CN1295616A - Mage家族肿瘤相关抗原衍生物及其编码核酸序列 - Google Patents
Mage家族肿瘤相关抗原衍生物及其编码核酸序列 Download PDFInfo
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- CN1295616A CN1295616A CN99804604A CN99804604A CN1295616A CN 1295616 A CN1295616 A CN 1295616A CN 99804604 A CN99804604 A CN 99804604A CN 99804604 A CN99804604 A CN 99804604A CN 1295616 A CN1295616 A CN 1295616A
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Abstract
本发明涉及来自MAGE家族的几种新型蛋白质以及产生它们的方法,特别是涉及与免疫融合配偶体如脂蛋白D融合的MAGE蛋白。可以将这种抗原配制成疫苗,用于治疗一系列肿瘤。还提供了用于纯化MAGE蛋白的几种新颖的方法。
Description
本发明是关于包含肿瘤相关抗原的蛋白质衍生物,发现它具有癌症疫苗治疗效用。详细地说,本发明的衍生物包括含有一种抗原的融合蛋白,此抗原是由MAGE基因家族(例如MAGE-3,MAGE-1)编码的,并连接于一个提供T辅助表位的免疫融合配偶体例如来自流感嗜血杆菌B的蛋白D脂质化形式;还包括化学修饰的MAGE蛋白,其中抗原的二硫键被还原,所形成的巯基被阻断,以及还包括基因修饰的MAGE蛋白,提供了一种亲和性标记和/或基因修饰防止二硫桥形成。还描述了纯化MAGE蛋白,配制疫苗用于治疗一系列癌症的方法,所治疗的癌症包括,但不局限于黑素瘤,乳腺癌,膀胱癌,肺癌,NSCLC,头和扁平细胞癌,结肠癌,以及食道癌。
在黑素瘤细胞(包括恶性黑素瘤)以及包括NSCLC(非小细胞肺癌),头和颈扁平细胞癌,膀胱移行细胞癌和食道癌的其它一些癌细胞上,可显著地表达由MAGE基因家族编码的抗原,但是,在除了睾丸和胎盘之外的正常组织上不能检测出此抗原(Gaugler,1994;Weynants,1994;Patard,1995)。在69%的黑素瘤中可表达MAGE-3(Gaugler,1994),在44%的NSCLC(Yoshimatsu 1988),48%头和颈扁平细胞癌,34%膀胱移行细胞癌,57%食道癌,32%结肠癌,以及在24%乳腺癌中(Van Pel,1995;Inoue,1995;Fujie 1997;Nishimura 1997),也可检测出MAGE-3。表达MAGE蛋白的癌症被称为Mage相关肿瘤。
在应用黑素瘤细胞和自体淋巴细胞混合培养物的实验中,已巧妙地证实人黑素瘤细胞具有免疫原性。这些培养物经常会产生特异性的细胞毒T淋巴细胞(CTLs),这种细胞能够专一性地裂解自体黑素瘤细胞,但是不能裂解自体成纤维细胞,也不能裂解自体的EBV-转化的B淋巴细胞(Knuth,1984;Anichini,1987)。现在,在自体黑素瘤细胞上已鉴定了几种可由这些CTL克隆识别的抗原,包括MAGE家族的抗原。
在自体黑素瘤细胞上,由于它被特异性CTLs识别而被确定的第一种抗原被称为MZ2-E(Vanden Eynde,1989),并且此抗原是由基因MAGE-1编码的(Vander Brnggen,1991)。定向针对MZ2-E的CTLs可识别和裂解来自自体的以及来自其它病人的MZ2-E阳性黑素瘤细胞,只要这些细胞具有HLA.A1等位基因。
MAGE-1基因属于12个密切相关基因的家族,MAGE 1,MAGE 2,MAGE 3,MAGE 4,MAGE 5,MAGE 6,MAGE 7,MAGE 8,MAGE 9,MAGE 10,MAGE 11,MAGE 12,它们定位于染色体X上,并且在它们的编码序列中彼此共有64%-85%的同源性(De Plaen,1994)。有时它们被称为MAGE A1,MAGE A2,MAGE A3,MAGE A4,MAGE A5,MAGE A6,MAGEA7,MAGE A8,MAGE A9,MAGE A10,MAGE A11,MAGE A12(MAGE A家族)。另二类蛋白质也是MAGE家族的一部分,尽管相关性较远。它们是MAGE B和MAGE C家族。MAGE B家族包括MAGE B1(也称为MAGEXp1,和DAM 10),MAGE B2(也称为MAGE Xp2和DAM 6),BAGE B3和MAGE B4,MAGE C家族目前包括MAGE C1和MAGE C2。一般来说,可将MAGE蛋白定义为含有朝向蛋白质C-末端定位的核心序列标记(例如对于MAGE A1,一种309个氨基酸的蛋白质,其核心标记相当于氨基酸195-279)。
可按如下描述此核心标记的共有模式,其中X代表任何一种氨基酸,小写字体残基是保守的(所允许的保守性突变体),大写字体残基是完全保守的。
核心序列标记
LixvL(2x)I(3x)g(2x)apEExiWexl(2x)m(3-4x)Gxe(3-4x)gxp(2x)llt(3x)VqexYLxYxqVPxsxP(2x)yeFLWGprA(2x)Et(3
x)kv
保守性取代是熟知的,在序列对比计算机程序中一般是作为默认计分矩阵(default scoring metrices)被产生。这类程序包括PAM250(Dayhoft M.O.等,(1978),“蛋白质中的进化改变模型”,在“蛋白质序列和结构图集”5(3)M.O.Dayhoft(ed),345-352中),国家生物医学研究基地,Washington,和Blosum 62(Steven Henikoft and JorjaG,Henikoft(1992),“来自蛋白质模块的氨基酸取代矩阵”),Proc.Natl.Acad.Sci.USA 89(生物化学):10915-10919。
一般来说,在如下种类之内的取代是保守性取代,但是在各种类之间的取代被认为是非保守性的。这些种类是:
ⅰ)天冬氨酸/天冬酰胺/谷氨酸/谷氨酰胺
ⅱ)丝氨酸/苏氨酸
ⅲ)赖氨酸/精氨酸
ⅳ)苯丙氨酸/酪氨酸/色氨酸
ⅴ)亮氨酸/异亮氨酸/缬氨酸/甲硫氨酸
ⅵ)甘氨酸/丙氨酸
通常根据本发明,MAGE蛋白在此核心区内与MAGE A1的氨基酸195-279将有大约50%的等同性。
在MAGE-3蛋白上已鉴定了几个CTL表位。一个这样的表位MAGE-3 A1,是位于MAGE-3蛋白的氨基酸168和176之间的一个九肽序列,当它与MHC Ⅰ类分子HLA.A1相结合被呈递时,将构成一个对CTLs特异性的表位。最近,根据它们能够在黑素瘤细胞和自体淋巴细胞的混合培养物中建立起CTL应答,已在MAGE-3蛋白的肽序列上鉴定出另外二个CTL表位。这二个表位具有分别针对HLA.A2(Van derBruggen,1994)和HLA.B44(Herman,1996)等位基因的特异性结合基元。
本发明提供了一类MAGE蛋白衍生物。这类衍生物适合用于适宜治疗一系列肿瘤类型的治疗性疫苗制剂。
在本发明的一个实施方案中,该衍生物是一个融合蛋白,它含有一个连接于异源性配偶体的,来自MAGE蛋白家族的抗原。该蛋白质可能是化学缀合的,但是,可优选地作为重组融合蛋白被表达,使之与非融合蛋白相比较,能在表达系统中高水平地被产生。因此,此融合配偶体可能有助于提供T辅助表位(免疫融合配偶体),优选地是由人识别的T辅助表位。或者可能有助于以比天然重组蛋白更高的产率表达此蛋白质(表达增强子)。优选地,此融合配偶体将既是免疫融合配偶体,又是表达增强配偶体。
在本发明的优选形式中,免疫融合配偶体是由蛋白D产生的,蛋白D是革兰氏阴性菌流感嗜血杆菌B的一种表面蛋白(WO91/18926)。优选地,此蛋白D衍生物大约包含此蛋白质的起始1/3,具体地大约是第一个N末端的100-110个氨基酸。优选地此蛋白D衍生物是脂质化的。脂蛋白D融合配偶体的起始109个残基优选地被包含在N-端,以便为疫苗候选抗原提供附加的外源性T细胞表位,并且增加在大肠杆菌中的表达水平(因此也起表达增强子的作用)。此脂质尾部可确保抗原对抗原呈递细胞的最佳表现。
其它的融合配偶体包括来自流感病毒,NS1(血细胞凝集素)的非结构性蛋白。虽然可使用不同的片段,只要它们包含T-辅助表位,但是,一般是利用N-端的81个氨基酸。
在另一个实施方案中,免疫融合配偶体是称为LYTA的蛋白质。优选地是应用此分子的C-端部分。LYTA是由合成N-乙酰-L-丙氨酸酰胺酶,酰胺酶LYTA的肺炎链球菌产生的,(由lytA基因编码的{基因,43(1986),265-272页},一种可特异性降解肽聚糖骨架中某些键的自溶素)。LYTA蛋白的C-端结构域负责对胆碱,或者对某些胆碱类似物如DEAE的亲和性。已经利用此特性开发大肠杆菌C-LYTA表达质粒,用于表达融合蛋白。对于在其氨基端含有C-LYTA片段的杂交蛋白的纯化已有描述{生物技术学:10,(1992),795-798页}。如在此使用的,一个优选的实施方案利用了在残基178起始的C-末端中发现的Lyta分子的重复部分。特别优选的形式掺入了残基188-305。
上面提到的免疫融合配偶体还具有促进表达的优势。特别是以比天然重组MAGE蛋白较高的产率表达这种融合体。
在临床应用中,本发明者已显示这种构建物能够治疗黑素瘤。在一个病例中,给予二剂无佐剂的脂D 1/3 MAGE 3 His蛋白之后,使患有第Ⅳ期黑素瘤的病人消除了转移病灶。
因此,本发明在实施方案中提供了几种融合蛋白,它们含有连接于免疫融合配偶体的,来自MAGE家族的肿瘤相关抗原。优选的免疫融合配偶体是蛋白D或其片段,最优的是脂蛋白D。优选的MAGE蛋白是MAGE A1或MAGE A3。脂蛋白D部分优选的是含有起始1/3脂蛋白D。
本发明的蛋白质优选地是在大肠杆菌中被表达。在优选的实施方案中,此蛋白连同一个亲和性标记一起被表达,例如含有5-9个,优选地是6个组氨酸残基的组氨酸尾部。这些残基有助于纯化。
本发明还提供了一种编码本发明蛋白质的核酸。可以将这种序列插入适当的表达载体,以及用于DNA/RNA疫苗接种,或者在适当的宿主中表达。表达此核酸的微生物载体可被用作疫苗。这类载体包括例如,痘病毒,腺病毒,α-病毒,李斯特氏菌(listeria)和monarphage。
可应用标准的DNA合成技术合成编码本发明蛋白质的DNA序列,例如借助于D.M.roberts等在生物化学1985.24.5090-5098中描述的酶促连接反应,或借助于应用例如热稳定性聚合酶的PCR技术,或者借助于这些技术的组合形式。
可在含有所需三磷酸核苷dATP,dCTP,dGTP和dTTP的适当缓冲液中,在温度10°-37℃,一般在50μl或更小的体积内,应用DNA聚合酶如DNA聚合酶Ⅰ(Klenow片段),在体外进行DNA的酶促聚合反应。可在适当的缓冲液中,如0.05M Tris(pH7.4),0.01M MgCl2,0.01M二硫苏糖醇,1mM亚精胺,1mM ATP和0.1mg/ml牛血清白蛋白,在4℃到室温的温度下,一般以50ml或更小的体积,应用DNA连接酶如T4 DNA连接酶进行DNA片段的酶促连接反应。应用固相技术,通过常规的磷酸三酯,亚磷酸盐或亚磷酸酰胺化学过程,可进行DNA聚合体或片段的化学合成,在例如“基因片段的化学和酶促合成-实验室手册”(H.G.Garssen和A.lang编著),Verlag Chemie,Weinheim(1982)中,或者如下的其它学术刊物中描述了这种固相技术,例如M.J.Gait,H.W.D.Matthes,M.Singh,B.S.Sproat,and R.C.Titmas,核酸研究,1982,10,6243;B.S.Sporat,and W.Bannwarth,四面体通讯。1983,24.5771;M.D.Matteucci and M.H.Caruthers,四面体通讯-1980,21.719;M.D.Matteucci and M.H.Caruthers,美国化学协会杂志,1981,103,3185;SP.Adams等,美国化学协会杂志,1983,105,661;N.D.Sinha,J.Biernat,J.McMannus,and H.Koester,核酸研究,1984,12,4539;以及H.W.D.Matthes等,EMBO杂志,1984,3,801。
可借助于常规的重组技术实施本发明的方法,例如在Maniatis等,分子克隆一实验室手册:冷泉港,1982-1989中所述的重组技术。
具体地说,此方法可包括如下步骤:
ⅰ)制备能够在宿主细胞内表达此DNA聚合体的可复制或整合的表达载体,此DNA聚合体包含编码该蛋白质或其致免疫性衍生物的核苷酸序列;
ⅱ)用所述载体转化宿主细胞;
ⅲ)在使之能够表达该DNA聚合体,产生所述蛋白质的条件下培养此转化的宿主细胞;以及
ⅳ)回收这种蛋白质。
在此使用的术语“转化”意指将外来的DNA导入宿主细胞。例如应用在“基因工程”,S.M.Kingsman和A.J.Kingsman编著,Blackwell学术刊物,牛津,英格兰,1988中所述的常规技术,通过以适当的质粒或病毒载体转化,转染或感染可达到此目的。术语“转化的”或“转化体”,此后将用于所形成的,包含和表达所需外来基因的宿主细胞。
此表达载体是新颖的,也是本发明的组成部分。
根据本发明,在连接条件下制备这种可复制的表达载体,可通过切开与宿主细胞匹配的载体而提供具有完整复制子的线性DNA区段,并且使此线性区段与一个或几个DNA分子结合,此DNA分子与所述线性区段一起,编码所需产物,如编码本发明的蛋白质或其衍生物的DNA聚合物。
因此,可预先形成这种DNA聚合物,或者如果需要,可在构建载体的过程中形成。
载体的选择部分地由宿主细胞决定,它可以是原核细胞或真核细胞,但优选的是大肠杆菌或DHO细胞。适合的载体包括质粒,噬菌体,粘粒和重组病毒。
借助于例如上面引用的Maniatis等所述的步骤,以适当的酶对此DNA进行限制性酶切,聚合反应和连接反应,可实现常规地制备这种可复制的表达载体。
根据本发明,可通过在转化条件下以本发明可复制的表达载体转化宿主细胞,制备重组的宿主细胞。合适的转化条件是常规性的,例如在上面引证的Maniatis等文献中所描述的,或者在“DNA克隆”第二卷,D.M.Glover编著,IRL出版公司,1985中所描述的。
转化条件的选择取决于宿主细胞。因此,对于细菌宿主细胞如大肠杆菌,可先用CaCl2溶液(Cohen等,国家科学院学报,1973.69,2110)或者用含有RbCl,MnCl2,乙酸钾和甘油的混合溶液处理,然后用3-[N-吗啉代]-丙烷-磺酸,RbCl和甘油处理。对于培养的哺乳动物细胞,可通过将载体DNA钙共沉淀在此细胞上进行转化。本发明还延伸至用本发明的可复制表达载体转化的宿主细胞。
在使之能够表达DNA聚合物的条件下对转化的宿主细胞进行常规培养,如在例如上面引证的文献Maniatis等和“DNA克隆”中所描述的。因此,优选地是对此细胞提供营养物,并在50℃以下的温度培养。
根据宿主细胞和表达产物的定位(细胞内,或者分泌到培养基内或细胞周质内),用常规方法回收此产物。因此,例如当宿主细胞是细菌如大肠杆菌,可用物理,化学或酶促法将它裂解,然后从形成的裂解物中分离出蛋白质产物。当宿主细胞是哺乳动物细胞,一般可从营养培养基中,或者从无细胞的提取物中分离出此产物。常规的蛋白质分离技术包括选择性沉淀,吸附层析,以及包括应用单克隆抗体亲和柱的亲和层析。
本发明的蛋白质或者以可溶解的液体形式,或者以冷冻干燥的形式被提供。
预期每个人剂量包括1-1000μg的蛋白质,优选地是30-300μg。
本发明还提供了在药剂学允许的赋形剂内包含本发明蛋白质的药剂组合物。优选的疫苗组合物至少含有脂蛋白D-MAGE-3。这种疫苗还可任选地含有一种或几种其它的肿瘤相关抗原,例如属于MAGE和GAGE家族的其它成员。适合的其它肿瘤相关抗原包括MAGE-1,GAGE-1或酪氨酸酶蛋白。
在‘疫苗设计’(“亚单位和佐剂技术”(Powell M.F和Newman M.J编著),(1995),Plenum出版社,纽约)中概述了疫苗制剂。Fullerton,美国专利4,235,877描述了将它微囊化在脂质体内。
本发明的蛋白质优选地加入佐剂,配制成本发明的疫苗制剂。适合的佐剂包括铝盐如氢氧化铝凝胶(矾)或磷酸铝,但是还可能是钙、铁或锌盐,或者可能是酰化酪氨酸或酰化糖的不溶性悬液,阳离子或阴离子衍生化多糖,或多膦嗪,其它已知的佐剂包括含CpG的寡核苷酸。此寡核苷酸的特征在于CpG二核苷酸未被甲基化。这类寡核苷酸已被熟知,例如已在WO96/02555中被描述。
在本发明的制剂中优选的是,其佐剂组合物可诱导优选的TH1型免疫应答。适合的佐剂系统包括例如,单磷酰脂质A(monophosphoryl lipidA),优选的是3-脱氧-酰化单磷酰脂质A(3D-MPL)与一种铝盐的组合物。CpG寡核苷酸也可优选地诱导TH1应答。
一种增强的系统包括单磷酰脂质A和皂角苷衍生物的组合物,优选的是如WO94/00153中所公开的QS21和3D-MPL的组合物,或者是一种较低反应原性组合物,在此如在WO96/33739中所公开的,QS21已用胆固醇淬灭了。
一种特别有效的佐剂配方已在WO95/17210中描述了,包括在水包油乳液中的QS21 3D-MPL和生育酚,这是一个优选的配方。
因此,在本发明的一个实施方案中提供了一种疫苗,包含本发明的蛋白质,更优选的是加入单磷酰脂质A或其衍生物佐剂的脂蛋白D(或其衍生物)-MAGE-3。
优选地,此疫苗另外还含有皂角苷,更优选的是QS21。
优选地此制剂另外还含有一种水包油乳液和生育酚。本发明还提供了一种制备疫苗制剂的方法,包括将本发明的蛋白质与药剂学允许的赋形剂如3D-MPL一起混合。
本发明的一方面是提供了一种方法,用于纯化以重组技术产生的MAGE蛋白。此方法包括将此蛋白质溶解于例如强离液剂(StrongChaotropic agent)(例如尿素,盐酸胍)中,或者溶解于两性离子去垢剂(如Empigen BB-n-十二烷基-N,N-二甲基甘氨酸)中,还原此蛋白质分子内和分子间的二硫键,阻断所形成的巯基,以防氧化再偶联,以及使此蛋白质经历一次或几次层析步骤。
优选的阻断剂是烷基化剂。这类阻断剂包括,但不局限于α-卤酸或α-卤酰胺。例如可导致此蛋白质羧甲基化或羧酰胺化(脲基甲基化)的碘乙酸和碘乙酰胺。还可应用其它一些阻断剂,在文献中已有论述(参见例如,‘蛋白质’,第二卷,H.neurath,RL Hill和C-L Boeder编著,Academic出版社1976,或者用于蛋白质修饰的化学试剂,第Ⅰ卷,RL Lundblad和CM Noyes编著,CRC出版社,1985)。其它这种阻断剂的典型例子包括,N-乙基马来酰亚胺,氯乙酰磷酸盐,O-甲基异脲和丙烯腈。使用这种阻断剂是有益的,因为它可防止产物聚集,并可确保下游纯化过程的稳定性。
在本发明的一个实施方案中,选择阻断剂以便诱导稳定的共价不可逆衍生物(例如α-卤酸或α-卤酰胺)。但是,也可选择其它的阻断剂,使之在纯化过程之后可将阻断剂除去,以便释放出非衍生化的蛋白质。
具有衍生化自由巯基的MAGE蛋白是新型蛋白,构成了本发明的一个方面。特别是羧酰胺化的或羧甲基化的衍生物是本发明的优选实施方案。
在本发明的优选实施方案中,为本发明的蛋白质安装了一个亲和性尾部,例如CLYTA或聚组氨酸尾部。在这种情况下,在阻断步骤之后优选地是使蛋白质经历亲和层析。对于那些具有聚组氨酸尾部的蛋白质,可实施固定化金属离子亲和层析(IMAC)。可以是任何一种适合的金属离子。例如锌、镍、铁、镁或铜,但是优选的是锌或镍。IMAC缓冲液优选地含有两性离子去垢剂如Empigen BB(此后写作Empigen),因为这样将导致最后的产物内具有较低水平的内毒素。
如果产生了具有Clyta部分的蛋白质,那么可通过利用它对胆碱或胆碱类似物如DEAE的亲和性来纯化这些蛋白质。在本发明的实施方案中,为此蛋白质安装了聚组氨酸尾部和Clyta部分。通过简单的二步亲和层析纯化程序就可纯化这些蛋白质。
将参照下面的实施例对本发明作进一步描述:
实施例Ⅰ:
制备表达融合蛋白脂蛋白D-MAGE-3-His(LPD 1/3-MAGE-3-His或LpD MAGE-3-His)的重组大肠杆菌菌株。
1.大肠杆菌表达系统:
为了产生脂蛋白D,已将编码蛋白D的DNA克隆进入表达载体pMG81。此质粒利用来自λ-噬菌体DNA的信号,驱动转录和翻译插入的外来基因。此载体含有λPL启动子PL,操纵基因OL和二个利用位点(NutL和NutR),以便当提供N蛋白时解除转录的极性作用(Gross等,1985,分子细胞生物学,5∶1015)。将含有PL启动子的载体导入大肠杆菌溶原性宿主,以便使质粒DNA稳定。溶原性宿主菌株包含被整合进入基因组的复制缺损性λ-噬菌体DNA(Shatzman等,1983,见“基因表达的实验操作”,Inouya(编著),PP1-14,Academic出版社,NY)。λ-噬菌体DNA引导cI阻抑蛋白的合成,此阻抑蛋白结合了载体的OL阻抑蛋白,并阻止RNA聚合酶与PL启动子结合,从而转录插入的基因。表达菌株AR58的cI基因含有温度敏感性突变,使之可以通过温度改变来调节PL引导的转录,也就是说,提高培养物温度可使阻抑物失活并启动对外来蛋白质的合成。此表达系统使之可能对外来蛋白质的合成进行控制,特别是对于有可能对细胞有毒的外来蛋白质的合成(Shimataka和Rosenberg,1981,自然292:128)。
2.大肠杆菌AR58菌株。
用于产生LPD-MAGE-3-His蛋白的AR58溶原性大肠杆菌菌株,是标准的NIH大肠杆菌K12菌株N99(F-Su-galK2,lacZ-thr-)的一个衍生株。它含有缺损的溶原性λ-噬菌体(galE::TN10,1Kil-cI857 DH1)。Kil-表型可防止宿主大分子合成的关闭。cI857突变对cI阻抑物赋予温度敏感性损伤。DH1缺失除去了λ-噬菌体右侧的操纵子以及宿主的bio.uvr3和chlA基因座。通过用以前生长在SAS00衍生株(galE::TN10,1Kil-cI857 DH1)上的Pλ-噬菌体原种转导N99产生了AR58菌株。由于在邻接的galE基因中存在编码四环素抗性的TN10转座子,可用四环素选择将缺损性溶原菌导入N99。N99和SA500是从国立健康研究所Dr.Martin Rosenberg氏实验室得到的大肠杆菌K12菌株。
3.构建设计表达重组蛋白质LPD-MAGE-3-His的载体:
基本原理是,表达作为融合蛋白的MAGE3,应用脂质化蛋白D的N-端1/3作为在MAGE-3N端连接的融合配偶体,并且将几个组氨酸残基序列(His尾部)置于它的C-端。
蛋白D是一个脂蛋白(一个42 KDa的暴露在革兰氏阴性菌流感嗜血杆菌表面的免疫球蛋白D结合蛋白)。此蛋白作为一个前体被合成,它具有18个氨基酸残基信号序列,含有对细菌脂蛋白的共有序列(WO91/18926)。
当脂蛋白的信号序列在分泌过程中被加工时,Cys(在前体分子中的位置19)成为氨基末端残基,并且通过共价脂连接的和酰胺连接的脂肪酸而同时被修饰。
于是,连接于氨基端半胱氨酸残基的脂肪酸发挥膜固定点的作用。
将表达此融合蛋白的质粒设计成表达一个前体蛋白,此前体蛋白含有18个氨基酸的信号序列和被加工蛋白D的起始109个残基,二个无关的氨基酸(甲硫氨酸和天冬氨酸),MAGE-3的氨基酸残基2-314,二个起枢纽区作用的Gly残基,以便暴露出后面的7个His残基。
因此,该重组菌株产生432个氨基酸残基长度的被加工的脂质化His结尾的融合蛋白(见图1),在ID No 1中描述了其氨基酸序列,在ID No 2中描述了其编码序列。
4.为产生LPD-MAGE-3-His融合蛋白的克隆方案(载体pRIT14477):
使用了含有编码序列MAGE-3基因(Gaugler.B等,1994)的cDNA质粒(从Ludwig研究所Dr.Thierry Boon获得),以及含有Lipo-D-1/3编码序列的N-端部分的载体pRIT 14586(按照图2中的草图制备)。此克隆方案包括如下步骤(图3)。
a)-应用有义寡核苷酸:5’gc gcc atg gat ctg gaa cag cgt agtcag cac tgc aag cct,以及反义寡核苷酸:5’gcg tct aga tta atg gtgatg gtg atg gtg atg acc gcc ctc ttc ccc ctc tct caa,PCR扩增在质粒cDNA MAGE3中存在的序列;此扩增将导致在N端出现如下的修饰:将起始的5个密码子改变成大肠杆菌应用密码子,在位置1以Asp密码子转换Pro密码子,在5’端安装NcoⅠ位点,以及最后加入二个2Gly密码子和7个His密码子,随后是C端的XbaⅠ位点。
b)-将上面扩增的外原片段克隆进入TA克隆载体,并且制备中间载体pRIT 14647。
c)-从质粒pRIT 14647切取NcoⅠ XbaⅠ片段,并将它克隆进入载体pRIT 14586。
d)-转化宿主菌株AR58。
e)-挑选和鉴定含有质粒pRIT 14477,表达LPD-MAGE-3-His融合蛋白的大肠杆菌菌株转化体。
实施例Ⅱ:
制备LPD 1/3-MAGE-3-His抗原:
1.菌株的培养和诱导-表达LPD 1/3-MAGE-3-His:
在2升的摇瓶内培养用质粒pRIT 14477转化的AR58细胞,每个摇瓶含有400ml以酵母浸膏(6.4g/L)和硫酸卡那霉素(50mg/L)补充的LY 12培养基。在摇床上30℃培育8±1小时之后,从每个摇瓶内取出少量样品作显微镜检查。将二个摇瓶中的内含物合并,对20升的发酵罐提供此接种物。
将此接种物(大约800ml)加入预先灭菌的20升(总容积)发酵罐中,其中含有7升以50mg/L硫酸卡那霉素补充的培养基。通过定时加入NH4OH(25% v/v)校准pH并保持在6.8,并将温度校准和保持在30℃。利用搅拌速度反馈控制法使通气速度校准和保持在12升/分钟,使溶解氧张力保持在50%的饱和度。保持发酵罐内500g/cm2(0.5巴)的过压状态。
通过对加入碳素注入液的控制实施分批加料培养法。以0.04ml/分钟的起始速度加入此注入液,在起初42小时中按指数递增,以便保持0.1/小时的生长速度。
42小时之后使发酵罐内的温度迅速升高至39℃,并且在诱导期内使加料速度保持恒定在0.005ml/g DCW/min,再持续22-23小时,在此时间内,LPD-MAGE-3-His的细胞内表达达到最高水平。
在整个生长/诱导期内按规定的时间间隔,以及在发酵的终点取出小份量(15ml)发酵液,以便跟踪微生物生长和细胞内产物表达的动力学,并且为微生物鉴定和纯度检测提供样品。
在发酵的终点,培养物的光密度是80-120(相当于48-72g DCW/L的细胞浓度),液体总量是大约12升。使培养物迅速冷却至6-10℃,并通过在4℃以5000xg的速度离心30分钟,从发酵培养物中分离出了ECK32细胞。将浓缩的ECK32细胞迅速贮存在塑料袋中,并立即在-80℃冷冻。
2.提取此蛋白质:
在4℃使冷冻的浓缩ECK32细胞融化,然后再悬浮于细胞裂解缓冲液中,达到最后的光密度60(相当于大约36g DCW/L的细胞浓度)。
借助于二次通过高压匀浆器(1000巴)使此细胞裂解。离心破裂的细胞悬液(10000g,4℃,30分钟),用TritonX100(1% w/v)+EDTA(1mM)洗此沉淀组份2次,随之用磷酸缓冲盐水(PBS)+Tween20(0.1% v/v)洗,最后再用PBS洗。在每次清洗步骤之间,以10000g,4℃离心悬浮液30分钟,弃去上清液,保留沉淀组份。
实施例Ⅲ:
对融合蛋白Lipo D-MAGE3的鉴定:
1.纯化:
应用下述的步骤顺序从细胞匀浆中纯化了LPD-MAGE-3-His:
a)-使来自细胞裂解物的洗过的沉淀组份溶液化,
b)-化学还原蛋白质分子内和分子间的二硫键,随之阻断巯基,以防氧化再偶联,
c)-微过滤此反应混合物,以便除去颗粒并减少内毒素,
d)-利用多组氨酸尾部和锌装填的螯合琼脂糖之间的亲和性相互作用,捕获和初步纯化LPD-MAGE-3-His,
e)-通过阴离子交换层析除去污染蛋白质。
使此纯化的LPD-MAGE-3-His经受几次精制步骤:
f)-应用Superdex 75,通过尺寸排阻层析法进行缓冲交换/尿素清除,
g)-作过滤处理,
h)-应用葡聚糖凝胶G25,通过尺寸排阻层析法进行缓冲交换/脱盐。
下面对每个步骤作更详细的描述:
1.1)-使细胞匀浆沉淀溶液化
在800ml盐酸胍(6M)和磷酸钠(0.1M.pH7.0)溶液中,使来自最后漂洗步骤(如上面所述)的沉淀组份在4℃再溶液化过夜。
1.2)-还原和羧甲基化
用氩气冲洗此溶液化材料(淡黄色混浊悬液),以便清除所有残留的氧气,并加入2-巯基乙醇贮备液(14M)使最后浓度为4.3M(这相当于每ml溶液中含0.44ml2-巯基乙醇).
将形成的溶液分成2份,转移至2个玻璃烧瓶内,在水浴中将二个烧瓶加热至95℃。在95℃15分钟之后,从水浴中取出烧瓶,使之冷却,将其中的溶液合并进入一个箔片加盖的烧杯(5L)内,置于冰上,加入固体碘乙酰胺并剧烈搅拌,使最后浓度为6M(这相当于每ml溶液内含有1.11g碘乙酰胺)。在黑暗中使此混合物在冰上保持1小时,确保碘乙酰胺完全溶解,然后通过加入大约1升氢氧化钠(5M)进行中和(继续剧烈搅拌,并继续监测pH),使最后的pH为7.5-7.8。
使所形成的混合物在黑暗中,再在冰上保持30分钟,此后将其pH再校准至pH7.5-7.8。
1.3)-微过滤
在装配有Minikros中空纤维滤芯(参考NO.M22M-600-01N;面积5600cm2,0.2μm)的Amicon Proflux M12切向流动滤器中对此混合物作微过滤。保留滤过液进行后续的层析纯化。
1.4)-金属(Zn2+)螯合层析(IMAC)
以被装填进入BPG100/500柱(Pharmacia Biotechnology,分类号18-1103-01)的螯合琼脂糖FF(Pharmacia Biotechnology,分类号17-0575-01)进行金属螯合层析。装填床的尺寸为:直径10cm;横切面积79cm2,床高度19cm,装填容量1500ml。空柱先用氢氧化钠(0.5M)清洁,然后用纯水洗。
在Buchner漏斗上(真空下),用纯水(8升)洗此支持载体(在20% v/v乙醇中),并借助于使至少15升ZnCl2溶液(0.1M)流过而以锌装填此支持载体,通过用10升纯水洗支持载体而除去过剩的锌,直至流出液的pH达到ZnCl2溶液的pH(5.0)。然后用4升含有盐酸胍(6M)和磷酸钠(0.1M.pH7.0)的溶液平衡此支持载体。
将来自微过滤,含有LPD-MAGE-3-His的滤出液与此支持载体混合(分批结合),然后装BPG柱,并填充含有盐酸胍(6M)和磷酸钠(0.1M.pH7.0)的溶液。
下面的金属螯合层析步骤是以60ml/分钟的冲洗流速度进行。先用含有盐酸胍(6M)和磷酸钠(0.1M,pH7.0)的溶液洗柱,然后用含有尿素(6M)和磷酸钠(0.1M,pH7.0)的溶液洗,直到柱洗出液达到在OD280nm的吸光度为O(基线)。
用二个柱体积的含有尿素(6M),磷酸钠(0.1M,pH7.0)和咪唑(0.5M)的溶液,洗脱出了半纯的LPD-MAGE-3-His蛋白组份。此组份的电导大约是16mS/cm。
1.5)阴离子交换层析
在继续阴离子交换层析之前,通过用含有尿素(6M)和Tris-HCl(20mM,pH8.0)的溶液稀释,将半纯LPD-MAGE-3-His蛋白组份的导电性降低至大约4ms/cm。
应用装填在BPG200/500柱(Pharmacia Biotechnology,分类号18-1103-11)内的Q-琼脂糖FF(Pharmecia Biotechnology,分类号。17-0510-01)进行阴离子交换层析。装填床的尺寸为:直径10cm;横切面积314cm2;床高度9cm;装填容量2900ml。
装柱(连同20% v/v乙醇),并用9升纯水,以70ml/分钟的冲洗流速度洗柱。用3升氢氧化钠(0.5M)清洁装填的柱床,再用30升纯水洗,然后用6升含有尿素(6M)和Tris-HCl(20mM,pH8.0)的溶液平衡。将稀释的半纯LPD-MAGE-3-His对柱上加样,然后用9升含有尿素(6M),Tris-HCl(20mM,pH8.0),EDTA(1mM)和Tween(0.1%)的溶液洗脱,直至洗脱液的吸光度(280纳米)降到0。
再用6升含有尿素(6M)和Tris-HCl(20mM,pH8.0)的溶液作进一步的洗脱。
用含有尿素(6M),Tris-HCl(20mM,pH8.0)和NaCl(0.25M)的溶液从柱上将纯化的LPD-MAGE-3-His洗脱出。
1.6)-尺寸排阻层析
通过尺寸排阻层析达到了从纯化的LPD-MAGE-3-His中除去尿素和缓冲交换二个目的。应用装填在XK50/100柱(PharmaciaBiotechnology,分类号。18-8753-01)中的Superdex 75(PharmaciaBiotechnology,分类号。17-1044-01)进行这种层析。装填床的尺寸为:直径5cm;横切面积19.6cm2;床高度90cm;装填容量1800ml。
在乙醇(20%)中装柱,并用5升纯水,以20ml/分钟的流出速度洗柱。用2升氢氧化钠(0.5M)清洗柱床,用5升纯水洗,然后用5升含有Tween 80(0.1% v/v)的磷酸缓冲盐水平衡。
将纯化的LPD-MAGE-3-His组份(每次脱盐运转的最大量为500ml)在柱上加样,使洗出流速为20ml/分钟。用3升含有Tween 80(0.1% v/v)的PBS从柱中洗脱出脱盐的纯LPD-MAGE-3-His。
含有LPD-MAGE-3-His的组份在柱床的空隙容积中被洗脱出。
1.7)过滤处理
使来自尺寸排阻层析的批量LPD-MAGE-3-His在层流通风橱(级别10.000)中通过0.22μm孔径的膜进行过滤。将此批量滤液在-80℃冷冻,贮存直至脱盐步骤。
1.8)-脱盐层析
因为最后批量产品的重量克分子渗透压浓度应该小于400mOsM,所以需要进行进一步的缓冲交换步骤,以便降低盐浓度。这可通过应用装填在BPG100/950柱(Pharmacia Biotechnology,分类号。18-1103-03)中的葡聚糖凝胶G25(Pharmacia Biotechnology,分类号。17-0033-02)的脱盐层析步骤来实现。装填床的尺寸为:直径10cm,横切面积78.6cm2,床高度85cm,装填容量6500ml。
用7升纯水使葡聚糖凝胶G25水合,使其在4℃膨胀过夜。然后随同纯水将此凝胶装填于柱中,使洗出流速为100ml/分钟。
用6升氢氧化钠(0.5M)清洗柱床,然后用10升含有磷酸钠(10mM,pH6.8),NaCl(20mM)和Tween80(0.1% v/v)的溶液平衡。
将纯化的LPD-MAGE-3-His组份(每次脱盐运转的最大量为1500ml)在柱上加样,使洗出流速为100ml/分钟。使在柱床的空隙容积中被洗脱出的脱盐的纯LPD-MAGE-3-His组份通过0.22μm孔径的膜过滤除菌,并贮存在-80℃。
将最后的批量蛋白质融化至+4℃,然后被等量分装至小药瓶内,并在乳糖赋形剂(3.2%)中冷冻干燥。
2.在考马斯染色的SDS-聚丙烯酰胺凝胶上分析:
在还原状态的12.5%丙烯酰胺凝胶上,通过SDS-PAGE对LPD-MAGE-3-His纯化的抗原进行了分析。
加样蛋白质50μg用于考马斯兰染色,5μg用于硝酸银染色。分析了临床组96K19和指示组96J22。可见一条相当于60KDa分子量的主要电泳带。还可见二条较小的大约45KDa和35KDa的附加电泳带。
3.蛋白质印迹(Western Blot)分析法:
应用小鼠单克隆抗体,借助于蛋白质印迹试验,对通过SDS-PAGE分析LPD-MAGE-3-His显示的肽进行了鉴定。这些抗体是应用MAGE-3-His蛋白(这种蛋白质不含LPD-MAGE-3-His的LPD部分)的纯制剂在组织内形成的。
根据它们对蛋白质印迹分析的适配性,选择了二个单克隆抗体制剂(Mab 22和mab 54),并用于大批量释放的鉴定试验,图4显示用Mab 32和54染色之后对组分96K19和96J22获得的电泳带图形。在12.5%的SDS-PAGE上对600ng蛋白质进行了分析,并被转移至尼龙膜上,与Mab32和54(60μg/ml)反应,最后用偶联于过氧化物酶的抗-小鼠抗体显示。
借助于这二个单克隆抗体,揭示了由SDS-PAGE检测的60KDa和30KDa肽。
实施例Ⅳ:
1.应用LPD-MAGE-3-His蛋白的疫苗制剂:
用于这些实验的疫苗是从编码脂蛋白D1/3-MAGE-3-His的重组DNA产生,在来自菌株AR58的大肠杆菌中被表达,加入佐剂或不加入佐剂。佐剂的配方包括在水包油乳剂中的3-脱氧酰基化单磷酰脂质A(3D-MPL)和QS21的混合物。在WO95/17210中以前已描述过佐剂系统SBAS2。
3D-MPL:是一种以革兰氏阴性菌明尼苏达沙门氏菌的脂多糖(LPS)衍生的免疫刺激物。MPL已被脱酰基化,并在其脂质A部分缺乏磷酸基。这种化学处理显著地降低了其毒性,然而保留了其免疫刺激特性(Ribi,1986)。Ribi Immunochemistry对SB-Biologicals生产和供应MPL。在Smith Kline Beecham Biologicals完成的实验已表明,与各种赋形剂组合的3D-MPL,其体液免疫性和TH1型细胞免疫性都大大提高了。
QS21:是一种从南美Quillaja Saponaria Molina树的树皮中提取的天然皂角苷分子。为了从树皮粗提取物中分离出单一的皂角苷而建立的纯化技术,使之可能分离出特定的皂角苷QS21,这是一种三萜苷,已证明与其原始成分相比较具有较强的佐剂活性和较低的毒性。已表明QS21可激活将CTLs局限于几种亚单位Ags的MHCⅠ类,并且可刺激Ag特异性淋巴细胞增殖(Kensil,1992)。Aquila(正规的剑桥生物技术公司)对SB-Biologicals生产和供应QS21。
在SmithKline Beecham Biologicals完成的实验已表明,在体液和TH1型细胞免疫应答中,MPL和QS21的组合物具有清楚的协同作用。
水包油乳剂由用两种油(生育酚和角鲨烯)制成的有机相和作为乳化剂的含有Tween 80的PBS构成的水相组成。该乳剂包含5%角鲨烯、5%生育酚、0.4% Tween 80,平均粒径为180nm,并被称为SB62(参见WO95/17210)。
在SmithKline Beecham Biologicals完成的实验已证明,对3D-MPL/QS21(SBAS2)添加这种O/W乳剂,将进一步增加3D-MPL/QS21针对各种亚单位抗原的免疫刺激特性。
2.乳剂SB62(2倍浓缩的)的制备:
将Tween 80溶解于磷酸缓冲盐水(PBS)中,形成在PBS中的2%溶液。为了提供100ml二倍浓缩的乳剂,将5g DL-α-生育酚和5ml角鲨烯彻底搅拌均匀。加入90ml PBS/Tween溶液并彻底混匀。然后使所形成的乳剂通过一个注射器,并最后用M110S微液化机使之微液化。形成的油滴具有大约180nm的大小。
3.制备脂蛋白D 1/3-MAGE-3-His QS21/3D-MPL水包油(SBAS2)制剂:
在水包油乳剂中配制作为MPL和QS21组合物的佐剂。将此制剂输运进入0.7ml的小药瓶内,以便与冷冻干燥的抗原混合(小药瓶内含有30-300μg抗原)。
用此佐剂或者仅用PBS重建溶解冷冻干燥的LPD-MAGE-3-His制剂之后,可得到最终的疫苗。
通过以PBS代替此蛋白质,制备了无抗原的佐剂对照。
4.疫苗抗原:融合蛋白脂蛋白D 1/3-MAGE-3-His:
脂蛋白D是暴露在革兰氏阴性菌流感嗜血杆菌表面的脂蛋白。
作为融合配偶体掺入被加工蛋白D起始109个残基的包函体,以便提供具有T-细胞表位的疫苗抗原。除了LPD部分之外,此蛋白还含有二个无关的氨基酸(Met和Asp),MAGE-3的氨基酸残基2-314,二个起枢纽区作用,以便暴露后面7个His残基的Gly残基。
实施例Ⅴ:
1.LPD-MAGE-3-His对小鼠和猴的免疫原性:
为了测定人MAGE-3蛋白的抗原性和免疫原性,对二个不同品系的小鼠(C 57BL/6和Balb/c)注射了待选用的疫苗,这二个品系在它们的基因背景和MHC等位基因有所不同。对于二个小鼠品系,理论上预测潜在的MHCⅠ类和MHCⅡ类肽基元序列为LPD-MAGE-3-His融合蛋白的MAGE部分。
a)-免疫方案:
用在SBAS2中配制的或不在其中配制的5μg LPD-MAGE-3-His,以用于人时的1/10浓度,对每个品系的5只小鼠,间隔2周在脚垫内注射2次。
b)-增殖测定:
在第二次注射的2周之后,通过碾磨来自此小鼠的脾或腘淋巴结制备淋巴细胞。将2×105个细胞一式三份置于96孔培养板中,并用不同浓度(1-0.1μg/ml)的His-MAGE-3照这样,或者包被在乳胶微珠上,在体外对此细胞反复制激72小时。
与仅注射SBAS-2制剂或PBS的小鼠淋巴细胞增殖反应相比较,注射LPD-MAGE-3-His蛋白的C57BL/6或Balb/C小鼠,对其脾细胞(见图5和7)和淋巴结细胞(见图6和8)都观测到提高的MAGE-3特异性淋巴细胞增殖活性。
而且,小鼠用在佐剂SBAS2中的LPD-MAGE-3-His免疫之后,对于来自此小鼠的淋巴细胞,获得了显著较高的增殖反应(见图6和8)。
c)-结论:
LPD-MAGE-3-His对小鼠是致免疫性的,并且通过使用SBAS2佐剂制剂,可使这种免疫原性增加。
2.抗体应答:
a)-免疫方案:
用PBS或SBAS2,或者5μg LPD-MAGE-3-His或5μg LPD-MAGE-3-His+SBAS2,通过以2周的间隔脚垫内注射2次免疫Balb/C或C57BL/6小鼠。分别以3只动物和5只动物用于对照组和试验组。
b)-间接ELISA试验:
第二次注射之后二周,采集各个动物的血清,用于间接ELISA试验。将2μg/ml的纯化His MAGE3用作包被抗原。在PBS+1%新生牛血清中37℃饱和1小时后。将此小鼠血清在饱和缓冲液内作系列稀释(从1/1000开始),并在4℃温育过夜,或者在37℃温育90分钟。在PBS/Tween20,0.1%中漂洗之后,用生物素化的羊抗-小鼠总IgG(1/1000)或羊抗-小鼠IgGl,IgG2a,IgG2b抗血清(1/500)作第二抗体。在37℃温育90分钟之后,加入偶联于过氧化物酶的链亲和素,并用TMB(四甲基联苯胺过氧化物)作底物。10分钟之后,通过加入H2SO4 0.5M终止反应,并测定光密度(O.D)。
c)-结果:
图9对不同组小鼠(N=5只/组)之间的血清相对平均中点滴度进行比较,此滴度是达到曲线中点所需的平均稀释度。
这些结果表明,对于所测定的二个品系小鼠,在单纯注射2次LPD-MAGE-3-His之后都可测定到微弱的抗体应答,但是,在有SBAS2存在情况下注射LPD-MAGE-3-His时,可产生较高的抗-MAGE-3抗体浓度。因此,只要二次注射LPD-MAGE-3-His+SBAS2,间隔2周,就足以产生所观测到的高水平抗体应答。
当与C57BL/6小鼠所得到的反应相比较,对于Balb/c小鼠可观测到较强的抗体应答,这可以用这二个品系之间单倍型或背景不同来解释,尽管对于C57BL/6小鼠,与单独注射LPD-MAGE-3-His相比较,注射LPD-MAGE-3-His+SBAS2之后,也达到较高的抗体滴度。
在图10和11上可看到对不同组小鼠免疫接种之后的Ig亚类-特异性抗-MAGE-3应答,图中给出了血清平均中点稀释度的比较值。
即使是来自用LPD-MAGE-3-His在佐剂SBAS2中免疫接种的小鼠的任何一个血清样品,都未检测出IgA或IgM。
相反,对于单用LPD-MAGE-3-His免疫接种的小鼠,其血清中的总IgG水平轻微升高,对于用在SBAS2中的LPD-MAGE-3-His注射的动物,其血清中IgG水平显著地升高。
对不同IgG-亚类的浓度分析表明,在小鼠中诱发了混合的抗体应答,因为与单用抗原(Ag)或佐剂注射的小鼠相比较,用加佐剂的Ag免疫接种的小鼠,其所有被测定的IgG亚类(IgG1,IgG2a,IgG2b)的水平都比较高。
但是,用存在SBAS2的LPD-MAGE-3免疫接种之后,这种混合抗体应答的特性似乎取决于小鼠的品系,因为在Balb/c和C57BL/6小鼠的血清中,分别发现IgG1和IgG2b占优势。
3.脂蛋白D 1/3 MAGE-3-His+SBAS2佐剂对猕猴的免疫原性
选用3组猕猴(MacacaMulatta),每组5只动物。RTS,S和GP120用作阳性对照。
分组:
第一组右腿:RTS.S/SBAS2
左腿:GP120/SBAS2
第二组右腿:RTS.S/SB26T
左腿:GP120/SB26T
第三组右腿:Lipo D 1/3 MAGE-3-His/sbas2
在0天使动物接受疫苗注射,在28天和84天作加强接种,然后采血测定它们对MAGE-3和蛋白D成分的抗体应答。在右腿后部肌肉内快速浓注给予疫苗(0.5ml)。
每14天采集小量血液样品一次从股静脉采集3ml血液样品,未加入肝素,允许其凝结至少1小时,然后在室温下以2500rpm离心10分钟。
分离出血清,在-20℃冷冻,送交借助于特定的ELISA试验测定抗体水平。
将96孔微量培养板(maxisorb Nunc)用5μg His MAGE 3或蛋白D在4℃包被过夜。在37℃用PBS NCS1%饱和1小时之后,加入系列稀释的兔血清(从1/10开始),在37℃作用1小时30分钟,用PBS Tween洗3次之后,加入生物素化的抗-兔血清(Amersharm ref RPN 1004lot88)(1/5000)。洗培养板,加入过氧化物酶偶联的链亲和素(1/5000),在37℃作用30分钟。洗涤之后,加入50μl TMB(Bio Rad)作用7分钟,用0.2M H2SO4终止反应,在450nm测定光密度(O.D)。借助于Softmaxpro计算中点稀释度。
抗体应答:
每14天采集小量血液样品一次,以便通过ELISA试验跟踪对MAGE-3抗体应答的动力学。结果表明,一次注射LPD 1/3 MAGE-3-His+SBAS2之后,MAGE-3特异性总Ig滴度较低,而对相同的猴注射第2次和第3次Lipo D 1/3 MAGE-3-His+佐剂之后,在5只动物中的3只看到明显的增强反应。对于不良的应答者即使注射3次之后仍然为阴性。在第Ⅱ或第Ⅲ次注射后28天(post Ⅱ or post Ⅲ),抗体滴度已返回到基线水平。测定这些抗体的亚类主要是IgG而不是IgM。转换至IgG暗示,T辅助应答已被触发。蛋白D特异性抗体应答虽然较弱,但是正好与MAGE-3抗体应答平行。
实施例Ⅵ
1.LPD-MAGE-3-His
以类似的方法制备了LPD-MAGE-1-His。其氨基酸和DNA序列被描绘在SEQUENCE ID NOS3和4中。以类似于LPD-MAGE-1-His蛋白的方式纯化了所形成的蛋白质。简单地说,将此细胞培养物制成匀浆,并在0.5% Empigen去垢剂存在下用4M盐酸胍和0.5M β-巯基乙醇处理。将此产物过滤,并用0.6M碘乙酰胺处理其滤出液。使此羧酰胺化组份经受IMAC(锌-螯合-琼脂糖FF)层析处理。首先用含有4M盐酸胍和磷酸钠(20mM,pH7.5)以及0.5% Empigen的溶液平衡并洗柱,然后用以磷酸钠(20mM,pH7.5)0.5% Empigen缓冲液配制的含有4M尿素的溶液洗柱。以同样的缓冲液洗脱此蛋白质,但是此缓冲液含有递增浓度的咪唑(20mM,400mM和500mM)。
用4M尿素稀释此洗出液。用存在0.5% Empigen的20mM磷酸缓冲液(pH7.5)配制的4M尿素平衡和洗Q-琼脂糖柱。用同样的但不含去垢剂的缓冲液实施第二次洗柱。用同样的,但含有递增浓度咪唑(150mM,400mM,1M)的缓冲液洗脱此蛋白质。对洗出液进行了超过滤。
实施例Ⅶ
为了产生NS1-MAGE-3-His构建表达质粒pRIT 14426并转化宿主菌株AR58:
蛋白质设计:
在图12中描绘了对将在大肠杆菌中被表达的融合蛋白NS1,-MAGE-3-His的设计。
所形成蛋白质的一级结构具有在ID NO 5中列出的序列。
在大肠杆菌表达质粒中,将对应于上述蛋白质设计的编码序列(IDNO.6)安排在λpL启动子的控制之下。
为了产生NS1-MAGE-3-His融合蛋白的克隆方案:
起始材料是从Ludwig研究所Dr.Tierry Boon得到的cDNA质粒和载体PMG81,DNA质粒含有MAGE-3基因编码序列,载体PMG81含有来自流感病毒(Influenza)NS1(非结构蛋白)编码区的81aa。
图13中草拟的克隆方案包括如下步骤:
a)应用如下有义寡核苷酸和反义寡核苷酸PCR扩增在质粒cDNAMAGE-3中呈递的序列:
有义寡核苷酸:5’gc gcc atg gat ctg gaa cag cgt agt cag cactgc aag cct,
反义寡核苷酸:5’gcg tct aga tta atg gtg atg gtg atg gtg atgacc gcc ctc ttc ccc ctc tct caa,
这种扩增在N端导致了如下的修饰:
起始5个密码子改变成大肠杆菌应用密码子,在位置1用天冬氨酸密码子代替脯氨酸密码子,在5’端安装了一个NcoⅠ位点,最后加入了2个甘氨酸密码子和7个组氨酸密码子,随后是C端的XbaⅠ位点。
b)将上面扩增的片段克隆进入外源的TA克隆载体,并制备过渡性载体pRIT 14647。
c)从质粒pRIT14647切取NCoⅠ XbaⅠ片段,并克隆进入载体pRITPMG81。
d)转化宿主菌株AR58。
e)选择并鉴定含有表达NS1-MAGE-3-His融合蛋白的质粒pRIT14426(见图14)的大肠杆菌菌株转化体。
鉴定重组NS1-MAGE-3-His(pRIT14426):
使细菌在30℃生长在以50μg/ml卡那霉素补充的LB培养基上。当此培养物达到O.D=0.3(在620nm)后,通过将温度升高至42℃进行加热诱导。
诱导4小时后,收获细胞,再悬浮于PBS中,通过在French压碎机中碎压3次使细胞裂解(借助于崩解作用)。离心(100000g,60分钟)之后,通过SDS-PAGE分析沉淀的上清液和总提取物。在考马斯B1染色的凝胶中可见蛋白质,在此融合蛋白相当于大肠杆菌总蛋白的大约1%。此重组蛋白显示为一条单独的电泳带,具有44.9K的近似MW。应用抗-NS1单克隆抗体通过蛋白质印迹分析法对此融合蛋白进行了鉴定。
实施例Ⅷ:
纯化NS1-MAGE-3-His(大肠杆菌)用于兔/小鼠免疫:
纯化步骤:
以如下纯化步骤用于纯化此抗原:
细胞裂解+离心
↓
抗原溶液化+离心
↓
Ni2+-NTA琼脂糖
↓
浓缩
↓
制备室(Prep cell)
↓
TCA沉淀和PBS溶液化。
a.裂解
借助于Rannie(匀浆器)在203ml 50mM PO4 pH7缓冲液中使细菌细胞(23g)裂解,然后在JA 20转子中以15000rpm离心此裂解液30分钟。
弃去上清液。
b.抗原溶液化
将1/3的沉淀物在34ml 100mM PO4-6M GuHCl pH7中在4℃再溶液化O/N。在JA20转子中以15000rpm离心30分钟之后,弃去沉淀,上清液通过IMAC进一步纯化。
c.亲和层析:Ni2+-NTA琼脂糖(Qiagen)
柱容量:15ml(16mm×7.5cm)
装填缓冲液:0.1M PO4-6M GuHCl pH7
样品缓冲液:同上
洗柱缓冲液:0.1M PO4-6M GuHCl pH7
0.1M PO4-6M尿素pH7
洗脱:在以6M尿素补充的0.1M PO4 pH7缓冲液中的咪唑梯度液(0→250mM)。
流速:2ml/min。
a.浓缩:
合并IMAC洗出液抗原阳性的组份(160ml),在Amicon搅拌室内通过Filtron膜(ω-型,截止分子量10000)浓缩至5ml。此时当用SDS-PAGE测定时纯度是大约70%。
b.制备电泳(Prep Cell Biorad)
在0.8ml还原性样品缓冲液中将2.4ml浓缩的样品煮沸,并在10%丙烯酰胺凝胶上加样。以用4%SDS补充的Tris-甘氨酸缓冲液pH8.3洗脱此抗原,合并NS1-MAGE-3-His阳性的组份。
a.TCA沉淀:
对此抗原作TCA沉淀,在JA20转子内以15000rpm离心20分钟之后弃去上清液。将此沉淀物在PBS缓冲液pH7.4中再溶液化。
此蛋白质冷冻/融化后可溶解于PBS,存放于37℃3小时未发现有任何降解,当借助于SDS(12.5%PAGE)测定时,此蛋白质具有大约50000道尔顿的近似分子量。
实施例Ⅸ:
制备表达融合蛋白CLYTA-MAGE-1-His尾部的大肠杆菌菌株。
1.构建表达质粒pRIT14613和转化宿主菌株AR58:
蛋白质设计:
在图15中描绘了对将在大肠杆菌中被表达的融合蛋白CLYTA-MAGE-1-His的设计。
所形成蛋白质的一级结构具有在序列ID.NO.7中列出的序列。
在大肠杆菌表达质粒中,将对应于上述蛋白质设计的编码序列(见SEQUENCE 1D NO.8)安排在λPL启动子的控制之下。
克隆:
起始材料是载体PCUZ1和载体pRIT14518。载体PCRZ1含有来自肺炎链球菌LytA编码区的117个C-端密码子,在此载体pRIT14518中,以前我们已经亚克隆了MAGE-1基因cDNA,此cDNA来自从Ludwig研究所Dr.Thierry Boon得到的质粒。
为表达CLYTA-MAGE-1-His蛋白的克隆方案(见图16中的示意图16)包括下列步骤:
2.制备CLYTA-MAGE-1-His编码序列的组件:
a)第一步是PCR扩增,指定使NdeⅠ-AflⅢ限制性位点位于CLYTA序列的二侧。应用质粒PCUZ1a作模板以及如下寡核苷酸作引物进行此PCR扩增,有义寡核苷酸为:5’tta aac cac acc tta agg agg ata taacat atg aaa ggg gga att gta cat tea gac。反义寡核苷酸为:5’gccaga cat gtc caa ttc tgg cct gtc tgc cag。这将导致扩增378个核苷酸长度的CLYTA序列。
b)第二步是使CLYTA序列连接于MAGE-1-His序列,产生此融合蛋白的编码序列。此步骤包括切取NdeⅠ-AflⅢ Clyta片段,并插入预先用NdeⅠ和NcoⅠ(NcoⅠ和AflⅢ兼容性的)限制性酶切开的载体pRIT14518中,产生质粒pRIT14613。
c)转化宿主菌株AR58。
d)选择并鉴定含有质粒pRIT14613的大肠杆菌转化体(KAN抗性的)(见图16)。
1.对重组蛋白cLYTA-MAGE-1-His(pRIT14613)的鉴定:
使细菌在30℃生长在以50μg/ml卡那霉素补充的LB培养基上。当此培养物达到OD=0.3(在620nm)后,通过升高温度至38℃进行加热诱导。
诱导4小时后,收获细胞,再悬浮于PBS中,通过一次投射(oneshot)使细胞裂解(借助于崩解作用)。离心之后,通过SDS-PAGE分析沉淀的上清液和总提取物。在考马斯B1染色的凝胶中可见蛋白质,在此融合蛋白相当于大肠杆菌总蛋白的大约1%。此重组蛋白显示为一条单独的电泳带,具有大约49KD的近似MW。应用抗-MAGE-1多克隆抗体通过蛋白质印迹分析法对此融合蛋白进行了鉴定。
重建由长λPL启动子(对萘啶酮酸诱导有用的)和CLYTA-MAGE-1编码序列(pRIT14614)组成的表达单元:
由质粒pRIT DVA6制备了含有长PL启动子和部分CLYTA序列的EcoRⅠ-NCO1限制性片段,并将它插入质粒pRIT14613的EcoRⅠ-NCO1位点之间。
得到了重组质粒pRIT14614。
将编码融合蛋白CLYTA-MAGE-1-His的重组质粒pRIT14614(见图17)用于转化大肠杆菌AR120。选择卡那霉素抗性候选菌株,并进行了鉴定。
对重组蛋白的鉴定:
使细菌在30℃生长在以50mg/ml卡那霉素补充的LB培养基上。当此培养物达到CD=400(在620nm)时,加入萘啶酮酸至60mg/ml的最终浓度。
诱导4小时后,收获细胞,再悬浮于PBS中,并借助于崩解作用(崩解CLS“one shot”型)使细胞裂解。离心之后,通过SDS-PAGE分析沉淀的上清液和总提取物。在考马斯兰染色的凝胶中可见蛋白质,在此融合蛋白相当于大肠杆菌总蛋白的大约1%。应用兔抗-Mage-1多克隆抗体,通过蛋白质印迹分析法对此融合蛋白进行了鉴定。此重组蛋白显示为一条单独的电泳带,具有大约49KD的近似MW。
实施例Ⅹ:
CLYTA-MAGE-3-His
A:肿瘤排斥性重组抗原:融合蛋白CLYTA-MAGE-3-His;在此,C-lyt A融合配偶体导致表达一个可溶性蛋白,起亲和性标记的作用,并且提供有用的T-辅助细胞。
制备表达融合蛋白CLYTA-MAGE-3-His尾部的大肠杆菌菌株。
构建表达质粒pRIT14646并转化宿主菌株AR120:
蛋白质设计:
在图18中描绘了对将在大肠杆菌中被表达的融合蛋白Clyta-Mage-3-His的设计。
所形成蛋白质的一级结构具有在序列ID NO.9中描绘的序列,其编码序列被描绘在序列ID NO.10中。
在大肠杆菌表达质粒中,将对应于上述蛋白质设计的编码序列安排在λPL启动子的控制之下:
克隆:
起始材料是载体PCUZ1和载体pRIT14426,载体PCUZ1含有在“基因”43(1986)P.265-272中论述的,来自肺炎链球菌LytA编码区的117个C-端密码子,在载体pRIT14426中,以前我们已经亚克隆了MAGE-3基因cDNA,此cDNA来自从Ludwig研究所Dr.Tierry Boon得到的质粒。
为表达CLYTA-MAGE-3-His蛋白的克隆方案(见示意图19)包括如下步骤:
1.-制备CLYTA-MAGE-3-His编码序列的组件:
1.1第一步是PCR扩增,指定使Af1Ⅱ和AflⅢ限制性位点位于CLYTA序列的二侧。应用质粒PCUZ1作模板以及如下寡核苷酸作引物进行此PCR扩增,有义寡核苷酸为:5’tta aac cac acc tta agg agg atataa cat atg aaa ggg gga att gta cat tca gac,反义寡核苷酸为:5’ccc aca tgt cca gac tgc tgg cca att ctg gcc tgt ctg cca gtg。这将导致扩增427个核苷酸长度的CLYTA序列。将上面的扩增片段克隆进入外源(Invitrogen)的TA克隆载体,以便产生过渡性载体pRIT14661。
1.2第二步是使CLYTA序列连接于MAGE-3-His序列,产生此融合蛋白的编码序列。此步骤包括切取AflⅡ-AflⅢ Clyta片段,并插入预先用AflⅡ和NcoⅠ(NcoⅠ和AflⅡ兼容性的)限制性酶切开的载体pRIT14426中,产生质粒pRIT14662。
2.-重建由长λPL启动子(对萘啶酮酸诱导有用的)和CLYTA-Mage-3编码序列组成的表达单元:
由质粒pRIT14662制备了含有短PL启动子和CLYTA-MAGE-3-His编码序列的BglⅡ-XbaⅠ限制性片段,并将它插入质粒TCM67的BglⅡ-XbaⅠ位点之间(质粒TCM67是含有氨苄青霉素抗性和长λPL启动子的PBR322衍生物,在国际专利申请PCT/EP92/01827中已有论述)。获得了质粒pRIT14607。
将编码融合蛋白Clyta-mage-3-His的重组质粒pRIT14607用于转化大肠杆菌AR120(Mott等1985,国家科学院学报82:88)。选择并鉴定了氨苄青霉素抗性的候选菌株。
3.-制备质粒pRIT 14646:
最后构建了类似于pRIT 14607但具有卡那霉素选择作用的质粒(pRIT14646)。
对重组蛋白质的鉴定:
使细菌在30℃生长在以50mg/ml卡那霉素补充的LB培养基上。当此培养物达到OD=400(在600nm)时,加入萘啶酮酸至60?g/ml的最终浓度。
诱导4小时之后,收获细胞,再悬浮于PBS中,并借助于崩解作用(崩解CLS“one shot”型)使细胞裂解。离心之后,通过SDS-PAGE分析沉淀的上清液和总提取物。在考马斯兰染色的凝胶中可见蛋白质,在此融合蛋白相当于大肠杆菌总蛋白质的大约1%。应用兔抗Mage-3多克隆抗体,通过蛋白质印迹分析法对此融合蛋白进行了鉴定。此重组蛋白显示为一条单独的电泳带,具有大约58KD的近似MW。
实施例Ⅺ
重组蛋白CLYTA-MAGE-3-His的纯化:
在20升的发酵罐内,在分批加料的条件下30℃培养重组菌AR120(pRIT14646)。通过加入萘啶酮酸至最终浓度60?g/ml,诱导重组蛋白的表达。在发酵终点收获细胞,以60OD/600的密度,使之二次通过French压力破裂器(20000psi)而被裂解,在4℃将裂解的细胞以15000g离心20分钟。将含有此重组蛋白的上清液对DEAE琼脂糖DL6B树胶交换柱(pharmacia)加样,此交换柱已用0.3M NaCl,20mM Tris HCl pH7.6的缓冲液A预先平衡。用缓冲液A洗柱之后,以在缓冲液A中配制的2%胆碱洗脱融合蛋白。将应用抗-Mage-3抗体,通过蛋白质印迹分析揭示的阳性抗原组份合并在一起。将DEAE洗脱的抗原加入0.5%Empigen BB(一种二性离子去垢剂)和0.5M NaCl,然后在以0.5% EmpigenBB,0.5M NaCl,50mM磷酸缓冲液pH7.6(缓冲液B)平衡的金属离子亲和性层析柱上加样。
用缓冲液B洗此IMAC柱,直至在280nm的吸光度达到基线值。为了实施清除去垢剂,以不含Empigen BB的缓冲液B(缓冲液C)第二次洗柱。然后用在缓冲液C中配制的0-250mM的咪唑梯度液洗脱抗原。
合并0.090-0.250 M的咪唑组份,在10KDa的Filtron ω-膜上浓缩,然后对PBS缓冲液透析。
结论:
我们已证实,融合蛋白LPD-MAGE-3-His对小鼠是致免疫的,且该免疫原性(增殖性应答和抗体应答)可通过使用上述佐剂被进一步增强。通过衍生化形成二硫键的巯基可以提高纯化作用。
我们还证实,通过用存在佐剂的LPD-MAGE-3-His作疫苗接种可触发更强的抗体应答。在C57 BL/6小鼠血清中发现的优势同种型是IgG2b暗示产生了TH1型免疫应答。
对于人,临床安排一个病人用未加入佐剂的LPD-MAGE-3-His制剂治疗后清除了黑素瘤。
序列表
(1)一般信息(ⅰ)申请人:SmithKline Beecham Biologicals(ⅱ)发明名称:疫苗(ⅲ)序列数:10(ⅳ)通讯地址:
(A)收信人:SmithKline Beecham
(B)街道:2 New Horizons Court, Great West Road,B
(C)城市:Middx
(D)洲:
(E)国家:UK
(F)邮编:TW8 9EP(ⅴ)计算机可读形式:
(A)媒体类型:软盘
(B)计算机:IBM兼容机
(C)操作系统:DOS
(D)软件:FastSEQ for Windows Version 2.0(ⅵ)目前申请数据:
(A)申请号:
(B)申请日期:
(C)分类:(ⅶ)在先申请数据:
(A)申请号:
(B)申请日期:(ⅷ)代理人/代理商信息:
(A)姓名:Dalton,Marcus J
(B)注册号:
(C)参照/摘要书号:B45126(ⅸ)电讯信息:
(A)电话:0181 9756348
(B)光传真:0181 9756177
(C)电报:
(2)SEQ ID NO:1信息:(ⅰ)序列特征:
(A)长度:452个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:1:Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu1 5 10 15Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys20 25 30Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 35 40 45Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp50 55 60Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val65 70 75 80Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe85 90 95Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr100 105 110Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met115 120 125Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu130 135 140Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr145 150 155 160Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr165 170 175Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro180 185 190Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser195 200 205Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr210 215 220Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val225 230 235 240Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro245 250 255Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Ash Trp Gln Tyr260 265 270Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val275 280 285Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile290 295 300Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn305 310 315 320Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile325 330 335Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu340 345 350Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp355 360 365Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu370 375 380Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp385 390 395 400Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His405 410 415Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His420 425 430Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Ser Gly Gly His His His435 440 445His His His450
(2)SEQ ID No:2信息:(ⅰ)序列特征:
(A)长度:1353个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:2:ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCGTTTGCA 180CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360ATGACAGAAA ACTTTGAAAC CATGGATCTG GAACAGCGTA GTCAGCACTG CAAGCCTGAA 420GAAGGCCTTG AGGCCCGAGG AGAGGCCCTG GGCCTGGTGG GTGCGCAGGC TCCTGCTACT 480GAGGAGCAGG AGGCTGCCTC CTCCTCTTCT ACTCTAGTTG AAGTCACCCT GGGGGAGGTG 540CCTGCTGCCG AGTCACCAGA TCCTCCCCAG AGTCCTCAGG GAGCCTCCAG CCTCCCCACT 600ACCATGAACT ACCCTCTCTG GAGCCAATCC TATGAGGACT CCAGCAACCA AGAAGAGGAG 660GGGCCAAGCA CCTTCCCTGA CCTGGAGTCC GAGTTCCAAG CAGCACTCAG TAGGAAGGTG 720GCCGAATTGG TTCATTTTCT GCTCCTCAAG TATCGAGCCA GGGAGCCGGT CACAAAGGCA 780GAAATGCTGG GGAGTGTCGT CGGAAATTGG CAGTATTTCT TTCCTGTGAT CTTCAGCAAA 840GCTTCCAGTT CCTTGCAGCT GGTCTTTGGC ATCGAGCTGA TGGAAGTGGA CCCCATCGGC 900CACTTGTACA TCTTTGCCAC CTGCCTGGGC CTCTCCTACG ATGGCCTGCT GGGTGACAAT 960CAGATCATGC CCAAGGCAGG CCTCCTGATA ATCGTCCTGG CCATAATCGC AAGAGAGGGC 1020GACTGTGCCC CTGAGGAGAA AATCTGGGAG GAGCTGAGTG TGTTAGAGGT GTTTGAGGGG 1080AGGGAAGACA GTATCTTGGG GGATCCCAAG AAGCTGCTCA CCCAACATTT CGTGCAGGAA 1140AACTACCTGG AGTACCGGCA GGTCCCCGGC AGTGATCCTG CATGTTATGA ATTCCTGTGG 1200GGTCCAAGGG CCCTCGTTGA AACCAGCTAT GTGAAAGTCC TGCACCATAT GGTAAAGATC 1260AGTGGAGGAC CTCACATTTC CTACCCACCC CTGCATGAGT GGGTTTTGAG AGAGGGGGAA 1320GAGGGCGGTC ATCACCATCA CCATCACCAT TAA 1353
(2)SEQ ID NO:3信息:(ⅰ)序列特征:
(A)长度:1341个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:3:ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCGTTTGCA 180CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360ATGACAGAAA ACTTTGAAAC CATGGGCTCT CTGGAACAGC GTAGTCTGCA CTGCAAGCCT 420GAGGAAGCCC TTGAGGCCCA ACAAGAGGCC CTGGGCCTGG TGTGTGTGCA GGCTGCCACC 480TCCTCCTCCT CTCCTCTGGT CCTGGGCACC CTGGAGGAGG TGCCCACTGC TGGGTCAACA 540GATCCTCCCC AGAGTCCTCA GGGAGCCTCC GCCTTTCCCA CTACCATCAA CTTCACTCGA 600CAGAGGCAAC CCAGTGAGGG TTCCAGCAGC CGTGAAGAGG AGGGGCCAAG CACCTCTTGT 660ATCCTGGAGT CCTTGTTCCG AGCAGTAATC ACTAAGAAGG TGGCTGATTT GGTTGGTTTT 720CTGCTCCTCA AATATCGAGC CAGGGAGCCA GTCACAAAGG CAGAAATGCT GGAGAGTGTC 780ATCAAAAATT ACAAGCACTG TTTTCCTGAG ATCTTCGGCA AAGCCTCTGA GTCCTTGCAG 840CTGGTCTTTG GCATTGACGT GAAGGAAGCA GACCCCACCG GCCACTCCTA TGTCCTTGTC 900ACCTGCCTAG GTCTCTCCTA TGATGGCCTG CTGGGTGATA ATCAGATCAT GCCCAAGACA 960GGCTTCCTGA TAATTGTCCT GGTCATGATT GCAATGGAGG GCGGCCATGC TCCTGAGGAG 1020GAAATCTGGG AGGAGCTGAG TGTGATGGAG GTGTATGATG GGAGGGAGCA CAGTGCCTAT 1080GGGGAGCCCA GGAAGCTGCT CACCCAAGAT TTGGTGCAGG AAAAGTACCT GGAGTACCGG 1140CAGGTGCCGG ACAGTGATCC CGCACGCTAT GAGTTCCTGT GGGGTCCAAG GGCCCTCGCT 1200GAAACCAGCT ATGTGAAAGT CCTTGAGTAT GTGATCAAGG TCAGTGCAAG AGTTCGCTTT 1260TTCTTCCCAT CCCTGCGTGA AGCAGCTTTG AGAGAGGAGG AAGAGGGAGT CGGCGGTCAT 1320CACCATCACC ATCACCATTA A 1341
(2)SEQ ID NO:4信息:(ⅰ)序列特征:
(A)长度:466个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:4:Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu1 5 10 15Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys20 25 30Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro35 40 45Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp50 55 60Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val65 70 75 80Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe85 90 95Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr100 105 110Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met115 120 125Gly Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu130 135 140Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Thr145 150 155 160Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr165 170 175Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe180 185 190Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser195 200 205Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser210 215 220Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe225 230 235 240Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met245 250 255Leu Glu Ser Val Ile Lys Ash Tyr Lys His Cys Phe Pro Glu Ile Phe260 265 270Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys275 280 285Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly290 295 300Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr305 310 315 320Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His325 330 335Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr340 345 350Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr355 360 365Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp370 375 380Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala385 390 395 400Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala405 410 415Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu420 425 430Glu Glu Glu Gly Val Gly Gly His His His His His His His435 440 445
(2)SEQ ID NO:5信息:(ⅰ)序列特征:
(A)长度:404个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:5:Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile50 55 60Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr65 70 75 80Met Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu85 90 95Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala100 105 110Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val115 120 125Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser130 135 140Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp145 150 155 160Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser165 170 175Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys180 185 190Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu195 200 205Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln210 215 220Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu225 230 235 240Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr245 250 255Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp260 265 270Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile275 280 285Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu290 295 300Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly305 310 315 320Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu325 330 335Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu340 345 350Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His355 360 365His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu370 375 380His Glu Trp Val Leu Arg Glu Gly Glu Glu Gly Gly His His His His385 390 395 400His His His
(2)SEQ ID NO:6信息:(ⅰ)序列特征:
(A)长度:1212个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:6:ATGGATCCAA ACACTGTGTC AAGCTTTCAG GTAGATTGCT TTCTTTGGCA TGTCCGCAAA 60CGAGTTGCAG ACCAAGAACT AGGTGATGCC CCATTCCTTG ATCGGCTTCG CCGAGATCAG 120AAATCCCTAA GAGGAAGGGG CAGCACTCTT GGTCTGGACA TCGAGACAGC CACACGTGCT 180GGAAAGCAGA TAGTGGAGCG GATTCTGAAA GAAGAATCCG ATGAGGCACT TAAAATGACC 240ATGGATCTGG AACAGCGTAG TCAGCACTGC AAGCCTGAAG AAGGCCTTGA GGCCCGAGGA 300GAGGCCCTGG GCCTGGTGGG TGCGCAGGCT CCTGCTACTG AGGAGCAGGA GGCTGCCTCC 360TCCTCTTCTA CTCTAGTTGA AGTCACCCTG GGGGAGGTGC CTGCTGCCGA GTCACCAGAT 420CCTCCCCAGA GTCCTCAGGG AGCCTCCAGC CTCCCCACTA CCATGAACTA CCCTCTCTGG 480AGCCAATCCT ATGAGGACTC CAGCAACCAA GAAGAGGAGG GGCCAAGCAC CTTCCCTGAC 540CTGGAGTCCG AGTTCCAAGC AGCACTCAGT AGGAAGGTGG CCGAATTGGT TCATTTTCTG 600CTCCTCAAGT ATCGAGCCAG GGAGCCGGTC ACAAAGGCAG AAATGCTGGG GAGTGTCGTC 660GGAAATTGGC AGTATTTCTT TCCTGTGATC TTCAGCAAAG CTTCCAGTTC CTTGCAGCTG 720GTCTTTGGCA TCGAGCTGAT GGAAGTGGAC CCCATCGGCC ACTTGTACAT CTTTGCCACC 780TGCCTSGGCC TCTCCTACGA TGGCCTGCTG GGTGACAATC AGATCATGCC CAAGGCAGGC 840CTCCTGATAA TCGTCCTGGC CATAATCGCA AGAGAGGGCG ACTGTGCCCC TGAGGAGAAA 900ATCTGGGAGG AGCTGAGTGT GTTAGAGGTG TTTGAGGGGA GGGAAGACAG TATCTTGGGG 960GATCCCAAGA AGCTGCTCAC CCAACATTTC GTGCAGGAAA ACTACCTGGA GTACCGGCAG 1020GTCCCCGGCA GTGATCCTGC ATGTTATGAA TTCCTGTGGG GTCCAAGGGC CCTCGTTGAA 1080ACCAGCTATG TGAAAGTCCT GCACCATATG GTAAAGATCA GTGGAGGACC TCACATTTCC 1140TACCCACCCC TGCATGAGTG GGTTTTGAGA GAGGGGGAAG AGGGCGGTCA TCACCATCAC 1200CATCACCATT AA 1212
(2)SEQ ID NO:7信息:(ⅰ)序列特征:
(A)长度:445个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:7:Met Lys Gly Gly Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys1 5 10 15Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr20 25 30Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp35 40 45Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp50 55 60Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val65 70 75 60Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met85 90 95Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr100 105 110Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Leu Asp Met Gly115 120 125Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu Glu130 135 140Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Thr Ser145 150 155 160Ser Ser Ser Pro Leu val Leu Gly Thr Leu Glu Glu Val Pro Thr Ala165 170 175Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe Pro180 185 190Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser Ser195 200 205Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser Leu210 215 220Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe Leu225 230 235 240Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met Leu245 250 255Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe Gly260 265 270Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys Glu275 280 285Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly Leu290 295 300Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr Gly305 310 315 320Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His Ala325 330 335Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr Asp340 345 350Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr Gln355 360 365Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp Ser370 375 380Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala Glu385 390 395 400Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala Arg405 410 415Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu Glu420 425 430Glu Glu Gly Val Gly Gly His His His His His His His435 440 445
(2)SEQ ID NO:8信息:(ⅰ)序列特征:
(A)长度:1338个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:8:ATGAAAGGGG GAATTGTACA TTCAGACGGC TCTTATCCAA AAGACAAGTT TGAGAAAATC 60AATGGCACTT GGTACTACTT TGACAGTTCA GGCTATATGC TTGCAGACCG CTGGAGGAAG 120CACACAGACG GCAACTGGTA CTGGTTCGAC AACTCAGGCG AAATGGCTAC AGGCTGGAAG 180AAAATCGCTG ATAAGTGGTA CTATTTCAAC GAAGAAGGTG CCATGAAGAC AGGCTGGGTC 240AAGTACAAGG ACACTTGGTA CTACTTAGAC GCTAAAGAAG GCGCCATGGT ATCAAATGCC 300TTTATCCAGT CAGCGGACGG AACAGGCTGG TACTACCTCA AACCAGACGG AACACTGGCA 360GACAGGCCAG AATTGGACAT GGGCTCTCTG GAACAGCGTA GTCTGCACTG CAAGCCTGAG 420GAAGCCCTTG AGGCCCAACA AGAGGCCCTG GGCCTGGTGT GTGTGCAGGC TGCCACCTCC 480TCCTCCTCTC CTCTGGTCCT GGGCACCCTG GAGGAGGTGC CCACTGCTGG GTCAACAGAT 540CCTCCCCAGA GTCCTCAGGG AGCCTCCGCC TTTCCCACTA CCATCAACTT CACTCGACAG 600AGGCAACCCA GTGAGGGTTC CAGCAGCCGT GAAGAGGAGG GGCCAAGCAC CTCTTGTATC 660CTGGAGTCCT TGTTCCGAGC AGTAATCACT AAGAAGGTGG CTGATTTGGT TGGTTTTCTG 720CTCCTCAAAT ATCGAGCCAG GGAGCCAGTC ACAAAGGCAG AAATGCTGGA GAGTGTCATC 780AAAAATTACA AGCACTGTTT TCCTGAGATC TTCGGCAAAG CCTCTGAGTC CTTGCAGCTG 840GTCTTTGGCA TTGACGTGAA GGAAGCAGAC CCCACCGGCC ACTCCTATGT CCTTGTCACC 900TGCCTAGGTC TCTCCTATGA TGGCCTGCTG GGTGATAATC AGATCATGCC CAAGACAGGC 960TTCCTGATAA TTGTCCTGGT CATGATTGCA ATGGAGGGCG GCCATGCTCC TGAGGAGGAA 1020ATCTGGGAGG AGCTGAGTGT GATGGAGGTG TATGATGGGA GGGAGCACAG TGCCTATGGG 1080GAGCCCAGGA AGCTGCTCAC CCAAGATTTG GTGCAGGAAA AGTACCTGGA GTACCGGCAG 1140GTGCCGGACA GTGATCCCGC ACGCTATGAG TTCCTGTGGG GTCCAAGGGC CCTCGCTGAA 1200ACCAGCTATG TGAAAGTCCT TGAGTATGTG ATCAAGGTCA GTGCAAGAGT TCGCTTTTTC 1260TTCCCATCCC TGCGTGAAGC AGCTTTGAGA GAGGAGGAAG AGGGAGTCGG CGGTCATCAC 1320CATCACCATC ACCATTAA 1338
(2)SEQ ID NO:9信息:(ⅰ)序列特征:
(A)长度:454个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:9:Met Lys Gly Gly Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys1 5 10 15Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr20 25 30Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp35 40 45Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp50 55 60Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val65 70 75 80Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met85 90 95Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr100 105 110Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Leu Ala Ser Met115 120 125Leu Asp Met Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu130 135 140Gly Leu Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala145 150 155 160Pro Ala Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val165 170 175Glu Val Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro180 185 190Gln Ser Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro195 200 205Leu Trp Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly210 215 220Pro Ser Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser225 230 235 240Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala245 250 255Arg Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn260 265 270Trp Gln Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu275 280 285Gln Leu Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His 290 295 300Leu Tyr Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu305 310 315 320Gly Asp Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu325 330 335Ala Ile Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp340 345 350Glu Glu Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile355 360 365Leu Gly Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn370 375 380Tyr Leu Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu385 390 395 400Phe Leu Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val405 410 415Leu His His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro420 425 430Pro Leu His Glu Trp Val Leu Arg Glu Gly Glu Glu Gly Gly His His435 440 445His His His His His450
(2)SEQ ID NO:10信息:(ⅰ)序列特征:
(A)长度:1362个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性(ⅱ)分平类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:10:ATGAAAGGGG GAATTGTACA TTCAGACGGC TCTTATCCAA AAGACAAGTT TGAGAAAATC 60AATGGCACTT GGTACTACTT TGACAGTTCA GGCTATATGC TTGCAGACCG CTGGAGGAAG 120CACACAGACG GCAACTGGTA CTGGTTCGAC AACTCAGGCG AAATGGCTAC AGGCTGGAAG 180AAAATCGCTG ATAAGTGGTA CTATTTCAAC GAAGAAGGTG CCATGAAGAC AGGCTGGGTC 240AAGTACAAGG ACACTTGGTA CTACTTAGAC GCTAAAGAAG GCGCCATGGT ATCAAATGCC 300TTTATCCAGT CAGCGGACGG AACAGGCTGG TACTACCTCA AACCAGACGG AACACTGGCA 360GACAGGCCAG AATTGGCCAG CATGCTGGAC ATGGATCTGG AACAGCGTAG TCAGCACTGC 420AAGCCTGAAG AAGGCCTTGA GGCCCGAGGA GAGGCCCTGG GCCTGGTGGG TGCGCAGGCT 480CCTGCTACTG AGGAGCAGGA GGCTGCCTCC TCCTCTTCTA CTCTAGTTGA AGTCACCCTG 540GGGGAGGTGC CTGCTGCCGA GTCACCAGAT CCTCCCCAGA GTCCTCAGGG AGCCTCCAGC 600CTCCCCACTA CCATGAACTA CCCTCTCTGG AGCCAATCCT ATGAGGACTC CAGCAACCAA 660GAAGAGGAGG GGCCAAGCAC CTTCCCTGAC CTGGAGTCTG AGTTCCAAGC AGCACTCAGT 720AGGAAGGTGG CCAAGTTGGT TCATTTTCTG CTCCTCAAGT ATCGAGCCAG GGAGCCGGTC 780ACAAAGGCAG AAATGCTGGG GAGTGTCGTC GGAAATTGGC AGTACTTCTT TCCTGTGATC 840TTCAGCAAAG CTTCCGATTC CTTGCAGCTG GTCTTTGGCA TCGAGCTGAT GGAAGTGGAC 900CCCATCGGCC ACGTGTACAT CTTTGCCACC TGCCTGGGCC TCTCCTACGA TGGCCTGCTG 960GGTGACAATC AGATCATGCC CAAGACAGGC TTCCTGATAA TCATCCTGGC CATAATCGCA 1020AAAGAGGGCG ACTGTGCCCC TGAGGAGAAA ATCTGGGAGG AGCTGAGTGT GTTAGAGGTG 1080TTTGAGGGGA GGGAAGACAG TATCTTCGGG GATCCCAAGA AGCTGCTCAC CCAATATTTC 1140GTGCAGGAAA ACTACCTGGA GTACCGGCAG GTCCCCGGCA GTGATCCTGC ATGCTATGAG 1200TTCCTGTGGG GTCCAAGGGC CCTCATTGAA ACCAGCTATG TGAAAGTCCT GCACCATATG 1260GTAAAGATCA GTGGAGGACC TCGCATTTCC TACCCACTCC TGCATGAGTG GGCTTTGAGA 1320GAGGGGGAAG AGGGCGGTCA TCACCATCAC CATCACCATT AA 1362
参考文献:-Anichini A.,Fossati G.,Parmiani G.今日免疫学,8:385(1987)。-De Plaen E.,Arden K.,Traversari C.,et al.免疫遗传学,40:360(1994)。-Gaugler B.,Van den Eynde B.,van der Bruggen P.,et al.实验医学杂志,179:921(1994)。-Herman J.,van der Bruggen P.,Immanuel F.,et al.免疫遗传学,43:377(1996)。-Inoue H.,Mori M.,Li J.,et al.国际癌杂志,63:523(1995)。-Kensil C.R.,Soltysik S.,Patel U.,et al.in:Channock R.M.,Ginsburg H.S.,Brown F.,et al.,(eds.),疫苗92,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.),36-40:(1992)。-Knuth A.,Danowski B.,Oettgen H.F.,et al.美国国家科学院学报,81:3511(1984).-Patard J.J.,Brasseur F.,Gil-Diez s.,et al.国际癌杂志,64:60(1995)。-Ribi E.,et al.in:Levine L.,Bonventre P.F.,Morello J.,et al.(eds).,美国微生物学协会,Washington DC,微生物学1986,9-13;(1986)。-Van den Eynde B.,Hainaut P.,Hrin M.et al.国际癌杂志,44:634(1989)。-Van der Bruggen P.,Traversari C.,Chomez P.,et al.科学,254:1643(1991)。-Van der Bruggen P.,Bastin J.,Gajewski T.,et al.欧洲免疫学杂志,24:3038(1994).-Van Pel A.,van der Bruggen P.,Coulie P.G.,et al.,免疫学评论,145:229(1995)。-Weynants P.,Leth B.,Brasseur F.,et al.国际癌杂志,56:826(1994)。-Nishimura S,Fujita M,Terata N,Tani T,Kodama M,Itoh K,Nihon RinshoMeneki Gakkai Kaishi 1997,四月,20(2):95-101。-Fuijie T et al,肿瘤学纪事1997四月,8(4):369-2。
Claims (20)
1.一种肿瘤相关性抗原衍生物,其来自MAGE家族。
2.根据权利要求1的抗原,其中该衍生物是连接于免疫融合配偶体或表达增强配偶体的MAGE蛋白。
3.根据权利要求1或2的抗原,其中的衍生物包含一个亲和性标记。
4.根据权利要求1-3中任何之一的抗原,它包含衍生化的自由巯基。
5.根据权利要求4的抗原,它是一个羧酰胺化或羧甲基化的衍生物。
6.一种根据权利要求2,3,4或5的蛋白质,其中的融合配偶体是来自流感嗜血杆菌B的蛋白D或其片段,来自流感病毒的NS1蛋白或其片段,或者是来自肺炎链球菌的Lyta或其片段。
7.根据权利要求2,3,4或5的蛋白质,其中的融合配偶体是来自流感嗜血杆菌B的蛋白D的脂质化形式或其片段。
8.根据权利要求1-7的蛋白质,其中的MAGE蛋白是选自如下种类:MAGE A1,MAGE A2,MAGE A3,MAGE A4,MAGE A5,MAGE A6,MAGEA7,MAGE A8,MAGE A9,MAGE A10,MAGE A11,MAGE A12,MAGE B1,MAGE B2,MAGE B3和MAGE B4,MAGE C1,MAGE C2。
9.一种核酸序列,编码在此要求专利保护的蛋白质。
10.一种载体,包含权利要求9的核酸。
11.一种宿主,是用权利要求10的载体转化的。
12.一种疫苗,含有根据权利要求1-8中任何之一的蛋白质,或者根据权利要求9的核酸。
13.根据权利要求12的疫苗,另外还包含一种佐剂,和/或免疫刺激细胞因子或趋化因子。
14.根据权利要求12或13的疫苗,其中该蛋白质是以水包油或油包水乳剂作赋形剂提供的。
15.根据权利要求13或14的疫苗,其中的佐剂包括3D-MPL,QS21或CpG寡核苷酸。
16.在此要求专利保护的疫苗,另外还包含一种或几种其它抗原。
17.在此要求专利保护的疫苗,是用于医药。
18.在此要求专利保护的蛋白质或核酸在制造一种用于免疫治疗患有黑素瘤或其它MAGE-相关肿瘤的病人的疫苗中的用途。
19.一种纯化MAGE蛋白或其衍生物的方法,包括还原二硫键,用封阻基团封闭所形成的自由巯基,以及使所形成的衍生物经受一个或几个层析纯化步骤。
20.一种用于生产疫苗的方法,包括借助于权利要求19的方法纯化MAGE蛋白或其衍生物的几个步骤,以及将所形成的蛋白质配制成疫苗。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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GBGB9802543.0A GB9802543D0 (en) | 1998-02-05 | 1998-02-05 | Vaccine |
GB9802543.0 | 1998-02-05 | ||
GBGB9802650.3A GB9802650D0 (en) | 1998-02-06 | 1998-02-06 | Vaccine |
GB9802650.3 | 1998-02-06 | ||
PCT/EP1999/000660 WO1999040188A2 (en) | 1998-02-05 | 1999-02-02 | Tumor-associated antigen derivatives from the mage family, and nucleic acid sequences encoding them, used for the preparation of fusion proteins and of compositions for vaccinations |
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CN1295616A true CN1295616A (zh) | 2001-05-16 |
CN1227360C CN1227360C (zh) | 2005-11-16 |
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KR (2) | KR100824105B1 (zh) |
CN (1) | CN1227360C (zh) |
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AT (4) | ATE505542T1 (zh) |
AU (1) | AU737337B2 (zh) |
BR (1) | BR9907691B1 (zh) |
CA (2) | CA2584482C (zh) |
CY (4) | CY1105685T1 (zh) |
CZ (2) | CZ298347B6 (zh) |
DE (3) | DE69929310T2 (zh) |
DK (4) | DK1659179T3 (zh) |
ES (2) | ES2255248T3 (zh) |
HK (4) | HK1033838A1 (zh) |
HU (1) | HU228467B1 (zh) |
IL (4) | IL137442A0 (zh) |
NO (2) | NO328507B1 (zh) |
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PT (4) | PT1584685E (zh) |
SA (1) | SA99200126B1 (zh) |
SI (4) | SI1659179T1 (zh) |
TR (1) | TR200002284T2 (zh) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN105181966A (zh) * | 2015-09-02 | 2015-12-23 | 南通大学附属医院 | 一种mage-a9的用途 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102251034A (zh) * | 2011-07-05 | 2011-11-23 | 北京大学人民医院 | 基于mage-c2/ct10基因辅助诊断多发性骨髓瘤患者的定量检测试剂盒 |
CN105181966A (zh) * | 2015-09-02 | 2015-12-23 | 南通大学附属医院 | 一种mage-a9的用途 |
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