CN1249781A - 登革病毒的前m/m蛋白的表位、合成肽 - Google Patents
登革病毒的前m/m蛋白的表位、合成肽 Download PDFInfo
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Abstract
本发明涉及5种登革2型病毒前M/M蛋白的合成肽,相应于3-31,45-67,57-92,69—93及103-124位氨基酸序列。在小鼠体内评价了抗肽免疫应答。也获得了包括前M/M蛋白区域的重组融合蛋白。证实在前M/M蛋白的肽中存在人及小鼠体内B细胞表位。肽3-31和103—124激发抗四种登革病毒血清型的中和抗体。病毒特异性增殖应答在用非缀合肽3-31及57-92免疫的小鼠体内证实。用缀合肽3-31,57-92及69-93免疫的小鼠当其被登革2型病毒攻击时处于被保护状态。因此,登革2型病毒前M/M蛋白质中顺序表位的存在,及其在抗黄病毒的免疫应答中的关联被证实。
Description
技术领域
本发明属于生物工程学领域,并涉及重组DNA技术,尤其涉及编码登革病毒血清型2的前M/M蛋白的合成肽及含有登革病毒血清型2和4的前M/M蛋白表位的嵌合蛋白的生产。
本技术目的是鉴别与所有登革病毒血清型交叉反应的前M/M中和及保护性表位,以获得用于人体免疫接种的免疫原。
现有技术
登革病毒属于黄病毒科,黄病毒属(Westaway,E.G.et al,1985,黄病毒科,Intervirol.24 p.183),其是一种具有一正极性单RNA链作为遗传物质的包膜病毒,该遗传物质编码通过细胞及病毒蛋白酶在共转导及转导后加工的多蛋白。
在病毒膜内有两种结构蛋白:E(包膜)和M(膜),还有一些其它结构蛋白:形成等轴核衣壳的C(衣壳)。另外已鉴定出至少7种非结构蛋白(NS1,NS2a,NS2b,NS3,NS4a,NS4b,NS5)。
糖蛋白E及NS1能独立地提供抗登革病毒同源血清型的主动及被动保护,而相关表位的高度构象复杂性仍被保持。由此重组真核细胞系已被主要选出作为这些蛋白质的免疫评价体系,如痘苗病毒(Bray,M.et.al.1989,用表达带或不带非结构蛋白NS1的登革-4结构蛋白的重组痘苗病毒免疫的小鼠被保护不患致命的登革病毒脑炎,J.Virol,63 p2853)及杆状病毒(Zhang.Y.M.et al.1988,用杆状病毒重组子表达的登革结构蛋白和非结构蛋白NS1免疫小鼠诱导对登革病毒脑炎的抗性,J.Virol.62 p3027)。
小蛋白M(8kDa)以名为前M(大约22kDa)的糖基化的前体合成,其在感染细胞释放病毒之前或之后被晚期内切蛋白裂解(Murray,J.M.et al.1993,登革病毒2型蛋白prM和C-prM的加工,J.Gen.Virol.74 p175)。大概由细胞蛋白酶进行的裂解似乎发生在后高尔基体酸性小泡内,被使此小泡低pH去稳定的制剂抑制(Randolph,v.B.et al.1990,嗜酸性胺抑制黄病毒prM蛋白的蛋白裂解加工,Virol.174 p450)。前片段在体外只在细胞外介质中发现,其在体内的去处仍是未知数(Murray,J.M.et al.1993,登革病毒2型蛋白prM和C-prM的加工,J.Gen.Virol.74 p.175)。据认为在黄病毒胞吐期间,前M/M的功能是避免E的融合膜区域被环境的酸性PH活化(Randolph.V.B.et al.1990,嗜酸性胺抑制黄病毒prM蛋白的蛋白裂解加工,Virol 174 p.450);如果该事件发生,则病毒的释放将被阻止。事实上已确定前M与E在未成熟胞内病毒颗粒内相互作用(Wengler,G.y Wengler,G.1989,细胞相关的西尼罗黄病毒由E+前M蛋白异二聚体包被,该异二聚体在病毒释放期间由蛋白裂解破坏和重构,J.Virol.63 p.2521),且E的天然构象只在前M存在下可获得(Konishi,E.y Mason,P.W.1993,日本脑炎病毒包膜糖蛋白的正确成熟需要与前膜蛋白共合成,J.Virol.67 p.1672)。另外已经释放的在其膜内只有前M的病毒颗粒通常呈现出比完全成熟的病毒颗粒低的感染性(Wengler,G.y Wengler,G.1989,细胞相关的西尼罗黄病毒由E+前M蛋白异二聚体包被,该异二聚体在病毒释放期间由蛋白裂解破坏和重构,J.Virol.63 p.2521),其中尽管存在M及前M,但前者是主要的。
当前M及M在重组痘苗病毒中被表达时,其提供一主动保护作用,但不是与前片段一起发生(Brag,M.y Lai,C.-J.1991.登革病毒前膜和膜蛋白激发保护性免疫应答,Virol.185 p.5057),另外,前M或M与糖蛋白E组合在相同重组痘苗病毒中提供的保护水平一般高于那些由每个蛋白单独所达水平。相似地,在鼠体内抗前M/M的某些抗体能被动地保护(Kaufman,B.M.et al,1989.登革病毒prM糖蛋白的单克隆抗体保护小鼠免受致死性登革感染,Am J.Trop.Med.&Hyg.41 p.576)
合成肽的应用使得能证实根据空间构象的抗原性的分子基础及所涉及抗原的免疫学性质[Arnon,R.y Sda,-M.1985.合成疫苗:现状及未来,Ann.Inst.Pasteur/Immunol 136 D.271-282]。作为抗登革病毒疫苗亚单位的合成肽使得在最终配制品中只包括不引起免疫扩增的保护表位(Halstead,S.B.y O’Ruourke,E.J.1977.登革病毒和单核巨噬细胞,I.非中和抗体的感染促进作用,J.Exp.Med.14 p.201;Halstead,S.B.1979,被动传递的抗体对猕猴中登革感染的体内促进作用,J.Infect.Dis.140 p.527),或包括四种血清型的保护性肽。E和NS1的抗原决定簇的鉴定已成功进行。但对同样重要的前M/M蛋白没有类似的研究,这也是本文的结果是这一方向的第一步的原因。
在E.coli中表达黄病毒蛋白前M、M及E的尝试还未尽善尽美(Chambers,T.J.et al.1990,黄热病毒蛋白在感染细胞中的产生:不连续的多蛋白种类的鉴定及使用区域特异性多克隆抗血清对裂解动力学的分析,Virol.177 p.159;Yan,B.-S.et al.1994,截短潜在的膜结合区克服了在大肠杆菌中表达丙型肝炎病毒蛋白E1的困难,J.Virol.Meths 49 p.343)。显而易见地,这些蛋白质在C-末端的疏水区域是导致低的或不可测异源表达水平的原因(Yah,B.-S.et al.1994.截短潜在的膜结合区克服了在大肠杆菌中表达丙型肝炎病毒蛋白E1的困难,J.Virol.Meths.49 p-343)。
那些蛋白质(以及NS1)在E.coli中的表达一般已经通过与其它细菌蛋白如β-半乳糖苷酶(Cane,P.A.y Gould,E.A.1988,通过用细菌合成的非结构蛋白(NS1)片段免疫降低黄热病小鼠神经毒力,J.Gen.Virol.69 p.1241)、TRPE(Megret,F.et al.1992.利用重组融合蛋白和单克隆抗体确定登革病毒包膜糖蛋白上的线性和不连续抗原位点,Virol.187 p.480)及金黄色葡萄球菌的A蛋白(Murray,J.M.etal.1993.登革病毒2型蛋白prM和C-prM的加工,J.Gen.Virol.74p.175)融合而获得。在这些融合蛋白中,大多数相关构象表位缺失,这是因为尽管产生的抗病毒的抗血清能识别全病毒,但它们不能中和它也不能抑制其凝血性质(Megret,F.et al.1992.利用重组融合蛋白和单克隆抗体确定登革病毒包膜糖蛋白上的线性和不连续抗原位点,Virol.187 p.480)。但最近报导显示:融合蛋白的可溶性及作为结果应用非变性方法将其纯化可保护大多数其具有的中和表位(Seif,S.A.et al.1995.在重组大肠杆菌系统中表达的日本脑炎病毒E蛋白的C-末端上的中和表位的更精细定位,Vaccine 13 p.1515)及保护性表位(Srivastowa.A.K.et al.1995.用在大肠杆菌中制备的登革病毒2型E和NS1融合蛋白免疫的小鼠被保护不受致死性登革病毒感染Vaccine 13 p.1251)。
在前M/M情况下,其前功能域有6个参与3个二硫键的半胱氨酸,及在天冬酰胺69内的N-糖基化位点。E和NS1的结构甚至更复杂,其包含6个二硫键及几个N-糖基化位点。但M的小胞外域明显没有那些构象复杂性,因为其没有半胱氨酸,且其天然构型无糖基化。
异源片段在拓扑结构较多或较少了解的免疫原性蛋白质允许区域内的插入及这些融合物的免疫作用是合成肽应用的互补选择。两种策略都能确定顺序B细胞及T细胞表位的存在。这些表位的生物学重要性能通过实验评价以判定在某些疫苗制备物中是否可包括它们。
发明详述
来自登革2型病毒前M/M蛋白、包括58%氨基酸序列(97/166AA)的5种肽被化学合成。它们是3-31;45-67;57-92;69-93及103-124,其被依次称为B19-6;B20-2;B19-5;B20-1;B20-3。
将与载体蛋白缀合或未缀合的肽接种在Balb/c小鼠体内。用缀合肽免疫后所得血清经降低噬斑数的体外中和及ELISA测试。我们还研究了在免疫小鼠体内抗登革病毒2型攻击的主动保护作用。
在用非缀合肽免疫的小鼠体内,抗登革2型病毒的抗体应答经ELISA评价,且T淋巴细胞的增殖反应也同时评价。
还获得了融合蛋白,且由肽(1-42及92-133)所覆盖的4个区域中的2个被插入其中,并在E.coli中表达。用这些融合物的免疫接种将互补用合成肽所得结果。
在小鼠及人中B细胞表位的存在被证实,是由于经ELISA肽被免疫的小鼠的抗体及具有登革病毒的临床及血清诊断的病人血清识别。肽19-6和20-3能诱导抗四种登革病毒血清型的中和抗体的产生。
病毒特异性增殖应答在经非缀合肽19-6及19-5免疫的小鼠体内证实。用缀合肽19-6、20-1及19-5免疫的小鼠示出当它们经登革2型病毒攻击时具有统计学上有效的保护水平。
这样在登革2型病毒前M/M蛋白中顺序表位的存在及其在抗这些黄病毒的免疫应答中的相关性被证实。
实施例1登革病毒前M/M蛋白的抗原区及T细胞表位的预测
不同理论方法用于预测D2病毒前M/M蛋白中的抗原区。这些区域是那些可能被所得抗病毒蛋白抗体所识别及产生识别初始蛋白的抗体的区域。一些预测T细胞表位的方法被应用。
五种具有可能的B-及T细胞表位(4个在前M,1个在M)的初始肽被发现,对这些蛋白质的抗原结构的研究及可能具有免疫学重要性的肽的实验检测的研究基于此发现。
1.1人抗原性的预测
用于预测抗原性的方法基于氨基酸序列,是由于登革病毒前M/M蛋白的三维结构未经实验测定,且与已知三维结构蛋白在序列水平没有明显类似。
1981年在古巴分离的登革2型病毒A15毒株(Kouri G.et al.1986.古巴的出血性登革病流行史,Bull.P.A.H.O 20 p.24)用于完成本实施例。根据以下标准选择潜在抗原区:
a)根据不同预测方法,基于亲水性(Hoop,T.P.y Woods.K.R.1981.从氨基酸序列预测蛋白抗原决定簇,Proc.Natl.Acad.Sci USA 78 p.3824;Parker,J.M.R.et al.1986.从HPLC肽保留数据得出的新亲水性:预测的表面残基与抗原性和X-射线衍生的可及位点的相关性,Biochemistry 25 p.5425),柔性(Karplus,P.A.y Schultz,G.E.1985.蛋白质中的链柔性预测,选择肽抗原的工具,Naturwissenschaften 72p.212)及可及性(Emini,E.A.et al.1985.由病毒特异性合成肽诱导甲型肝炎病毒中和抗体,J.Virol 55 p.836)的高抗原倾向的区域。
b)根据使用PHD的二级结构的预测的具有形成环及转角的高可能性的区域(Rost,B.y Sander,C.1993.蛋白质二级结构预测精度高于70%,J.Mol.Biol 232 p.584;Rost,B.y Sander,C.1994.结合进化信息和神经网络预测蛋白质二级结构,Proteins 19 p.55;Rost,B.y Sander,C.1994.蛋白质家族的溶剂可及性的保守及预测,Proteins 20 p.216)。
c)包括或不包括就其它黄病毒而言的插入/抑制的高变异性区域,及在登革病毒中应用或未用的其它黄病毒中糖基化的潜在区域。
a)抗原性分布图
图1示出当赋予前M和M区段4种与抗原性相关的氨基酸特性时所得分布图。
在前区域内在具有6-9,16-21,28-31,42-47,58-65及82-91残基的区域具有高亲水性及可及性值。出乎意料的在相应于跨膜螺旋的41-76残基间巨大疏水区的存在被认为未暴露于免疫系统。在M的小胞外域(残基1-40),主要亲水性/可及性区域在13-31氨基酸之间,尤其在其起始处(AA13-16)。
b)二级结构的预测
图2示出根据PHD程序的二级结构的预测及前M及M区段的可及性。预测结果示出许多潜在抗原区(根据图1的分布图)倾向于与在蛋白质表面的暴露残基形成环/β-转角。预测在M蛋白41-76位氨基酸之间区域形成跨膜螺旋,这与此区域疏水性相吻合,并提示M蛋白的抗原性肽主要在胞外域(1-40)。
c)登革病毒及其它黄病毒前M及M蛋白的序列对比,变异性及糖基化对比
通常未暴露于溶剂的区域在同源蛋白质家族内具有较大保守性,因而较高变异性的区域具有较高被暴露的概率。
对于病毒,变异性也是免疫学压力一种逃避机制;当然不能排除一些保守区域也许是抗原性的或在表面可能有保守区域。
对4种血清型登革病毒的15个分离株的前M及M区的序列分析示出:至少69%的残基是严格保守的。较重要的可变残基在前M的28-30,55-59,69-72及80-83位,及在M的27-30位。通常这些区带与图1的抗原性分布图的绝大部分相吻合。
在多于30个黄病毒分离物中这些区域的序列的比较示出前M的1-33区是高变异性的,并带有倾向于插入/抑制(在8和30位)的可能存在的环及N-糖基化的一些潜在位点。相反,在前M的33-91区域是低变异性的;有一些部位在所有黄病毒中都是严格保守的,如形成3个二硫键的6个半胱氨酸,在40-65区域的至少5个酸性残基,及87-91碱性序列,在此之后在成熟病毒释放之前或期间内发生蛋白酶裂解(图3)。
抗原性登革复合物中保守的残基Asn-69具有复合物前M/M蛋白的唯一N-糖基化。但在黄病毒家族,该区域位于高变异性的可能暴露的环内。同时,与其它黄病毒内潜在N-糖基化位点(如JE,SLE,MVE,YF中AA14及LI,LAN,YF,TBE中AA32)相吻合的登革病毒前M/M残基是靠近被认为是抗原性区的β-转角。
1.2T细胞表位的预测
经两种独立方法进行预测:Rothbard及Taylor模式方法(Rothbard,J.B.y Taylor,W.R.1988.T细胞表位中共有的序列模式,EMBO.J.7.P.93)及有形成α-螺旋结构[AMPHI 7及11]倾向的片段的确定(Margalit,H.et al.1987.从初级序列预测免疫优势辅助T细胞抗原位点,J.Immunol.138 p.2213)。结果示于图4。
1.3为相关表位的鉴别而预计的肽
中性及保护性肽的确定通常对更有效疫苗的研制是非常重要的,且来自高抗原性倾向区域的肽对其鉴别非常有用,尤其那些线型性质的肽。
表1示出一系列包含倾向于具有D2病毒前M/M蛋白的B及T细胞表位(根据一些用于此实施例的预测方法)的区域的肽。如果该预测的正确性经实验证实,则每个区域的免疫学重要表位将经过设计每个区域中的肽而准确排列。
表1.登革病毒前M/M蛋白中的预计的抗原性肽
代码 | 序列 | 区域 |
B19-6 | LTTRNGEPHMIVMRQEKGKSLLFKTGDGV | 3-31 |
B20-2 | CEDTITYKCPLLRQNEPEDIDCW | 45-67 |
B19-5 | RQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRS | 57-92 |
B20-1 | NSTSTWVTYGTCTTTGEHRREKRSV | 69-93 |
B20-3 | LETRTETWMSSEGAWKHAQRIE | 103-124 |
实施例2.寡肽及寡核苷酸的化学合成
2.1寡肽的合成
所有的肽都用Boc-策略以固相在p-甲基二苯甲基胺树脂(resinMBHA,BACHEM,瑞士)上合成。
保护性氨基酸经BACHEM提供,氨基酸链反应基团的保护基是:Arg(Tos),ASP(OBzl),Cys(4-Me-Bzl),Glu(OBzl),Lys(2-Cl-Z),Trp(CHO),Tyr(Cl2-Bzl)。所用的Asn,Gln及Pro在侧链中没有保护基。
Boc-氨基酸保护基的去掉是用在二氯甲烷中的37.5%三氟乙酸进行的。二异丙基碳二亚胺(DIC)的原位活化用于每个残基的偶联反应,Asn,Gln除外,这两个氨基酸用DIC及在H,N-二甲基甲酰胺中的1-羟基苯并三唑活化。
树脂的最终脱保护及肽释放在特殊设备中进行,所用步骤已知为Low-High HF。
在步骤的第一部分(Low HF),保护性的树脂系统用HF(25%):DMS(65%):p-甲酚(10%)在0℃处理120分钟,在含Trp肽情况下,将该混合物用HF(25%):DMS(60%):EDT(10%):p-甲酚(5%)替换。然后将树脂-肽用二乙基乙醚、二氯甲烷及2-丙酮洗数次,并真空干燥。
在步骤的第二部分(High HF),将树脂-肽用HF(90%):苯甲醚(10%),在0℃处理60分钟。
将粗产物用乙醚洗,然后用30%在水中的乙酸萃取,最后冻干。
将肽在BAKER C-18(4.6×100mm)柱中经BP-HPLC,并在JEOL HX-110HF设备中以FAB作为电离方法经质谱测定加以鉴定。
登革病毒前M/M蛋白中氨基酸序列及其定位示于表1。
2.2寡核苷酸的合成
根据亚磷酰胺方法在Gene Assembler Plus设备中自动合成寡核苷酸。
6种寡核苷酸的序列示于表2。
表2:在每个末端内产生的便于后期操作的XbaI及EcoRI位点分别以下划线及双下划线示出,修饰的蛋白质读框由碱基三联体表示。
实施例3:肽与载体蛋白偶联及免疫方案3.1肽与BSA的偶联肽的偶联以如下方式进行:
寡核苷酸位置 | 核苷酸序列 |
1)5′pre DEN-2 | 5’-TTT CTA GAT TTC CAT TTA ACC ACA CGT T-3’ |
2)3′pre DEN-2 | 5’-T TTC TAG ACC AAG GTC CAT GGC CAT GAG-3’ |
3)5′M DEN-2 | 5’-TTT CTA GAA TCA GTG GCA CTC GTT CCA CAT G-3’ |
4)3′M DEN-2 | 5’-T TTC TAG AAA GCC TGG ATG TCT CAA GAT CCA-3’ |
5)5′M DEN-4 | 5’-TTT CTA GAT TCA GTA GCT TTA ACA CCA C-3’ |
6)3′M DEN-4 | 5’-T TGA ATT CGC GAA TCT TGG GTT TCT GAG-3’ |
1.BSA的活化:边摇动边将以5μg/μl在二甲基甲酰胺中的80μl双功能试剂间-马来酰亚胺苯甲酰基-N-羟基琥珀酰亚胺酯(MBS)滴加入2.8mg牛白蛋白组分V(BSA)在250μl PBS中的溶解物中。然后将其在室温保持振荡30分钟,并将混合物经过PD10柱。
2.肽与活化的BSA偶联:将1mg肽溶于300μl PBS中的溶解物边摇动边滴加入活化的BSA溶液中。将其在室温保持3小时,并用Lowry方法检测浓度。
3.2免疫方案:
与BSA连接的肽的免疫方案如下:
将4-6周龄的雄性Balb/c小鼠用50μg肽-BSA缀合物经腹膜内免疫。另外,进行两种免疫方案,一种用BSA而另一种用PBS。共进行4次接种,每次相隔15天。在第一次剂量中使用弗氏完全佐剂,而其它次用弗氏不完全佐剂。在最后一次接种7天后从后眶静脉取血样。
将每个方案所得血清置于-20℃至后期使用。
实施例4体外蚀斑减少中和试验
根据Morens所述进行中和技术(Morens,D.M.et al.1985.在BHK-21细胞中经半微量方法进行的登革病毒的简化的蚀斑减少中和测定:BHK悬浮试验与标准蚀斑减少中和的比较。J.Clin.Microbiol.22p.250)。
制备抗肽血清和抗BSA对照物及阴性血清的1/10至1/640的稀释液。将每个血清的稀释液与具有15-20PFU/50μl的病毒(登革2型病毒A15株)稀释液相接触。
将混合物在37℃温育1小时。将共50μl的每种混合物分成三份加入24孔培养盘中的BHK-21细胞中,然后在37℃在CO2温育器中温育4小时。接着加入0.5ml含羧甲基纤维素的培养基,并考虑使用的病毒血清型再温育几天。在这些天之后,进行染色及计数由病毒产生的裂解蚀斑。
在每种情况下效价以可获得50%蚀斑数的减少的稀释度表示。结果示于表3。
表3抗19-6及20-3的抗肽血清的PRNT
抗肽 | 每种血清的中和效价 | |||
D1 | D2 | D3 | D4 | |
B19-6 | 1/100 | 1/180 | 1/60 | 1/160 |
B20-3 | 1/110 | 1/80 | 1/80 | 1/80 |
实施例5 T细胞表位的鉴定
在前M的肽中T细胞表位的存在可通过对游离肽(非缀合肽)免疫小鼠体内引起的抗肽抗体应答的研究加以评价。当与首次用来实验的动物体内应答相比,接触过抗原的动物在对加强剂量抗原的应答中证实产生较高血清抗体。这些结果证实在这些肽中存在B细胞表位,并示出这些序列含有T细胞表位,其能体内刺激Th活性以增进抗体应答效价。
脾T淋巴细胞的病毒特异性增殖应答在肽免疫的BALB/C小鼠中被证实。来自19-6及19-5免疫小鼠的T细胞当其与登革2型病毒培养时,在体外母细胞化发育分析中增殖。但20-2肽不能引起抗病毒的有效增殖应答。其可能含有T细胞隐蔽表位,在肽游离形式中被识别,但不似在天然感染中优势免疫表位呈递及病毒加工的结果。(图5)
实施例6 保护性分析
将用1/2500稀释度(相当于100LD50致死量)活的小鼠适应登革2型病毒(A15株)经颅内注射最后一次免疫7天后的小鼠进行实验。对小鼠观测21天以上,观测其发病率及死亡率。用Fisher’s检验测统计学显著性。肽免疫的动物及对照动物的存活百分率示于图6,由肽19-5,19-6及20-1引起的保护水平为统计学显著的(p<0.05)。
实施例7 间接ELISA检测抗肽抗体
人血清:
将肽19-6,20-1,20-2,20-3以10μg/ml在包被缓冲液中的浓度固定于培养板中,然后在4℃温育过夜。加入用PBS-Tween稀释200倍的血清。最后加入人/过氧化物酶全抗免疫球蛋白缀合物,接着加入底物(邻苯二胺,H2O2,0.05M磷酸盐柠檬酸缓冲液,PH5)。在ELISA读数器中在492nm进行读数,并测定每种肽的截断值。
所用血清来自经用抗登革全抗体的血凝抑制技术(Clarke,D.H.yCasals,J.1958,节肢动物携带的病毒的血凝及血凝抑制技术,Am.T.Trop Med.Hyg.7,p.561)及抑制作用的ELISA(Vazquez,S.,Femandez,R.1989,Utilizacion de un metodo de ELISA de Inhibicion enel diagnostico serologico de dengue,Rev.Cub.Med.Trop.41(1)p18-26)经血清学诊断为登革病毒临床感染的患者。
该研究包括在古巴1981年,巴拿马1994年及哥斯达黎加1994年发生的流行的118例患者血清。登革2型病毒从这些病例中分离出,另外血清型1和4在哥斯达黎加分离出;它们根据在首次及二次感染情况下抑制血凝抗体的效价来分类。
46.6%的血清对所用的4种肽是阳性的。获得对肽B19-6,20-1,20-2及20-3的阳性率分别为56.8%,79.6%,77.1%及83.1%。
经样本/截断值的光密度系数计算的每种肽的反应性平均指数是1.07,1.52,1.57及1.49。
小鼠血清:
如上所述使用间接ELISA,但使用与过氧化物酶缀合的抗小鼠Ig。抗肽血清中所得抗体效价通常为1/10000以上。
实施例8前M/M片段在脑膜炎奈瑟氏球菌P64k蛋白质中的插入
在此实施例中,登革2型病毒(A15株)及登革4型(814669株)的前M/M蛋白的表达片段(Zhao,B.et al.1986.全长登革4型病毒DNA序列的克隆:编码结构蛋白的基因的分析,Virol.155 p.77)插入在我们已预先鉴定的脑膜炎奈瑟氏球菌蛋白质P64k中(Silva,R.et al.1992.编码脑膜炎奈瑟氏球菌的外膜蛋白的核苷酸序列及所述蛋白在疫苗制备物中的应用,欧洲专利0 474 313,1997),其已被证实在一些动物模型中具有高免疫原性。此外,P64k在E.coli中的表达水平达到细菌总蛋白30%以上。
二聚体性质的P64k蛋白(64kDa)在每个亚单位中有两个功能区:一个具有结合脂肪酸活性(1-100),另一个具有硫辛酰胺脱氢酶活性(117-594)。二者均经X射线晶体学被鉴定为相对独立的构象区(Li de La Sierra,I.et al.1994.来自脑膜炎奈瑟氏球菌的重组外膜蛋白的晶体学及初步X-射线研究,J.Mol.Biol.235 P.1154;Li dela Sierra,I.et al.1997.来自脑膜炎奈瑟氏球菌的表面抗原的硫辛酰胺脱氢酶区域的分子结构,J.Mol.Biol 269 p.129)。
选择前者(在45位氨基酸内)进行前M/M的1-42及92-133片段的插入,这是由于此小区域更暴露且似乎不参与二聚体形成。这提示针对天然P64k,如果插入位点在117-594位区域,嵌合蛋白的球状结构变化较小,另外,其直接参与二聚体的形成。
将编码氨基酸44-53(TLETDKATMD)的区域,其包括与用于融合蛋白生产的P64k基因的结合脂肪酸区域,初步改变为TLDLEMD。进行该修饰以避免P64k被原发肝硬化患者血清识别,这种患者具有抗在线粒体脱氢硫辛酰胺乙酰基转移酶中存在的同源表位的自身抗体(Tuaillon,N.et al.1992.由原发肝硬化中抗M2自身抗体特异识别的二氢硫辛酰胺乙酰转移酶的硫辛酰合成十八肽,J.Immunol.148 p.445)。
以下阐述了产生两个克隆的策略:
将Pre-2,M-2及M-4片段经PCR扩增,分别使用寡核苷酸1和2,3和4及5和6组合(见表2),并用pD-5质粒做模板。此pD-5质粒包括一克隆入pBluescript载体(Stratagene)中的登革2型病毒(A15株)前M/M基因的拷贝。将在每种情况下所得DNA条带(120bp)用XbaI(Pre-2及M-2)或XbaI/EcoR I(M-4)消化,并将其克隆入在克隆入载体pM-92中的P64k基因的135-145位人工产生的相应位点。此外,在已提及的XbaI及EcoR I位点中含有M-2和M-4条带的嵌合克隆经三重连接反应产生。具有正确方向插入子的重组克隆经限制性分析及DNA测序加以鉴定。
将由Pre-2(pD31),M-2(pD30),M-2/M-4(pD33)及M-4(pD34)的克隆产生的融合蛋白在E.coli MM294(Fˉend Al hsdR17(rk -mk +)sup E44 thi-1 relAl?RfbDl?SpoTl?)菌株中在色氨酸操纵子的启动子(ptrp)控制下表达。所有都获得预期大小,并且表达水平在细菌总蛋白质的30%以上,但PD31蛋白质显示高度不稳定性(图7)。所有融合蛋白在ELISA(数据未显示)及Western印迹(图8)中均被抗P64k的一些鼠单克隆抗体识别,其中检测到在全细胞提取物中明显的降解。这些蛋白质的氨基酸序列示于序列表。
用经非变性手段半纯化的PD33及PD34融合蛋白免疫小鼠在ELISA中具有抗其的高效价(高达1/100000),并同时获得在ELISA中抗合成肽的效价高达1/4000的抗体。
附图描述
图1:登革病毒前M(A)和M(B)蛋白的亲水性,可及性及柔性分布图。
图2:前M(A)及M(蛋白质)的二级结构及可及性预测。AA:氨基酸。PHD sec:二级结构预测(E=β,H=螺旋,L=环)。P-3 acc:可及性预测(e=暴露,b=未暴露)。Sub sec(Sub acc):二级结构预测(可及性)灵验性为82.4%(70%)的残基。
图3:前M及M蛋白的变异性分布图。变异性考虑3套黄病毒序列而被计算出。Dengue:15个登革分离物的序列。MTV:由蚊子转递的黄病毒包括登革病毒、Kunji,西尼罗病毒,墨累山谷脑炎病毒及圣路易斯脑炎病毒。黄病毒:30个以上不同黄病毒分离物的序列:(MTV+黄热病毒,Langat,跳跃病病毒及蜱传脑炎病毒)。
图4:前M(A)及M(B)蛋白质T细胞表位的预测:AMPHI7(11):7(11)个残基的两亲区段预测,阳性残基是两亲性区段潜在抗原性的中心氨基酸。RT4(5):4(5)个残基抗原性分布图预测,阳性残基是那些符合分布图的残基。
图5:肽免疫小鼠19-6,19-5及20-2的脾T细胞对登革2型病毒抗原的增殖应答(在10,20及40μg/ml浓度)。
图6:肽免疫的和对照动物中的存活百分率。由肽19-5,19-6及20-1诱导的保护水平是统计学显著的。
图7:用融合蛋白及P64k蛋白(pM-92质粒)转化的MM294E.coli菌株的10%SDS-PAGE。泳道:1-未转化的MM294菌株,2-pM-92/MM294,3-pD-30/MM294,4-pD-31/MM294,5-pD-33/MM294,6-pD-34/MM294。
图8:应用融合蛋白及P64k蛋白(pM-92质粒)转化的MM294E.coli菌株的ACM114的Western印迹。泳道:1-未转化的MM294菌株,2-pM-92/MM294,3-pD-30/MM294,4-pD-31/MM294,5-pD-33/MM294,6-pD-34/MM294。
序列表(1)一般信息:
(i)申请人:
(A)名称:遗传和生物技术工程中心
(B)街道:Ave.31 entre 158 y 190,Playa
(C)城市:哈瓦那
(E)国家:古巴
(F)邮政编码(ZIP):10600
(G)电话:53 7 218466
(H)传真:53 7 218070/336008
(A)名称:佩德罗库利热带医学研究所
(B)街道:Autopista Novia del Mediodia Km 6,La Lisa
(C)城市:哈瓦那
(E)国家:古巴
(F)邮政编码(ZIP):11100
(G)电话:53 7 220633
(H)传真:53 7 335061
(ii)发明名称:登革病毒的前M/M蛋白表位,合成肽及其应用
(iii)序列数:9
(iv)计算机阅读形式:
(A)媒介类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,Version#1.30(EPO)
(v)在先申请资料:
(A)申请号:CU 13/97
(B)申请日:1997年1月15日(2)SEQ ID NO:1的信息:
(i)序列特征:
(A)长度:29个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:肽
(iii)假设:是
(iv)反义:否
(v)片段类型:内部(vi)原始来源:
(A)生物体:登革病毒
(B)毒株:登革-2
(C)具体分离物:A-15(xi)序列描述:SEQ ID NO:1Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val Met Arg Gln Glu1 5 10 15Lys Gly Lys Ser Leu Leu Phe Lys Thr Gly Asp Gly Val
20 25(2)SEQ ID NO:2的信息:
(i)序列特征:
(A)长度:23个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:肽
(iii)假设:是
(iv)反义:否
(v)片段类型:内部
(vi)原始来源:
(A)生物体:登革病毒
(B)毒株:登革-2
(C)具体分离物:A-15
(xi)序列描述:SEQ ID NO:2Cys Glu Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn Glu1 5 10 15Pro Glu Asp Ile Asp Cys Trp
20(2)SEQ ID NO:3的信息:
(i)序列特征:
(A)长度:36个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:肽
(iii)假设:是
(iv)反义:否
(v)片段类型:内部
(vi)原始来源:
(A)生物体:登革病毒
(B)毒株:登革-2
(C)具体分离物:A-15
(xi)序列描述:SEQ ID NO:3Arg Gln Asn Glu Pro Glu Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser1 5 10 15Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Thr Gly Glu His Arg Arg
20 25 30Glu Lys Arg Ser
35(2)SEQ ID NO:4的信息:
(i)序列特征:
(A)长度:25个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:肽
(iii)假设:是
(iv)反义:否
(v)片段类型:内部
(vi)原始来源:
(A)生物体:登革病毒
(B)毒株:登革-2
(C)具体分离物:A-15
(xi)序列描述:SEQ ID NO:4Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Thr Gly1 5 10 15Glu His Arg Arg Glu Lys Arg Ser Val
20 25(2)SEQ ID NO:5的信息:
(i)序列特征:
(A)长度:22个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:肽
(iii)假设:是
(iv)反义:否
(v)片段类型:内部
(vi)原始来源:
(A)生物体:登革病毒
(B)毒株:登革-2
(C)具体分离物:A-15
(xi)序列描述:SEQ ID NO:5Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys1 5 10 15His Ala Gln Arg Ile Glu
20(2)SEQ ID NO:6的信息:
(i)序列特征:
(A)长度:635个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:是
(iv)反义:否
(vi)原始来源:
(A)生物体:融合蛋白
(vii)现有来源:
(B)克隆:PD31
(xi)序列描述:SEQ ID NO:6Met Ala Leu Val Glu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn1 5 10 15Val Asp Ile Ile Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val
20 25 30Asp Asp Thr Leu Ile Thr Leu Asp Leu Asp Phe His Leu Thr Thr Arg
35 40 45Asn Gly Glu Pro His Met Ile Val Ser Arg Gln Glu Lys Gly Lys Ser
50 55 60Leu Leu Phe Lys Thr Gly Asp Gly Val Asn Met Cys Thr Leu Met Ala65 70 75 80Met Asp Leu Gly Leu Glu Met Asp Val Pro Ala Glu Val Ala Gly Val
85 90 95Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly
100 105 110Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala
115 120 125Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro
130 135 140Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp145 150 155 160Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala
165 170 175Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr
180 185 190Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu
195 200 205Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn
210 215 220Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala225 230 235 240Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met
245 250 255Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu
260 265 270Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu
275 280 285Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys
290 295 300Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu305 310 315 320Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val
325 330 335Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met
340 345 350Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met
355 360 365Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp
370 375 380Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys385 390 395 400Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu
405 410 415Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val
420 425 430Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala
435 440 445Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met
450 455 460Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln465 470 475 480Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu
485 490 495Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly
500 505 510Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu
513 520 525Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp
530 535 540Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr545 550 555 560Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile
565 570 575Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile
580 585 590Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His
595 600 605Pro Thr Leu Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly
610 615 620Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys625 630 635(2)SEQ ID NO:7的信息:
(i)序列特征:
(A)长度:635个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:是
(iv)反义:否
(vi)原始来源:
(A)生物体:融合蛋白
(vii)现有来源:
(B)克隆:PD30
(xi)序列描述:SEQ ID NO:7Met Ala Leu Val Glu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn1 5 10 15Val Asp Ile Ile Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val
20 25 30Asp Asp Thr Leu Ile Thr Leu Asp Leu Glu Ser Val Ala Leu Val Pro
35 40 45His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser
50 55 60Glu Gly Ala Trp Lys His Ala Gln Arg Ile Glu Thr Trp Ile Leu Arg65 70 75 80His Pro Gly Phe Leu Glu Met Asp Val Pro Ala Glu Val Ala Gly Val
85 90 95Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly
100 105 110Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala
115 120 125Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro
130 135 140Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp145 150 155 160Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala
165 170 175Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr
180 185 190Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu
195 200 205Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn
210 215 220Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala225 230 235 240Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met
245 250 255Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu
260 265 270Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu
275 280 285Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys
290 295 300Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu305 310 315 320Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val
325 330 335Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met
340 345 350Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met
355 360 365Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp
370 375 380Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys385 390 395 400Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu
405 410 415Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val
420 425 430Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala
435 440 445Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met
450 455 460Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln465 470 475 480Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu
485 490 495Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly
500 505 510Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu
515 520 525Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp
530 535 540Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr545 550 555 560Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile
565 570 575Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile
580 585 590Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His
595 600 605Pro Thr Leu Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly
610 615 620Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys625 630 635(2)SEQ ID NO:8的信息:
(i)序列特征:
(A)长度:677个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:是
(iv)反义:否
(vi)原始来源:
(A)生物体:融合蛋白
(vii)现有来源:
(B)克隆:PD34
(xi)序列描述:SEQ ID NO:8Met Ala Leu Val Glu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn1 5 10 15Val Asp Ile Ile Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val
20 25 30Asp Asp Thr Leu Ile Thr Leu Asp Leu Asp Phe His Leu Thr Thr Arg
35 40 45Asn Gly Glu Pro His Met Ile Val Ser Arg Gln Glu Lys Gly Lys Ser
50 55 60Leu Leu Phe Lys Thr Gly Asp Gly Val Asn Met Cys Thr Leu Met Ala65 70 75 80Met Asp Leu Gly Ser Val Ala Leu Val Pro His Val Gly Met Gly Leu
85 90 95Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His
100 105 110Ala Gln Arg Ile Glu Thr Trp Ile Leu Arg His Pro Gly Phe Leu Glu
115 120 125Met Asp Val Pro Ala Glu Val Ala Gly Val Val Lys Glu Val Lys Val
130 135 140Lys Val Gly Asp Lys Ile Ser Glu Gly Gly Leu Ile Val Val Val Glu145 150 155 160Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala Glu Ala Ala Ala Ala Pro
165 170 175Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro Ala Pro Gln Ala Ala Gln
180 185 190Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp Val Val Val Leu Gly Gly
195 200 205Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala Ala Ala Asp Glu Gly Leu
210 215 220Lys Val Ala Ile Val Glu Arg Tyr Lys Thr Leu Gly Gly Val Cys Leu225 230 235 240Asn Val Gly Cys Ile Pro Ser Lys Ala Leu Leu His Asn Ala Ala Val
245 250 255Ile Asp Glu Val Arg His Leu Ala Ala Asn Gly Ile Lys Tyr Pro Glu
260 265 270Pro Glu Leu Asp Ile Asp Met Leu Arg Ala Tyr Lys Asp Gly Val Val
275 280 285Ser Arg Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Ser Arg Lys Val
290 295 300Asp Val Ile Gln Gly Asp Gly Gln Phe Leu Asp Pro His His Leu Glu305 310 315 320Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu Gln Ala Ala Pro Thr Gly
325 330 335Glu Lys Lys Ile Val Ala Phe Lys Asn Cys Ile Ile Ala Ala Gly Ser
340 345 350Arg Val Thr Lys Leu Pro Phe Ile Pro Glu Asp Pro Arg Ile Ile Asp
355 360 365Ser Ser Gly Ala Leu Ala Leu Lys Glu Val Pro Gly Lys Leu Leu Ile
370 375 380Ile Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr Val Tyr Ser Thr385 390 395 400Leu Gly Ser Arg Leu Asp Val Val Glu Met Met Asp Gly Leu Met Gln
405 410 415Gly Ala Asp Arg Asp Leu Val Lys Val Trp Gln Lys Gln Asn Glu Tyr
420 425 430Arg Phe Asp Asn Ile Met Val Asn Thr Lys Thr Val Ala Val Glu Pro
435 440 445Lys Glu Asp Gly Val Tyr Val Thr Phe Glu Gly Ala Asn Ala Pro Lys
450 455 460Glu Pro Gln Arg Tyr Asp Ala Val Leu Val Ala Ala Gly Arg Ala Pro465 470 475 480Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala Gly Val Ala Val Thr Asp
485 490 495Arg Gly Phe Ile Glu Val Asp Lys Gln Met Arg Thr Asn Val Pro His
500 505 510Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln Pro Met Leu Ala His Lys
515 520 525Ala Val His Glu Gly His Val Ala Ala Glu Asn Cys Ala Gly His Lys
530 535 540Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly Val Ala Tyr Thr Ser Pro545 550 555 560Glu Val Ala Trp Val Gly Glu Thr Glu Leu Ser Ala Lys Ala Ser Gly
565 570 575Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp Ala Ala Ser Gly Arg Ala
580 585 590Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr Lys Leu Ile Phe Asp Ala
595 600 605Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile Val Gly Pro Asn Gly Gly
610 615 620Asp Met Ile Gly Glu Val Cys Leu Ala Ile Glu Met Gly Cys Asp Ala625 630 635 640Ala Asp Ile Gly Lys Thr Ile His Pro His Pro Thr Leu Gly Glu Ser
645 650 655Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys Thr Asp Leu Pro
660 665 670Pro Gln Lys Lys Lys
675(2)SEQ ID NO:9的信息:
(i)序列特征:
(A)长度:635个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:是
(iv)反义:否
(vi)原始来源:
(A)生物体:融合蛋白
(vii)现有来源:
(B)克隆:PD33
(xi)序列描述:SEQ ID NO:9Met Ala Leu Val Glu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn1 5 10 15Val Asp Ile Ile Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val
20 25 30Asp Asp Thr Leu Ile Thr Leu Asp Leu Glu Ser Val Ala Leu Thr Pro
35 40 45His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met Ser Ser
50 55 60Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile Leu Arg65 70 75 80Asn Pro Arg Phe Leu Glu Met Asp Val Pro Ala Glu Val Ala Gly Val
85 90 95Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly
100 105 110Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala
115 120 125Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro
130 135 140Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp145 150 155 160Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala
165 170 175Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr
180 185 190Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu
195 200 205Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn
210 215 220Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala225 230 235 240Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met
245 250 255Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu
260 265 270Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu
275 280 285Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys
290 295 300Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu305 310 315 320Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val
325 330 335Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met
340 345 350Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met
355 360 365Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp
370 375 380Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys385 390 395 400Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu
405 410 415Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val
420 425 430Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala
435 440 445Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met
450 455 460Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln465 470 475 480Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu
485 490 495Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly
500 505 510Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu
515 520 525Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp
530 535 540Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr545 550 555 560Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile
565 570 575Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile
580 585 590Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His
595 600 605Pro Thr Leu Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly
610 615 620Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys625 630 635
Claims (11)
1、属于登革病毒前M/M蛋白的合成肽或模拟化合物,特征在于包括3-31,57-92,69-93及103-124位氨基酸区域,且包括至少一种与任何登革病毒血清型交叉反应的表位。
2、如权利要求1的包括3-31,57-92,69-93及103-124位氨基酸区域的属于登革病毒前M/M蛋白的合成肽,特征在于是序列表中分别以肽19-6,19-5,20-1及20-3表示的肽。
3、抗黄病毒的诊断试验或药物配制品,特征在于包括如权利要求1或2所述的肽或其一部分,与或不与蛋白质或其它载体缀合,不依赖于所使用的佐剂或赋形剂。
4、黄病毒特异性抗体或其部分,特征在于其识别如权利要求1或2提及的序列。
5、抗黄病毒疫苗或治疗制备物,或诊断性试验,特征在于包括权利要求4所述抗体或其部分。
6、特征在于包含登革2和4型病毒前M/M蛋白表位的遗传构建物,包含与载体蛋白融合的1-42和92-134位氨基酸区域。
7、如权利要求6的遗传构建物,特征在于其与脑膜炎奈瑟氏球菌P64k蛋白融合,在序列表中描述为PD31(Pre-2),PD30(M-2),PD34(M-4)及PD33(M-2/M-4)。
8、如权利要求6或7的遗传构建物,特征在于含有至少一个黄病毒前M/M蛋白表位。
9、诊断性试验或药物配制品,特征在于含有至少一个如权利要求6、7或8的遗传构建物的产物或其部分。
10、抗体或其部分,特征在于可识别权利要求6或7提及的序列。
11、抗黄病毒的药物配制品或诊断试验,特征在于含有权利要求10所述的抗体或其部分。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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CU1997013A CU22683A1 (es) | 1997-01-15 | 1997-01-15 | Epítopes de la proteína pre-m/m del virus del dengue, péptidos sintéticos, proteínas quiméricas y sus usos |
CU13/1997 | 1997-01-15 | ||
CU13/97 | 1997-01-15 |
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CN1249781A true CN1249781A (zh) | 2000-04-05 |
CN1198933C CN1198933C (zh) | 2005-04-27 |
Family
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CNB988032031A Expired - Fee Related CN1198933C (zh) | 1997-01-15 | 1998-01-13 | 登革病毒的前m/m蛋白的表位、合成肽 |
Country Status (15)
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US (1) | US6383488B1 (zh) |
EP (1) | EP0966535B1 (zh) |
JP (1) | JP3647048B2 (zh) |
CN (1) | CN1198933C (zh) |
AT (1) | ATE284444T1 (zh) |
AU (1) | AU743414B2 (zh) |
BR (1) | BR9807486A (zh) |
CA (1) | CA2277816A1 (zh) |
CU (1) | CU22683A1 (zh) |
DE (1) | DE69828032T2 (zh) |
ES (1) | ES2235310T3 (zh) |
HK (1) | HK1025999A1 (zh) |
MY (1) | MY120971A (zh) |
NO (1) | NO993468L (zh) |
WO (1) | WO1998031814A1 (zh) |
Cited By (5)
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CN102409063A (zh) * | 2001-12-04 | 2012-04-11 | 巴法里安诺迪克有限公司 | 黄病毒ns1亚单位疫苗 |
WO2018077208A1 (en) * | 2016-10-27 | 2018-05-03 | The Mighty Ocean International Co., Ltd. | Dengue virus-like particle, antibody against dengue virus, and composition comprising the same |
CN108610416A (zh) * | 2008-10-13 | 2018-10-02 | 生物医学研究所 | 登革热病毒中和抗体及其用途 |
CN110734500A (zh) * | 2013-06-26 | 2020-01-31 | 北卡罗来纳-查佩尔山大学 | 用于登革病毒疫苗的方法与组合物 |
CN116082499A (zh) * | 2021-11-08 | 2023-05-09 | 东莞市朋志生物科技有限公司 | 一种抗登革ns1蛋白的抗体及其应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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CU23245A1 (es) | 2001-07-16 | 2007-10-17 | Inst De Medicina Tropical Pedr | CADENAS QUIMéRICAS CODIFICANTES PARA PROTEINAS INDUCTORAS DE EFECTOS CONTRA VIRUS. PREPARADOS UTILIZANDO PROTEINAS QUIMéRICAS |
EP1454988A1 (en) * | 2003-03-03 | 2004-09-08 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Infectious flavivirus pseudo-particles containing functional prM-E envelope proteins |
US7281274B2 (en) * | 2003-10-16 | 2007-10-09 | Lmp Media Llc | Electronic media distribution system |
CA2576798C (en) * | 2004-07-27 | 2014-09-09 | Gwong-Jen J. Chang | Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes and methods for their use |
CU23578A1 (es) * | 2005-09-16 | 2010-09-30 | Ct Ingenieria Genetica Biotech | Proteína de la cápsida del virus dengue inductora de respuesta protectora y composición vacunal |
CU23630A1 (es) | 2006-10-30 | 2011-02-24 | Ct Ingenieria Genetica Biotech | Moléculas peptídicas quiméricas con propiedades antivirales contra los virus de la familia flaviviridae |
WO2009024534A2 (en) * | 2007-08-17 | 2009-02-26 | Intercell Ag | Japanese encephalitis virus (jev) and tick-borne encephalitis virus (tbev) peptides stimulating human t cell responses |
CU20080028A6 (es) * | 2008-02-29 | 2011-02-24 | Ct Ingenieria Genetica Biotech | Compuestos químicos obtenidos in silico para la preparación de composiciones farmacéuticas para atenuar o inhibir la infección por virus dengue y otros flavivirus |
CN101921310B (zh) * | 2009-06-15 | 2014-03-12 | 温州医学院 | 登革病毒特异性hla-a2限制性表位肽及应用 |
WO2011133112A1 (en) * | 2010-04-20 | 2011-10-27 | National University Of Singapore | Cell penetrating peptide derived from the premembrane protein of flavivirus |
WO2012159187A2 (en) * | 2011-05-26 | 2012-11-29 | Universidade Federal Do Rio De Janeiro - Ufrj | Denv-derived peptides and methods for the inhibition of the flavivirus replication |
CU24188B1 (es) * | 2012-12-27 | 2016-07-29 | Ct De Ingeniería Genética Y Biotecnología | Composición vacunal contra el virus dengue |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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AU6093296A (en) * | 1995-06-07 | 1996-12-30 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Infectious dengue 2 virus pdk-53 as quadravalent vaccine |
-
1997
- 1997-01-15 CU CU1997013A patent/CU22683A1/es unknown
-
1998
- 1998-01-13 WO PCT/CU1998/000001 patent/WO1998031814A1/es active IP Right Grant
- 1998-01-13 CA CA002277816A patent/CA2277816A1/en not_active Abandoned
- 1998-01-13 AU AU56499/98A patent/AU743414B2/en not_active Ceased
- 1998-01-13 JP JP53348698A patent/JP3647048B2/ja not_active Expired - Fee Related
- 1998-01-13 CN CNB988032031A patent/CN1198933C/zh not_active Expired - Fee Related
- 1998-01-13 EP EP98900843A patent/EP0966535B1/en not_active Expired - Lifetime
- 1998-01-13 BR BR9807486A patent/BR9807486A/pt not_active Application Discontinuation
- 1998-01-13 DE DE69828032T patent/DE69828032T2/de not_active Expired - Fee Related
- 1998-01-13 US US09/341,833 patent/US6383488B1/en not_active Expired - Fee Related
- 1998-01-13 ES ES98900843T patent/ES2235310T3/es not_active Expired - Lifetime
- 1998-01-13 AT AT98900843T patent/ATE284444T1/de not_active IP Right Cessation
- 1998-01-15 MY MYPI98000163A patent/MY120971A/en unknown
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1999
- 1999-07-14 NO NO993468A patent/NO993468L/no not_active Application Discontinuation
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2000
- 2000-08-22 HK HK00105273A patent/HK1025999A1/xx not_active IP Right Cessation
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102409063A (zh) * | 2001-12-04 | 2012-04-11 | 巴法里安诺迪克有限公司 | 黄病毒ns1亚单位疫苗 |
CN108610416A (zh) * | 2008-10-13 | 2018-10-02 | 生物医学研究所 | 登革热病毒中和抗体及其用途 |
CN108610416B (zh) * | 2008-10-13 | 2022-01-14 | 生物医学研究所 | 登革热病毒中和抗体及其用途 |
CN110734500A (zh) * | 2013-06-26 | 2020-01-31 | 北卡罗来纳-查佩尔山大学 | 用于登革病毒疫苗的方法与组合物 |
WO2018077208A1 (en) * | 2016-10-27 | 2018-05-03 | The Mighty Ocean International Co., Ltd. | Dengue virus-like particle, antibody against dengue virus, and composition comprising the same |
CN110050064A (zh) * | 2016-10-27 | 2019-07-23 | 赵黛瑜 | 登革热类病毒颗粒、登革热病毒抗体、及含其的组合物 |
US11046754B2 (en) | 2016-10-27 | 2021-06-29 | Day-Yu Chao | Dengue virus-like particle, antibody against dengue virus, and composition comprising the same |
TWI769185B (zh) * | 2016-10-27 | 2022-07-01 | 海樂源有限公司 | 登革熱類病毒顆粒、抗登革熱病毒抗體、及含其之組合物 |
CN116082499A (zh) * | 2021-11-08 | 2023-05-09 | 东莞市朋志生物科技有限公司 | 一种抗登革ns1蛋白的抗体及其应用 |
CN116082499B (zh) * | 2021-11-08 | 2023-10-27 | 东莞市朋志生物科技有限公司 | 一种抗登革ns1蛋白的抗体及其应用 |
Also Published As
Publication number | Publication date |
---|---|
ES2235310T3 (es) | 2005-07-01 |
EP0966535B1 (en) | 2004-12-08 |
US6383488B1 (en) | 2002-05-07 |
ATE284444T1 (de) | 2004-12-15 |
NO993468L (no) | 1999-09-14 |
BR9807486A (pt) | 2000-03-21 |
EP0966535A1 (en) | 1999-12-29 |
DE69828032T2 (de) | 2005-12-01 |
DE69828032D1 (de) | 2005-01-13 |
CN1198933C (zh) | 2005-04-27 |
JP2001508457A (ja) | 2001-06-26 |
AU5649998A (en) | 1998-08-07 |
MY120971A (en) | 2005-12-30 |
JP3647048B2 (ja) | 2005-05-11 |
AU743414B2 (en) | 2002-01-24 |
HK1025999A1 (en) | 2000-12-01 |
NO993468D0 (no) | 1999-07-14 |
CU22683A1 (es) | 2001-07-20 |
WO1998031814A1 (es) | 1998-07-23 |
CA2277816A1 (en) | 1998-07-23 |
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