
325 330 335
Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile
340 345 350
Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg
355 360 365
Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr
370 375 380
Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser
385 390 395 400
Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val
405 410 415
Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala
420 425 430
Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg
435 440 445
Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His
450 455 460
Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His G1y Arg Tyr Val Pro
465 470 475 480
Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn
485 490 495
Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser
500 505 510
Ala Asn Leu Met Ala Ala Ala Tyr Val His Ser Asp Gly Ser Tyr Pro
515 520 525
Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser
530 535 540
Ser Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn
545 550 555 560
Trp Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys
565 570 575
Ile Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr
580 585 590
Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu
595 600 605
Gly Ala Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr
610 615 620
Glu Gly Val Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr
625 630 635 640
Gly Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu
645 650 655
<210>51
<211>2037
<212>DNA
<213>人工序列
<220>
<223>编码与人MUC-1融合的肺炎链球菌C-LytA P2辅助表位
C-Lyta的DNA
<400>51
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60
gtccaaatgg cggccgctta cgtacattcc gacggctctt atccaaaaga caagtttgag 120
aaaatcaatg gcacttggta ctactttgac agttcaggct atatgcttgc agaccgctgg 180
aggaagcaca cagacggcaa ctggtactgg ttcgacaact caggcgaaat ggctacaggc 240
tggaagaaaa tcgctgataa gtggtactat ttcaacgaag aaggtgccat gaagacaggc 300
tgggtcaagt acaaggacac ttggtactac ttagacgcta aagaaggcgc catgcaatac 360
atcaaggcta actctaagtt cattggtatc actgaaggcg tcatggtatc aaatgccttt 420
atccagtcag cggacggaac aggctggtac tacctcaaac cagacggaac actggcagac 480
aggccagaaa tgacaccggg cacccagtct cctttcttcc tgctgctgct cctcacagtg 540
cttacagttg ttacaggttc tggtcatgca agctctaccc caggtggaga aaaggagact 600
tcggctaccc agagaagttc agtgcccagc tctactgaga agaatgctgt gagtatgacc 660
agcagcgtac tctccagcca cagccccggt tcaggctcct ccaccactca gggacaggat 720
gtcactctgg ccccggccac ggaaccagct tcaggttcag ctgccacctg gggacaggat 780
gtcacctcgg tcccagtcac caggccagcc ctgggctcca ccaccccgcc agcccacgat 840
gtcacctcag ccccggacaa caagccagcc ccgggctcca ccgccccccc agcccacggt 900
gtcacctcgg ccccggacac caggccgccc ccgggctcca ccgccccccc agcccacggt 960
gtcacctcgg ccccggacac caggccgccc ccgggctcca ccgcgcccgc agcccacggt 1020
gtcacctcgg ccccggacac caggccggcc ccgggctcca ccgccccccc agcccatggt 1080
gtcacctcgg ccccggacaa caggcccgcc ttggcgtcca ccgcccctcc agtccacaat 1140
gtcacctcgg cctcaggctc tgcatcaggc tcagcttcta ctctggtgca caacggcacc 1200
tctgccaggg ctaccacaac cccagccagc aagagcactc cattctcaat tcccagccac 1260
cactctgata ctcctaccac ccttgccagc catagcacca agactgatgc cagtagcact 1320
caccatagca cggtacctcc tctcacctcc tccaatcaca gcacttctcc ccagttgtct 1380
actggggtct ctttcttttt cctgtctttt cacatttcaa acctccagtt taattcctct 1440
ctggaagatc ccagcaccga ctactaccaa gagctgcaga gagacatttc tgaaatgttt 1500
ttgcagattt ataaacaagg gggttttctg ggcctctcca atattaagtt caggccagga 1560
tctgtggtgg tacaattgac tctggccttc cgagaaggta ccatcaatgt ccacgacgtg 1620
gagacacagt tcaatcagta taaaacggaa gcagcctctc gatataacct gacgatctca 1680
gacgtcagcg tgagtgatgt gccatttcct ttctctgccc agtctggggc tggggtgcca 1740
ggctggggca tcgcgctgct ggtgctggtc tgtgttctgg ttgcgctggc cattgtctat 1800
ctcattgcct tggctgtctg tcagtgccgc cgaaagaact acgggcagct ggacatcttt 1860
ccagcccggg atacctacca tcctatgagc gagtacccca cctaccacac ccatgggcgc 1920
tatgtgcccc ctagcagtac cgatcgtagc ccctatgaga aggtttctgc aggtaatggt 1980
ggcagcagcc tctcttacac aaacccagca gtggcagcca cttctgccaa cttgtag 2037
<210>52
<211>678
<212>PRT
<213>人工序列
<220>
<223>与人MUC-1融合的肺炎链球菌C-LytA P2辅助表位C-Lyta
<400>52
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Met Ala Ala Ala Tyr Val His Ser Asp Gly
20 25 30
Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr
35 40 45
Phe Asp Ser Ser Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr
50 55 60
Asp Gly Asn Trp Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly
65 70 75 80
Trp Lys Lys Ile Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala
85 90 95
Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp
100 105 110
Ala Lys Glu Gly Ala Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile
115 120 125
Gly Ile Thr Glu Gly Val Met Val Ser Asn Ala Phe Ile Gln Ser Ala
130 135 140
Asp Gly Thr Gly Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp
145 150 155 160
Arg Pro Glu Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu
165 170 175
Leu Leu Thr Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser
180 185 190
Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val
195 200 205
Pro Ser Ser Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu
210 215 220
Ser Ser His Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp
225 230 235 240
Val Thr Leu Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr
245 250 255
Trp Gly Gln Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly
260 265 270
Ser Thr Thr Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys
275 280 285
Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala
290 295 300
Pro Asp Thr Arg Pro Pro Pro Gly Ser Thr Ala Pro Pro Ala His Gly
305 310 315 320
Val Thr Ser Ala Pro Asp Thr Arg Pro Pro Pro Gly Ser Thr Ala Pro
325 330 335
Ala Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly
340 345 350
Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg
355 360 365
Pro Ala Leu Ala Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala
370 375 380
Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr
385 390 395 400
Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser
405 410 415
Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser
420 425 430
Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro Leu
435 440 445
Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser
450 455 460
Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser
465 470 475 480
Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile
485 490 495
Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu
500 505 510
Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu
515 520 525
Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe
530 535 540
Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser
545 550 555 560
Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly
565 570 575
Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val
580 585 590
Leu Val Ala Leu Ala Ile Val Tyr Leu Ils Ala Leu Ala Val Cys Gln
595 600 605
Cys Arg Arg Lye Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp
610 615 620
Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg
625 630 635 640
Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser
645 650 655
Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala
660 665 670
Ala Thr Ser Ala Asn Leu
675
Claims (27)
1.一种融合伴侣蛋白,其包含胆碱结合域和异源通用T辅助表位。
2.一种如权利要求1所述的融合伴侣蛋白,其中所述的胆碱结合域来源于LytA的C末端。
3.一种如权利要求2所述的融合伴侣蛋白,其中C-LytA或其衍生物包含SEQ ID NO:1至6中任意序列的至少4个重复。
4.一种如权利要求1至3中任意一项所述的融合伴侣蛋白,其中胆碱结合域选自包含如下的组:
a)如SEQ ID NO:7所示的LytA的C-末端域;或
b)SEQ ID NO:8的序列;或
c)包括与SEQ ID NO:1至6中任意序列具有至少85%同一性,优选至少90%同一性,更优选至少95%同一性,最优选至少97-99%同一性的氨基酸序列的肽序列;或
d)包括SEQ ID NO:7或SEQ ID NO:8的氨基酸序列中的至少15,20,30,40,50或100个连续氨基酸的氨基酸序列的肽序列。
5.如权利要求1至4中任意一项所述的融合伴侣蛋白,其还进一步包括异源蛋白。
6.如权利要求5所述的融合蛋白,其中所述的异源蛋白与融合伴侣化学结合。
7.如权利要求5或6所述的融合蛋白,其中所述的异源蛋白来源于选自如下的有机体:人免疫缺陷性病毒HIV-1,人单纯疱疹病毒,巨细胞病毒,轮状病毒,EB病毒,水痘带状疱疹病毒,肝炎病毒如肝炎B病毒,肝炎A病毒,肝炎C病毒和肝炎E病毒,呼吸道合胞体病毒,副流感病毒,麻疹病毒,流行性腮腺炎病毒,人***瘤病毒,黄病毒或流感病毒,奈瑟氏菌属,莫拉氏菌属,博代氏杆菌属,分枝杆菌属,包括结核分枝杆菌;埃希氏菌属,包括肠毒素大肠埃希菌;沙门氏菌属,李斯特氏菌属,螺杆菌属,葡萄球菌属,包括金黄葡萄球菌,表皮葡萄球菌;疏螺旋体属,衣原体,包括砂眼衣原体,肺炎衣原体;疟原虫,包括恶性疟原虫;弓形虫,念珠菌。
8.一种如权利要求5或6所述的融合蛋白,其中所述异源蛋白为肿瘤相关蛋白或组织特异性蛋白或其免疫原性片段。
9.一种如权利要求8所述的融合蛋白,其中所述的异源蛋白或其片段选自:MAGE 1,MAGE3,MAGE 4,PRAME,BAGE,LAGE 1,LAGE2,SAGE,HAGE,XAGE,PSA,PAP,PSCA,prostein,P501S,HASH2,Cripto,B726,NY-BR1.1,P510,MUC-1,***酶,STEAP,酪氨酸酶,端粒酶,存活素,CASB616,P53,或her 2 neu。
10.如权利要求6至9中任意一项所述的融合蛋白,其进一步包括具有至少4个组氨酸残基的亲和标签。
11.编码如权利要求1至10所述的蛋白的核酸序列。
12.包含权利要求11的核酸序列的表达载体。
13.用权利要求11所述的核酸序列或权利要求12所述的表达载体转化的宿主。
14.一种免疫原性组合物,其包括如权利要求1至10中任意一项所述的蛋白或权利要求11所述的DNA序列和药学上可接受的赋形剂。
15.如权利要求14所述的免疫原性组合物,其另外还包括TH-1诱导佐剂。
16.一种如权利要求15所述的免疫原性组合物,其中所述的TH-1诱导佐剂选自包含如下的佐剂组:3D-MPL,QS21,QS21和胆固醇的混合物,CpG寡核苷酸或两种或多种所述佐剂的混合物。
17.制备如权利要求14至16中任意一项所述的免疫原性组合物的方法,其包括将权利要求6至10中任意一项所述的融合蛋白或权利要求11所述的编码的多核苷酸与合适的佐剂、稀释剂或其他药学上可接受的载体混合。
18.一种生产如权利要求1至10中任意一项所述的融合蛋白的方法,其包括在足够所述融合蛋白产生的条件下培养宿主细胞和从培养基中回收融合蛋白。
19.如权利要求1至10中任意一项所述的蛋白或如权利要求11所述的DNA序列,其用于药物。
20.如权利要求1至10中任意一项所述的蛋白或如权利要求11所述的DNA序列在生产能在病人中引起免疫应答的免疫原性组合物中的应用。
21.如权利要求20所述的应用,其中所述的免疫应答通过顺序施用如下物质而引发:i)所述的蛋白,随后为所述的DNA序列;或ii)所述的DNA序列,随后为所述的蛋白。
22.如权利要求21所述的应用,其中所述的DNA序列被覆盖至生物可降解的珠粒上或经粒子轰击途径来传递。
23.如权利要求21或权利要求22所述的应用,其中所述的蛋白中添加了佐剂。
24.如权利要求1至10中任意一项所述的蛋白或如权利要求11所述的DNA序列在生产用于免疫治疗患有癌症或易患癌症的病人的免疫原性组合物中的应用。
25.如权利要求24所述的应用,其中所述的癌症为***癌,结肠癌,肺癌,乳腺癌或黑素瘤。
26.通过施用安全和有效量的如权利要求12所述的组合物来治疗患有癌症的病人的方法。
27.如权利要求26所述的方法,其中所述的癌症为***癌,结肠直肠癌,肺癌,乳腺癌或黑素瘤。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0213365.0 | 2002-06-11 | ||
GBGB0213365.0A GB0213365D0 (en) | 2002-06-11 | 2002-06-11 | Novel compounds |
GB0300914A GB0300914D0 (en) | 2003-01-15 | 2003-01-15 | Vaccines |
GB0300914.9 | 2003-01-15 | ||
PCT/EP2003/006096 WO2003104272A1 (en) | 2002-06-11 | 2003-06-06 | Immunogenic compositions |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1675237A true CN1675237A (zh) | 2005-09-28 |
CN100360558C CN100360558C (zh) | 2008-01-09 |
Family
ID=29738082
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB038194872A Expired - Fee Related CN100360558C (zh) | 2002-06-11 | 2003-06-06 | 免疫原性组合物 |
Country Status (28)
Country | Link |
---|---|
US (1) | US20060147477A1 (zh) |
EP (1) | EP1511768B1 (zh) |
JP (1) | JP2006512047A (zh) |
KR (1) | KR20050010040A (zh) |
CN (1) | CN100360558C (zh) |
AR (1) | AR040178A1 (zh) |
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AU (1) | AU2003246411A1 (zh) |
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CO (1) | CO5631450A2 (zh) |
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DK (1) | DK1511768T3 (zh) |
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HK (1) | HK1074846A1 (zh) |
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PE (1) | PE20040554A1 (zh) |
PL (1) | PL374534A1 (zh) |
PT (1) | PT1511768E (zh) |
RU (1) | RU2004135541A (zh) |
TW (1) | TW200400970A (zh) |
UY (1) | UY27840A1 (zh) |
WO (1) | WO2003104272A1 (zh) |
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CN108164586A (zh) * | 2018-01-22 | 2018-06-15 | 西南医科大学 | 合成多肽及其应用 |
CN110922455A (zh) * | 2019-12-28 | 2020-03-27 | 重庆艾力彼生物科技有限公司 | 铜绿假单胞菌疫苗重组蛋白rePilA-FliC及制备方法和应用 |
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CN114729375A (zh) * | 2019-09-18 | 2022-07-08 | 国家科学研究中心 | 源自噬菌体t5的纳米颗粒的生产和功能化以及治疗用途 |
CN117003890A (zh) * | 2023-08-09 | 2023-11-07 | 成都新诺明生物科技有限公司 | 一种含有P2和两个Fc的gE融合蛋白及其制备方法和应用 |
CN117003890B (zh) * | 2023-08-09 | 2024-06-04 | 成都新诺明生物科技有限公司 | 一种含有P2和两个Fc的gE融合蛋白及其制备方法和应用 |
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WO2010037408A1 (en) | 2008-09-30 | 2010-04-08 | Curevac Gmbh | Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof |
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SI2528621T1 (sl) * | 2010-01-27 | 2017-01-31 | Glaxosmithkline Biologicals S.A., | Modificirani tuberkolozni antigeni |
EA023725B1 (ru) | 2010-03-23 | 2016-07-29 | Новартис Аг | Соединения (липопептиды на основе цистеина) и композиции в качестве агонистов tlr2, применяемые для лечения инфекционных, воспалительных, респираторных и других заболеваний |
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EP1311673A2 (en) * | 2000-03-27 | 2003-05-21 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of prostate cancer |
-
2003
- 2003-06-06 US US10/517,420 patent/US20060147477A1/en not_active Abandoned
- 2003-06-06 PL PL03374534A patent/PL374534A1/xx not_active Application Discontinuation
- 2003-06-06 DK DK03757056T patent/DK1511768T3/da active
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- 2003-06-06 WO PCT/EP2003/006096 patent/WO2003104272A1/en active IP Right Grant
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- 2003-06-06 JP JP2004511340A patent/JP2006512047A/ja active Pending
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- 2003-06-06 CN CNB038194872A patent/CN100360558C/zh not_active Expired - Fee Related
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- 2003-06-06 EP EP03757056A patent/EP1511768B1/en not_active Expired - Lifetime
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- 2003-06-09 AR ARP030102046A patent/AR040178A1/es not_active Application Discontinuation
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- 2003-06-09 PE PE2003000573A patent/PE20040554A1/es not_active Application Discontinuation
- 2003-06-09 UY UY27840A patent/UY27840A1/es unknown
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108164586A (zh) * | 2018-01-22 | 2018-06-15 | 西南医科大学 | 合成多肽及其应用 |
CN108164586B (zh) * | 2018-01-22 | 2021-03-26 | 西南医科大学 | 合成多肽及其应用 |
CN114729375A (zh) * | 2019-09-18 | 2022-07-08 | 国家科学研究中心 | 源自噬菌体t5的纳米颗粒的生产和功能化以及治疗用途 |
CN110922455A (zh) * | 2019-12-28 | 2020-03-27 | 重庆艾力彼生物科技有限公司 | 铜绿假单胞菌疫苗重组蛋白rePilA-FliC及制备方法和应用 |
CN110922455B (zh) * | 2019-12-28 | 2023-01-13 | 重庆艾力彼生物科技有限公司 | 铜绿假单胞菌疫苗重组蛋白rePilA-FliC及制备方法和应用 |
CN113278078A (zh) * | 2021-05-25 | 2021-08-20 | 西南医科大学 | 多肽序列及其应用 |
CN117003890A (zh) * | 2023-08-09 | 2023-11-07 | 成都新诺明生物科技有限公司 | 一种含有P2和两个Fc的gE融合蛋白及其制备方法和应用 |
CN117003890B (zh) * | 2023-08-09 | 2024-06-04 | 成都新诺明生物科技有限公司 | 一种含有P2和两个Fc的gE融合蛋白及其制备方法和应用 |
Also Published As
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AU2003246411A1 (en) | 2003-12-22 |
IS7547A (is) | 2004-11-23 |
PL374534A1 (en) | 2005-10-31 |
NZ537125A (en) | 2006-08-31 |
TW200400970A (en) | 2004-01-16 |
MY139557A (en) | 2009-10-30 |
IL165372A0 (en) | 2006-01-15 |
JP2006512047A (ja) | 2006-04-13 |
CA2487831A1 (en) | 2003-12-18 |
DE60313163D1 (de) | 2007-05-24 |
HK1074846A1 (en) | 2005-11-25 |
PT1511768E (pt) | 2007-07-16 |
NO20045148L (no) | 2005-03-01 |
US20060147477A1 (en) | 2006-07-06 |
MXPA04012443A (es) | 2005-04-19 |
EP1511768B1 (en) | 2007-04-11 |
RU2004135541A (ru) | 2005-10-10 |
DK1511768T3 (da) | 2007-08-20 |
ATE359295T1 (de) | 2007-05-15 |
AR040178A1 (es) | 2005-03-16 |
PE20040554A1 (es) | 2004-09-30 |
BR0311732A (pt) | 2005-03-01 |
UY27840A1 (es) | 2003-12-31 |
CN100360558C (zh) | 2008-01-09 |
KR20050010040A (ko) | 2005-01-26 |
ES2285174T3 (es) | 2007-11-16 |
CO5631450A2 (es) | 2006-04-28 |
EP1511768A1 (en) | 2005-03-09 |
DE60313163T2 (de) | 2008-01-03 |
WO2003104272A1 (en) | 2003-12-18 |
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