CN1890261A - Nmb0928蛋白及其在药物制剂中的用途 - Google Patents
Nmb0928蛋白及其在药物制剂中的用途 Download PDFInfo
- Publication number
- CN1890261A CN1890261A CNA2004800358798A CN200480035879A CN1890261A CN 1890261 A CN1890261 A CN 1890261A CN A2004800358798 A CNA2004800358798 A CN A2004800358798A CN 200480035879 A CN200480035879 A CN 200480035879A CN 1890261 A CN1890261 A CN 1890261A
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- Prior art keywords
- pharmaceutical preparation
- nmb0928
- albumen
- preparation
- vaccine
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Abstract
本发明涉及新的疫苗抗原的用途,所述新的疫苗抗原用于预防性或治疗性抵抗细菌、病毒、癌症或其它来源的疾病。本发明的技术目的是开发能增加已有疫苗的保护性谱,从而扩展其抵抗不同病原体的制剂。为达到该目的,分离了NMB0928蛋白并鉴定其为能够诱导杀菌活性的脑膜炎奈瑟氏球菌的外膜制剂的成分。此外,克隆并表达了编码蛋白NMB0928的基因,纯化了所述蛋白和随后在动物生物模型中评估了其免疫原性。来自同源基因的序列的高度的保守性表明,当其以不同途径提供时,显示了其诱导交叉免疫应答的抗原的较高价值。本发明的所得制剂适用于药物工业,作为供人使用的疫苗制剂。
Description
本发明涉及医学领域,特别是涉及开发预防性或治疗性应用的新的疫苗制剂,所述新的疫苗制剂可提高抗来自不同来源的疾病的疫苗抗原的免疫应答的质量。
脑膜炎奈瑟氏球菌(Neisseria meningitidis),其唯一已知的宿主是人的革兰氏阴性双球菌,是流行性脑脊髓膜炎(meningococcameningitis)病原体(causal agent)。通常该细菌发现于正常人群中的一些无症状携带者中,因为该生态位是其微生物分离的最常见来源。
在世界范围内,小于两岁的儿童是对感染性流行性脑脊髓膜炎比较易感的群体,但青少年和正常成人群体也可受到感染。
对大多数受感染的个体,未治疗的脑膜炎球菌病具有致命的发展过程,而接种疫苗可通过早在细菌定居期阻止事件发生来预防该病症。
为了在普通人群中诱导对该疾病的防护,已发展了数个策略来获得能够满足所需要求的疫苗。为了该目的,已考虑使用荚膜抗原,因为其免疫学特异性可分类成该微生物的血清群。这些血清群中的五个群已被确定为导致了全球范围内的大部分脑膜炎球菌病的临床病例。在亚撒哈拉非洲(sub-Saharan Africa),血清群A是流行病的主要原因。在大多数情况下,在发达国家,血清群B和C与发病相关联。血清群Y和W135在大多数疾病的复发病例中较为普遍,并且其在美国的一些地区较为流行,在过去数年中有显著增长。从该数据可看到将荚膜多糖作为疫苗候选物使用、研究和测定的原因是很显然的。基于荚膜多糖的提供抗血清群A、C、Y和W-135的保护作用的四价疫苗在美国已获许可。接种后引发的抗体是血清群特异的(Rosenstein N.等人2001.Menningococcal disease.N.Engl.J.Med,344,1378-1388)。
血清群B,与其它血清群不同,一直是地方性和流行性脑膜炎球菌病的重要原因,并且这主要是由于完全缺少有效的针对其的疫苗。已注意到B荚膜多糖的免疫原性较差,而且对于基于该化合物的疫苗存在理论上的风险,即因为其结构与存在于人神经系统胎儿结构中寡糖链相似,从而诱导免疫耐受性和自身免疫(Finne J.等人1987.AnIgG monoclonal antibody to group B meningococci cross-reactswith developmentally regulated polysialic acid units ofglycoproteins in neural and extraneural tisues.J.Immunol,138:4402-4407)。因此,抗血清群B的疫苗的开发集中在亚荚膜抗原的使用上。
外膜蛋白和泡囊(vesicle)疫苗
在70年代,产生基于外膜蛋白的疫苗的最初偿试是基于通过去污剂排除外膜蛋白制剂中的LPS(Frasch CE和Robbins JD.1978.Protection against group B meningococcal disease.III.Immunogenicity of serotype 2 vaccines and specificity ofprotection in a guinea pig model.J Exp Med 147(3):629-44)。然后沉淀外膜蛋白(OMP)以产生悬浮在氯化钠中的聚集体。尽管在动物中产生了很有前景的结果,但这些疫苗在成人或青少年中都不诱导杀菌性抗体(Zollinger WD,等人1978.Safety and immunogenicityof a Neisseria menningitidis type 2 protein vaccine in animalsand humans.J.Infect.Dis.137(6):728-39),这些疫苗的欠佳表现主要归因于伴随沉淀作用而产生的三级结构的丧失。因此,下一个合理的步骤是制备外膜泡囊形式的具有其天然构象的蛋白的疫苗(Zollinger WD,等人1979.complex of meningococcal group Bpolysaccharide and type 2 outer membrane protein immunogenicin man.J.Clin.Invest.63(5):836-48,Wang LY和Frasch CE.1984.Development of a Neisseria meningitidis group B serotype 2bprotein vaccine and evaluation in a mouse model.Infect Immun46(2):408-14136)。
这些外膜泡囊疫苗的免疫原性显著超过OMP聚集体,而且通过吸附至佐剂氢氧化铝,免疫原性显示进一步被加强(Wang LY和FraschCE.1984.Neisseria meningitidis group B serotype 2b proteinvaccine and evaluation in a mouse model.Infect Immun.46(2):408-14136)。
已使用不同制剂的可溶性外膜泡囊疫苗进行了许多功效试验。在1980年代为应对在古巴(Sierra GV等人1991.Vaccine againstgroup B Neisseria meningitidis:protection trial and massvaccinationre sults in Cuba.NIPH Ann Dis.14(2):195-210)和挪威(Bjune G,等人1991.Effect of outer membrane vesicle vaccineagainst group B meningococcal disease in Norway.Lancet.338(8775):1093-6)暴发的疾病分别开发了两个研究最广泛深入的疫苗。由古巴Finlay研究院生产的OMV疫苗(商标为VA-MENGOC-BC)制备于具有血清群C多糖的株系B:4:P1.19,15和高分子量OMP的制剂,并且被吸附至氢氧化铝(Sierra GV等人1991.Vaccine againstgroup B Neisseria meningitidis:protection trial and massvaccination results in Cuba.NIPH Ann Dis.14(2):195-210)。该疫苗为快速减退古巴的流行病作出了贡献(Rodriguez AP,等人Theepidemioiogical impact of antimeningococcal B vaccination inCuba.1999.Mem Inst Oswaldo Cruz.94(4):433-40)。
由挪威国家公共卫生研究院(NIPH)生产的疫苗类似地最初打算在由来自ET-5克隆(B:15:P1.7,16)的另一种生物导致的高度地方性疾病时期使用。其也是产生自吸附至氢氧化铝的纯化的外膜泡囊的单价疫苗(BjuneG,等人1991.Effect of outer membrane vesiclevaccine against group B meningococcal disease in Norway.Lancet.338(8775):1093-6)。
外膜泡囊疫苗似乎有效地提供了充分天然构象的外膜蛋白,使得至少在青少年和成人中产生功能性细菌抗体。产生的抗体应答也已显示增加脑膜炎球菌的调理素性吞噬作用(opsonophagocytosis)。疫苗的精确配制(即OMP含量、LPS含量和佐剂的存在或不存在)对免疫原性具有显著的影响(Lehmann AK,等人1991.Immunizationagainst serogroup B meningococci.Opsonin response in vaccineesas measured by chemiluminescence.APMIS.99(8):769-72,Gomez JA,等人1998.Effect of a djuvants in the isotypes and bactericidalactivity of antibodies against the transferrin-bindingproteins of Neisseria meningitidis.Vaccine.16(17):1633-9,Steeghs L,等人1999.Immunogenicity of Outer MembraneProteins in a Lipopolysaccharide-Deficient Mutant of Neisseriameningitidis:Influence of Adjuvants on the Immune Response.Infect Immun.67(10):4988-93)。
疾病分离物的抗原性特征谱也在快速地改变,因而如果不改变疫苗组成以反映地方流行病学,只作用有限数目的经选择的株系的疫苗在数年内可能会变得无效。
目前,OMV疫苗使用范围超过任何一种其它的血清群B疫苗,并且在由单个PorA型引起的疾病暴发的情况下具有潜在的用途。
在株系间产生交叉反应的免疫原还需要充分确定。来自Finlay研究院和NIPH疫苗试验的接种后血清的研究显示抗PorA(P1,1类血清亚型蛋白)和OpcA(另一种主要的OMP,以前称为Opc)二者的抗体(Wedege E,等人1998.Immune Responses against Major OuterMembrane Antigens of Neisseria meningitidis in Vaccinees andControls Who Contracted Meningococcal Disease during theNorwegian Serogroup B Protection Trial.Infect Immun.66(7):3223-31),在介导血清杀菌活性(具有PorA mosl免疫原性的)上都是非常重要的,这些抗原都显示明显的株系与株系之间的差异。
PorA蛋白的重要性和该蛋白中明显的变异性水平使得更加关注通过单链(单价)基于OMV的疫苗提供的保护可能是血清亚型限制性的(即,依赖于PorA型),所述蛋白在暴发之间和期间在接种后(和病后)主要杀菌活性所针对的表位上进行持续的变异(Jelfs J,等人2000.Sequence Variation in the porA Gene of a Clone of Neisseriameningitidis during Epidemic Spread Clin Diagn Lab Immunol.7(3):390-5)。
为克服该潜在的问题,在荷兰的RIVM开发了含有来自6个不同流行病原分离物的PorA蛋白的OMV疫苗(Van Der Ley P和PoolmanJT.1992.Construction of a multivalent meningococcal vaccinestrain based on the class 1 outer membrane protein.Infect Immun.60(8):3156-61,Claassen I,等人 1996.Production,characterization and control of a Neisseria meningitidishexavalent class 1 outer membrane protein containing vesiclevaccine.Vaccine.14(10):1001-8)。在该情况下,从两个已良好表征的H44/76株系的变体提取疫苗泡囊,所述株系已通过基因工程改造表达三个分别的PorA蛋白。
对广谱抗原的搜寻
很明显,外膜蛋白(OMP)可诱导抗血清群B疾病的功能性免疫应答,但由于外膜蛋白的表面暴露区域的不均匀性导致至今未开发出具有广谱性保护作用的疫苗。由外膜泡囊(OMV)疫苗诱导的适中的交叉免疫性已推动了对外膜抗原(或抗原群)的搜寻,所述抗原诱导功能性抗体并且存在于所有脑膜炎球菌(meningococcal)株系上。这些抗原,如果其存在于所有株系而与血清群无关,就可能形成真正的广谱性脑膜炎球菌疫苗的基础,该疫苗可消除多糖接种后产生的病原株系上的荚膜转换的潜在问题。
当免疫显性PorA蛋白的变异性限制其用作广谱疫苗变得明显时,对许多其他主要的外膜蛋白的疫苗的潜能进行了考虑,这些蛋白中的数个蛋白处在进一步的开发之中。已被考虑的蛋白包括第5类蛋白(OpcA)、NspA和铁调节蛋白(TbpA和B、FbpA、FetA)。TbpB和TbpA形成转铁蛋白结合复合物的部分。最近的工作显示TbpA在结合铁上具有比TbpB更大的功能作用(Pintor M,等人1998.Ahalysis of TbpAand TbpB functionality in defective mutants of Neisseriameningitidis.J Med Microbiol 47(9):757-60)并且是比TbpB更有效的免疫原。
通过新的技术,使用来自不同脑膜炎球菌株系的外膜蛋白制剂组合免疫小鼠,已发现高度保守的小的外膜蛋白(Martin D,等人1997.Highly Conserved Neisseria meningitidis Surface ProteinConfers Protection against Experimental Infection.J Exp Med185(7):1173-83)。将来自小鼠的B细胞用于产生杂交瘤,然后就抗多个脑膜炎球菌株系的交叉反应性筛选所述杂交瘤。发现一个高度交叉反应性的单克隆抗体结合被称作NspA的22kDa的外膜蛋白。使用重组NspA蛋白的免疫显示在小鼠中诱导抗来自血清群A-C株系的交叉反应性杀菌应答。接种疫苗也保护小鼠免受致死的脑膜炎球菌性感染(Martin D,等人1997.Higly Conserved Neisseria meningitidisSurface Protein Confers Protection against ExperimentalInfection.J Exp Med 185(7):1173-83)。在遗传上多样性的脑膜炎球菌株系之间的NspA序列的比较表明所述蛋白是高度保守的(97%的同源性)(Cadieux N,等人1999.Bactericidal andCross-Protective Activities of a Monoclonal Antibody Directedagainst Neisseria meningitidis NspA Outer Membrane Protein.Infect Immun 67(9):4955-9)。
使用抗NspA单克隆抗体通过ELISA法在99.2%的来自血清群A-C的受试株系中检测到NspA的存在(Martin D,等人1997.HighlyConserved Neisseria meningitidis Surface Protein ConfersProtection against Experimental Infection.J Exp Med 185(7):1173-83)。这些单克隆抗体已显示抗许多脑膜炎球菌株系的杀菌性并且能够在小鼠模型中减少脑膜炎球菌菌血症(Cadieux N,等人1999.Bactericidal and Cross-Protective Activities of a MonoclonalAntibody Directed against Neisseria meningitidis NspA OuterMembrane Protein.Infect Immun 67(9):4955-9)。尽管该数据似乎表明NspA是个能够跨血清群界限起保护作用的很有前景的疫苗候选物,但来自小鼠的多克隆抗重组NspA血清不和大约35%的病原性血清群B脑膜炎球菌株系的表面结合,尽管在这些生物中存在nspA基因(Moe GR等人1999.Differences in Surface Expression ofNspA among Neisseria meningitidis Group B Strains.Infect Immun67(11):5664-75)。
抗原呈递和疫苗制剂
更早期的工作已显示抗原呈递的形式可能是极其重要的。膜结合蛋白上的表位通常依赖于正确的三级结构的维持,而这反过来通常依赖于疏水性的膜结合结构域。已显示,外膜蛋白的制剂只有当以泡囊形式呈递时才能在人体中引起免疫性(Zollinger WD,等人1979.complex of meningococcal group B polysaccharide and type 2 outermembrane protein immunogenic in man.J Clin Invest 63(5):836-48,Zollinger WD,等人1978.Safety and immunogenicity ofa Neisseria meningitidis type 2 protein vaccine in animals andhumans.J Infect Dis 137(6):728-39)。
单个蛋白的疫苗已在本领域内使用了数十年并且通常表现出良好的稳定性。如果为了可使抗原保持膜结合而要求以泡囊的形式进行呈递,那么可能难以保证稳定性和可重复性。外膜泡囊的免疫原性和反应原性可随着在纯化过程中除去的蛋白和LPS的量的改变而变化。但在OMV疫苗生产中,在泡囊生产上已积累了大量的经验,当前生产的疫苗要接受全面的质量控制。完全合成的脂质体泡囊的构建可以允许进一步优化和标准化这些疫苗(Christodoulides M,等人1998.Immunization with recombinant class 1 outer-membrane proteinfrom Neisseria meningitidis:influence of liposomes andadjuvants on antibody avidity,recognition of native proteinand the induction of a bactericidal immune response againstmeningococci.Microbiology 144(Pt11):3027-37)。换句话说,已将外膜蛋白以泡囊或作为纯的表达蛋白的形式呈递,产生的抗体应答适中。目前主要的偿试已集中在导致全身性IgG的产生的脑膜炎球菌疫苗的肌内注射。然而,在经鼻上皮侵入宿主的脑膜炎球菌疾病中,分泌性IgA的产生也可能是很重要的。
脑膜炎奈瑟氏球菌的基因组序列
在2000年期间阐明和公开了MC58(血清群B脑膜炎球菌)(Tettelin H,等人2000.complete Genome Sequence of Neisseriameningitidis Serogroup B strain MC58.Science 287 (5459):1809-15172)和Z2491(血清群A菌株)(Parkhill J,等人2000.complete DNA sequence of a serogroup A strain of Neisseriameningitidis Z2491.Nature 404(6777):502-6173)的基因组序列。经注释的基因序列的可利用性应当对脑膜炎球菌疫苗研究产生极大的影响。虽然MC58基因组测序正在进行中,但Pizza等人开始鉴定经预测为编码膜结合的、表面暴露的或外运的蛋白的开放阅读框架。他们鉴定了570个这样的ORF,通过聚合酶链式反应对其进行扩增并将其克隆入大肠杆菌(Escherichia coli)中,使被编码的蛋白以His标记的融合蛋白或谷胱甘肽S转移酶融合蛋白表达(Pizza M,等人2000.Identification of Vaccine Candidates Against SerogroupB Meningococcus by Whole-Genome Sequencing.Science 287(5459):1816-20)。61%(350个)的经选择的OFR被成功地表达,没有表达的ORF通常是含有超过一个疏水跨膜结构域(可能排除了许多外膜结合蛋白)的ORF。纯化重组蛋白并用于接种小鼠。然后通过酶联免疫吸附(ELISA)测定法和流式细胞术就与多脑膜炎球菌株系的表面结合以及通过使用血清杀菌测定法就抗两个株系的杀菌活性对免疫血清进行评价。最终基于在所有三个测定中都呈阳性应答的基础上选择了7个蛋白以作进一步研究。使用许多这些蛋白和佐剂组合的试验性疫苗制剂在小鼠中已显示诱导相当高的抗同源脑膜炎球菌株系(MC58)的杀菌滴度,但没有一个蛋白诱导的SBA滴度和MC58外膜泡囊疫苗诱导的一样高(Giuliani MM,等人2000.Proceedings 12th IPNC.p.22)。另一方面,存在一些这样的证据,即在小鼠中,这些蛋白的组合可比单个蛋白表现出更高的免疫源性(Santini L.等人 2000.Proceedings 12th IPNC.p.25)。在本工作中被排除(可能因为蛋白表达的失败或其免疫学特性的修饰)的大量开放阅读框架,也可能具有疫苗潜能和需要作进一步研究。
当理解了单个抗原对脑膜炎奈瑟氏球菌的发病机理的贡献后,可更有效地选择疫苗组分。抗原自身可产生有效的疫苗候选物或,可选择地,可考虑将经减毒的突变体作为疫苗的成分。
在该教导下,使用在几种病原性微生物中具有高度序列保守性的疫苗候选物可提供对多种其(即这些微生物)可能引起疾病的解决方案,如果这些候选物通过免疫系统的作用诱导便利的应答。
本发明追求的技术目标是开发这样的疫苗制剂,即其能够增加和/或拓宽抵抗不同病原体或抵抗广泛的单个病原体变体的经诱导的免疫应答,这些病原体是癌症、细菌、病毒或任何其它来源的病原体。
发明描述
在本发明的工作目标中,第一次报道将NMB0928蛋白用作具有抗脑膜炎球菌疾病或由奈瑟氏菌属的成员引起的任何感染的治疗性或预防性特性的疫苗制剂的成分。
本发明的新的特点在于以前未报道过的,在制剂中使用具有新的特性的NMB0928蛋白能够诱导广谱性保护作用的全身性和粘膜性免疫应答,这是由于该蛋白在不同的脑膜炎奈瑟氏球菌(Neisseriameningitidis)和淋病奈瑟氏球菌(Neisseria gonorrhoeae)分离株中保守的特性导致的。
附图概述
图1.用于NMB0928蛋白克隆和表达的克隆载体pM100。pTrip,色氨酸启动子;N-term P64k,P-64k N-末端片段;T4 Terminator,T4噬菌体转录终止子。
图2.pM100载体中NMB0928基因的核苷酸序列的最终构建体。
图3.从细胞破碎获得的级分的SDS-PAGE分析;泳道1,上清液;泳道2,细胞沉淀。
图4.始于破碎沉淀的NMB0928蛋白的溶解过程的SDS-PAGE分析:(A)泳道1,破碎沉淀;泳道2,用含有3M尿素的1×TE缓冲液洗涤后的沉淀;泳道3,由洗涤产生的可溶性级分;(B)泳道1,用含有6M尿素的1×TE缓冲液溶解的上清液;泳道2,溶解沉淀。
图5.抗重组NMB0928蛋白的抗体(IgG)水平,在经鼻内或腹腔内途径用相同抗原免疫小鼠后获得所述抗体。显示ELISA的结果,结果表示为使免疫前血清的值加倍的最高稀释度的倒数。
图6.存在于脑膜炎奈瑟氏球菌OMV中的NMB0928蛋白的识别,使用来自用所述重组蛋白免疫的小鼠的血清通过Western印迹法来识别:箭头表示对应于经免疫鉴定的NMB0928蛋白的带。
图7.在经鼻内途径用抗原NMB0928重组蛋白免疫的小鼠中,在粘膜水平上抗NMB0928重组蛋白的IgA抗体应答。结果表示为使免疫前血清的值加倍的最高稀释度的倒数。(A)唾液中的I gA抗体应答。(B)肺清洗液中的IgA抗体应答。
图8.使用BLAST程序在NMB0928蛋白(“查询”)和来自不同血清群的脑膜炎奈瑟氏球菌的基因组中经注释的序列(“目标”)之间进行同源检索的结果。
图9.不同的脑膜炎奈瑟氏球菌株系中NMB0928蛋白的识别,通过抗NMB0928重组抗原产生的血清来识别。在图中只显示当经腹膜内途径使用半纯化的蛋白时获得的结果,然而在其余情况下也观察到类似的行为。结果表示为使免疫前血清的值加倍的最高稀释度的倒数。
图10.在年幼大鼠模型中,在抗脑膜炎球菌感染的被动保护实验中的血清之间的比较,所述血清是经腹膜内途径施用通过两种方法获得的蛋白免疫产生的。
图11.NMB0928蛋白和一组非关联抗原的识别,通过产生的mAb(mAb E45-8-15、2G23-12)来识别。P1,I类蛋白脑膜炎奈瑟氏球菌株系B:4:P1.15;P64K,来自脑膜炎奈瑟氏球菌的丙酮酸脱氢酶的E3亚基;T.T,破伤风类毒素(tetanus toxoid);HBsAg,B型肝炎表面抗原。
图12.NMB0928蛋白的识别,通过来自脑膜炎球菌疾病的存活者的人恢复期血清来识别。使用健康供体的血清作为对照。结果表示为在ELISA型测定法中的吸光度(492nm)。
图13.来自动物血清的JY1抗肽滴度,所述动物用游离肽(JY1)、重组蛋白(NMB0928)或缀合物JY1-NMB0928来免疫。
实施例
实施例1
血清群B脑膜炎奈瑟氏球菌外膜泡囊制剂中的NMB0928蛋白的检测
为了研究存在于血清群B脑膜炎奈瑟氏球菌(株系B:4:P1.19,15)外膜泡囊中的蛋白,按照其他地方描述的方法(Grg A,等人1985.Electrophoresis 6:599-604)进行双相电泳。随后使用胰蛋白酶(Promega,Madison,WI,U.S.)对胶上提取的蛋白进行酶促消化。通过使用微型柱(Zip Tips,Millipore,MA,U.S.)将消化后产生的肽提取至溶液中。为进行质谱分析用60%乙腈、1%甲酸然后立即使用nanotips(Protana,Denmark)将肽从微柱中洗脱。
在配备有电离源(nanoESI)的具cuadrupole和飞行时间混合质谱仪(QTof-2TM,Manchester,United Kingdom)中进行测量。使用0.02秒的扫描间隔,在0.98秒内在400-2000w/z范围内获得质谱测量法的数据。使用MassLynx程序(3.5版本,Micromass)获取数据和处理数据。
使用ProFound程序(Zhang W和Chait BT.2000.ProFound:anexpert system for protein identification using massspectrometric peptide mapping information.Anal Chem72:2482-2489.
http://prowl.rockefeller.edu/cgi-bin/ProFound)进行基于质谱数据的蛋白质鉴定。考虑到要遇到的可能修饰:甲硫氨酸的氧化、半胱氨酸的脱酰胺和羧基酰胺甲基化,对SwissProt数据库(http://www.ebi.ac.uk/swissprot/)和NCBI(
http://www.ncbi.nlm.nih.gov/)中的基因和衍生的蛋白质序列进行检索。
使用MASCOT程序(Perkins DN,等人1999.Probability-basedprotein identification by searching sequence databases usingmass spectrometry data.Electrophoresis 20:3551-3567.http://www.matrixscience.com/)进行基于质谱的蛋白质鉴定。检索参数包括半胱氨酸修饰以及氧化和脱酰胺作用。
从结果(从存在于外膜泡囊制剂中的蛋白的鉴定中获得的结果)的分析,选择NMB0928蛋白(来自其的一个肽已通过质谱进行了鉴定)就其作为可能的疫苗候选物进行估测。
实施例2
NMB0928基因编码的产物为脑膜炎奈瑟氏球菌的脂蛋白-34的鉴定
为鉴定NMB0928蛋白,使用BLAST程序(Altschul SF,等人1990.Basic local alignment search tool.J Mol Biol 215:403-410,http://www.ncbi.nlm.nih.gov/BLAST/)在NCBI数据库中进行序列同源性检索。该方法的结果表明其除了与奈瑟氏菌属的其他血清群中的相应蛋白同源外,还与几种微生物中的蛋白同源,所述蛋白包括由1991年鉴定的来自大肠肝菌的nlpB基因编码的脂蛋白-34。已证明该蛋白被分离成外膜蛋白脂质体(Bouvier J,Pugsley A.P andStragier,P.1991.Agene for new lipoprotein in the dapA-purCinterval of the E.coli chromosome.J Bacteriol173(17):5523-31)。
该蛋白在几种微生物属的基因组中的保守性已使其作为直向同源蛋白群包含于NCBI[gnl|CDD|12651,COG3317,NlpB,未表征的脂蛋白(细胞包膜生物发生,外膜)]报道的保守区域中,这表明对所有这些微生物存在共同的系统发生祖先。
使用MBGD数据库(Uchiyama,I.2003.MBGD:microbial genomedatabase for comaprative análisis.Nucleic Acids Res.31,58-62.)分析这些基因的相邻部分,发现了在基因组织上的显著相似性,从而将NMB0928蛋白鉴定为脑膜炎奈瑟氏球菌的脂蛋白-34(NlpB)。
实施例3
编码来自脑膜炎奈瑟氏球菌的NMB0928蛋白的NMB0928基因在大肠肝菌中的克隆和表达
为了克隆和表达NMB0928基因,使用pM-100克隆载体。该载体允许使用不同的限制性酶进行克隆和允许在大肠杆菌中以包函体的形式产生异源蛋白的高表达水平。
pM-100载体(图1)具有下列元件:色氨酸启动子、编码来自P64kDa的N端片段(来自脑膜炎奈瑟氏球菌株系B:4:P1.19,15)的47个氨基酸的稳定性序列的基因区段、噬菌体T4转录终止子的序列和赋予对青霉素的抗性的作为选择标记的基因的序列。
按照编码NMB 0928蛋白(实施例1)的核苷酸序列设计两个引物(7740和7741)来扩增来自株系B:4:P1.19,15的基因组DNA的该基因的片段,不具有编码预计的信号肽的序列。
BgIII
7740:5′GCAGATCTTGGCAGCAAAACCGAAC3′
(Seq.ID.No.1)
EcoRV
7741:5′ATG
GATATCCTCAGCTCGAGATGGAG3′
(Seq.ID.No.2)
为预测信号肽,使用SignalP World Wide Web server(
http://www.cbs.dtu.dk/services/SignalP-2.0)。
在使用引物7740和7741对NMB0928基因(RandallK,等人1988.Science 42394:487-491)进行PCR扩增后,使用BglII和EcoRV限制性酶消化PCR产物,并克隆入之前已被消化的pM-100克隆载体。最终的构建体显示于图2,NMB0928蛋白以融合至P64kDa蛋白的Nt-区段的融合蛋白的形式表达。使用ALFexpress II自动测序仪(TermoSequenaseTM CyTM 5Dye Terminador Kit,Amersham Biosciences)和分别结合P64稳定子和T4转录终止子的序列的寡核苷酸1573(Seq.ID.No.8)和6795(Seq.ID.No.9)对经克隆的NMB0928基因进行测序。此处产生的质粒对其以后的用途称为pM-242。
为了表达NMB0928基因,通过化学方法使用pM-242质粒(图2)转化GC366大肠杆菌株系。在加有1%甘油、1%酪蛋白、0.1mM CaCl2、1mM MgSO4和50ug/mL氨苄青霉素的基本培养基(M9)中进行表达实验(Miller JH.1972.Experiments in Molecular Genetics,ColdSpring Harbor Laboratory Press,NEW York,USA)。将细菌培养物在37℃和250rpm下温育12小时。将生长的培养物离心并对细胞沉淀物进行超声破碎(IKA LABORTECHNIK)。通过SDS-PAGE(Laemmli UK.1970.Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4.Nature 277:680)和考马斯亮蓝R-250染色分析来自沉淀物的级分和上清液。通过凝胶光密度测定法(LKBBromma 2202 Ultrascan laser densitometer;Amersham PharmaciaBiotech,United Kingdom)测量表达百分率。从沉淀部分获得NMB0928蛋白,约为该部分的总蛋白含量的60%(图3)。用含有2M尿素的1xTE缓冲液(10mM Tris-羟甲基氨基甲烷、1mM乙二氨四乙酸,pH 8)洗涤沉淀,一些杂质进入上清液中而NMB0928蛋白仍保留在沉淀中(图4A)。然后,用含有6M尿素的1xTE缓冲液溶解沉淀,涉及的蛋白进入了可溶性部分,该部分对1xTE缓冲液进行透析,产生具有70%纯度的最终制剂,如可在图4B中所看到的。
实施例4
在经腹膜内和鼻内途径用NMB0928蛋白免疫后诱导的免疫应答的评估
为评估NMB0928蛋白的免疫原性,设计免疫实验并在小鼠中进行免疫实验,其中给药以两种不同的方法制备的相同的蛋白。第一方法在于从聚丙烯酰酰胺凝胶上提取条带(Castellanos L,等人1996.Aprocedure for protein elution from reverse-stainedpolyacrylamide gels applicable at the low picomole level:Analternative route to the preparation of low a bundance proteinsfor microanalysis.Electroforesis 17:1564-1572),第二种方法在实施例3中提及并且产物被称为半纯化的蛋白。
将分成每组8只的4组雌性Balb/C小鼠(8-10周龄)用这些制剂免疫。经鼻内或腹膜内途径进行三次免疫,两次免疫间隔15天。用弗氏佐剂乳化经腹膜内施用的蛋白。表1描述了各组的构成:
表1:用于免疫的Balb/C小鼠的组
组 | 从凝胶中提取的蛋白 | 半纯化蛋白 | 途径 |
1 | 50μg | -- | i.n |
2 | -- | 50μg | i.n |
3 | 10μg | -- | i.p |
4 | -- | 10μg | i.p |
通过ELISA方法,在第三次接种后采集的血清样品中确定抗重组蛋白和存在于细菌中的同源蛋白的抗体滴度(IgG)。图5显示了各个动物中抗重组蛋白的抗体滴度。在第二次接种后确定抗体水平,尽管其在第三次接种后更高。此外,进行了通过Western印迹法的免疫鉴定,在所述方法中,各蛋白条带被识别。经腹膜内途径免疫的组具有显著高于经鼻内途径产生的滴度。
由于组内方差的非齐性(non-homogeneity),为进行结果的统计分析,根据巴特利特检验,使用Kruskal-Wallis的方差的非参数分析。使用Dunn的多重比较检验比较各处理的平均值。
用重组蛋白免疫后获得的血清识别存在于CU385株系的外膜蛋白(OMP)制剂中的天然蛋白。这些结果显示于图6中。
为分析粘膜应答,对唾液样品和肺洗涤物进行测定。图7只显示经鼻内途径免疫的组。在接受半纯化的蛋白的组内观察到IgA滴度的增加。
实施例5
在不同脑膜炎奈瑟氏球菌株系中编码NMB0928蛋白的基因的序列的表征。
为分析在奈瑟氏菌属的病原性种中编码NMB0928蛋白的基因的序列保守性,使用BLAST程序(Altschul SF,等人1990.Basic localalignment search tool.J Mol Biol 215:403-410.http://www.ncbi.nlm.nih.gov/BLAST/)对脑膜炎奈瑟氏球菌(血清群A、B和C)和淋病奈瑟氏球菌的基因组(在NCBI数据库中注释为NC003116.1,
NC003112.1,NC003221,NC002946 SANGER 135720|Contigl)进行检索。图8显示了那些在各个被分析的基因组中产生有效比对的序列的序列比对结果。在血清群A和C中这些序列与获得的编码NMB0928蛋白的基因的序列(Seq.ID.No.3)具有98%的同一性,在血清群B中这些基因与编码NMB0928蛋白的基因序列具有99%的同一性,在淋病奈瑟球菌中这些序列与编码NMB0928蛋白的基因序列具有96%的同源性。此外,在三个古巴人的分离物中确定了被涉及的基因的序列(Seq.ID.No.5-7),所述分离物属于血清群B(B:4:P1.19,15),并使用ClustalX程序(
http://www.ebi.ac.uk/clustalw/)进行序列比对。比对结果显示在被分析的株系间的NMB0928基因的核苷酸序列上存在极大的保守性。
考虑到前面提到的序列间存在的高度相似性,将NMB0928蛋白用作疫苗候选者将可能产生具有抗脑膜炎球菌疾病的广谱性保护作用(由于交叉反应的结果)的有效免疫应答。
实施例6
通过用NMB0928蛋白免疫Balb/C小鼠诱导的具有广谱作用的免疫应答的表征
为估计使用NMB0928蛋白的免疫是否诱导与其它奈瑟氏球菌属株系产生广泛交叉反应的应答,进行ELISA分析。使用7个属于不同血清型和血清亚型的奈瑟氏球菌属株系的完整细胞包被聚苯乙烯平板。将该平板用抗NMB0928蛋白的、混合的血清温育,如实施例4中所描述的,所述血清通过两种免疫途径获得。
图9显示使用经腹膜内途径施用的半纯化蛋白引起的抗该蛋白的血清获得的结果。如所观察到的,免疫血清识别存在于不同株系中的蛋白,其水平与在CU385株系中发现的水平相似。在该测定中其他血清具有相似的行为。
实施例7
在幼年大鼠模型中,由对于NMB0928蛋白特异性的鼠科动物血清诱导的抗同源和异源株系的保护作用
为确定获得的抗血清的功能性活性,在幼年大鼠模型中对针对脑膜炎球菌感染的保护性进行测定。将24只大鼠(5-6天龄)分组,每组6只大鼠。
确定经腹膜内途径施用的血清是否保护大鼠免受由一小时后经相同途径接种的细菌(株系CU385)引起的感染。混合各组血清并稀释10倍(在无菌PBS中),然后将其接种幼年大鼠。四小时后,对动物取样并对其血液中存活的细菌计数。
为解释结果,进行方差分析(Anova),接着进行Dunnet’s多重比较检验,其中将受试组和阴性对照进行比较。如图10中所看到的,接受了抗血清的组。
用从古巴患者分离的株系M982和120/90感染幼年大鼠来进行相似的测定,所述株系的血清学分类与株系B385同源。此外,使用来自血清群C的株系233(C:2a:P1.5)和来自血清群B的株系H44/76(B:15:P1.7,16)进行攻击实验。在所有情况中,抗血清保护幼年大鼠免受脑膜炎球菌感染。
实施例8
能够介导抗脑膜炎奈瑟氏球菌的杀菌活性的抗NMB0928蛋白的单克隆抗体的产生
为产生特异性抗NMB0928蛋白的单克隆抗体(mAb)和研究介导抗脑膜炎奈瑟氏球菌的同源和异源株系的杀菌活性的功能性能力,用纯度高于70%的NMB0928蛋白制剂(实施例3)进行免疫方案。在Balb/C(H-2d,雌性,5-6周龄)中进行免疫,如下使用4次免疫:在第0、15和30天按常规免疫,对每只小鼠经皮下施用用弗氏佐剂乳化的10μg NMB0928抗原(总体积100μl);在第50天,经腹膜内途径对每只小鼠施用10μg在磷酸缓冲盐水(140mM NaCl、270mM KCl、1.5mM KH2PO4、6.5mM Na2HPO4×2H2O、pH 7.2)中的抗原。在第0和第45天进行抽血。
将具有最高滴度(使用NMB0928蛋白作为包被抗原(实施例3),通过间接ELISA测量的滴度)的来自动物的脾细胞与X63 Ag8 653小鼠骨髓瘤细胞融合。根据标准的方法分离和筛选所得的杂交瘤(Gavilondo JV.1995.Anticuerpos Monoclonales:Teoría yPráctica,Elfos Scientiae,La Habana,Cuba)。
使用5μg/ml的各抗原和相同浓度的各待测mAb,通过间接ELISA法对由杂交瘤分泌的针对NMB0928蛋白的抗体的反应性以及它们的交叉反应性非关联抗原进行检测。图11显示在该实验中获得的结果,总共获得了2个特异性识别NMB0928蛋白并且既不和对应于P64K的N末端的氨基酸序列反应也不和其他的被测定的非关联抗原反应的阳性克隆(mAb E45-8-15和2G23-12)。
为确定产生的抗NMB0928蛋白的mAb介导抗脑膜炎奈瑟氏球菌的同源和异源株系的杀菌性应答的能力,进行杀菌性检测。杀菌性抗体滴度表示为能够杀死50%或更多细菌的受试抗体的最高稀释度的倒数,mAb 2G23-12具有高于1∶128的抗同源株系B:4:P1.19,15和高于1∶64的抗异源株系B:15:P1.7,16和C:2a:P1.5的杀菌性滴度。
实施例9
抗NMB0928蛋白的鼠科动物免疫应答的靶区域的表征
为了在蛋白中鉴定被产生的抗重组抗原的鼠科动物抗血清更经常识别的区域,进行SPOTScan测定法。在纤维素膜上合成成套函盖蛋白质序列的重叠肽,用以1∶100稀释的混合血清温育。通过用缀合抗鼠科动物G免疫球蛋白的碱性磷酸酶进行温育,接着通过加入含有底物溴氯吲哚磷酸的溶液来检测抗原-抗体反应。
无论何种用于免疫的制剂,都观察到蛋白内几个共同的抗原性区域。然而,在用弗氏佐剂佐化的蛋白免疫的组中,存在更加广泛的识别模式。
实施例10
通过人血清对NMB0928蛋白的识别
在该通过ELISA法进行的研究中,使用来自康复期的个体的人血清收集物。用通过制备性电泳获得的NMB0928蛋白(5μg/ml)包被板。在用3%于含有Tween-20的PBS中的脱脂奶粉封闭板后,在相同的溶液中稀释(1∶50)血清并在所述板中进行温育。如已广泛报道的进行免疫测定。健康供体血清用作阴性对照。此外,将来自用抗乙型肝炎的重组疫苗接种的个体的混合血清用作非关联对照(数据未显示)。
图12显示在该测定中用5个康复期患者的血清获得的结果。可以看出人血清识别所述蛋白,这表明在脑膜炎球菌感染期间所述蛋白进行表达并且其具有免疫原性。
实施例11
NMB0928蛋白作为肽的载体
为证明重组NMB0928蛋白的载体能力,将其缀合至15聚体(15mer)的合成肽,所述肽来源于来自HIV-1、JY1分离株的gp120蛋白的V3区域。通过戊二醛方法进行缀合。以3次免疫的方案将游离的JY1肽、重组NMB0928蛋白和缀合物JY1-NMB0928给药成年小鼠,其中免疫原用弗式佐剂进行乳化。在第三次免疫后两周,从经免疫的动物获取血清样品,并通过ELISA法分析样品以确定抗肽抗体滴度。为进行该测定,用游离肽(20μg/ml)包被板并继续进行免疫测定,如前面所描述的。实验的结果(图13)显示了NMB0928蛋白的载体能力,即在其缀合后能够显著地加强抗JY1肽的抗体应答。
序列表
<110>Center for Genetic Engineering and Biotechnology
<120>NMB0928蛋白及其在药物制剂中的用途
<130>NMB0928
<140>CU 2003/0286
<141>2003-12-02
<150>CU 2003/0286
<151>2003-12-02
<160>9
<170>PatentIn Ver.2.1
<210>1
<211>25
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸7740
<400>1
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<210>2
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸7741
<400>2
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<210>3
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<213>脑膜炎奈瑟氏球菌(Neisseria meningitidis)
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gcctgctccg gcagcaaaac cgaacagccc aagctcgact accaaagccg gtcgcaccgc 180
ctgatcaaac ttgaagtccc acctgatttg aacaaccccg accaaggcaa cctctaccgc 240
ctgcctgccg gttcgggcgc cgtccgcgcc agcgatttgg aaaaacgccg cacacccgcc 300
gtccaacagc ctgccgatgc cgaagtattg aaaagcgtca aaggtgtccg cctcgagcgc 360
gacggcagcc aacgctggct cgttgtcgac ggcaagtctc ctgccgaaat ctggccgctc 420
ctgaaagcct tttggcagga aaacggcttc gacatcaaat ccgaagaacc cgccatcgga 480
caaatggaaa ccgagtgggc ggaaaaccgc gccaaaatcc cccaagacag cttgcgccgc 540
ctcttcgaca aagtcggctt gggcggcatc tactccaccg gcgagcgcga caaattcatc 600
gtccgtatcg aacagggcaa aaacggcgtt tccgacatct tcttcgccca caaagccatg 660
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cccaacctcg aagccgcttt cctgacgcgc tttatgcaat atttgggcgt tgacggacag 780
caggcggaaa acgcatcggc aaaaaaacct acccttcccg ccgccaacga aatggcgcgt 840
atcgaaggca aaagcctgat tgtctttggc gactacggca gaaactggcg gcgcaccgtg 900
ctcgccctcg accgcatcgg gctgaccgtc gtcggtcaaa acaccgaacg ccacgccttc 960
ctggttcaaa aagccccgaa cgaaagcaat gcagttaccg aacaaaaacc cggcctgttc 1020
aaacgcctgc tgggcaaagg caaagcggag aaacctgccg aacagccgga actgattgtc 1080
tatgcagaac ctgtcgccaa cggctcgcgc atcgtcctgc tcaacaaaga cggcagcgca 1140
tatgccggca aagacgcatc cgcattattg ggcaaactcc attccgaact gcgttaa 1197
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Ser His Arg Leu Ile Lys Leu Glu Val Pro Pro Asp Leu Asn Asn Pro
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Asp Gln Gly Asn Leu Tyr Arg Leu pro Ala Gly Ser Gly Ala Val Arg
35 40 45
Ala Ser Asp Leu Glu Lys Arg Arg Thr Pro Ala Val Gln Gln Pro Ala
50 55 60
Asp Ala Glu Val Leu Lys Ser Val Lys Gly Val Arg Leu Glu Arg Asp
65 70 75 80
Gly Ser Gln Arg Trp Leu Val Val Asp Gly Lys Ser pro Ala Glu Ile
85 90 95
Trp Pro Leu Leu Lys Ala phe Trp Gln Glu Asn Gly Phe Asp Ile Lys
100 105 110
Ser Glu Glu Pro Ala Ile Gly Gln Met Glu Thr Glu Trp Ala Glu Asn
115 120 125
Arg Ala Lys Ile Pro Gln Asp Ser Leu Arg Arg Leu Phe Asp Lys Val
130 135 140
Gly Leu Gly Gly Ile Tyr Ser Thr Gly Glu Arg Asp Lys Phe Ile Val
145 150 155 160
Arg Ile Glu Gln Gly Lys Asn Gly Val Ser Asp Ile Phe Phe Ala His
165 170 175
Lys Ala Met Lys Glu Val Tyr Gly Gly Lys Asp Lys Asp Thr Thr Val
180 185 190
Trp Gln Pro Ser Pro Ser Asp Pro Asn Leu Glu Ala Ala Phe Leu Thr
195 200 205
Arg Phe Met Gln Tyr Leu Gly Val Asp Gly Gln Gln Ala Glu Asn Ala
210 215 220
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Asn Thr Glu Arg His Ala Phe Leu Val Gln Lys Ala Pro Asn Glu Ser
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Asn Ala Val Thr Glu Gln Lys Pro Gly Leu Phe Lys Arg Leu Leu Gly
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Lys Gly Lys Ala Glu Lys Pro Ala Glu Gln Pro Glu Leu Ile Val Tyr
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Ala Glu Pro Val Ala Asn Gly Ser Arg Ile Val Leu Leu Asn Lys Asp
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Gly Ser Ala Tyr Ala Gly Lys Asp Ala Ser Ala Leu Leu Gly Lys Leu
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His Ser Glu Leu Arg
355
<210>5
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ggttcgggcg ccgtccgcgc cagcaatttg gaaaaacgcc gcacacccac cgtccaacag 180
cctgccgatg ccgaagtatt gaaaagcgtc aaaggtgtcc gcctcgagcg cgacggcagc 240
caacgctggc tcgttgtcga cggcaagtct cctgccgaaa tctggccgct cctgaaagcc 300
ttttggcagg aaaacggctt cgacatcaaa tccgaagaac ccgccatcgg acaaaaggaa 360
accgagtggg cggaaaaccg cgccaaaatc ccccaagaca gcttgcgccg cctcttcgac 420
aaagtcggct tgggcggcat ctactccacc ggcgagcgcg acaaattcat cgtccgtatc 480
gaacagggca aaaacggcgt ttccgacatc ttcttcgccc acaaagccat gaaagaagtg 540
tacggcggca aagacaaaga cacgaccgta tggcagccct ccccgtccga tcccaacctc 600
gaagccgctt tcctgacgcg ctttatgcaa tatttgggcg ttgacggaca gcaggcggaa 660
aacgcatcgg caaaaaaacc tacccttccc gccgccaacg aaatggcgcg tatcgaaagc 720
aaaagcctga ttgtctttgg cgactacggc agaaactggc ggcgcaccgt gctcgccctc 780
gaccgcatcg ggctgaccgt cgtcggtcaa aacaccgaac gccacgcctt cctggctcaa 840
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<210>6
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<400>6
ggcagcaaaa ccgaacagcc caagctcgac taccaaagcc ggtcgcaccg cctgatcaaa 60
cttgaagtcc cacctgattt gaacaacccc gaccaaggca acctctaccg cctgcctgcc 120
ggttcgggcg ccgtccgcgc cagcgatttg gaaaaacgcc gcacacccac cgtccaacag 180
cctgccgatg ccgaagtatt gaaaagcgtc aaaggtgtcc gcctcgagcg cgacggcagc 240
caacgctggc tcgttgtcga cggcaagtct cctgccgaaa tctggccgct cctgaaagcc 300
ttttggcagg aaaacggctt cgacatcaaa tccgaagaac ccgccatcgg acaaaaggaa 360
accgagtggg cggaaaaccg cgccaaaatc ccccaagaca gcttgcgccg cctcttcgac 420
aaagtcggct tgggcggcat ctactccacc ggcgagcgcg acaaattcat cgtccgtatc 480
gaacagggca aaaacggcgt ttccgacatc ttcttcgccc acaaagccat gaaagaagtg 540
tacggcggca aagacaaaga cacgaccgta tggcagccct ccccgtccga tcccaacctc 600
gaagccgctt tcctgacgcg ctttatgcaa tatttgggcg ttgacggaca gcaggcggaa 660
aacgcatcgg caaaaaaacc tacccttccc gccgccaacg aaatggcgcg tatcgaaagc 720
aaaagcctga ttgtctttgg cgactacggc agaaactggc ggcgcaccgt gctcgccctc 780
gaccgcatcg ggctgaccgt cgtcggtcaa aacaccgaac gccacgcctt cctggctcaa 840
aaagccccga acgaaagcaa tgcagttacc gaacaaaaac ccggcctgtt caaacgcctg 900
ctgggcaaag gcaaagcgga gaaacctgcc gaacagccgg aactgattgt ctatgcagaa 960
cctgtcgcca acgcctcgcg catcgtcctg ctcaacaaag acggcagcgc atatgccggc 1020
aaagacgcat ccgcattatt gggcaaactc cattccga 1058
<210>7
<211>1058
<212>DNA
<213>脑膜炎奈瑟氏球菌
<400>7
ggcagcaaaa ccgaacagcc caagctcgac taccaaagcc ggtcgcaccg cctgatcaaa 60
cttgaagtcc cacctgattt gaacaacccc gaccaaggca acctctaccg cctgcctgcc 120
ggttcgggcg ccgtccgcgc cagcaatttg gaaaaacgcc gcacacccac cgtccaacag 180
cctgccgatg ccgaagtatt gaaaagcgtc aaaggtgtcc gcctcgagcg cgacggcagc 240
caacgctggc tcgttgtcga cggcaagtct cctgccgaaa tctggccgct cctgaaagcc 300
ttttggcagg aaaacggctt cgacatcaaa tccgaagaac ccgccatcgg acaaaaggaa 360
accgagtggg cggaaaaccg cgccaaaatc ccccaagaca gcttgcgccg cctcttcgac 420
aaagtcggct tgggcggcat ctactccacc ggcgagcgcg acaaattcat cgtccgtatc 480
gaacagggca aaaacggcgt ttccgacatc ttcttcgccc acaaagccat gaaagaagtg 540
tacggcggca aagacaaaga cacgaccgta tggcagccct ccccgtccga tcccaacctc 600
gaagccgctt tcctgacgcg ctttatgcaa tatttgggcg ttgacggaca gcaggcggaa 660
aacgcatcgg caaaaaaacc tacccttccc gccgccaacg aaatggcgcg tatcgaaagc 720
aaaagcctga ttgtctttgg cgactacggc agaaactggc ggcgcaccgt gctcgccctc 780
gaccgcatcg ggctgaccgt cgtcggtcaa aacaccgaac gccacgcctt cctggctcaa 840
aaagccccga acgaaagcaa tgcagttacc gaacaaaaac ccggcctgtt caaacgcctg 900
ctgggcaaag gcaaagcgga gaaacctgcc gaacagccgg aactgattgt ctatgcagaa 960
cctgtcgcca acgcgtcgcg catcgtcctg ctcaacaaag acggcagcgc atatgccggc 1020
aaagacgcat ccgcattatt gggcaaactc cattccga 1058
<210>8
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸1573
<400>8
ttccatggta gataaaagaa tggctttag 29
<210>9
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸6795
<400>9
aactgcaggc ttgtaaaccg ttttgtg 27
Claims (31)
1.称为NMB0928的脑膜炎奈瑟氏球菌(N.meningitidis)的蛋白,其特征在于其是能够在受体生物中产生抗感染的保护性应答的抗原和具有示于序列表中Seq.ID.No.4的氨基酸序列,所述感染是由来自奈瑟氏球菌属的细菌引起的。
2.权利要求1的称为NMB0928的蛋白,其特征在于其由示于序列表中Seq.ID.No.3的NMB0928基因编码。
3.权利要求2的NMB0928基因,其特征在于具有示于序列表中Seq.ID.No.3的碱基序列和编码权利要求1的蛋白。
4.通过重组技术或化学合成获得的蛋白或肽,其特征在于具有NMB0928蛋白的序列并能够在受体生物中产生抗感染的保护性应答,所述感染由来自权利要求1的奈瑟氏球菌属细菌引起。
5.药物制剂,其特征在于含有权利要求1、2和4的蛋白或肽或根据权利要求1,2和4通过天然方式产生的权利要求1的蛋白。
6.权利要求5的药物制剂,其特征在于其是能够在受体生物体中产生抗感染的保护性应答的疫苗,所述感染是由来自奈瑟氏球菌属的细菌引起的。
7.权利要求5和6的药物制剂,其特征在于其是能够在受体生物体中产生抗感染的保护性应答的疫苗,所述感染是由脑膜炎奈瑟氏球菌引起的。
8.权利要求5和6的药物制剂,其特征在于其是能够在受体生物体中产生抗感染的保护性应答的疫苗,所述感染是由淋病奈瑟氏球菌(Neisseria gonorrhoeae)引起的。
9.权利要求5、6、7和8的药物制剂,其特征在于其是预防性或治疗性制剂。
10.权利要求5、6、7和8的药物制剂,其特征在于其是含有一种或几种不同抗原特征的抗原的组合制剂,所述抗原是通过重组方式、合成方式获得的或通过天然方式产生的。
11.权利要求5、6、7和8的药物制剂,其特征在于含有多糖抗原。
12.权利要求5、6、7、8和9的药物制剂,其特征在于制剂成分之一是脑膜炎奈瑟氏球菌的荚膜多糖。
13.权利要求9的药物制剂,其特征在于含有一种多糖蛋白缀合物,所述多糖部分对应于细菌的多糖。
14.权利要求5、6、7和8的药物制剂,其特征在于含有一种或几种经灭活的微生物。
15.权利要求5、6、7和8的药物制剂,其特征在于含有肽抗原。
16.权利要求5和6的药物制剂,其特征在于含有激素。
17.权利要求5和6的药物制剂,其特征在于含有生长因子。
18.权利要求5至17的药物制剂,其特征在于其是经肠胃外途径给药的制剂。
19.权利要求5至17的药物制剂,其特征在于其是经粘膜途径给药的制剂。
20.权利要求5至17的药物制剂,其特征在于其是经口途径给药制剂。
21.权利要求5至20的药物制剂,其特征在于其是免疫刺激剂或免疫强化剂制剂。
22.权利要求5至21的药物制剂,其特征在于其含有NMB0928抗原的肽或片段。
23.权利要求5至21的药物制剂,其特征在于其含有NMB0928抗原的模拟表位。
24.经基因修饰的生物,其特征在于含有单独的或包含于另外的基因序列中的权利要求3的基因或其部分。
25.权利要求24的药物制剂,其特征在于含有经基因修饰的活的、减毒的生物或其制剂。
26.药物制剂,其特征在于含有由权利要求24的生物表达的蛋白,并且能够在受体生物中产生抗感染的保护性应答,所述感染由来自奈瑟氏球菌属的细菌引起。
27.药物制剂,其特征在于含有权利要求1、2和4的、作为不同特征抗原的载体的蛋白或肽。
28.药物组分,其特征在于含有权利要求1和2的NMB0928蛋白或其片段,并且能够单独地或在其他组分存在的情况下使得可以在人中检测脑膜炎球菌病。
29.药物组分,其特征在于含有权利要求3的基因或其片段,并且能够单独地或在其他组分存在的情况下使得可以在人中检测脑膜炎球菌病。
30.权利要求1和2的NMB0928蛋白或其片段在生物传感器或其他药物或生物技术应用中的用途。
31.权利要求3的NMB0928基因或其片段在生物传感器或其他药物或生物技术应用中的用途。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CU2003/0286 | 2003-12-03 | ||
CU2002008620030286A CU23236A1 (es) | 2003-12-03 | 2003-12-03 | PROTEINA NMB0928 Y SU USO EN FORMULACIONES FARMACéUTICAS P |
Publications (2)
Publication Number | Publication Date |
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CN1890261A true CN1890261A (zh) | 2007-01-03 |
CN100549027C CN100549027C (zh) | 2009-10-14 |
Family
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CNB2004800358798A Expired - Fee Related CN100549027C (zh) | 2003-12-03 | 2004-12-02 | Nmb0928蛋白及其在药物制剂中的用途 |
Country Status (16)
Country | Link |
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US (1) | US20070128230A1 (zh) |
EP (1) | EP1693379B1 (zh) |
KR (1) | KR20060124625A (zh) |
CN (1) | CN100549027C (zh) |
AR (1) | AR046937A1 (zh) |
AT (1) | ATE456573T1 (zh) |
AU (1) | AU2004294377A1 (zh) |
BR (1) | BRPI0417334A (zh) |
CA (1) | CA2547537A1 (zh) |
CU (1) | CU23236A1 (zh) |
DE (1) | DE602004025377D1 (zh) |
NO (1) | NO20063017L (zh) |
NZ (1) | NZ547521A (zh) |
RU (1) | RU2335505C2 (zh) |
WO (1) | WO2005054282A1 (zh) |
ZA (1) | ZA200604492B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US11147866B2 (en) | 2016-09-02 | 2021-10-19 | Sanofi Pasteur Inc. | Neisseria meningitidis vaccine |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CU23549A1 (es) * | 2005-12-29 | 2010-07-20 | Ct Ingenieria Genetica Biotech | Composiciones farmacéuticas que contienen la proteína nma0939 |
CU23572A1 (es) * | 2006-03-31 | 2010-09-30 | Ct Ingenieria Genetica Biotech | Composición farmacéutica que comprende la proteína nmb0938 |
CU23575A1 (es) * | 2006-03-31 | 2010-09-30 | Ct Ingenieria Genetica Biotech | Composición farmacéutica que comprende la proteína nmb0606 |
Family Cites Families (5)
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DE4219821A1 (de) * | 1992-06-17 | 1993-12-23 | Boehringer Mannheim Gmbh | Spezifischer Nachweis von Neisseria Gonorrhoeae |
CU22559A1 (es) * | 1996-01-17 | 1999-05-03 | Ct Ingenieria Genetica Biotech | Sistema de expresión de antígenos heterologos en e. coli como proteínas de fusión |
NZ508366A (en) * | 1998-05-01 | 2004-03-26 | Chiron Corp | Neisseria meningitidis antigens and compositions |
MXPA02004283A (es) * | 1999-10-29 | 2002-10-17 | Chiron Spa | Peptidos antigenicos neisseriales. |
GB9928196D0 (en) * | 1999-11-29 | 2000-01-26 | Chiron Spa | Combinations of B, C and other antigens |
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2003
- 2003-12-03 CU CU2002008620030286A patent/CU23236A1/es unknown
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- 2004-12-01 AR ARP040104482A patent/AR046937A1/es unknown
- 2004-12-02 DE DE602004025377T patent/DE602004025377D1/de active Active
- 2004-12-02 NZ NZ547521A patent/NZ547521A/xx unknown
- 2004-12-02 WO PCT/CU2004/000016 patent/WO2005054282A1/es active Application Filing
- 2004-12-02 AU AU2004294377A patent/AU2004294377A1/en not_active Abandoned
- 2004-12-02 CN CNB2004800358798A patent/CN100549027C/zh not_active Expired - Fee Related
- 2004-12-02 AT AT04802608T patent/ATE456573T1/de not_active IP Right Cessation
- 2004-12-02 US US10/580,888 patent/US20070128230A1/en not_active Abandoned
- 2004-12-02 KR KR1020067011597A patent/KR20060124625A/ko not_active Application Discontinuation
- 2004-12-02 BR BRPI0417334-1A patent/BRPI0417334A/pt not_active IP Right Cessation
- 2004-12-02 RU RU2006123414/13A patent/RU2335505C2/ru not_active IP Right Cessation
- 2004-12-02 EP EP04802608A patent/EP1693379B1/en active Active
- 2004-12-02 CA CA002547537A patent/CA2547537A1/en not_active Abandoned
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11147866B2 (en) | 2016-09-02 | 2021-10-19 | Sanofi Pasteur Inc. | Neisseria meningitidis vaccine |
US11707514B2 (en) | 2016-09-02 | 2023-07-25 | Sanofi Pasteur Inc. | Neisseria meningitidis vaccine |
Also Published As
Publication number | Publication date |
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WO2005054282A1 (es) | 2005-06-16 |
AU2004294377A1 (en) | 2005-06-16 |
RU2006123414A (ru) | 2008-01-10 |
CN100549027C (zh) | 2009-10-14 |
ZA200604492B (en) | 2007-05-30 |
ATE456573T1 (de) | 2010-02-15 |
US20070128230A1 (en) | 2007-06-07 |
CU23236A1 (es) | 2007-09-26 |
NO20063017L (no) | 2006-09-01 |
AR046937A1 (es) | 2006-01-04 |
EP1693379B1 (en) | 2010-01-27 |
NZ547521A (en) | 2009-07-31 |
RU2335505C2 (ru) | 2008-10-10 |
BRPI0417334A (pt) | 2007-03-27 |
DE602004025377D1 (de) | 2010-03-18 |
KR20060124625A (ko) | 2006-12-05 |
CA2547537A1 (en) | 2005-06-16 |
EP1693379A1 (en) | 2006-08-23 |
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