CN1215167C - 幽门螺杆菌活疫苗 - Google Patents
幽门螺杆菌活疫苗 Download PDFInfo
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- CN1215167C CN1215167C CNB971995516A CN97199551A CN1215167C CN 1215167 C CN1215167 C CN 1215167C CN B971995516 A CNB971995516 A CN B971995516A CN 97199551 A CN97199551 A CN 97199551A CN 1215167 C CN1215167 C CN 1215167C
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Abstract
本发明涉及新的重组活疫苗,该疫苗能提供抵抗幽门螺杆菌感染的保护性免疫,本发明还涉及筛选用于最适疫苗的幽门螺杆菌抗原的方法。
Description
本发明涉及新的重组活疫苗,该疫苗提供了抵抗幽门螺杆菌感染的保护性免疫,并涉及筛选最适疫苗的幽门螺杆菌抗原的方法。
螺杆菌是与胃炎,胃溃疡和胃癌的发展相关的革兰氏阴性细菌病原体。几个螺杆菌种类定居于胃,最主要的是幽门螺杆菌,H.heilmanii和猫螺杆菌。虽然幽门螺杆菌是与人感染相关的最常见的种类,但是也已经发现H.heilmanii和猫螺杆菌也感染人。在第三世界国家以及在工业化国家观察到高的幽门螺杆菌感染率。在幽门螺杆菌中叙述的所有毒力因子中,已知脲酶对于在悉生猪和裸鼠的定居是必需的。脲酶是由两个结构亚基(UreA和UreB)组成的酶。前面的研究已经表明利用重组UreB加霍乱毒素口服免疫能够保护小鼠不受猫螺杆菌和幽门螺杆菌的胃定居(Michetti等人,胃肠病学107(1994),1002-1011)。但是,通过口服重组UreB抗原,在几种情况下只能获得不完全的保护。其它显示给出部分保护的幽门螺杆菌抗原是87千道尔顿液泡细胞毒素VacA(Cover和Blaser,生物化学杂志,267(1992),10570;Marchetti等人,科学267(1995),1655)和13和58千道尔顿热休克蛋白HspA和HspB(Ferrero等人,美国国家科学院院报92(1995),6499)。
减毒病原体,例如细菌,如沙门氏菌已知是有效的活疫苗。减毒的沙门氏菌作为好的疫苗在人中的效力的第一个证据来自利用化学诱变的伤寒沙门氏菌Ty21a菌株的研究,(Germanier和Furer,传染病杂志,141(1975),553-558),这一菌株在成人志愿者中(Gilman等人,传染病杂志,136(1977),717-723),后来在埃及的儿童中成功地进行了大型现场试验(Whadan等人,感染疾病杂志,145(1982),292-295)。口服给药Ty21a疫苗能够在三年的观察过程中保护96%接种的埃及儿重。从那时起,新的减毒沙门氏菌活载体疫苗已经开发出来(Hone等人,疫苗9(1991),810-816),其中掺入染色体的完全确定的突变产生了能够在口服给药后诱导强免疫应答的非毒性菌株(Tacket等人,疫苗10(1992),443-446和Tacket等人,感染免疫60(1992),536-541)。与前面的失活的大批伤寒疫苗相比,活减毒沙门氏菌疫苗的其它优点包括它的安全性,容易给药,长时间保护和没有不利反应(Levine等人,伤寒热疫苗。见Plotkin S.A.,Mortimer E.A.Jr(编辑)疫苗。费城:WB Saunders(1988),333-361)。
伤寒沙门氏菌突变体已经广泛用于递送抗原,因为可以使用小鼠作为动物模型,该模型确信是模仿了伤寒沙门氏菌在人中的感染。最通常使用的伤寒沙门氏菌的减毒方法包括影响芳香氨基酸的合成的位点特异性诱变。将这样的菌株命名为aro突变体,如在利用这些构建体的动物模型和人试验中证实的,该突变体具有微不足道的致病性(Hoiseth和Stocker,自然291(1981),238-239;Tacket等人(1992),出处同上)。活沙门氏菌疫苗递送异源抗原的潜在免疫原性已经得到利用。在减毒沙门氏菌中表达特异抗原已经使鼠获得抵抗几种细菌病原体的保护。能够表达螺杆菌抗原和保护接种动物的重组活疫苗的用途还没有叙述。减毒的活疫苗在治疗螺杆菌感染中的用途也还不明显。其原因是在螺杆菌感染的过程中诱导了针对病原体本身的强免疫应答,但是,这不会导致保护性免疫。所以,非常惊人的是当使用重组减毒细菌细胞作为抗原载体时获得了保护性免疫应答,该载体能够表达编码螺杆菌抗原的DNA分子。显然,表达螺杆菌抗原的重组减毒细菌细胞能够产生的针对异源螺杆菌抗原的免疫应答与螺杆菌本身针对自身的同源抗原的性质不同。非保护性免疫应答就这样惊人地转化成抵抗螺杆菌感染的保护性免疫应答。这种预料不到的发现使利用重组减毒病原体,例如细菌细胞,特别是沙门氏菌,作为筛选保护性抗原的载体,在任何抵抗螺杆菌感染的疫苗中以这种方式应用已鉴定的保护性抗原,和使用重组减毒细菌作为保护性抗原的载体用于在人和其它动物中针对螺杆菌感染的免疫成为可能。
因此,本发明的主题是重组减毒病原体,它含有至少一个编码螺杆菌抗原的异源核酸分子,其中所述的病原体能够表达所述的核酸分子或者能够导致在靶细胞中表达所述的核酸。优选地,该核酸分子是DNA分子。
减毒的病原体是微生物菌株,该菌株能够引起感染和优选地引起针对真正病原体的有效免疫保护,但其本身不再是致病性的。减毒病原体可以是细菌,病毒,真菌或寄生虫。优选地,它是细菌,例如沙门氏菌,如鼠伤寒沙门氏菌或伤寒沙门氏菌,霍乱弧菌(Mekalanos等人,自然306(1983),551-557),志贺氏菌种类如弗氏志贺氏菌(Sizemore等人,科学,270(1995),299-302;Mounier等人,EMB0 J.11(1992),1991-1999),利斯特氏菌如单核细胞增生利斯特氏菌(Milon和Cossart,微生物学趋势3(1995),451-453),大肠杆菌,链球菌,如格式链球菌(Medaglini等人,美国国家科学院院报92(1995)6868-6872),或分枝杆菌,如卡介苗(Flynn,细胞分子生物学,40增刊1(1994),31-36)。更优选地,病原体是减毒肠杆菌,如霍乱弧菌,弗氏志贺氏菌,大肠杆菌或沙门氏菌。最优选地,减毒病原体是沙门氏菌细胞,例如沙门氏菌aro突变体细胞。但是,减毒病原体可以是病毒,例如减毒疫苗病毒,腺病毒或痘病毒。
插入病原体的核酸分子编码螺杆菌抗原,优选地是猫螺杆菌,H.heilmanii或幽门螺杆菌抗原,更优选地是幽门螺杆菌抗原。螺杆菌抗原可以是天然螺杆菌多肽,其免疫反应片段,或天然多肽或其片段的免疫反应变体。另外,螺杆菌抗原可以是刺激天然螺杆菌抗原的三维结构的保护性碳水化合物或肽模拟物。从在细菌细胞表面上呈递的肽库可以获得肽模拟物(参见,PCT/EP96/01130)。当然,转化细胞也可以含有编码不同螺杆菌抗原的几个DNA分子。
编码螺杆菌抗原的核酸分子可以定位于染色体外载体,例如质粒上,和/或整合进病原体的细胞染色体中。当将病原体用作疫苗时,染色体整合通常是优选的。
减毒细菌可以用于直接在细菌细胞中转录和翻译所述的核酸分子或递送所述的核酸分子到感染的靶细胞,以致通过真核靶细胞机制转录和/或翻译该DNA分子。这一间接的细菌接种过程,本文命名为遗传接种,已经利用志贺氏菌作为载体成功地使用了(Sizemore,D.R.,Branstrom,A.A.和Sadoff,J.C.(1995)减毒志贺氏菌作为DNA递送载体用于DNA介导的免免疫,科学,270:299-302)。
在本发明的优选实施方案中,螺杆菌抗原是脲酶,脲酶亚基或免疫反应变体,或其片段或其多肽模拟物。在本发明的另一优选的实施方案中,螺杆菌抗原是来自螺杆菌的分泌性多肽,免疫反应变体或其片段或其肽模拟物。在国际专利申请PCT/EP96/02544中已经公开了鉴定编码这样的分泌性多肽,特别是编码粘附素的螺杆菌基因的方法,该专利申请引入本文作为参考。这一方法包括
a)在含有与分泌活性的标记偶联的诱导型转座子的宿主有机体中制备幽门螺杆菌DNA的基因库,
b)诱导转座子插入到幽门螺杆菌的DNA中和
c)通过标记进行含有分泌性基因的克隆的选择,和可选择地进一步
d)通过含有具有分泌活性的基因的克隆的DNA进行幽门螺杆菌的再转化,其中通过在染色体中整合该DNA产生等基因幽门螺杆菌突变体菌株,和
e)进行检测粘附缺陷性幽门螺杆菌突变体菌株的选择。
通过上面的方法得到的抗原的适当例子选自包括抗原AlpA,AlpB,免疫反应变体或其片段或其肽模拟物的组。在国际专利申请PCT/EP96/02545和PCT/EP96/04124中已经公开了抗原AlpA和AlpB的核酸和氨基酸序列,这些专利申请引入本文作为参考。另外,在SEQ ID NO:1和2中显示了AlpB的核酸和氨基酸序列,在SEQ ID NO:3和4中显示了AlpA的核酸和氨基酸序列。
但是,同样可以想象的是例如通过自溶释放可以利用在该表面上呈递的细胞内抗原,并获得免疫保护。
根据本发明的螺杆菌抗原在重组病原体中的呈递可以以不同的方式完成。以组成型,诱导型或相变异型方式可以在重组病原体中合成该一种或多种抗原。如关于螺杆菌抗原的组成或诱导合成,可以参考Sambrook等人(分子克隆,实验室手册,冷泉港实验室出版社)已经叙述已知的表达系统。
特别优选地,该抗原在相变异型表达系统中呈递。在EP-B-0 565 548中公开了这种用于在杂合活疫苗中生产和呈递外源抗原的相变异型表达系统,该专利引入本文作为参考。在这样的相变异型表达系统中,编码螺杆菌抗原的核酸分子是在表达信号的控制下的,该信号在病原体中基本上是无活性的,并且能够通过核酸引起的自发重新组构,例如在病原体中的DNA重新组构机制,例如,特异的DNA倒位方法,特异的DNA缺失方法,特异的DNA复制方法或特异的突出链错配机制进行活化。
具有相变异型表达系统的重组细胞能够形成两个A和B亚群,其中通过在重组核酸中自发重新组构划分为所述的亚群,其中所述的亚群A能够感染和本身免疫活化,而亚群B,是从亚群A再生的,产生至少一种异源螺杆菌抗原,并且与所述的附加抗原免疫相关地起作用。
通过核酸重新组构可以直接完成,或者可以通过控制编码螺杆菌抗原的基因的表达的蛋白质编码基因的活化间接完成编码螺杆菌抗原的表达信号的活化。间接活化表示系统允许通过级联系统产生螺杆菌抗原,这是因为,例如,由DNA重新组构直接控制的基因编码特异于在螺杆菌基因前面的启动子,或以另一种特异方式诱导螺杆菌基因表达的基因调节物的RNA聚合酶。在本发明的特别优选的实施方案中,编码螺杆菌抗原的基因的表达信号是噬菌体启动子,例如,T3,T7或SP6启动子,表达信号的活化是由导致在病原体中产生相应的噬菌体RNA聚合酶的核酸重新组构引起的。
可以调节相变异型表达系统以提供螺杆菌抗原的预选的表达水平。这可以通过修饰表达信号的核苷酸序列完成,该表达信号通过核酸重新组构机制,和/或通过进一步插入遗传调节元件活化。
以细胞内,以及细胞外的方式在本发明的病原体中可以产生螺杆菌抗原。例如,自体转运蛋白系统如IgA-蛋白酶系统(例如,参见EP-A-0254090)或大肠杆菌AIDA-1粘附素系统(Benz等人,分子微生物学,6(1992),1539)适于作为细胞外分泌系统。其它适当的外膜转运蛋白系统是RTX毒素转运蛋白,例如,大肠杆菌溶血素转运蛋白系统(Hess等人,美国国家科学院院报,93(1996),11458-11463)。
除了编码螺杆菌抗原的核酸分子,本发明的病原体可以含有第二个异源核酸,例如DNA分子,该分子编码定量或定性地影响免疫应答的免疫调节多肽。这样的免疫调节多肽的例子是免疫刺激肽,细胞因子,如IL-2,IL-6或IL-12,趋化因子,毒素,如伤寒毒素B或粘附素。
本发明同时涉及含有如上所述的作为活化剂的重组减毒病原体的药物组合物,可选择地和药物上可接受的稀释剂,载体和佐剂在一起。优选地,组合物是活疫苗。接种途径根据接种载体的选择而变化。以单剂量或间隔重复进行给药。适当的剂量取决于各种参数如疫苗载体本身,或给药途径。通常,每次接种的剂量含有约106到1012个细胞(CFU),优选地约108到1010个细胞(CFU)。可以选择对粘膜表面给药(例如,眼,鼻内,口服,胃,肠,直肠,阴道或尿道)或通过肠胃外的途径(例如,皮下,皮内,肌肉内,静脉内或腹膜内)。制备活疫苗的方法包括配制含有药物上可接受的稀释剂,载体和/或佐剂的药物有效量的减毒病原体。
药物组合物可以任何适当的形式提供,例如适当液体载体如水或牛奶中的悬浮液,胶囊,片剂等等。在本发明的优选实施方案中,该组合物是冷冻干燥产物,在使用之前将它们悬浮于液体载体中。
另外,本发明涉及制备上面定义的重组减毒病原体的方法,包括步骤a)在减毒病原体中插入编码螺杆菌抗原的核酸分子,其中得到的重组病原体,例如,转化的细菌细胞能够表达所述核酸分子或能够导致在靶细胞中表达所述的核酸分子和b)在适当的条件下培养所述的重组减毒病原体。如果病原体是细菌细胞,编码螺杆菌抗原的核酸分子可以定位于染色体外的质粒上。但是,也可能将该核酸分子插入在病原体的染色体中。
另外,本发明涉及鉴定螺杆菌抗原的方法,该抗原在哺乳动物宿主中产生保护性免疫应答,该方法包括步骤:a)提供在减毒病原体中的螺杆菌的表达基因库,和b)筛选基因库的克隆的在哺乳动物宿主中获得抵抗螺杆菌感染的保护性免疫的能力。优选地,这一鉴定方法发生在相变异型表达系统中,使所有螺杆菌抗原的稳定表达成为可能。然后,重组克隆可以作为“库”用于测试动物如小鼠的口服免疫。然后,这些克隆作为保护性抗原的潜力通过利用螺杆菌,例如小鼠适应的幽门螺杆菌菌株攻击感染可以确定。所以,直接选择最适幽门螺杆菌疫苗抗原的可能性是存在的。
本发明将进一步通过下面的图和序列表说明。
图1:图解说明了脲酶表达载体pYZ97,在该载体上,编码脲酶亚基UreA和UreB的基因定位于T7启动子10的转录控制下。在T7启动子和脲酶基因之间存在核糖体结合位点(RBS)。另外,该质粒连续地展示了复制原点(ori),β-内酰胺酶抗性基因(bla)和4T7终止子。
除了通过T7启动子表达,通过隐启动子也可以引起脲酶A和脲酶B亚基的组成型低水平表达。该启动子定位于质粒pYZ97上的T7启动子上游。
图2:显示了质粒pYZ97上用于脲酶表达的转录调节区的核苷酸序列和脲酶亚基A的氨基酸序列的开头。
图3:图解说明了T7 RNA聚合酶(T7RNAP)表达盒pYZ88,pYZ84和pYZ114,这些表达盒可以整合进入细菌染色体。
在高表达盒pYZ88中,λPL启动子以相反方向定位于T7RNAP基因的上游。温度敏感性阻遏物cI 857(cI)的基因是在这一启动子的控制下的。噬菌体fd(fdT)的终止子定位于cI基因的上游。gin基因(Mertens,EMBOJ.3(1984),2415-2421)编码DNA重新组构机制的控制酶。编码tRNA Arg的DNA序列定位于gin基因的下游。
在A相中,负责T7RNAP基因的表达的PL启动子定向于cI857基因和gin基因的方向。结果是在28℃的允许温度下形成活性阻遏物,从而减少从PL启动子的转录。在较高的温度下,PL启动子的转录提高,因为阻遏物至少部分地在这样的外部影响下失活。在转录中依赖温度的提高也导致下面gin基因的表达的相应的提高,这是因为控制酶催化了PL启动子的倒位和在B相的转换,其中T7RNAP基因得到表达。
在高表达系统pYZ88中,另一个fdT转录终止子定位于卡那霉素抗性基因(km)和这一基因的启动子之间。以这种方式,通常减少T7RNAP表达的反向定位于T7RNAP基因的反义RNA的合成减少了。这导致了T7RNAP的高表达。
在中等表达系统pYZ84中,转录终止子(fdT)定位于PL启动子和T7RNAP基因的开端之间。以这种方式,减少了T7RNAP mRNA的表达。另外,反义RNA影响了T7RNAP翻译。所以,只发生了中等表达。
在低表达系统pYZ114中,另外在PL中导入了100bp的缺失(ΔPL)。以这种方式,高度减少了PL启动子的活性,这导致了较低的T7RNAP表达,所以减少了UreA/B基因表达。在这一构建体中,已经观察到隐启动子对pYZ97的影响。
图4:显示了在接种的小鼠的肠液中抗幽门螺杆菌的抗体的ELISA结果。
图5:显示了在接种的小鼠的血清中抗幽门螺杆菌的抗体的ELISA的结果。
图6:显示了在H.pyroli攻击后,接种的小鼠的胃组织中脲酶的活性。
SEQ ID NO:1和2显示了来自幽门螺杆菌的粘附素基因AlpB的核苷酸序列,和它们所编码的多肽的氨基酸序列。
SEQ ID NO:3和4显示了来自幽门螺杆菌的粘附素基因AlpA的核苷酸序列和它们所编码的蛋白质的氨基酸序列。
SEQ ID NO:5和6显示了质粒pYZ97上用于脲酶表达的转录调节区的核苷酸序列和脲酶亚基A的氨基酸序列的开头。
实验部分
实施例1
ureA和ureB基因的克隆
从阿姆斯特丹大学分离和由Jos van Putten博士提供的临床样品P1(以前的69A)的染色体DNA已经遗传克隆了编码脲酶的结构基因,ureA和ureB。通过PCR途径,利用引物对YZ019(5’-GGAATTCCATATGAAACTGACTCCCAAAGAG-3’)和RH132(5’-CTGCAGTCGACTAGAAAATGCTAAAGAG-3’)扩增分离这些基因。从GenBank(登记号M60398,X57132)推测引物的序列。引物YZ019的DNA序列覆盖了公开的序列的核苷酸2659-2679,并且进一步含有翻译调节序列(下游盒;Sprengart,M.L.等人,1990,核酸研究,18:1719-1723)和NdeI的切割位点。引物RH132的DNA序列覆盖了公开的序列的核苷酸5071-5088和切割位点SalI。扩增产物的大小是2.4kbp,包含没有来自螺杆菌染色体的原始转录起始和终止序列的ureA和ureB基因的全部编码区。利用NdeI和SalI消化纯化的PCR片段并插入T7表达质粒pYZ57的相应的克隆位点,以便产生质粒pYZ97。
最初,pYZ57起源于质粒pT7-7,Tabor叙述了这些(1990,分子生物学的当前方案,16.2.1-16.2.11,Greene出版和Wiley-Interscience,纽约)。在pT7-7骨架的不同位点通过下面的步骤导入两个终止子片段:(1)串联的T7终止子。从pEP12切出2.2kbp EcoRI/HindIII片段(Brunschwig和Darzins,1992,基因,111:35-41)并且将纯化的片段与预消化的pBA连接(Mauer,J.等人,1997,细菌学杂志,179:794-804)。利用HindIII和ClaI消化连接产物。随后,将得到的2.2kbp HindIII/ClaI片段插入预消化的pT7-7。(2)T1终止子。从质粒pDS3EcoRV(H.Bujard博士;提供ZMBH,Heidelberg)切出230bp HpaI/NdeI片段。然后,利用Klenow进一步处理该片段产生平端。将纯化的rrnBT1片段插入前面的利用BglII预消化,随后通过Klenow处理成为平端的pT7-7衍生物。图1叙述了用于在鼠伤寒沙门氏菌中表达编码脲酶亚基UreA和UreB的脲酶基因的完整的载体pYZ97。如图1所示,通过T7启动子10可以控制脲酶基因。核糖体结合位点(RBS)定位于T7启动子和脲酶基因之间。另外,该质粒展示了复制原点(ori)和β-内酰胺酶抗性基因(bla)。
除了由T7启动子控制的表达,脲酶A和B亚基的组成型适中水平表达确实发生于沙门氏菌RNA聚合酶驱动的启动子。该启动子定位于质粒pYZ97上T7启动子的上游。为了详细的分子分析,将纯化的pYZ97的BglII/HindIII片段亚克隆进入pCR-ScriptTM SK(+)试剂盒(Stratagene)并进行DNA测序。序列数据证明了在它们的整个序列中的各种元件(参见图2和SEQ ID NO:5和6):部分ureA基因,下游盒,RBS,T7启动子和T1终止子(rrnBT1)。序列分析也公开了在T1终止子区和T7启动子之间的区域,沙门氏菌RNA聚合酶启动子定位于在该区域中。序列数据表明这一组成型启动子的定位在核苷酸222-245之间,这已经从结构预测中推测出来(Lisser和Margalit,1993,核酸研究,21:1507-1516)。
实施例2
通过活疫苗给药进行免疫保护
材料和方法
细菌菌株:利用鼠伤寒沙门氏菌SL3261活载体疫苗菌株作为重组幽门螺杆菌脲酶质粒构建体的受体。鼠伤寒沙门氏菌SL3261是起源于鼠伤寒沙门氏菌SL1344野生型菌株的aroA的转座突变体。鼠伤寒沙门氏菌SL3261是非毒力菌株,在口服给药后,给予小鼠抵抗野生型鼠伤寒沙门氏菌感染的保护(Hoiseth和Stocker(1981)出处同上)。鼠伤寒沙门氏菌SL3261和其衍生物分别含有脲酶表达质粒pYZ97(染色体外)和T7RNAP表达盒pYZ88,pYZ84或pYZ114(整合进入染色体),如表1所示。利用Luria肉汤或琼脂在28℃进行细菌生长。利用在血清平板上37℃生长的幽门螺杆菌野生型菌株进行攻击实验。
小鼠的免疫:在用于实验之前在动物实验室喂养从Interfauna(德国Tuttlingen)购买的四星期大的Balb/c小鼠2星期。在眼窝后从所有的小鼠取150微升血以便获得免疫前血清。在免疫后1星期和3星期从所有免疫的小鼠重复眼窝后取血。
8组,每组包括5只小鼠的组(包括对照)用于这一研究(表2)。A组,天然对照组,既没有利用沙门氏菌免疫也没有利用野生型幽门螺杆菌攻击。其余的组都进行了口服免疫。B组,阴性对照组,未接受沙门氏菌,但利用幽门螺杆菌进行了攻击。利用沙门氏菌疫苗菌株免疫和利用幽门螺杆菌攻击来自C组和G组的小鼠。最后的H组接受重组脲酶B与伤寒毒素并且同时受到攻击。
在免疫之前,不给小鼠固体食物并且4小时没有水使之过夜。利用不锈钢导管口服给予100微升3%碳酸氢钠,中和胃pH。然后,来自组B的小鼠接受100微升PBS和来自C-G组的小鼠接受1.0×1010CFU的沙门氏菌100微升体积。来自H组的小鼠接受四次100微升的重组幽门螺杆菌脲酶B加伤寒毒素的混合物,每星期一次剂量。在每次免疫后,再给小鼠水和食物。
幽门螺杆菌攻击:在第一次口服免疫后四星期,利用幽门螺杆菌攻击来自B-H组的小鼠。在攻击前4小时使小鼠在没有固体食物和没有水的情况下过夜。利用不锈钢导管给小鼠口服100微升3%碳酸氢钠,接着口服5.0×109CFU/毫升剂量的幽门螺杆菌。在攻击后,恢复小鼠的水和食物。
从小鼠收集血液和组织:在第一次免疫后12个星期,使小鼠没有食物过夜,随后处死以便分析保护性和免疫应答。在颈错位处死之前,利用甲氧荧烷麻醉小鼠用于心脏末端取血。在无菌条件下,小心取出每个小鼠的脾和胃,分别置于冰上的无菌容器中直到进一步加工。获取大小肠,以便进一步分离肠液。
加工胃和测量脲酶活性:通过组织中活性脲酶的存在测量小鼠胃中幽门螺杆菌的定居程度。根据供应商的说明利用Jatrox测试(Rohm-PharmaGmbH,Weiterstadt,德国)。暴露胃粘膜并利用PBS洗涤,将一半胃窦部分立即置于含有底物的Eppendorf管内以便测量脲酶活性。在将试管室温温育4小时后测量550纳米的吸光值。剩余的胃组织储藏在-20℃以便进一步处理。从天然小鼠的胃获得的脲酶活性值用于建立基线以表明没有幽门螺杆菌感染和因此的保护,天然小鼠没有经过免疫或攻击。
表1
表达鼠伤寒沙门氏菌疫苗菌株的UreA和UreB
菌株 | 脲酶表达 | 来源 |
鼠伤寒沙门氏菌SL3261 | 阴性 | Hoiseth和Stocker |
鼠伤寒沙门氏菌SL3262pYZ97 | 组成型低 | 本研究 |
鼠伤寒沙门氏菌SL3261∷pYZ88pYZ97 | T7诱导的高表达 | 本研究 |
鼠伤寒沙门氏菌SL3261∷pYZ84pYZ97 | T7诱导的中等表达 | 本研究 |
鼠伤寒沙门氏菌SL3261∷pYZ114pYZ97 | T7诱导的低表达 | 本研究 |
表2
用于免疫的小鼠组
组 | 免疫原 | 口服免疫次数 |
A | 没有 | 0 |
B | PBS口服免疫 | 1 |
C | 鼠伤寒沙门氏菌S3261 | 1 |
D | 鼠伤寒沙门氏菌S3261 pYZ97 | 1 |
E | 鼠伤寒沙门氏菌S3261∷pYZ88pYZ97 | 1 |
F | 鼠伤寒沙门氏菌S3261∷pYZ84pYZ97 | 1 |
G | 鼠伤寒沙门氏菌S3261∷pYZ114pYZ97 | 1 |
H | 脲酶B加伤寒毒素 | 4 |
结果:
在对照小鼠(B和C组)中,观察到100%的幽门螺杆菌的感染。在利用重组减毒病原体免疫的小鼠(D,E,F,G组)中,观察到0%到60%的感染(100%到40%的保护)。免疫保护与体液抗UreA和UreB应答不相关,这表明除了体液免疫,细胞免疫是抵抗幽门螺杆菌感染的保护的关键。结果表明以由鼠伤寒沙门氏菌减毒菌株递送的UreA和UreB口服免疫小鼠能有效地诱导高水平的抵抗幽门螺杆菌定居的保护。
在利用重组脲酶B加伤寒毒素免疫的小鼠中,在所述的实验条件下观察到比当给予本发明的重组减毒病原体时高得多的脲酶活性水平。
表3已经说明了脲酶测试的结果。
表3 组 小鼠 E550nm,4h E4h-E对照 E校正*3 稀释度
A 1 0,085 -0,022 -0,066 200μl+400μl
A 2 0,091 -0,016 -0,048 200μl+400μl
A 3 0,116 0,009 0,027 200μl+400μl
A 4 0,099 -0,008 -0,024 200μl+4000μl
A 5 0,101 -0,006 -0,018 200μl+400μl
对照 0,107 0 0 200μl+400μl
B 1 0,394 0,292 0,876 200μl+400μl
B 2 0,464 0,362 1,086 200μl+400μl
B 3 0,329 0.227 0,681 200μl+400μl
B 4 0,527 0,425 1,275 200μl+400μl
B 5 0,462 0,36 1,08 200μl+400μl
对照 0,102 0 0 200μl+400μl
C 1 0,248 0,145 0,435 200μl+400μl
C 2 0,369 0,266 0,798 200μl+400μl
C 3 0,209 0,106 0,318 200μl+400μl
C 4 0,219 0,116 0,348 200μl+400μl
C 5 0,24 0,137 0,411 200μl+400μl
对照 0,103 0 0 200μl+400μl
D 1 0,143 0,002 0,004 300μl+300μl
D 2 0,156 0,015 0,03 300μl+300μl
D 3 0,142 0,001 0,002 300μl+300μl
D 4 0,114 -0,027 -0,054 300μl+300μl
D 5 0,133 -0,008 -0,016 300μl+300μl
对照 0,141 0 0 300μl+300μl
E 1 0,127 0,027 0,081 200μl+400μl
E 2 0,094 -0,006 -0,018 200μl+400μl
E 3 0,099 -0,001 -0,003 200μl+400μl
E 4 0,161 0,061 0,183 200μl+400μl
E 5 0,198 0,098 0.294 200μl+400μl
对照 0,1 0 0 20μl+400μl
F 1 0,166 0,025 0,05 300μl+300μl
F 2 0,145 0,004 0,008 300μl+300μl
F 3 0,166 0,025 0,05 300μl+300μl
F 4 0,154 0,013 0,026 300μl+300μl
F 5 0,301 0,16 0.32 300μl+300μl
对照 0,141 0 0 300μl+30μl
G 1 0,084 -0,019 -0,057 200μl+400μl
G 2 0,087 -0,016 -0,048 200μl+400μl
G 3 0,269 0,166 0,498 200μl+400μl
G 4 0,085 -0,018 -0,054 200μl+400μl
G 5 0,092 -0,011 -0,033 200μl+400μl
对照 0,103 0 0 200μl+400μl
H 1 0,638 0,531 1,593 200μl+400μl
H 2 0,282 0,175 0,525 200μl+400μl
H 3 0,141 0,034 0,102 200μl+400μl
H 4 0,135 0,028 0,084 200μl+400μl
H 5 0,171 0,064 0,192 200μl+400μl
对照 0,107 0 0 200μl+400μl
实施例3
各种表达ureA/ureB亚基的重组鼠伤寒沙门氏菌菌株的构建和分子分析
用于免疫实验的鼠伤寒沙门氏菌菌株的说明
鼠伤寒沙门氏菌SL3261(pYZ97)(构建体A):将鼠伤寒沙门氏菌SL3261活疫苗载体菌株用作重组脲酶质粒构建体pYZ97的受体。
鼠伤寒沙门氏菌SL3261∷YZ系列(pYZ97)(构建体B):
这些载体菌株是鼠伤寒沙门氏菌SL3261的衍生物,该衍生物已经装备了T7 RNA聚合酶(T7RNAP)表达盒,图3中有图解说明。这些表达盒编码T7RNAP的基因,该基因以如Yan等人的以前的发明中公开的两相方式(开/关)表达(“用于在杂合活疫苗中产生和呈递外源抗原的两相系统”,PCT/EP91/02478)。可以将盒整合进入细菌的染色体,并给在开位置的细胞提供最适量的T7RNAP以便活化依赖T7RNAP的表达质粒如pYZ97。
YZ84盒的原理是置于片段上的可倒位的λPL启动子,该片段通过噬菌体Mu反向重组酶Gin颠倒(Yan和Meyer,1996,生物技术杂志,44:197-201)。根据PL启动子的定向,表达了gin基因(关位置)或T7RNAP基因(开位置)。YZ84包括了下面的调节元件:(1)温度敏感性cItsλ阻遏物(cI),该阻遏物在28℃阻遏PL启动子并在37℃解离。(2)噬菌体fd终止子(fdT)减弱gin基因的表达以便在可倒位的片段上获得PL启动子适度的颠率。
两相表达系统能够高度表达外源抗原,如脲酶亚基A和B。已知高度表达外源抗原降低了沙门氏菌载体的存活性,所以减弱了免疫应答和随后的保护潜力。已经表明两相系统具有提高重组沙门氏菌的存活力的天然能力,该重组沙门氏菌表达大量的外源抗原。在构建体B中,ureA和ureB基因的表达主要是在强T7启动子的控制下的,导致脲酶亚基的高生产。如果T7RNAP表达盒是在关位置,并且没有T7RNAP存在,沙门氏菌启动子在适度范围内组成性地表达ureA和ureB基因。
分析用于免疫实验的各种鼠伤寒沙门氏菌菌株产生的ureA/B亚基
首先通过SDS-聚丙烯酰胺凝胶分析沙门氏菌构建体A和B对UreA和UreB的表达。在用100微克/毫升氨苄青霉素补充的液体Luria肉汤中从过夜培养物开始在37℃培养重组菌株。在对数生长期通过离心收集细菌,将细胞沉淀再悬浮于10毫摩尔Tris-HCl和10毫摩尔EDTA(pH8.0)中,将细胞密度在所有探测中调节到标准A590=1.0。将细菌悬浮液与等体积的SDS样品缓冲液(Sambrook,J.等人,1989,分子克隆:实验室手册,第二版,冷泉港实验室出版社,冷泉港,纽约)混合并煮沸5分钟。将20微升悬浮液加载到两个SDS-10%聚丙烯酰胺凝胶上;利用考马斯蓝染料染色一个凝胶,另一个电印迹到硝酸纤维素膜上,并进一步加工以用于免疫印迹分析。在10(v/w)%脱脂牛奶Tris缓冲盐水(TBS)(TrisHCl 100毫摩尔,NaCl 150毫摩尔,pH7.2)中,在室温下封闭携带转移蛋白质的硝酸纤维素膜45分钟。在TBS-0.05(v/v)%Tween-20中洗涤3次后,将兔抗UreB抗体(AK201)在5(w/v)%脱脂牛奶-TBS中的1∶2000稀释液加到条带上,在4℃温育过夜。从利用通过亲和层析纯化的重组脲酶B亚基免疫的兔中获得血清。利用0.05(v/v)%在PBS中的Tween-20洗涤膜3次10分钟,并进一步在5(w/v)%脱脂牛奶-TBS中与山羊抗兔IgG抗体辣根过氧化物酶缀合物在室温温育45分钟。在利用上面的0.2(v/v)%Tween-20洗涤3次后,利用ECL试剂盒(Amersham,德国)根据供应商的指导显色膜。
构建体A:在构建体A(鼠伤寒沙门氏菌菌株SL3261(pYZ97)的全细胞溶胞产物的考马斯染色的凝胶中观察到67kDa和30kDa的蛋白质;这些大小分别与UreB和UreA的大小非常相关。这些蛋白质在含有鼠伤寒沙门氏菌SL3261菌株的对照泳道中没有。利用兔抗UreB抗体免疫印迹分析同样的蛋白质样品证明在考马斯染色的凝胶中观察到的67千道尔顿蛋白质为UreB。通过在37℃分别温育2,6和11小时在不同生长期也检测了来自鼠伤寒沙门氏菌菌株SL3261(pYZ97)的ureB的表达。在所有生长期包括在静止期都观察到ureB的表达;虽然,在生长早期观察到更高的表达。利用菌株SL3261(pYZ97)获得的结果表明UreA和UreB蛋白质对于沙门氏菌是非毒性的,并且在37℃在细菌生长的任何时期都能够被表达。
个构建体的比较揭示了构建体B是更有效的生产者,而构建体A中等表达了ureA和ureB。在构建体B的细菌生长过程中,ureA和ureB在更长的时期恒定地高表达,甚至在没有抗生素选择的情况下。这证明了在与构建体A的比较中构建体B的生产力是罕见的。
简要地说,我们的数据表明在鼠伤寒沙门氏菌中可以表达来自幽门螺杆菌的UreA和UreB,而没有引起对细菌的不利影响,因此当通过沙门氏菌载体传递时,在动物保护实验中上面两种亚基是适当的。
质粒稳定性
质粒稳定性对于保证基因编码的抗原的稳定表达是必要的,这些基因已经克隆进入该质粒。
体外质粒稳定性。将质粒pYZ97上存在的和无质粒鼠伤寒沙门氏菌菌株SL3261中没有的氨苄青霉素抗性标记用作质粒稳定性的指示物。如上所述,在LB液体培养基中,在28℃培养鼠伤寒沙门氏菌菌株SL3261达到100代(Summers,D.K.和D.J.Sherrat.1984,细胞36:1097-1103)。每10代,通过在普通的LB琼脂平板和用100微克/毫升氨苄青霉素补充的LB琼脂平板上涂布等数目的细菌稀释液,从沙门氏菌的菌落形成单位(CFU)的总数确定氨苄青霉素抗性CFU的数目。
体内质粒稳定性。在小鼠口服5.0×109CFU的鼠伤寒沙门氏菌SL3261(pYZ97)感染后2和7天,通过检测来自小鼠脾的总CFU和氨苄青霉素抗性CFU分析体内质粒稳定性。如上所述,口服沙门氏菌感染小鼠。感染后2天和7天,在甲氧荧烷麻醉下处死小鼠,无菌摘除脾进一步加工。在培养皿中将脾切成小块,与1毫升冰冷的ddH2O混合,通过18量规的针头几次悬浮脾细胞。然后,在有或没有100微克/毫升氨苄青霉素的LB琼脂平板上涂布细胞悬浮液。在37℃过夜温育平板,在第二天计数菌落。
在利用一个口服剂量的5.0×109CFU的鼠伤寒沙门氏菌SL3261(pYZ97)感染小鼠后,分析体内质粒稳定性。在感染后2和7天取出小鼠的脾,涂布在LB琼脂平板上检测总CFU和氨苄青霉素抗性CFU。在48小时后,从脾分离2.0×104氨苄青霉素抗性CFU(表4)。在免疫后7天,CFU计数下降到56,但再次,所有的都是氨苄青霉素抗性的。数据表明在体外和体内条件下在沙门氏菌中质粒pYZ97是稳定的,是适于评估作为抵抗幽门螺杆菌定居小鼠胃的保护性抗原的脲酶亚基的。感染后7天,沙门氏菌菌株SL3261的低回收率证明减毒这一菌株允许它安全地用于将脲酶递送进入小鼠。
表4
从小鼠脾回收鼠伤寒沙门氏菌SL3261pYZ菌株和评估体内pYZ97质粒稳定性
感染后时间 | 总CFUa | Ampr CFUb百分数 |
2天7天 | 2.0×10456 | 100100 |
a在利用5.0×109CFU的鼠伤寒沙门氏菌SL3261(pYZ97)口服接种2天和7天后,从小鼠脾中在没有抗生素的LB平板上分离的鼠伤寒沙门氏菌CFU数目。
b从小鼠脾中分离的CFU总数中氨苄青霉素抗性CFU的百分数
表5
在利用表达UreA和UreB的沙门氏菌免疫的小鼠的胃窦中检测脲酶活性和链霉素抗性幽门螺杆菌
小鼠组 | 数目 | 脲酶活性a | CFUb |
天然对照组PBS对照组SL3261pYZ97c | 555 | 0.058+0.0040.427+0.0590.057+0.006 | 0±02.7×03+1.0×10362.6+97.3 |
a脲酶活性是平均值±标准误差。
b通过在200微克/毫升链霉素补充的血清平板上涂布一段胃窦进行了链霉素抗性幽门螺杆菌P76菌株的CFU的确定。根据菌落形态,脲酶活性,和光学显微镜检测识别出幽门螺杆菌。数值对应于CFU±标准误差。
c如上面材料和方法中所述,利用表达来自幽门螺杆菌的ureA和ureB的鼠伤寒沙门氏菌SL3261(pYZ97)免疫的小鼠。
实施例4
在幽门螺杆菌小鼠模型中利用各种表达ureA/ureB亚基的重组鼠伤寒沙门氏菌菌株的保护实验
用于该实验的幽门螺杆菌菌株的说明
如Haas等人的发明中公开的(“Verfahern zur Identifizierungsekretorischer Gene aus Helicobacter pylori”;PCT/EP96/02544),脲酶缺陷型幽门螺杆菌P11菌株是利用TnMax5小转座子通过转座子穿梭诱变产生的P1的衍生物。在ureA基因的3’末端插入TnMax5导致ureA和ureB的表达缺陷因为两个基因是转录偶联的。
J.G.Fox博士(MIT,波士顿,MA)最初从猫分离物中确立了小鼠适应的幽门螺杆菌P49菌株。如P.Nedenskov-Sorensen所述(1990,传染病杂志,161:365-366),幽门螺杆菌P76菌株是通过与来自链霉素抗性幽门螺杆菌菌株的染色体DNA同源重组产生的P49的链霉素抗性衍生物。
在适当时,在200微克/毫升链霉素补充的血清平板上,在微需氧的环境(5%O2,85%N2,和10%CO2)中,在37℃培养所有的幽门螺杆菌菌株。
对小鼠的预防免疫实验
进行免疫实验测试沙门氏菌递送的UreA和B保护小鼠免受幽门螺杆菌的胃定居的能力。总共,已经进行了5个独立的免疫实验。每个实验包括5组,每组5只小鼠:(1)天然对照组是既没有用沙门氏菌免疫也没有用野生型幽门螺杆菌P49或者链霉素抗性衍生菌株P76攻击的小鼠;(2)PBS对照组是未免疫的小鼠,这些小鼠接受了PBS并口服幽门螺杆菌进行攻击;(3)沙门氏菌对照组是利用减毒的鼠伤寒沙门氏菌SL3261菌株单独免疫和用幽门螺杆菌攻击的小鼠;和(5)疫苗组是利用表达UreA和UreB的适当重组鼠伤寒沙门氏菌构建体(A+B)免疫和利用幽门螺杆菌攻击的小鼠。
在免疫之前,在没有固体食物并且4小时没有水的情况下让小鼠过夜。利用不锈钢导管口服给予100微升3%碳酸氢钠中和胃pH。在胃中和后,来自PBS对照组的小鼠马上接受了100微升PBS,来自沙门氏菌对照组和沙门氏菌疫苗组的小鼠分别接受了5.0×109CFU的鼠伤寒沙门氏菌菌株SL3261和各种重组构建体,总体积为100微升。在免疫后恢复小鼠的水和食物。
在口服免疫4星期后,利用1.0×109CFU的幽门螺杆菌攻击来自PBS对照组,沙门氏菌对照组和疫苗组的小鼠。在攻击之前4小时使小鼠在没有固体食物和没有水的情况下过夜。利用不锈钢导管口服给予小鼠100微升3%碳酸氢钠,然后给予口服剂量为1.0×109CFU/毫升的幽门螺杆菌P49或P76。在攻击后恢复小鼠的水和食物。
实施例5
在幽门螺杆菌小鼠模型中表达ureA/ureB亚基的各种重组鼠伤寒沙门氏菌菌株免疫分析保护实验
从小鼠收集血液和肠液用于血清学分析
利用血清和肠液评估所有小鼠的抗体应答。在免疫之前和免疫后3星期,螺杆菌感染之前从眼窝后收集150微升血液。在沙门氏菌免疫后11星期(攻击感染后6星期),通过甲氧荧烷麻醉下的心脏末端穿刺进行最后取血。如上所述(Elson,C.O.等人1984免疫方法杂志,67:101-108)作微小的改进在实验结束时取小鼠的小肠并加工。简要地说,通过2毫升含有0.1毫克/毫升大豆胰蛋白酶抑制剂(西格玛)的50毫摩尔EDTApH7.5(Riedel),除去肠的内容物。用0.15摩尔NaCl将体积调节到5毫升。剧烈涡旋样品,在2,500rpm下离心10分钟(Heraeus,德国),利用50微升在95%乙醇中的100毫摩尔苯甲基磺酰氟(PMSF)(Serva)补充上清液,接着以13,000rpm,在4℃离心20分钟(Hermes)。利用50微升100毫摩尔PMSF和50微升2%叠氮化纳(Merck)补充上清液,并在加入250微升7%牛血清白蛋白(Biomol)之前在冰上保持15分钟。在-20℃冷冻样品直到进一步使用。
通过ELISA分析小鼠血清和肠粘膜中的抗脲酶抗体
已知口服沙门氏菌免疫能够引发IgA抗体应答。通过ELISA评估利用鼠伤寒沙门氏菌构建体A+B免疫的小鼠和对照小鼠中抵抗脲酶亚基的IgA应答。通过在超声波处理器(Branson,Danbury,Conn.)上间隔5秒处理5次总共45秒,在磷酸盐缓冲盐水中制备幽门螺杆菌P1和它的脲酶缺陷型突变体衍生菌株P11的可溶性提取物。以13,000rpm在4℃离心这一悬浮液10分钟除去完整的细胞。在利用BioRad试剂盒确定蛋白质含量之后将上清液用作抗原。利用在碳酸钠一碳酸氢钠缓冲液pH9.6中的50微克/毫升抗原的50微升等分试样包被96孔的微滴平板(Nunc,德国)并在4℃温育过夜。利用Tris~缓冲剂一盐水(TBS)中的1.0(w/v)%脱脂牛奶在室温封闭这些孔45分钟,利用TBS-0.05% Tween-20洗涤3次。这些测试进行了一式三份,利用了50微升在加入孔中的0.5(w/v)%脱脂牛奶一TBS中分别稀释1∶100或1∶2的血清或消化道洗液,在4℃保存过夜。然后,利用TBS-0.05% Tween20洗涤孔3次,在所有孔中加入1∶3000的山羊抗小鼠IgA辣根过氧化物酶缀合物(西格玛)稀释液,在4℃温育过夜。利用在乙酸钠缓冲液中的邻苯二胺底物和过氧化氢在37℃温育30分钟产生颜色反应。利用10N H2SO4终止反应,在ELISA读数器(Digiscan,Asys Hitech GmbH,澳大利亚)中确定A492。
粘膜抗体:(构建体A)在实验结束时(幽门螺杆菌攻击后6星期)从处死的小鼠中取肠液,利用幽门螺杆菌野生型(P1)和粘膜缺陷型突变体菌株(P11)的总细胞提取物测试抗脲酶抗体的存在。如图4所示,在免疫的小鼠中对野生型幽门螺杆菌提取物的IgA抗体应答约比未免疫的或天然小鼠高10倍。在所有小鼠组中对脲酶缺陷型幽门螺杆菌突变体的粘膜IgA抗体应答都是非常低的,表明在免疫小鼠中大多数肠IgA抗体应答是针对脲酶的。
血清抗体:(构建体A)在免疫之前,免疫后3星期(攻击之前)和免疫后10星期(利用幽门螺杆菌攻击后6星期)检测抵抗野生型和脲酶缺陷型幽门螺杆菌的血清IgA抗体水平。如图5组A所示,利用表达脲酶的鼠伤寒沙门氏菌构建体A免疫的小鼠中抗野生型幽门螺杆菌抗体的水平与免疫前血清相比在免疫后3星期时高约20倍,在免疫后10星期高34倍。在所有小鼠组中包括利用沙门氏菌构建体A免疫的小鼠中抵抗脲酶缺陷针对幽门螺杆菌菌株的血清IgA抗体应答在3和10星期时都是低的(图5,组B),表明在免疫的小鼠中大多数IgA抗体应答是针对脲酶亚基的。在未免疫的小鼠中在10星期时观察到抵抗野生型幽门螺杆菌的低血清抗体应答,表明提早3星期给予幽门螺杆菌攻击足以在这些小鼠中诱导特异性抗体应答。在利用鼠伤寒沙门氏菌SL3261免疫3星期的小鼠(沙门氏菌对照组)中对野生型幽门螺杆菌的IgA应答适当提高,这可以通过沙门氏菌中能够诱导抵抗幽门螺杆菌的交叉反应抗体的抗原的存在来解释。相反,在免疫的小鼠中抗体对脲酶缺陷型幽门螺杆菌菌株的应答与未免疫的小鼠的抗体应答一样低(图5,组B)。这一结果表明大多数在免疫小鼠中观察到的抗体应答是针对脲酶的。在来自给予PBS或利用鼠伤寒沙门氏菌SL3261免疫的小鼠的10星期的血清中观察到针对脲酶阴性突变体的低抗体应答,表明观察到的抗体应答是由于针对攻击过程中提早3星期给予这些小鼠的幽门螺杆菌抗原的特异性免疫应答。在利用表达或不表达脲酶的沙门氏菌免疫的小鼠中,在3星期时观察到针对脲酶缺陷型幽门螺杆菌菌株的低抗体应答,但在给予PBS的小鼠中缺乏这种抗体应答。这证明在来自沙门氏菌和幽门螺杆菌的蛋白质之间存在交叉反应的表位。(构建体B):利用构建体B系列免疫的小鼠的血清学分析获得同样的结果表明通过重组沙门氏菌更高地产生抗原没有明显提高抗体应答。
通过免疫印迹分析在小鼠血清中分析抗脲酶抗体
分析诱导针对幽门螺杆菌的小鼠特异性免疫应答所必需的来自鼠伤寒沙门氏菌的UreA和UreB的表达。通过在利用沙门氏菌构建体A免疫的小鼠和对照小鼠的血清中,寻找抗UreA和抗UreB抗体进行了体内UreA和UreB表达的鉴定。从野生型幽门螺杆菌菌株P1制备幽门螺杆菌全细胞抗原。从3个血清平板回收细菌,再悬浮于PBS,在5,000g离心10分钟收集。在10毫摩尔Tris-HCl和10毫摩尔EDTA(pH8.0)中再悬浮细胞沉淀,在所有探测中将细胞密度调节到标准A590=1.0。将细菌悬浮液与等体积的SDS样品缓冲液(Sambrook,1989)混合,煮沸5分钟。将20微升沉淀加载到SDS-10%聚丙烯酰胺凝胶上。将蛋白质电印迹到硝酸纤维素膜上,切成条带,将条带在室温在10(v/w)%脱脂牛奶Tris-缓冲剂-盐水(TBS)(TrisHCl 100毫摩尔,NaCl 150毫摩尔,pH7.2)中封闭45分钟。在TBS-0.05(v/v)%吐温-20中洗涤3次后,在条带中加入5(w/v)%脱脂牛奶-TBS中的小鼠血清的1∶80稀释液,并在4℃温育过夜。从未免疫的小鼠和利用沙门氏菌免疫的小鼠中获得血清。在洗涤3次后,利用在5(w/v)%脱脂牛奶-TBS中稀释1∶3000的山羊抗小鼠IgG辣根过氧化物酶缀合物(西格玛)温育条带。根据供应商的说明将ECL化学荧光检测试剂盒(Amersham,德国)用于显色印迹。
在利用幽门螺杆菌攻击之前,在免疫后3星期获得免疫和非免疫小鼠的血清,并对表达UreA和UreB的野生型幽门螺杆菌P1菌株的全细胞溶胞产物测试。通过来自利用构建体A免疫的小鼠的血清识别大小分别相应于UreB和UreA的67千道尔顿和30千道尔顿的蛋白质。在利用来自非免疫小鼠或只利用沙门氏菌免疫的小鼠的血清测试的条带中没有观察到这些带,表明由沙门氏菌疫苗菌株表达的脲酶能够诱导对野生型幽门螺杆菌菌株的UreA和UreB的特异性抗体应答。在构建体B中获得了同样的结果。
实施例6
在幽门螺杆菌小鼠模型中确定在用各种表达ureA/ureB亚基的鼠伤寒沙门氏菌菌株预处理的小鼠中幽门螺杆菌的定居
胃的加工和脲酶活性的测定
脲酶测试:通过在小鼠胃的窦部分中测试脲酶活性进行对幽门螺杆菌定居胃的保护的分析。脲酶活性的测定是非常可靠,敏感和特异的方法以便测试幽门螺杆菌感染的存在(在消化道溃疡疾病中对幽门螺杆菌的NIH同感发育,1994。在消化道溃疡疾病中的幽门螺杆菌,JAMA.272:65)并且常规用于临床设置(Kawanishi,M.,S.等人1995。胃肠病学杂志,30:16-20;Kamija,S.等人1993。欧洲流行病学杂志,9:450-452;Conti-Nibali,S等人1990。美国胃肠病学杂志,85:1573-1575)和动物研究(Gottfried,M.R.等人1990。美国胃肠病学杂志,85:813-818)中。根据供应商的说明使用Jatrox-测试(Rohn-Pharma GmbH,Weiterstadt,德国)。在利用沙门氏菌免疫后11星期,处死小鼠和在无菌条件下小心摘除胃。将胃置于无菌容器中的冰冷PBS中,利用无菌的刀片切成小的弯曲切口暴露粘膜。利用PBS漂洗胃除去食物残渣并解剖,使窦区与原体区分离。立即将胃的窦部分的一半置于含有来自Jatrox-测试的500微升脲酶底物的Eppendorf管内。将胃样品在室温下温育4小时,测量在550纳米的吸光值(A550)。用从没有经过免疫或攻击的天然小鼠的胃中获得的脲酶活性值确定基线。基线相应于来自5个测试的天然小鼠的胃的平均脲酶活性值加线这一平均值的标准误差的2倍。比基线高的脲酶活性值认为是幽门螺杆菌定居阳性,低于基线的值认为是幽门螺杆菌定居阴性。
培养实验:在补充200微升/毫克链霉素的血清平板上涂布利用链霉素抗性幽门螺杆菌菌株P76攻击的小鼠中获得的胃的窦区的左侧部分,并在标准条件下温育。在温育3天后,根据菌落形态,显微镜检测,和脲酶活性鉴定细菌为幽门螺杆菌。从每个小鼠胃的样品中确定在平板上生长的幽门螺杆菌的菌落形成单位(CFU)的数目。
脲酶测试(构建体A和B):在麻醉后,处死利用约5.0×109CFU的沙门氏菌免疫的和利用1.0×109CFU的幽门螺杆菌菌株P49攻击的小鼠,以及对照小鼠,取出一段胃的窦区用于测定脲酶活性。如图6所示,利用沙门氏菌构建体A递送的UreA和B免疫的小鼠100%具有低于基线的脲酶活性,表明没有幽门螺杆菌的定居。相反,100%的非免疫小鼠(PBS)和单独利用鼠伤寒沙门氏菌菌株SL3261免疫的小鼠具有的脲酶活性测量值远远高于基线表明幽门螺杆菌定居于胃。将没有经过免疫或攻击的小鼠的天然组用于设定脲酶活性的基线。
构建体B系列的沙门氏菌具有的脲酶活性值高于基线表明幽门螺杆菌攻击菌株定居于胃。但是,在这一组中的脲酶活性像对照一样是较低的,表明利用沙门氏菌构建体B系列免疫的小鼠的部分保护状态(图6)。两个沙门氏菌构建体,A和B,介导同样的抗体应答但是ureA和ureB的表达不同。我们由此得出结论,表达的脲酶抗原的量是与获得最适保护相关的。
构建体A:为了使幽门螺杆菌的胃定居与脲酶活性相关,通过用沙门氏菌构建体A口服免疫小鼠并利用链霉素抗性幽门螺杆菌P76菌株攻击它们进行了新的保护实验。脲酶活性值与鉴定的幽门螺杆菌的CFU的数目相关。在5只利用表达脲酶的沙门氏菌免疫的小鼠的2只中,没有检测到幽门螺杆菌CFU,在所有5只免疫的小鼠中的CFU的平均数只有62。相反,在非免疫小鼠中CFU的数目是2,737,这相应于高44倍的定居。这些数据表明利用表达脲酶的沙门氏菌免疫的小鼠能够消除或明显减少幽门螺杆菌对小鼠胃的定居。
序列表
(1)一般信息:
(I)申请人:
(A)姓名:Max-Planck-Gesellschaft zur Foerderung der
Wissenschaften e.V.Berlin
(B)街道:Hofgartenstr.2
(C)城市:Muenchen
(E)国家:德国
(F)邮编(ZIP):80539
(ii)发明名称:幽门螺杆菌活疫苗
(iii)序列数目:6
(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-D0S/MS-DOS
(D)软件:PantentIn Release#1.0,#1.30版(EPO)
(2)SEQ ID NO:1的信息:
(i)序列特征:
(A)长度:1557碱基对
(B)类型:核酸
(C)链型:两种
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(vi)原始来源:
(A)生物体:幽门螺杆菌
(vii)直接来源:
(B)克隆:alpB
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1.1554
序列描述:SEQ ID NO:1:
ATG ACA CAA TCT CAA AAA GTA AGA TTC TTA GCC CCT TTA AGC CTA GCG
48
Met Thr Gln ser Gln Lys Val Arg Phe Leu Ala Pro Leu Ser Leu Ala
1 5 10 15
TTA AGC TTG AGC TTC AAT CCA GTG GGC GCT GAA GAA GAT GGG GGC TTT
96
Leu Ser Leu Ser Phe Asn Pro Val Gly Ala Glu Glu Asp Gly Gly Phe
20 25 30
ATG ACC TTT GGG TAT GAA TTA GGT CAG GTG GTC CAA CAA GTG AAA AAC
144
Met Thr Phe Gly Tyr Glu Leu Gly Gln Val Val Gln Gln Val Lys Asn
35 40 45
CCG GGT AAA ATC AAA GCC GAA GAA TTA GCC GGC TTG TTA AAC TCT ACC
192
Pro Gly Lys Ile Lys Ala Glu Glu Leu Ala Gly Leu Leu Asn ser Thr
50 55 60
ACA ACA AAC AAC ACC AAT ATC AAT ATT GCA GGC ACA GGA GGC AAT GTC
240
Thr Thr Asn Asn Thr Asn Ile Asn Ile Ala Gly Thr Gly Gly Asn Val
65 70 75 80
GCC GGG ACT TTG GGC AAC CTT TTT ATG AAC CAA TTA GGC AAT TTG ATT
288
Ala Gly Thr Leu Gly Asn Leu Phe Met Asn Gln Leu Gly Asn Leu Ile
85 90 95
GAT TTG TAT CCC ACT TTG AAC ACT AGT AAT ATC ACA CAA TGT GGC ACT
336
Asp Leu Tyr Pro Thr Leu Asn Thr Ser Asn Ile Thr Gln Cys Gly Thr
100 105 110
ACT AAT AGT GGT AGT AGT AGT AGT GGT GGT GGT GCG GCC ACA GCC GCT
384
Thr Asn Ser Gly Ser Ser Ser Ser Gly Gly Gly Ala Ala Thr Ala Ala
115 120 125
GCT ACT ACT AGC AAT AAG CCT TGT TTC CAA GGT AAC CTG GAT CTT TAT
432
Ala Thr Thr Ser Asn Lys Pro Cys Phe Gln Gly Asn Leu Asp Leu Tyr
130 135 140
AGA AAA ATG GTT GAC TCT ATC AAA ACT TTG AGT CAA AAC ATC AGC AAG
480
Arg Lys Met Val Asp Ser Ile Lys Thr Leu Ser Gln Asn Ile Ser Lys
145 150 155 160
AAT ATC TTT CAA GGC AAC AAC AAC ACC ACG AGC CAA AAT CTC TCC AAC
528
Asn Ile Phe Gln Gly Asn Asn Asn Thr Thr Ser Gln Asn Leu Ser Asn
165 170 175
CAG CTC AGT GAG CTT AAC ACC GCT AGC GTT TAT TTG ACT TAC ATG AAC
576
Gln Leu Ser Glu Leu Asn Thr Ala Ser Val Tyr Leu Thr Tyr Met Asn
180 185 190
TCG TTC TTA AAC GCC AAT AAC CAA GCG GGT GGG ATT TTT CAA AAC AAC
624
Ser Phe Leu Asn Ala Asn Asn Gln Ala Gly Gly Ile Phe Gln Asn Asn
195 200 205
ACT AAT CAA GCT TAT GGA AAT GGG GTT ACC GCT CAA CAA ATC GCT TAT
672
Thr Asn Gln Ala Tyr Gly Asn Gly Val Thr Ala Gln Gln Ile Ala Tyr
210 215 220
ATC CTA AAG CAA GCT TCA ATC ACT ATG GGG CCA AGC GGT GAT AGC GGT
720
Ile Leu Lys Gln Ala Ser Ile Thr Met Gly Pro Ser Gly Asp Ser Gly
225 230 235 240
GCT GCC GCA GCG TTT TTG GAT GCC GCT TTA GCG CAA CAT GTT TTC AAC
768
Ala Ala Ala Ala Phe Leu Asp Ala Ala Leu Ala Gln His Val Phe Asn
245 250 255
TCC GCT AAC GCC GGG AAC GAT TTG AGC GCT AAG GAA TTC ACT AGC TTG
816
Ser Ala Asn Ala Gly Asn Asp Leu Ser Ala Lys Glu Phe Thr Ser Leu
260 265 270
GTG CAA AAT ATC GTC AAT AAT TCT CAA AAC GCT TTA ACG CTA GCC AAC
864
Val Gln Asn Ile Val Asn Asn Ser Gln Asn Ala Leu Thr Leu Ala Asn
275 280 285
AAC GCT AAC ATC AGC AAT TCA ACA GGC TAT CAA GTG AGC TAT GGC GGG
912
Asn Ala Asn Ile Ser Asn Ser Thr Gly Tyr Gln Val Ser Tyr Gly Gly
290 295 300
AAT ATT GAT CAA GCG CGA TCT ACC CAA CTA TTA AAC AAC ACC ACA AAC
960
Asn Ile Asp Gln Ala Arg Ser Thr Gln Leu Leu Asn Asn Thr Thr Asn
305 3l0 315 320
ACT TTG GCT AAA GTT AGC GCT TTG AAT AAC GAG CTT AAA GCT AAC CCA
1008
Thr Leu Ala Lys Val Ser Ala Leu Asn Asn Glu Leu Lys Ala Asn Pro
325 330 335
TGG CTT GGG AAT TTT GCC GCC GGT AAC AGC TCT CAA GTG AAT GCG TTT
1056
Trp Leu Gly Asn Phe Ala Ala Gly Asn Ser Ser Gln Val Asn Ala Phe
340 345 350
AAC GGG TTT ATC ACT AAA ATC GGT TAC AAG CAA TTC TTT GGG GAA AAC
1104
Asn Gly Phe Ile Thr Lys Ile Gly Tyr Lys Gln Phe Phe Gly Glu Asn
355 360 365
AAG AAT GTG GGC TTA CGC TAC TAC GGC TTC TTC AGC TAT AAC GGC GCG
1152
Lys Asn Val Gly Leu Arg Tyr Tyr Gly Phe Phe Ser Tyr Asn Gly Ala
370 375 380
GGC GTG GGT AAT GGC CCT ACT TAC AAT CAA GTC AAT TTG CTC ACT TAT
1200
Gly Val Gly Asn Gly Pro Thr Tyr Asn Gln Val Asn Leu Leu Thr Tyr
385 390 395 400
GGG GTG GGG ACT GAT GTG CTT TAC AAT GTG TTT AGC CGC TCT TTT GGT
1248
Gly Val Gly Thr Asp Val Leu Tyr Asn Val Phe Ser Arg Ser Phe Gly
405 410 415
AGT AGG AGT CTT AAT GCG GGC TTC TTT GGG GGG ATC CAA CTC GCA GGG
1296
Ser Arg Ser Leu Asn Ala Gly Phe Phe Gly Gly Ile Gln Leu Ala Gly
420 425 430
GAT ACT TAC ATC AGC ACG CTA AGA AAC AGC TCT CAG CTT GCG AGC AGA
1344
Asp Thr Tyr Ile Ser Thr Leu Arg Asn Ser Ser Gln Leu Ala Ser Arg
435 440 445
CCT ACA GCG ACG AAA TTC CAA TTC TTG TTT GAT GTG GGC TTA CGC ATG
1392
Pro Thr Ala Thr Lys Phe Gln Phe Leu Phe Asp Val Gly Leu Arg Met
450 455 460
AAC TTT GGT ATC TTG AAA AAA GAC TTG AAA AGC CAT AAC CAG CAT TCT
1440
Asn phe Gly Ile Leu Lys Lys Asp Leu Lys Ser His Asn Gln His Ser
465 470 475 480
ATA GAA ATC GGT GTG CAA ATC CCT ACG ATT TAC AAC ACT TAC TAT AAA
1488
Ile Glu Ile Gly Val Gln Ile Pro Thr Ile Tyr Asn Thr Tyr Tyr Lys
485 490 495
GCT GGC GGT GCT GAA GTG AAA TAC TTC CGC CCT TAT AGC GTG TAT TGG
1536
Ala Gly Gly Ala Glu Val Lys Tyr Phe Arg Pro Tyr Ser Val Tyr Trp
500 505 510
GTC TAT GGC TAC GCC TTC TAA
1557
Val Tyr Gly Tyr Ala Phe
515
(2)SEQ ID NO;2的信息:
(I)序列特征:
(A)长度:518个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2:
Met Thr Gln Ser Gln Lys Val Arg Phe Leu Ala Pro Leu Ser Leu Ala
1 5 10 15
Leu Ser Leu Ser Phe Asn Pro Val Gly Ala Glu Glu Asp Gly Gly Phe
20 25 30
Met Thr Phe Gly Tyr Glu Leu Gly Gln Val Val Gln Gln Val Lys Asn
35 40 45
Pro Gly Lys Ile Lys Ala Glu Glu Leu Ala Gly Leu Leu Asn Ser Thr
50 55 60
Thr Thr Asn Asn Thr Asn Ile Asn Ile Ala Gly Thr Gly Gly Asn Val
65 70 75 80
Ala Gly Thr Leu Gly Asn Leu Phe Met Asn Gln Leu Gly Asn Leu Ile
85 90 95
Asp Leu Tyr Pro Thr Leu Asn Thr Ser Asn Ile Thr Gln Cys Gly Thr
100 105 110
Thr Asn Ser Gly Ser Ser Ser Ser Gly Gly Gly Ala Ala Thr Ala Ala
115 120 125
Ala Thr Thr Ser Asn Lys Pro Cys Phe Gln Gly Asn Leu Asp Leu Tyr
130 135 140
Arg Lys Met Val Asp Ser Ile Lys Thr Leu Ser Gln Asn Ile Ser Lys
145 150 155 160
Asn Ile Phe Gln Gly Asn Asn Asn Thr Thr Ser Gln Asn Leu Ser Asn
165 170 175
Gln Leu Ser Glu Leu Asrn Thr Ala Ser Val Tyr Leu Thr Tyr Met Asn
180 185 190
Ser Phe Leu Asn Ala Asn Asn Gln Ala Gly Gly Ile Phe Gln Asn Asn
195 200 205
Thr Asn Gln Ala Tyr Gly Asn Gly Val Thr Ala Gln Gln Ile Ala Tyr
210 215 220
Ile Leu Lys Gln Ala Ser Ile Thr Met Gly Pro Ser Gly Asp Ser Gly
225 230 235 240
Ala Ala Ala Ala Phe Leu Asp Ala Ala Leu Ala Gln His Val Phe Asn
245 250 255
Ser Ala Asn Ala Gly Asn Asp Leu Ser Ala Lys Glu Phe Thr Ser Leu
260 265 270
Val Gln Asn Ile Val Asn Asn Ser Gln Asn Ala Leu Thr Leu Ala Asn
275 280 285
Asn Ala Asn Ile Ser Asn Ser Thr Gly Tyr Gln Val Ser Tyr Gly Gly
290 295 300
Asn Ile Asp Gln Ala Arg Ser Thr Gln Leu Leu Asn Asn Thr Thr Asn
305 310 315 320
Thr Leu Ala Lys Val Ser Ala Leu Asn Asn Glu Leu Lys Ala Asn Pro
325 330 335
Trp Leu Gly Asn Phe Ala Ala Gly Asn Ser Ser Gln Val Asn Ala Phe
340 345 350
Asn Gly Phe Ile Thr Lys Ile Gly Tyr Lys Gln Phe Phe Gly Glu Asn
355 360 365
Lys Asn Val Gly Leu Arg Tyr Tyr Gly Phe Phe Ser Tyr Asn Gly Ala
370 375 380
Gly Val Gly Asn Gly Pro Thr Tyr Asn Gln Val Asn Leu Leu Thr Tyr
385 390 395 400
Gly Val Gly Thr Asp Val Leu Tyr Asn Val Phe Ser Arg Ser Phe Gly
405 410 415
Ser Arg Ser Leu Asn Ala Gly Phe Phe Gly Gly Ile Gln Leu Ala Gly
420 425 430
Asp Thr Tyr Ile Ser Thr Leu Arg Asn Ser Ser Gln Leu Ala Ser Arg
435 440 445
Pro Thr Ala Thr Lys Phe Gln Phe Leu Phe Asp Val Gly Leu Arg Met
450 455 460
Asn Phe Gly Ile Leu Lys Lys Asp Leu Lys Ser His Asn Gln His Ser
465 470 475 480
Ile Glu Ile Gly Val Gln Ile Pro Thr Ile Tyr Asn Thr Tyr Tyr Lys
485 490 495
Ala Gly Gly Ala Glu Val Lys Tyr Phe Arg Pro Tyr Ser Val Tyr Trp
500 505 510
Val Tyr Gly Tyr Ala Phe
515
(2)SEQ ID NO:3的信息:
(I)序列特征:
(A)长度:1557碱基对
(B)类型:核酸
(C)链型:两种
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(vi)原始来源:
(A)生物体:幽门螺杆菌
(vii)直接来源:
(B)克隆:alpA
(ix)特征:
(A)名称/关键词:CDS
(B)定位:1.1554
(xi)序列描述:SEQ ID NO:3:
ATG ATA AAA AAG AAT AGA ACG CTG TTT CTT AGT CTA GCC CTT TGC GCT
48
Met Ile Lys Lys Asn Arg Thr Leu Phe Leu Ser Leu Ala Leu Cys Ala
520 525 530
AGC ATA AGT TAT GCC GAA GAT GAT GGA GGG TTT TTC ACC GTC GGT TAT
96
Ser Ile Ser Tyr Ala Glu Asp Asp Gly Gly Phe Phe Thr Val Gly Tyr
535 540 545 550
CAG CTC GGG CAA GTC ATG CAA GAT GTC CAA AAC CCA GGC GGC GCT AAA
144
Gln Leu Gly Gln Val Met Gln Asp Val Gln Asn Pro Gly Gly Ala Lys
555 560 565
AGC GAC GAA CTC GCC AGA GAG CTT AAC GCT GAT GTA ACG AAC AAC ATT
192
Ser Asp Glu Leu Ala Arg Glu Leu Asn Ala Asp Val Thr Asn Asn Ile
570 575 580
TTA AAC AAC AAC ACC GGA GGC AAC ATC GCA GGG GCG TTG AGT AAC GCT
240
Leu Asn Asn Asn Thr Gly Gly Asn Ile Ala Gly Ala Leu Ser Asn Ala
585 590 595
TTC TCC CAA TAC CTT TAT TCG CTT TTA GGG GCT TAC CCC ACA AAA CTC
288
Phe Ser Gln Tyr Leu Tyr Ser Leu Leu Gly Ala Tyr Pro Thr Lys Leu
600 605 610
AAT GGT AGC GAT GTG TCT GCG AAC GCT CTT TTA AGT GGT GCG GTA GGC
336
Asn Gly Ser Asp Val Ser Ala Asn Ala Leu Leu Ser Gly Ala Val Gly
615 620 625 630
TCT GGG ACT TGT GCG GCT GCA GGG ACG GCT GGT GGC ACT TCT CTT AAC
384
Ser Gly Thr Cys Ala Ala Ala Gly Thr Ala Gly Gly Thr Ser Leu Asn
635 640 645
ACT CAA AGC ACT TGC ACC GTT GCG GGC TAT TAC TGG CTC CCT AGC TTG
432
Thr Gln Ser Thr Cys Thr Val Ala Gly Tyr Tyr Trp Leu Pro Ser Leu
650 655 660
ACT GAC AGG ATT TTA AGC ACG ATC GGC AGC CAG ACT AAC TAC GGC ACG
480
Thr Asp Arg Ile Leu Ser Thr Ile Gly Ser Gln Thr Asn Tyr Gly Thr
665 670 675
AAC ACC AAT TTC CCC AAC ATG CAA CAA CAG CTC ACC TAC TTG AAT GCG
528
Asn Thr Asn Phe Pro Asn Met Gln Gln Gln Leu Thr Tyr Leu Asn Ala
680 685 690
GGG AAT GTG TTT TTT AAT GCG ATG AAT AAG GCT TTA GAG AAT AAG AAT
576
Gly Asn Val Phe Phe Asn Ala Met Asn Lys Ala Leu Glu Asn Lys Asn
695 700 705 710
GGA ACT AGT AGT GCT AGT GGA ACT AGT GGT GCG ACT GGT TCA GAT GGT
624
Gly Thr Ser Ser Ala Ser Gly Thr Ser Gly Ala Thr Gly Ser Asp Gly
715 720 725
CAA ACT TAC TCC ACA CAA GCT ATC CAA TAC CTT CAA GGC CAA CAA AAT
672
Gln Thr Tyr Ser Thr Gln Ala Ile Gln Tyr Leu Gln Gly Gln Gln Asn
730 735 740
ATC TTA AAT AAC GCA GCG AAC TTG CTC AAG CAA GAT GAA TTG CTC TTA
720
Ile Leu Asn Asn Ala Ala Asn Leu Leu Lys Gln Asp Glu Leu Leu Leu
745 750 755
GAA GCT TTC AAC TCT GCC GTA GCC GCC AAC ATT GGG AAT AAG GAA TTC
768
Glu Ala Phe Asn Ser Ala Val Ala Ala Asn Ile Gly Asn Lys Glu Phe
760 765 770
AAT TCA GCC GCT TTT ACA GGT TTG GTG CAA GGC ATT ATT GAT CAA TCT
816
Asn Ser Ala Ala Phe Thr Gly Leu Val Gln Gly Ile Ile Asp Gln Ser
775 780 785 790
CAA GCG GTT TAT AAC GAG CTC ACT AAA AAC ACC ATT AGC GGG AGT GCG
864
Gln Ala Val Tyr Asn Glu Leu Thr Lys Asn Thr Ile Ser Gly Ser Ala
795 800 805
GTT ATT AGC GCT GGG ATA AAC TCC AAC CAA GCT AAC GCT GTG CAA GGG
912
Val Ile Ser Ala Gly Ile Asn Ser Asn Gln Ala Asn Ala Val Gln Gly
810 815 820
CGC GCT AGT CAG CTC CCT AAC GCT CTT TAT AAC GCG CAA GTA ACT TTG
960
Arg Ala Ser Gln Leu Pro Asn Ala Leu Tyr Asn Ala Gln Val Thr Leu
825 830 835
GAT AAA ATC AAT GCG CTC AAT AAT CAA GTG AGA AGC ATG CCT TAC TTG
1008
Asp Lys Ile Asn Ala Leu Asn Asn Gln Val Arg Ser Met Pro Tyr Leu
840 845 850
CCC CAA TTC AGA GCC GGG AAC AGC CGT TCA ACG AAT ATT TTA AAC GGG
1056
Pro Gln Phe Arg Ala Gly Asn Ser Arg Ser Thr Asn Ile Leu Asn Gly
855 860 865 870
TTT TAC ACC AAA ATA GGC TAT AAG CAA TTC TTC GGG AAG AAA AGG AAT
l104
Phe Tyr Thr Lys Ile Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn
875 880 885
ATC GGT TTG CGC TAT TAT GGT TTC TTT TCT TAT AAC GGA GCG AGC GTG
1152
Ile Gly Leu Arg Tyr Tyr Gly Phe Phe Ser Tyr Asn Gly Ala Ser Val
890 895 900
GGC TTT AGA TCC ACT CAA AAT AAT GTA GGG TTA TAC ACT TAT GGG GTG
1200
Gly Phe Arg Ser Thr Gln Asn Asn Val Gly Leu Tyr Thr Tyr Gly Val
905 910 915
GGG ACT GAT GTG TTG TAT AAC ATC TTT AGC CGC TCC TAT CAA AAC CGC
1248
Gly Thr Asp Val Leu Tyr Asn Ile Phe Ser Arg Ser Tyr Gln Asn Arg
920 925 930
TCT GTG GAT ATG GGC TTT TTT AGC GGT ATC CAA TTA GCC GGT GAG ACC
1296
Ser Val Asp Met Gly Phe Phe Ser Gly Ile Gln Leu Ala Gly Glu Thr
935 940 945 950
TTC CAA TCC ACG CTC AGA GAT GAC CCC AAT GTG AAA TTG CAT GGG AAA
1344
Phe Gln Ser Thr Leu Arg Asp Asp Pro Asn Val Lys Leu His Gly Lys
955 960 965
ATC AAT AAC ACG CAC TTC CAG TTC CTC TTT GAC TTC GGT ATG AGG ATG
1392
Ile Asn Asn Thr His Phe Gln Phe Leu Phe Asp Phe Gly Met Arg Met
970 975 980
AAC TTC GGT AAG TTG GAC GGG AAA TCC AAC CGC CAC AAC CAG CAC ACG
1440
Asn Phe Gly Lys Leu Asp Gly Lys Ser Asn Arg His Asn Gln His Thr
985 990 995
GTG GAA TTT GGC GTA GTG GTG CCT ACG ATT TAT AAC ACT TAT TAC AAA
1488
Val Glu Phe Gly Val Val Val Pro Thr Ile Tyr Asn Thr Tyr Tyr Lys
1000 1005 1010
TCA GCA GGG ACT ACC GTG AAG TAT TTC CGT CCT TAT AGC GTT TAT TGG
1536
Ser Ala Gly Thr Thr Val Lys Tyr Phe Arg Pro Tyr Ser Val Tyr Trp
1015 1020 1025 1030
TCT TAT GGG TAT TCA TTC TAA
1557
Ser Tyr Gly Tyr Ser Phe
1035
(2)SEQ ID NO:4的信息:
(i)序列特征:
(A)长度:518个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:4:
Met Ile Lys Lys Asn Arg Thr Leu Phe Leu Ser Leu Ala Leu Cys Ala
1 5 10 15
Ser Ile Ser Tyr Ala Glu Asp Asp Gly Gly Phe Phe Thr Val Gly Tyr
20 25 30
Gln Leu Gly Gln Val Met Gln Asp Val Gln Asn Pro Gly Gly Ala Lys
35 40 45
Ser Asp Glu Leu Ala Arg Glu Leu Asn Ala Asp Val Thr Asn Asn Ile
50 55 60
Leu Asn Asn Asn Thr Gly Gly Asn Ile Ala Gly Ala Leu Ser Asn Ala
65 70 75 80
Phe Ser Gln Tyr Leu Tyr Ser Leu Leu Gly Ala Tyr Pro Thr Lys Leu
85 90 95
Asn Gly Ser Asp Val Ser Ala Asn Ala Leu Leu Ser Gly Ala Val Gly
100 105 110
Ser Gly Thr Cys Ala Ala Ala Gly Thr Ala Gly Gly Thr Ser Leu Asn
115 120 125
Thr Gln Ser Thr Cys Thr Val Ala Gly Tyr Tyr Trp Leu Pro Ser Leu
130 135 140
Thr Asp Arg Ile Leu Ser Thr Ile Gly Ser Gln Thr Asn Tyr Gly Thr
145 150 155 160
Asn Thr Asn Phe Pro Asn Met Gln Gln Gln Leu Thr Tyr Leu Asn Ala
165 170 175
Gly Asn Val Phe Phe Asn Ala Met Asn Lys Ala Leu Glu Asn Lys Asn
180 185 190
Gly Thr Ser Ser Ala Ser Gly Thr Ser Gly Ala Thr Gly Ser Asp Gly
195 200 205
Gln Thr Tyr Ser Thr Gln Ala Ile Gln Tyr Leu Gln Gly Gln Gln Asn
210 215 220
Ile Leu Asn Asn Ala Ala Asn Leu Leu Lys Gln Asp Glu Leu Leu Leu
225 230 235 240
Glu Ala Phe Asn Ser Ala Val Ala Ala Asn Ile Gly Asn Lys Glu Phe
245 250 255
Asn Ser Ala Ala Phe Thr Gly Leu Val Gln GlyIle Ile Asp Gln Ser
260 265 270
Gln Ala Val Tyr Asn Glu Leu Thr Lys Asn Thr Ile Ser Gly Ser Ala
275 280 285
Val Ile Ser Ala Gly Ile Asn Ser Asn Gln Ala Asn Ala Val Gln Gly
290 295 300
Arg Ala Ser Gln Leu Pro Asn Ala Leu Tyr Asn Ala Gln Val Thr Leu
305 310 315 320
Asp Lys Ile Asn Ala Leu Asn Asn G1n Val Arg Ser Met Pro Tyr Leu
325 330 335
pro Gln Phe Arg Ala Gly Asn Ser Arg Ser Thr Asn Ile Leu Asn Gly
340 345 350
Phe Tyr Thr Lys Ile Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn
355 360 365
Ile Gly Leu Arg Tyr Tyr Gly Phe Phe Ser Tyr Asn Gly Ala Ser Val
370 375 380
Gly Phe Arg Ser Thr Gln Asn Asn Val Gly Leu Tyr Thr Tyr Gly Val
385 390 395 400
Gly Thr Asp Val Leu Tyr Asn Ile Phe Ser Arg Ser Tyr Gln Asn Arg
405 410 415
Ser Val Asp Met Gly Phe Phe Ser Gly Ile Gln Leu Ala Gly Glu Thr
420 425 430
Phe Gln Ser Thr Leu Arg Asp Asp Pro Asn Val Lys Leu His Gly Lys
435 440 445
Ile Asn Asn Thr His Phe Gln Phe Leu Phe Asp Phe Gly Met Arg Met
450 455 460
Asn Phe Gly Lys Leu Asp Gly Lys Ser Asn Arg His Asn Gln His Thr
465 470 475 480
Val Glu Phe Gly Val Val Val Pro Thr Ile Tyr Asn Thr Tyr Tyr Lys
485 490 495
Ser Ala Gly Thr Thr Val Lys Tyr Phe Arg Pro Tyr Ser Val Tyr Trp
500 505 510
Ser Tyr Gly Tyr Ser Phe
515
(2)SEQ ID NO:5的信息:
(i)序列特征:
(A)长度:656碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:两种
(ix)特征:
(A)名称/关键词:CDS
(B)定位:567..656
(xi)序列描述:SEQ ID NO:5:
AGATCTATGA ATCTATGATA TCAACACTCT TTTTGATAAA TTTTCTCGAG GTACCGAGCT
60
TGAGGCATCA AATAAAACGA AAGGCTCAGT CGAAAGACTG GGCCTTTCGT TTTATCTGTT
120
GTTTGTCGGT GAACGCTCTC CTGAGTAGGA CAAATCCGCC GGGAGCGGAT TTGAACGTTG
180
CGAAGCAACG GCCCGGAGGG TGGCGGGCAG GACGCCCGCC ATAAACTGCC ACAAGCTCGG
240
TACCGTTGAT CTTCCTATGG TGCACTCTCA GTACAATCTG CTCTGATGCG CTACGTGACT
300
GGGTCATGGC TGCGCCCCGA CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC
360
TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA
420
GGTTTTCACC GTCATCACCG AAACGCGCGA GGCCCAGCGC TTCGAACTTC TGATAGACTT
480
CGAAATTAAT ACGACTCACT ATAGGGAGAC CACAACGGTT TCCCTCTAGA AATAATTTTG
540
TTTAACTTTA AGAAGGAGAT ATACAT ATG AAA CTG ACT CCC AAA GAG TTA GAC
593
Met Lys Leu Thr Pro Lys Glu Leu Asp
520 525
AAG TTG ATG CTC CAC TAC GCT GGA GAA TTG GCT AAA AAA CGC AAA GAA
641
Lys Leu Met Leu His Tyr Ala Gly Glu Leu Ala Lys Lys Arg Lys Glu
530 535 540
AAA GGC ATT AAG CTT
656
Lys Gly Ile Lys Leu
545
(2)SEQ ID NO:6的信息:
(i)序列特征:
(A)长度:3O个氨基酸
(B)类型:氨基酸
(C)拓扑结构:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:6:
Met Lys Leu Thr Pro Lys Glu Leu Asp Lys Leu Met Leu His Tyr Ala
1 5 10 15
Gly Glu Leu Ala Lys Lys Arg Lys Glu Lys Gly Ile Lys Leu
20 25 30
Claims (9)
1.药物组合物,包括作为活性剂的免疫保护性活疫苗,该活疫苗是重组减毒沙门氏菌细胞,其包括编码螺杆菌脲酶、脲酶亚基或/和其免疫反应片段的异源核酸分子,其中所述的重组减毒沙门氏菌细胞能够表达所述的核酸分子或能够在靶细胞中表达所述的核酸分子。
2.根据权利要求1所述的组合物,其中沙门氏菌细胞是沙门氏菌aro突变体细胞。
3.根据权利要求1-2的任何一个所述的组合物,其中所述的编码螺杆菌脲酶、脲酶亚基或/和其免疫反应片段的核酸分子能够得到相变异表达。
4.根据权利要求3所述的组合物,其中所述的核酸分子编码螺杆菌脲酶、脲酶亚基或/和其免疫反应片段,是在表达信号的控制下的,表达信号在沙门氏菌细胞中基本上是无活化的,能够通过在沙门氏菌细胞中的核酸重新组构机制引起的核酸重新组构而活化。
5.根据权利要求4所述的组合物,其中表达信号是噬菌体启动子,活化是通过导致在沙门氏菌细胞中产生对应的噬菌体RNA聚合酶的DNA重新组构造成的。
6.根据权利要求1-5的任何一个所述的组合物,该组合物还包括药物上可接受的稀释剂、载体和佐剂。
7.制备活疫苗的方法,包括将根据权利要求1-5的任何一个的药物组合物以药物有效量与药物上可接受的稀释剂、载体和/或佐剂一起配制。
8.根据权利要求7所述的方法,包括制备重组减毒沙门氏菌细胞,包括步骤:
a)在减毒沙门氏菌细胞中插入编码螺杆菌脲酶、脲酶亚基或/和其免疫反应片段的核酸分子,其中获得重组减毒沙门氏菌细胞,其能够表达所述的核酸分子,或能够在靶细胞中导致所述的核酸分子的表达,和
b)在适当的条件下培养所述的重组减毒沙门氏菌细胞。
9.根据权利要求8所述的方法,其中所述的编码螺杆菌脲酶、脲酶亚基或/和其免疫反应片段的核酸分子定位于染色体外质粒上或插入染色体中。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96116337.5 | 1996-10-11 | ||
EP96116337A EP0835928A1 (en) | 1996-10-11 | 1996-10-11 | Helicobacter pylori live vaccine |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1246867A CN1246867A (zh) | 2000-03-08 |
CN1215167C true CN1215167C (zh) | 2005-08-17 |
Family
ID=8223286
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB971995516A Expired - Fee Related CN1215167C (zh) | 1996-10-11 | 1997-09-01 | 幽门螺杆菌活疫苗 |
Country Status (15)
Country | Link |
---|---|
EP (2) | EP0835928A1 (zh) |
JP (1) | JP2001510986A (zh) |
KR (1) | KR100510928B1 (zh) |
CN (1) | CN1215167C (zh) |
AT (1) | ATE392429T1 (zh) |
AU (1) | AU734635B2 (zh) |
BR (1) | BR9713254A (zh) |
CA (1) | CA2268033A1 (zh) |
DE (1) | DE69738641D1 (zh) |
IL (1) | IL129366A0 (zh) |
NO (1) | NO991692L (zh) |
NZ (1) | NZ335050A (zh) |
PL (1) | PL332575A1 (zh) |
TR (1) | TR199901450T2 (zh) |
WO (1) | WO1998016552A1 (zh) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6248551B1 (en) * | 1997-03-28 | 2001-06-19 | Institut Pasteur | Helicobacter aliphatic amidase AmiE polypeptides, and DNA sequences encoding those polypeptides |
US6585975B1 (en) * | 1998-04-30 | 2003-07-01 | Acambis, Inc. | Use of Salmonella vectors for vaccination against helicobacter infection |
CA2335487A1 (en) * | 1998-06-19 | 1999-12-23 | Merieux Oravax | Lt and ct in parenteral immunization methods against helicobacter infection |
WO2000003730A1 (en) * | 1998-07-16 | 2000-01-27 | Cheil Jedang Corporation | Urease based vaccine against helicobacter infection |
EP1278768B1 (en) * | 2000-04-27 | 2006-11-15 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for identifying helicobacter antigens |
BR112013015009B1 (pt) * | 2010-12-22 | 2020-11-10 | Intervet International B.V | vacina para proteger um animal contra uma doença clínica decorrente de uma infecção com bordetella bronchiseptica |
JP6225104B2 (ja) | 2011-04-08 | 2017-11-01 | タフツ メディカル センター インコーポレイテッドTufts Medical Center,Inc. | ペプデューシンの設計および使用 |
WO2017083618A1 (en) | 2015-11-13 | 2017-05-18 | Oasis Pharmaceuticals, LLC | Protease-activated receptor-2 modulators |
CN112048008B (zh) * | 2020-08-12 | 2021-06-15 | 河北医科大学第四医院(河北省肿瘤医院) | 一种幽门螺杆菌b细胞耐受表位及其制备的抗体 |
CN112143741B (zh) * | 2020-09-07 | 2022-09-20 | 中国科学院南海海洋研究所 | 一种生物膜基因岛GIVal43097及其切除方法 |
CN112143750B (zh) * | 2020-10-13 | 2022-12-27 | 宁夏医科大学 | 靶向m细胞的重组乳酸菌表面展示系统、重组乳酸菌疫苗及制备方法与应用 |
CN112190704B (zh) * | 2020-10-13 | 2022-11-15 | 宁夏医科大学 | M细胞靶向性重组乳酸菌疫苗及制备方法与应用 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5695983A (en) * | 1990-12-18 | 1997-12-09 | The General Hospital Corporation | Salmonella vaccines |
DE4041045A1 (de) * | 1990-12-20 | 1992-07-02 | Max Planck Gesellschaft | Zweiphasen-systeme fuer die produktion und praesentation von fremdantigen in hybriden lebendimpfstoffen |
FR2682122B1 (fr) * | 1991-10-03 | 1995-06-09 | Pasteur Institut | Nouveaux genes d'helicobacter pylori. leur utilisation pour la preparation de souches recombinantes de h. pylori. |
US5348867A (en) * | 1991-11-15 | 1994-09-20 | George Georgiou | Expression of proteins on bacterial surface |
JPH08509873A (ja) * | 1993-05-19 | 1996-10-22 | アンスティテュ・パストゥール | ヘリコバクター感染に対する免疫組成物、該組成物に用いられるポリペプチドおよび該ポリペプチドをコードする核酸配列 |
EP0654273A1 (en) * | 1993-11-18 | 1995-05-24 | Harry H. Leveen | Pharmaceutical product and method for treatment |
WO1996026740A1 (en) * | 1995-02-27 | 1996-09-06 | Enteron Limited Partnership | Methods and compositions for production of customized vaccines for diseases associated with antigens of microorganisms |
DE69637947D1 (de) * | 1995-04-28 | 2009-07-23 | Merieux Oravax Snc | Multimerer, rekombinanter urease impfstoff |
-
1996
- 1996-10-11 EP EP96116337A patent/EP0835928A1/en not_active Withdrawn
-
1997
- 1997-09-01 CN CNB971995516A patent/CN1215167C/zh not_active Expired - Fee Related
- 1997-09-01 IL IL12936697A patent/IL129366A0/xx unknown
- 1997-09-01 CA CA002268033A patent/CA2268033A1/en not_active Abandoned
- 1997-09-01 PL PL97332575A patent/PL332575A1/xx not_active IP Right Cessation
- 1997-09-01 DE DE69738641T patent/DE69738641D1/de not_active Expired - Lifetime
- 1997-09-01 WO PCT/EP1997/004744 patent/WO1998016552A1/en active IP Right Grant
- 1997-09-01 KR KR10-1999-7003168A patent/KR100510928B1/ko not_active IP Right Cessation
- 1997-09-01 TR TR1999/01450T patent/TR199901450T2/xx unknown
- 1997-09-01 BR BR9713254-3A patent/BR9713254A/pt not_active Application Discontinuation
- 1997-09-01 AT AT97940148T patent/ATE392429T1/de not_active IP Right Cessation
- 1997-09-01 NZ NZ335050A patent/NZ335050A/xx unknown
- 1997-09-01 EP EP97940148A patent/EP0931093B1/en not_active Expired - Lifetime
- 1997-09-01 JP JP51794498A patent/JP2001510986A/ja not_active Ceased
- 1997-09-01 AU AU42084/97A patent/AU734635B2/en not_active Ceased
-
1999
- 1999-04-09 NO NO991692A patent/NO991692L/no unknown
Also Published As
Publication number | Publication date |
---|---|
CN1246867A (zh) | 2000-03-08 |
DE69738641D1 (de) | 2008-05-29 |
BR9713254A (pt) | 1999-11-03 |
NZ335050A (en) | 2000-08-25 |
ATE392429T1 (de) | 2008-05-15 |
AU4208497A (en) | 1998-05-11 |
KR20000049090A (ko) | 2000-07-25 |
EP0931093A1 (en) | 1999-07-28 |
CA2268033A1 (en) | 1998-04-23 |
WO1998016552A1 (en) | 1998-04-23 |
EP0835928A1 (en) | 1998-04-15 |
PL332575A1 (en) | 1999-09-27 |
TR199901450T2 (xx) | 1999-10-21 |
AU734635B2 (en) | 2001-06-21 |
JP2001510986A (ja) | 2001-08-07 |
KR100510928B1 (ko) | 2005-08-31 |
NO991692L (no) | 1999-06-04 |
EP0931093B1 (en) | 2008-04-16 |
NO991692D0 (no) | 1999-04-09 |
IL129366A0 (en) | 2000-02-17 |
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