CN1148450C - 戊型肝炎巴基斯坦病毒株的重组蛋白及其在诊断方法和疫苗中的用途 - Google Patents
戊型肝炎巴基斯坦病毒株的重组蛋白及其在诊断方法和疫苗中的用途 Download PDFInfo
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Abstract
本发明公开了一种戊型肝炎的ORF2片段,其分子量约55千道尔顿,而且该蛋白具有戊型肝炎病毒的开放阅读框2蛋白的氨基酸112作为其氨基端。该ORF2片段具有很高的免疫原性,可以作为诊断免疫测定的抗原以及作为免疫原或疫苗来防止戊型肝炎的感染。本发明还公开了该非全长ORF2片段的编码序列、载体、宿主细胞、应用以及含有该片段的药物组合物。
Description
本发明是未审定的、1992年9月18日申请的、现已放弃的美国申请No.07/947,263的续展申请。
发明领域
本发明涉及肝炎病毒学领域。更具体地,本发明涉及来自肠传播的、巴基斯坦戊型肝炎病毒株SAR-55的重组蛋白,以及采用这些蛋白的诊断方法和疫苗应用。
发明背景
已经报道,在亚洲、非洲和中美洲有戊型肝炎流行病,一种肠传播的非甲型/非乙型肝炎(Balayan,M.S.(1987),
苏联医学回顾,章节E,病毒学回顾(SovietMedical Reviews,Section E,Virology Reviews),Zhdanov,0-V.M.(编辑),Chur,瑞士:
Harwood学术出版社,2卷,235-261;Purcell,R.G.,等人(1988)Zuckerman,A.J.(编辑),″病毒性肝炎和肝疾病″(Viral Hepatitis and Liver Disease),New York:Alan R.Liss,131-137;Bradley,D.W.(1990),
英国医学公告(British Medical Bulletin),46:442-461;Ticehurst,J.R.(1991)Hollinger,F.B.,Lemon,S.M.,Margolis,H.S.(编辑):″病毒性肝炎和肝疾病″(Viral Hepatitis and LiverDisease),Williams and Wilkins,Baltimore,501-513)。在戊型肝炎病毒(HEV)是地方病的国家中,偶发性的肝炎病例(推测是戊型肝炎)占报道肝炎的90%。需要开发在受感染个体的血清中检测出抗-HEV抗体的血清学测试方法是本领域中广泛意识到的,但是从受感染的人或动物中分泌出的HEV的浓度非常低,这使得不可能将这种HEV用作血清学测试的抗原源,尽管有少数在细胞培养中繁殖HEV的成功报道(Huang,R.T.等人,(1992),
普通病毒学杂志(J.Gen.Virol.),73:1143-1148),但是目前细胞培养不能有效地产生血清测试所需数量的抗原。
最近,世界范围内的为鉴别出与戊型肝炎关联的病毒基因组序列而进行的主要努力,已经导致克隆出数目有限的HEV株的基因组(Tam,A.W.等人(1991),病毒学(Virology),185:120-131;Tsarev,S.A.等人(1992),
美国科学院院报 (Proc.Natl.Acad.Sci.USA),89:559-563;Fry,K.E.等人(1992),
病毒基因 (Virus Genes),6:173-185)。对DNA序列的分析使研究者假设HEV基因组被分成3个开放阅读框(ORF),并且假设这些ORF编码完整的HEV蛋白。
来自缅甸(Myanmar)的HEV株的一部分基因组DNA序列公开于Reyes等人,1990,
科学(science),247:1335-1339。Tam等人(1991)和Reyes等人PCT专利申请WO91/15603(1991年10月17日出版)公开了HEV缅甸株的完整的核苷酸序列和推导的氨基酸序列。这些作者假设,在该毒株的序列中含有3个正向的开放阅读框(ORF)。
Ichikawa等人,1991,
微生物免疫学(Microbiil.Immunol.),35:535-543,公开了用来自HEV感染的猕猴血清筛选λgt11表达文库而分离出长度为240-320核苷酸的一系列克隆。一个克隆所表达的重组蛋白在大肠杆菌中被表达。该融合蛋白是由HEV Myanmar株的ORF-2的3′区域所编码的。
HEV墨西哥株和HEV缅甸株的ORF-2的3′区域中所编码的其他蛋白的表达,在Yarbough等人,1991,
病毒学杂志(J.Virology),65:5790-5797中有描述。这篇文章描述分离出来自HEV的2种cDNA克隆。这些克隆编码ORF-2的3′区域中的蛋白质。这些克隆在大肠杆菌中以融合蛋白形式表达。
Purdy等人,1992,
病毒学档案(Archives of Virology),123:335-349和Favorov等人,1992,
医学病毒学杂志(J.Of Medical Virology),36:246-250,公开了在大肠杆菌中表达来自缅甸株的更大的ORF-2蛋白片段。这些文献以及那些前面讨论的文献都仅公开了用细菌表达体系来表达一部分ORF-2基因。在本发明之前还没有公开全长ORF-2蛋白的成功表达。
将HEV的基因组结构和形态结构与其他病毒进行比较揭示,HEV与杯状病毒最接近。感兴趣的是,杯状病毒的结构蛋白是由其基因组的3′部分所编码(Neil,J.D.等人(1991)
病毒学杂志(J.Virol.),65:5440-5447;和Carter,M.J.等人(1992),
病毒学档案杂志(J.Arch.Virol.),122:223-235)而且尽管没有HEV基因组的3′端部分也编码结构蛋白的直接证据,但是在细菌细胞中3′基因组区域的某些小片段的表达导致产生与抗-HEV血清在ELISA和Western印迹法中反应的蛋白质(Yarborough,等人,(1991);Ichikawa等人(1991);Favorov等人(1992)和Dawson,G.J.等人(1992)
病毒学方法杂志(J.Virol.Meth);38:175-186)。然而ORF-2蛋白作为结构蛋白的功能在本发明之前还没有被证明。
由ORF-2基因片段所编码的小蛋白已经被用于免疫测定以检测动物血清中的抗HEV抗体。在血清学免疫测定中使用细菌表达的小蛋白作为抗原有多种潜在的缺点。首先,在细菌细胞中表达这些小蛋白常导致溶解度问题,并当粗制大肠杆菌裂解液被用作免疫测定中的抗原时会导致病人血清与大肠杆菌蛋白质的非特异性交叉反应(Purdy等人(1992))。其次,由于时间和成本的限制,将Western印迹法作为日常的传染病学中抗-HEV抗体的一线血清测试方法是不实际的。用来自HEV 3′端部分的小肽进行的ELISA仅能从已知的HEV感染病人中检测出41%的阳性。第三,已表明,对于许多病毒,包括小RNA病毒科(Picornaviridae)(它是与杯状病毒科最接近的科),主要的抗原和免疫原表位是高度构象化的(Lemon,S.M.等人(1991),Hollinger,F.B.,Lemon,S.M.,Margolis,H.S.(编辑):“病毒性肝炎和肝疾病”(Viral Hepatitis and Liver Disease),Williams and Wilkins,Baltimore,20-24)。出于这个原因,据信在真核体系中表达编码完整HEV基因的完整ORF,可导致产生可形成HEV-病毒样颗粒的蛋白。与上述的、仅代表HEV结构蛋白的部分的小蛋白相比,这种完整的ORF蛋白,会具有更接近天然衣壳蛋白的免疫学结构。所以,与目前使用的小蛋白相比,这些完整的ORF蛋白可以作为更具代表性的抗原和更有效的免疫原。
发明概述
本发明涉及分离的和基本纯化的人戊型肝炎病毒株SAR-55的制剂。
本发明还涉及分离的和基本纯化的人戊型肝炎病毒株SAR-55基因组RNA的制剂。
本发明还涉及人戊型肝炎病毒株SAR-55的cDNA。
本发明的一个目的是提供能够指导产生重组HEV蛋白的合成的核酸序列,以及等价的天然核酸序列。这种天然的核酸序列可从cDNA或基因组文库中分离出,即从这些文库中鉴别和分离出能指导HEV蛋白合成的基因。出于本申请的目的,核酸序列指RNA、DNA、cDNA或其任何合成的编码蛋白质的变异体。
本发明还涉及一种基于采用衍生自SAR-55 cDNA的引物而选择性扩增戊型肝炎基因片段,从而在生物样品中检测戊型肝炎的方法。
本发明还涉及使用衍生自SAR-55cDNA的单链反义多聚或寡聚核苷酸来抑制戊型肝炎基因的表达。
本发明还涉及分离的和基本纯化的、由SAR-55的HEV基因组编码的或由合成的核酸序列编码的HEV蛋白或其变异体,尤其涉及由至少一个HEV的完整开放阅读框所编码的重组蛋白。
本发明还涉及制备衍生自HEV基因组序列的重组HEV蛋白的方法,它通过克隆核酸和将cDNA插入表达载体,然后在宿主细胞中表达重组蛋白。
本发明还涉及将得到的重组HEV蛋白作为诊断剂和疫苗的用途。
本发明还包括在生物样品中检测对戊型肝炎特异的抗体的方法。这些方法可用于诊断由HEV引起的感染和疾病,还可用于监测疾病的进展情况。这些方法还可用于监测治疗哺乳动物HEV感染和疾病过程中治疗剂的功效。
本发明还涉及用于预防或治疗哺乳动物戊型肝炎的药物组合物。
附图说明
图1是用于表达HEV株SAR-55基因组的完整的ORF-2蛋白质的重组载体。
图2A和2B是十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE),其中用野生型杆状病毒或重组杆状病毒(含有编码ORF-2的基因)感染的昆虫细胞的细胞裂解液或者用考马斯蓝(A)染色,或者用HEV感染的黑猩猩血清进行Western印迹分析(B)。在图2A和2B中,泳道1含有未感染的SF-9细胞的全部细胞裂解液;泳道2含有用野生型杆状病毒感染的细胞的裂解液;泳道3含有用重组的杆状病毒感染的细胞的裂解液;和泳道4含有分子量参照物。
图3A-3A和3B分别显示了30nm和20nm病毒样颗粒的免疫电镜显微图(immunoelectron micrograph,IEM),它们是在重组感染的昆虫细胞中表达ORF-2蛋白而形成的。
图4显示了ELISA结果,其中从含有编码完整ORF-2的基因的昆虫细胞中表达出的重组ORF-2被用作抗原。在猕猴(cynomolgus monkey)用HEV的墨西哥株(Cyno-80A82、Cyno-9A97和Cyno 83)或巴基斯坦株(Cyno-374)接种后的不同时间,测定血清中抗HEV抗体的水平。
图5A-D显示了ELISA结果,其中从含有编码完整ORF-2的基因的昆虫细胞中表达出的重组ORF-2被用作抗原。在接种2只黑猩猩后,测定血清中抗HEV的IgG或IgM水平。
图6A-J显示了ELISA数据的比较,其中将重组的衍生自SAR-55的完整ORF-2蛋白用作抗原对将重组的衍生自HEV面甸株(Genelabs)的部分ORF-2蛋白用作抗原而获得ELISA数据。
图7A-J显示了用SAR-55 HEV的10%排泄物悬浮液的10倍连续稀释液(显示在各图上方的括号中)接种的猕猴中,抗HEV IgG ELISA和丙氨酸氨基转移酶(ALT)值。用于ELISA的重组抗原是:谷胱甘肽S-转移酶(GST);3-2(M),一种3-2表位(Yarbough等人,
(1991)病毒学杂志(J.Virol),65:5790-5797)和GST的融合蛋白;SG3(B),它是ORF-2的327个C末端氨基酸与GST的融合蛋白(Yarbough等人,(1993):Assay Development of diagnostic tests forHepatitis E in″International Symposium on Viral Hepatitis and Liver Disease.Scientific Program and Abstract Volume.″东京:VHFL p.87);和在昆虫细胞中直接表达的55KDa ORF-2产物。
图8A-E显示了用SAR-55HEV的10%排泄物悬浮液的10倍连续稀释液(显示在各图上方的括号中)接种的阳性猕猴中,抗HEV IgM ELISA和丙氨酸氨基转移酶(ALT)值。用于ELISA的重组抗原是:谷胱甘肽S-转移酶(GST);3-2(M),一种3-2表位(Yarbough等人,1991)和GST的融合蛋白;SG3(B),它是ORF-2的327个C末端氨基酸与GST的融合蛋白(Yarbough等人,1993);和在昆虫细胞中直接表达的55KDa ORF-2产物。
图9显示了2%琼脂糖凝胶的溴化乙锭染色结果,其中在凝胶上对SAR-55HEV的10%排泄物悬浮液的10倍连续稀释液(显示在各凝胶泳道上方)抽提物的PCR产物进行分离。PCR产物的预计长度为约640碱基对,在标为(M)的泳道中含有DNA大小的参照物。
图10显示在酵母中表达完整ORF-2蛋白或分子量更低的片段的p PIC9载体。
发明详述
本发明涉及分离的和基本纯化的、来自巴基斯坦的戊型肝炎病毒(hepatitis EVirus,HEV)株SAR-55。本发明还涉及克隆编码HEV蛋白质的病毒基因以及用表达系统表达重组蛋白。更具体地,本发明涉及衍生自SAR-55的开放阅读框(ORF)的克隆和表达。
本发明涉及分离的HEV蛋白质。本发明的HEV蛋白质较佳地是与天然的HEV蛋白质基本同源,最佳地是与天然HEV蛋白质在生物学上等价。如说明书和权利要求书中所用,“生物学上等价”指组份能够形成病毒样颗粒而且有免疫原性。一旦注射入哺乳动物,本发明的HEV蛋白质还可刺激产生保护性的抗体,这些抗体会保护哺乳动物免受野生型HEV的侵袭。如随后的说明书和权利要求书中所用,“基本同源”指与天然HEV蛋白质氨基酸序列的同源程度。与天然HEV蛋白质的同源程度较佳地为超过70%,更佳地超过90%,特别优选的是同源性超过90%的蛋白质。
较佳的HEV蛋白质是那些由ORF基因编码的蛋白质。特别感兴趣的是由HEV的ORF-2基因所编码的蛋白质,最感兴趣的是由HEV SAR-55株ORF-2基因所编码的蛋白质。优选的、由ORF-2基因编码的本发明蛋白质可形成病毒样颗粒。ORF-1、ORF-2和ORF-3蛋白质的氨基酸序列在下面分别示作SEQ ID NO.:1、SEQ ID NO.:2和SEQ ID NO.:3。
(SEQ ID NO.:1)
Met Glu Ala His Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala
1 5 10 15
Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn
20 25 30
Ala Val Val Val Arg Pro Phe Leu Ser His Gln Gln Ile Glu Ile
35 40 45
Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu
50 55 60
Val phe Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu
65 70 75
Glu Leu Tyr Cys Arg Ala Arg Ser Gly Arg Cys Leu Glu Ile Gly
80 85 90
Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Val His Arg
95 100 105
Cys Phe Leu Arg Pro Ala Gly Arg Asp Val Gln Arg Trp Tyr Thr
110 115 120
Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu
125 130 135
Arg Gly Leu Pro Ala Ala Asp Arg Thr Tyr CyS Phe Asp Gly Phe
140 145 150
Ser Gly Cys Asn Phe Pro Ala Glu Thr Gly Ile Ala Leu Tyr Ser
155 160 165
Leu His Asp Met Ser Pro Ser Asp Val Ala Glu Ala Met Phe Arg
170 175 180
His Gly Met Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu
185 190 195
Val Leu Leu Pro Pro Gly Thr Tyr Arg Thr Ala Ser Tyr Leu Leu
200 205 210
Ile His Asp Gly Arg Arg Val Val Val Thr Tyr Glu Gly Asp Thr
215 220 225
Ser Ala Gly Tyr Asn His Asp Val Ser Asn Leu Arg Ser Trp Ile
230 235 240
Arg Thr Thr Lys Val Thr Gly Asp His Pro Leu Val Ile Glu Arg
245 250 255
Val Arg Ala Ile Gly Cys His Phe Val Leu Leu Leu Thr Ala Ala
260 265 270
Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr
275 280 285
Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly Thr Pro Ser
290 295 300
Leu Phe Pro Thr Ser Cys Ser Thr Lys Ser Thr Phe His Ala Val
305 310 315
Pro Ala His Ile Trp Asp Arg Leu Met Leu Phe Gly Ala Thr Leu
320 325 330
Asp Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg
335 340 345
Gly Ile Ser Tyr Lys Val Thr Val Gly Thr Leu Val Ala Asn Glu
350 355 360
Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Val Ile Thr Ala
365 370 375
Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala
380 385 390
Ile Ser Lys Gly Met Arg Arg Leu Glu Arg Glu His Ala Gln Lys
395 400 405
Phe Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg
410 415 420
Asp Tyr Ile Pro Gly Arg Gln Leu Glu Phe Tyr Ala Gln Cys Arg
425 430 435
Arg Trp Leu Ser Ala Gly Phe His Leu Asp Pro Arg Val Leu Val
440 445 450
Phe Asp Glu Ser Ala Pro Cys His Cys Arg Thr Ala Ile Arg Lys
455 460 465
Ala Val Ser Lys Phe Cys Cys Phe Met Lys Trp Leu Gly Gln Glu
470 475 480
Cys Thr Cys Phe Leu Gln Pro Ala Glu Gly Val Val Gly Asp Gln
485 490 495
Gly His Asp Asn Glu Ala Tyr Glu Gly Ser Asp Val Asp Pro Ala
500 505 510
Glu Ser Ala Ile Ser Asp Ile Ser Gly Ser Tyr Val Val Pro Gly
515 520 525
Thr Ala Leu Gln Pro Leu Tyr Gln Ala Leu Asp Leu Pro Ala Glu
530 535 540
Ile Val Ala Arg Ala Gly Arg Leu Thr Ala Thr Val Lys Val Ser
545 550 555
Gln Val Asp Gly Arg Ile Asp Cys Glu Thr Leu Leu Gly Asn Lys
560 565 570
Thr Phe Arg Thr Ser Phe Val Asp Gly Ala Val Leu Glu Thr Ash
575 580 585
Gly Pro Glu Arg His Ash Leu Ser Phe Asp Ala Ser Gln Ser Thr
590 595 600
Met Ala Ala Gly Pro Phe Ser Leu Thr Tyr Ala Ala Ser Ala Ala
605 610 615
Gly Leu Glu Val Arg Tyr Val Ala Ala Gly Leu Asp His Arg Ala
620 625 630
Val Phe Ala Pro Gly Val Ser Pro Arg Ser Ala Pro Gly Glu Val
635 640 645
Thr Ala Phe Cys Ser Ala Leu Tyr Arg Phe Ash Arg Glu Ala Gln
650 655 660
Arg Leu Ser Leu Thr Gly Ash Phe Trp Phe His Pro Glu Gly Leu
665 670 675
Leu Gly Pro Phe Ala Pro Phe Ser Pro Gly His Val Trp Glu Ser
680 685 690
Ala Asn Pro Phe Cys Gly Glu Ser Thr Leu Tyr Thr Arg Thr Trp
695 700 705
Ser Glu Val Asp Ala Val Pro Ser Pro Ala Gln Pro Asp Leu Gly
710 715 720
Phe Thr Ser Glu Pro Ser Ile Pro Ser Arg Ala Ala Thr Pro Thr
725 730 735
Pro Ala Ala Pro Leu Pro Pro Pro Ala Pro Asp Pro Ser Pro Thr
740 745 750
Leu Ser Ala Pro Ala Arg Gly Glu Pro Ala Pro Gly Ala Thr Ala
755 760 765
Arg Ala Pro Ala Ile Thr His Gln Thr Ala Arg His Arg Arg Leu
770 775 780
Leu Phe Thr Tyr Pro Asp Gly Ser Lys Val Phe Ala Gly Ser Leu
785 790 795
Phe Glu Ser Thr Cys Thr Trp Leu Val Asn Ala Ser Asn Val Asp
800 805 810
His Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Tyr
815 820 825
Pro Ala Ser Phe Asp Ala Ala Ser Phe Val Met Arg Asp Gly Ala
830 835 840
Ala Ala Tyr Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala
845 850 855
Pro Asp Tyr Arg Leu Glu His Asn Pro Lys Arg Leu Glu Ala Ala
860 865 870
Tyr Arg Glu Thr Cys Ser Arg Leu Gly Thr Ala Ala Tyr Pro Leu
875 880 885
Leu Gly Thr Gly Ile Tyr Gln Val Pro Ile Gly Pro Ser Phe Asp
890 895 900
Ala Trp Glu Arg Ash His Arg Pro Gly Asp Glu Leu Tyr Leu Pro
905 910 915
Glu Leu Ala Ala Arg Trp Phe Glu Ala Asn Arg Pro Thr Cys Pro
920 925 930
Thr Leu Thr Ile Thr Glu Asp Val Ala Arg Thr Ala Ash Leu Ala
935 940 945
Ile Glu Leu Asp Ser Ala Thr Asp Val Gly Arg Ala Cys Ala Gly
950 955 960
Cys Arg Val Thr Pro Gly Val Val Gln Tyr Gln Phe Thr Ala Gly
965 970 975
Val Pro Gly Ser Gly Lys Ser Arg Ser Ile Thr Gln Ala Asp Val
980 985 990
Asp Val Val Val Val Pro Thr Arg Glu Leu Arg Asn Ala Trp Arg
995 1000 1005
Arg Arg Gly Phe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val
1010 1015 1020
Thr Gln Gly Arg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro
1025 1030 1035
Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ala Thr Val His
1040 1045 1050
Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His
1055 1060 1065
Ala Gly Leu Val Pro Ala Ile Arg Pro Asp Leu Ala Pro Thr Ser
1070 1075 1080
Trp Trp His Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu
1085 1090 1095
Ile Arg Gly Ala Tyr Pro Met Ile Gln Thr Thr Ser Arg Val Leu
1100 1105 1110
Arg Ser Leu Phe Trp Gly Glu Pro Ala Val Gly Gln Lys Leu Val
1115 1120 1125
Phe Thr Gln Ala Ala Lys Ala Ala Asn Pro Gly Ser Val Thr Val
1130 1135 1140
His Glu Ala Gln Gly Ala Thr Tyr Thr Glu Thr Thr Ile Ile Ala
1145 1150 1155
Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala
1160 1165 1170
Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val Ile Ile Asp
1175 1180 1185
Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser Asp Ala Ile Val
1190 1195 1200
Asn Asn Phe Phe Leu Ala Gly Gly Glu Ile Gly His Gln Arg Pro
1205 1210 1215
Ser Val Ile Pro Arg Gly Asn Pro Asp Ala Asn Val Asp Thr Leu
1220 1225 1230
Ala Ala Phe Pro Pro Ser Cys Glu Ile Ser Ala Phe His Glu Leu
1235 1240 1245
Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu
1250 1255 1260
Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Leu Pro Gln
1265 1270 1275
Glu Leu Thr Thr Cys Asp Ser Val Val Thr Phe Glu Leu Thr Asp
1280 1285 1290
Ile Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val
1295 1300 1305
Leu Ser Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr
1310 1315 1320
Asn Ala Ser His Ser Asp Val Arg Asp Ser Leu Ala Arg Phe Ile
1325 1330 1335
Pro Ala Ile Gly Pro Val Gln Val Thr Thr Cys Glu Leu Tyr Glu
1340 1345 1350
Leu Glu Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val
1355 1360 1365
Leu Glu Leu Asp Leu Cys Ser Arg Asp Val Ser Arg Ile Thr Phe
1370 1375 1380
Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala
1385 1390 1395
His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe
1400 1405 1410
Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Ala Ile
1415 1420 1425
Leu Ala Leu Leu Pro Gln Gly Val Phe Tyr Gly Asp Ala Phe Asp
1430 1435 1440
Asp Thr Val Phe Ser Ala Ala Val Ala Ala Ala Lys Ala Ser Met
1445 1450 1455
Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn
1460 1465 1470
Phe Ser Leu Gly Leu Glu Cys Ala Ile Met Glu Glu Cys Gly Met
1475 1480 1485
Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Ile Arg Ser Ala Trp
1490 1495 1500
Ile Leu Gln Ala Pro Lys Glu Ser Leu Arg Gly Phe Trp Lys Lys
1505 1510 1515
His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn
1520 1525 1530
Met Ala Val Ile Thr His Cys Tyr Asp Phe Arg Asp Leu Gln Val
1535 1540 1545
Ala Ala Phe Lys Gly Asp Asp Ser Ile Val Leu Cys Ser Glu Tyr
1550 1555 1560
Arg Gln Ser Pro Gly Ala Ala Val Leu Ile Ala Gly Cys Gly Leu
1565 1570 1575
Lys Leu Lys Val Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val
1580 1585 1590
Val Val Ala Pro Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe
1595 1600 1605
Ala Gly Arg Leu Thr Glu Lys Ash Trp Gly Pro Gly Pro Glu Arg
1610 1615 1620
Ala Glu Gln Leu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu
1625 1630 1635
Thr Asn Val Ala Gln Met Cys Val Asp Val Val Ser Arg Val Tyr
1640 1645 1650
Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Glu Met Leu Gln
1655 1660 1665
Ala Val Ala Asp Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro
1670 1675 1680
Val Leu Asp Leu Thr Asn Ser Ile Leu Cys Arg Val Glu
1685 1690
(SEQ ID NO:2)
Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro
1 5 10 15
Met Leu Pro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg
20 25 30
Gly Arg Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg
35 40 45
Val Asp Ser Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn
50 55 60
Pro Phe Ala Pro Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg
65 70 75
Val Arg Gln Pro Ala Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln
80 85 90
Ala Gln Arg Pro Ala Ala Ala Ser Arg Arg Arg Pro Thr Thr Ala
95 100 105
Gly Ala Ala Pro Leu Thr Ala Val Ala Pro Ala His Asp Thr Pro
110 115 120
Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln
125 130 135
Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Thr Gly
140 145 150
Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Ser Pro Leu Leu Pro
155 160 165
Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser
170 175 180
Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile Arg Tyr Arg
185 190 195
Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser
200 205 210
Phe Tyr Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn
215 220 225
Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
230 235 240
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn
245 250 255
Gln Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu
260 265 270
Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val
275 280 285
Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu
290 295 300
Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn
305 310 315
Thr Asn Thr Arg Val Ser Arg Tyr Ser Ser Thr Ala Arg His Arg
320 325 330
Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala
335 340 345
Ala Thr Arg Phe Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly
350 355 360
Val Gly Glu Ile Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu
365 370 375
Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser
380 385 390
Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn
395 400 405
Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln
410 415 420
Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Glu
425 430 435
Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp
440 445 450
Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu
455 460 465
Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr
470 475 480
Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser
485 490 495
Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val
500 505 510
Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro
515 520 525
Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro
530 535 540
Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala
545 550 555
Gly Tyr Pro Tyr ASn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu
560 565 570
Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr
575 580 585
Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val
590 595 600
Leu Ala Pro His Ser Val Leu Ala Leu Leu Glu Asp Thr Met Asp
605 610 615
Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys
620 625 630
Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala
635 640 645
Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu
650 655 660
(SEQ ID NO.:3)
Met Asn Asn Met Ser Phe Ala Ala Pro Met Gly Ser Arg Pro Cys
1 5 10 15
Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys
20 25 30
Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala Ala Val Val Gly
35 40 45
Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu
50 55 60
Ile Leu Ser Pro Ser Gln Ser Pro Ile Phe Ile Gln Pro Thr Pro
65 70 75
Ser Pro Pro Met Ser Pro Leu Arg Pro Gly Leu Asp Leu Val Phe
80 85 90
Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro
95 100 105
Ser Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly
110 115 120
Pro Arg Arg
三字符缩写按照20个天然存在的氨基酸的常规氨基酸速记。
优选的重组HEV蛋白质含有至少一个ORF蛋白质。可以制备其他由一个以上相同或不同ORF蛋白构成的重组蛋白,以改变蛋白质的生物特性。添加、置换或缺失个别氨基酸或个别氨基酸序列可能会提高HEV的生物活性,这在构思之中,
本发明还涉及一种核酸序列,它能够指导产生上述的HEV蛋白质或与HEV蛋白质基本同源的蛋白质。该核酸序列被称为SAR-55,它作为SEQ ID NO.:4示于下面,并且于1992年9月17日保藏在美国典型培养物保藏中心(ATCC)(ATCC保藏号75302)。
AGGCAGACCA CATATGTGGT CGATGCCATG GAGGCCCATC AGTTTATCAA 50
GGCTCCTGGC ATCACTACTG CTATTGAGCA GGCTGCTCTA GCAGCGGCCA 100
ACTCTGCCCT TGCGAATGCT GTGGTAGTTA GGCCTTTTCT CTCTCACCAG 150
CAGATTGAGA TCCTTATTAA CCTAATGCAA CCTCGCCAGC TTGTTTTCCG 200
CCCCGAGGTT TTCTGGAACC ATCCCATCCA GCGTGTTATC CATAATGAGC 250
TGGAGCTTTA CTGTCGCGCC CGCTCCGGCC GCTGCCTCGA AATTGGTGCC 300
CACCCCCGCT CAATAAATGA CAATCCTAAT GTGGTCCACC GTTGCTTCCT 350
CCGTCCTGCC GGGCGTGATG TTCAGCGTTG GTATACTGCC CCTACCCGCG 400
GGCCGGCTGC TAATTGCCGG CGTTCCGCGC TGCGCGGGCT CCCCGCTGCT 450
GACCGCACTT ACTGCTTCGA CGGGTTTTCT GGCTGTAACT TTCCCGCCGA 500
GACGGGCATC GCCCTCTATT CTCTCCATGA TATGTCACCA TCTGATGTCG 550
CCGAGGCTAT GTTCCGCCAT GGTATGACGC GGCTTTACGC TGCCCTCCAC 600
CTCCCGCCTG AGGTCCTGTT GCCCCCTGGC ACATACCGCA CCGCGTCGTA 650
CTTGCTGATC CATGACGGCA GGCGCGTTGT GGTGACGTAT GAGGGTGACA 700
CTAGTGCTGG TTATAACCAC GATGTTTCCA ACCTGCGCTC CTGGATTAGA 750
ACCACTAAGG TTACCGGAGA CCACCCTCTC GTCATCGAGC GGGTTAGGGC 800
CATTGGCTGC CACTTTGTCC TTTTACTCAC GGCTGCTCCG GAGCCATCAC 850
CTATGCCCTA TGTCCCTTAC CCCCGGTCTA CCGAGGTCTA TGTCCGATCG 900
ATCTTCGGCC CGGGTGGCAC CCCCTCCCTA TTTCCAACCT CATGCTCCAC 950
CAAGTCGACC TTCCATGCTG TCCCTGCCCA TATCTGGGAC CGTCTCATGT 1000
TGTTCGGGGC CACCCTAGAT GACCAAGCCT TTTGCTGCTC CCGCCTAATG 1050
ACTTACCTCC GCGGCATTAG CTACAAGGTT ACTGTGGGCA CCCTTGTTGC 1100
CAATGAAGGC TGGAACGCCT CTGAGGACGC TCTTACAGCT GTCATCACTG 1150
CCGCCTACCT TACCATCTGC CACCAGCGGT ACCTCCGCAC TCAGGCTATA 1200
TCTAAGGGGA TGCGTCGCCT GGAGCGGGAG CATGCTCAGA AGTTTATAAC 1250
ACGCCTCTAC AGTTGGCTCT TTGAGAAGTC CGGCCGTGAT TATATCCCCG 1300
GCCGTCAGTT GGAGTTCTAC GCTCAGTGTA GGCGCTGGCT CTCGGCCGGC 1350
TTTCATCTTG ACCCACGGGT GTTGGTTTTT GATGAGTCGG CCCCCTGCCA 1400
CTGTAGGACT GCGATTCGTA AGGCGGTCTC AAAGTTTTGC TGCTTTATGA 1450
AGTGGCTGGG CCAGGAGTGC ACCTGTTTTC TACAACCTGC AGAAGGCGTC 1500
GTTGGCGACC AGGGCCATGA CAACGAGGCC TATGAGGGGT CTGATGTTGA 1550
CCCTGCTGAA TCCGCTATTA GTGACATATC TGGGTCCTAC GTAGTCCCTG 1600
GCACTGCCCT CCAACCGCTT TACCAAGCCC TTGACCTCCC CGCTGAGATT 1650
GTGGCTCGTG CAGGCCGGCT GACCGCCACA GTAAAGGTCT CCCAGGTCGA 1700
CGGGCGGATC GATTGTGAGA CCCTTCTCGG TAATAAAACC TTCCGCACGT 1750
CGTTTGTTGA CGGGGCGGTT TTAGAGACTA ATGGCCCAGA GCGCCACAAT 1800
CTCTCTTTTG ATGCCAGTCA GAGCACTATG GCCGCCGGCC CTTTCAGTCT 1850
CACCTATGCC GCCTCTGCTG CTGGGCTGGA GGTGCGCTAT GTCGCCGCCG 1900
GGCTTGACCA CCGGGCGGTT TTTGCCCCCG GCGTTTCACC CCGGTCAGCC 1950
CCTGGCGAGG TCACCGCCTT CTGTTCTGCC CTATACAGGT TTAATCGCGA 2000
GGCCCAGCGC CTTTCGCTGA CCGGTAATTT TTGGTTCCAT CCTGAGGGGC 2050
TCCTTGGCCC CTTTGCCCCG TTTTCCCCCG GGCATGTTTG GGAGTCGGCT 2100
AATCCATTCT GTGGCGAGAG CACACTTTAC ACCCGCACTT GGTCGGAGGT 2150
TGATGCTGTT CCTAGTCCAG CCCAGCCCGA CTTAGGTTTT ACATCTGAGC 2200
CTTCTATACC TAGTAGGGCC GCCACACCTA CCCCGGCGGC CCCTCTACCC 2250
CCCCCTGCAC CGGATCCTTC CCCTACTCTC TCTGCTCCGG CGCGTGGTGA 2300
GCCGGCTCCT GGCGCTACCG CCAGGGCCCC AGCCATAACC CACCAGACGG 2350
CCCGGCATCG CCGCCTGCTC TTTACCTACC CGGATGGCTC TAAGGTGTTC 2400
GCCGGCTCGC TGTTTGAGTC GACATGTACC TGGCTCGTTA ACGCGTCTAA 2450
TGTTGACCAC CGCCCTGGCG GTGGGCTCTG TCATGCATTT TACCAGAGGT 2500
ACCCCGCCTC CTTTGATGCT GCCTCTTTTG TGATGCGCGA CGGCGCGGCC 2550
GCCTACACAT TAACCCCCCG GCCAATAATT CATGCCGTCG CTCCTGATTA 2600
TAGGTTGGAA CATAACCCAA AGAGGCTTGA GGCTGCCTAC CGGGAGACTT 2650
GCTCCCGCCT CGGTACCGCT GCATACCCAC TCCTCGGGAC CGGCATATAC 2700
CAGGTGCCGA TCGGTCCCAG TTTTGACGCC TGGGAGCGGA ATCACCGCCC 2750
CGGGGACGAG TTGTACCTTC CTGAGCTTGC TGCCAGATGG TTCGAGGCCA 2800
ATAGGCCGAC CTGCCCAACT CTCACTATAA CTGAGGATGT TGCGCGGACA 2850
GCAAATCTGG CTATCGAACT TGACTCAGCC ACAGACGTCG GCCGGGCCTG 2900
TGCCGGCTGT CGAGTCACCC CCGGCGTTGT CAGTACCAG TTTACCGCAG 2950
GTGTGCCTGG ATCCGGCAAG TCCCGCTCTA TTACCCAAGC CGACGTGGAC 3000
GTTGTCGTGG TCCCGACCCG GGAGTTGCGT AATGCCTGGC GCCGCCGCGG 3050
CTTCGCTGCT TTCACCCCGC ACACTGCGGC TAGAGTCACC CAGGGGCGCC 3100
GGGTTGTCAT TGATGAGGCC CCGTCCCTTC CCCCTCATTT GCTGCTGCTC 3150
CACATGCAGC GGGCCGCCAC CGTCCACCTT CTTGGCGACC CGAATCAGAT 3200
CCCAGCCATC GATTTTGAGC ACGCCGGGCT CGTTCCCGCC ATCAGGCCCG 3250
ATTTGGCCCC CACCTCCTGG TGGCATGTTA CCCATCGCTG CCCTGCGGAT 3300
GTATGTGAGC TAATCCGCGG CGCATACCCT ATGATTCAGA CCACTAGTCG 3350
GGTCCTCCGG TCGTTGTTCT GGGGTGAGCC CGCCGTTGGG CAGAAGCTAG 3400
TGTTCACCCA GGCGGCTAAG GCCGCCAACC CCGGTTCAGT GACGGTCCAT 3450
GAGGCACAGG GCGCTACCTA CACAGAGACT ACCATCATTG CCACGGCAGA 3500
TGCTCGAGGC CTCATTCAGT CGTCCCGAGC TCATGCCATT GTTGCCTTGA 3550
CGCGCCACAC TGAGAAGTGC GTCATCATTG ACGCACCAGG CCTGCTTCGC 3600
GAGGTGGGCA TCTCCGATGC AATCGTTAAT AACTTTTTCC TTGCTGGTGG 3650
CGAAATTGGC CACCAGCGCC CATCTGTTAT CCCTCGCGGC AATCCTGACG 3700
CCAATGTTGA CACCTTGGCT GCCTTCCCGC CGTCTTGCCA GATTAGCGCC 3750
TTCCATCAGT TGGCTGAGGA GCTTGGCCAC AGACCTGCCC CTGTCGCGGC 3800
TGTTCTACCG CCCTGCCCTG AGCTTGAACA GGGCCTTCTC TACCTGCCCC 3850
AAGAACTCAC CACCTGTGAT AGTGTCGTAA CATTTGAATT AACAGATATT 3900
GTGCATTGTC GTATGGCCGC CCCGAGCCAG CGCAAGGCCG TGCTGTCCAC 3950
GCTCGTGGGC CGTTATGGCC GCCGCACAAA GCTCTACAAT GCCTCCCACT 4000
CTGATGTTCG CGACTCTCTC GCCCGTTTTA TCCCGGCCAT TGGCCCCGTA 4050
CAGGTTACAA CCTGTGAATT GTACGAGCTA GTGGAGGCCA TGGTCGAGAA 4100
GGGCCAGGAC GGCTCCGCCG TCCTTGAGCT CGACCTTTGT AGCCGCGACG 4150
TGTCCAGGAT CACCTTCTTC CAGAAAGATT GTAATAAATT CACCACGGGG 4200
GAGACCATCG CCCATGGTAA AGTGGGCCAG GGCATTTCGG CCTGGAGTAA 4250
GACCTTCTGT GCCCTTTTCG GCCCCTGGTT CCGTGCTATT GAGAAGGCTA 4300
TCCTGGCCCT GCTCCCTCAG GGTGTGTTTT ATGGGGATGC CTTTGATGAC 4350
ACCGTCTTCT CGGCGGCTGT GGCCGCAGCA AAGGCATCCA TGGTGTTCGA 4400
GAATGACTTT TCTGAGTTTG ATTCCACCCA GAATAATTTT TCCTTGGGCC 4450
TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG GCTCATCCGC 4500
TTGTACCACC TTATAAGGTC TGCGTGGATT CTGCAGGCCC CGAAGGAGTC 4550
CCTGCGAGGG TTTTGGAAGA AACACTCCGG TGAGCCCGGC ACCCTTCTGT 4600
GGAATACTGT CTGGAACATG GCCGTTATCA CCCACTGTTA TGATTTCCGC 4650
GATCTGCAGG TGGCTGCCTT TAAAGGTGAT GATTCGATAG TGCTTTGCAG 4700
TGAGTACCGT CAGAGCCCAG GGGCTGCTGT CCTGATTGCT GGCTGTGGCC 4750
TAAAGTTGAA GGTGGATTTC CGTCCGATTG GTCTGTATGC AGGTGTTGTG 4800
GTGGCCCCCG GCCTTGGCGC GCTTCCTGAT GTCGTGCGCT TCGCCGGTCG 4850
GCTTACTGAG AAGAATTGGG GCCCTGGCCC CGAGCGGGCG GAGCAGCTCC 4900
GCCTCGCTGT GAGTGATTTT CTCCGCAAGC TCACGAATGT AGCTCAGATG 4950
TGTGTGGATG TTGTCTCTCG TGTTTATGGG GTTTCCCCTG GGCTCGTTCA 5000
TAACCTGATT GGCATGCTAC AGGCTGTTGC TGATGGCAAG GCTCATTTCA 5050
CTGAGTCAGT GAAGCCAGTG CTTGACCTGA CAAATTCAAT TCTGTGTCGG 5100
GTGGAATGAA TAACATGTCT TTTGCTGCGC CCATGGGTTC GCGACCATGC 5150
GCCCTCGGCC TATTTTGCTG TTGCTCCTCA TGTTTCTGCC TATGCTGCCC 5200
GCGCCACCGC CCGGTCAGCC GTCTGGCCGC CGTCGTGGGC GGCGCAGCGG 5250
CGGTTCCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT CAGCCCTTCG 5300
CAATCCCCTA TATTCATCCA ACCAACCCCT TCGCCCCCGA TGTCACCGCT 5350
GCGGCCGGGG CTGGACCTCG TGTTCGCCAA CCCGCCCGAC CACTCGGCTC 5400
CGCTTGGCGT GACCAGGCCC AGCGCCCCGC CGCTGCCTCA CGTCGTAGAC 5450
CTACCACAGC TGGGGCCGCG CCGCTAACCG CGGTCGCTCC GGCCCATGAC 5500
ACCCCGCCAG TGCCTGATGT TGACTCCCGC GGCGCCATCC TGCGCCGGCA 5550
GTATAACCTA TCAACATCTC CCCTCACCTC TTCCGTGGCC ACCGGCACAA 5600
ATTTGGTTCT TTACGCCGCT CCTCTTAGCC CGCTTCTACC CCTCCAGGAC 5650
GGCACCAATA CTCATATAAT GGCTACAGAA GCTTCTAATT ATGCCCAGTA 5700
CCGGGTTGCT CGTGCCACAA TTCGCTACCG CCCGCTGGTC CCCAACGCTG 5750
TTGGTGGCTA CGCTATCTCC ATTTCGTTCT GGCCACAGAC CACCACCACC 5800
CCGACGTCCG TTGACATGAA TTCAATAACC TCGACGGATG TCCGTATTTT 5850
AGTCCAGCCC GGCATAGCCT CCGAGCTTGT TATTCCAAGT GAGCGCCTAC 5900
ACTATCGCAA CCAAGGTTGG CGCTCTGTTG AGACCTCCGG GGTGGCGGAG 5950
GAGGAGGCCA CCTCTGGTCT TGTCATGCTC TGCATACATG GCTCACCTGT 6000
AAATTCTTAT ACTAATACAC CCTATACCGG TGCCCTCGGG CTGTTGGACT 6050
TTGCCCTCGA ACTTGAGTTC CGCAACCTCA CCCCCGGTAA TACCAATACG 6100
CGGGTCTCGC GTTACTCCAG CACTGCCCGT CACCGCCTTC GTCGCGGTGC 6150
AGATGGGACT GCCGAGCTCA CCACCACGGC TGCTACTCGC TTCATGAAGG 6200
ACCTCTATTT TACTAGTACT AATGGTGTTG GTGAGATCGG CCGCGGGATA 6250
GCGCTTACCC TGTTTAACCT TGCTGACACC CTGCTTGGCG GTCTACCGAC 6300
AGAATTGATT TCGTCGGCTG GTGGCCAGCT GTTCTACTCT CGCCCCGTCG 6350
TCTCAGCCAA TGGCGAGCCG ACTGTTAAGC TGTATACATC TGTGGAGAAT 6400
GCTCAGCAGG ATAAGGGTAT TGCAATCCCG CATGACATCG ACCTCGGGGA 6450
ATCCCGTGTA GTTATTCAGG ATTATGACAA CCAACATGAG CAGGACCGAC 6500
CGACACCTTC CCCAGCCCCA TCGCGTCCTT TTTCTGTCCT CCGAGCTAAC 6550
GATGTGCTTT GGCTTTCTCT CACCGCTGCC GAGTATGACC AGTCCACTTA 6600
CGGCTCTTCG ACCGGCCCAG TCTATGTCTC TGACTCTGTG ACCTTGGTTA 6650
ATGTTGCGAC CGGCGCGCAG GCCGTTGCCC GGTCACTCGA CTGGACCAAG 6700
GTCACACTTG ATGGTCGCCC CCTTTCCACC ATCCAGCAGT ATTCAAAGAC 6750
CTTCTTTGTC CTGCCGCTCC GCGGTAAGCT CTCCTTTTGG GAGGCAGGAA 6800
CTACTAAAGC CGGGTACCCT TATAATTATA ACACCACTGC TAGTGACCAA 6850
CTGCTCGTTG AGAATGCCGC TGGGCATCGG GTTGCTATTT CCACCTACAC 6900
TACTAGCCTG GGTGCTGGCC CCGTCTCTAT TTCCGCGGTT GCTGTTTTAG 6950
CCCCCCACTC TGTGCTAGCA TTGCTTGAGG ATACCATGGA CTACCCTGCC 7000
CGCGCCCATA CTTTCGATGA CTTCTGCCCG GAGTGCCGCC CCCTTGGCCT 7050
CCAGGGTTGT GCTTTTCAGT CTACTGTCGC TGAGCTTCAG CGCCTTAAGA 7100
TGAAGGTGGG TAAAACTCGG GAGTTATAGT TTATTTGCTT GTGCCCCCCT 7150
TCTTTCTGTT GCTTATTT 7168
核苷酸的缩写是本领域中标准使用的。
在一个方向上的序列按常规被称为正链(“+”链),因为它是RNA病毒的蛋白质编码链,即在上面被示作SEQ ID NO.:4的序列。
SAR-55开放阅读框的推导出的氨基酸序列是SEQ ID NO.:1、SEQ ID NO.:2和SEQ ID NO.:3。ORF-1从SEQ ID NO.:4的核苷酸28开始并延伸至核苷酸5078;ORF-2从SEQ ID NO.:4的核苷酸5147处开始并延伸至核苷酸1979;而ORF-3从SEQ ID NO.:4的核苷酸5106处开始并延伸至核苷酸368。
在构思之中的还有DNA序列的变异体,它们导致产生能够指导产生ORF-2蛋白类似物的DNA序列。如说明书和权利要求书中所用,“ORF-2蛋白类似物”指氨基酸序列与此处具体给出的序列基本上相同的蛋白质,其中此处所述序列中的一个或多个残基被生物学上等价的残基所替代,这样形成的蛋白质(即“类似物”)能够形成病毒颗粒而且是免疫原性的。应注意,上面给出的DNA序列代表了本发明的一个优选例子。由于遗传密码的简并性,所以可以制备许多种核苷酸,它们都是能够指导合成该ORF蛋白质或其类似物的DNA序列。因此,功能上与上述序列等价的DNA序列,或者功能上与指导合成ORF蛋白质类似物(按照上述的氨基酸序列产生的)的序列等价的DNA序列都被包括在本发明之内。
本发明涉及一种基于选择性地扩增戊型肝炎基因片段而在生物样品中检测戊型肝炎的方法。较佳地,这种方法采用一对单链引物,这一对引物来自DNA双链片段的相对链的非同源区域,而该DNA双链片段又来自在基因组中含有与SEQID NO.:4所示的SAR-55序列同源的区域的戊型肝炎病毒。这些引物可用于扩增选定核酸序列的方法,如在美国专利No.4,683,202中所述的方法。
本发明还涉及使用衍生自SAR-55 cDNA的单链反义多聚或寡聚核苷酸来抑制戊型肝炎基因的表达。这些反义的多聚或寡聚核苷酸可以是DNA或RNA。靶序列典型地是信使RNA,更佳地是加工或翻译RNA所需的信号序列。反义的多聚或寡聚核苷酸可偶连于聚阳离子比如聚赖氨酸,如Lemaitre,M.等人(1989)美国科学院院报84:648-652中所公开的那样;然后该偶联物可给哺乳动物施用,用量足以杂交并抑制信使RNA的功能。
本发明还涉及一种重组DNA方法,它用于制备HEV蛋白质,较佳地是含有至少一个ORF蛋白的蛋白质,最佳地是含有至少一个ORF-2蛋白的蛋白质。重组的ORF蛋白可以由一个ORF构成,或者由相同或不同的ORF蛋白组合而成。可使用天然的或合成的核酸序列来指导产生HEV蛋白。在本发明的一个例子中,该方法包括:
(a)制备能够指导宿主有机体产生HEV蛋白质的核酸序列;
(b)将核酸序列克隆入能够转入并在宿主有机体中复制的载体,该载体含有用于该核酸序列的可操作元件;
(c)将含有该核酸和可操作元件的载体转入能够表达蛋白质的宿主有机体;
(d)在适合扩增载体和表达蛋白质的条件下,培养宿主有机体;和
(e)收获蛋白质。
在本发明的另一例子中,公开了一种用于重组DNA合成由HEV核酸编码的蛋白质、较佳地由HEV的至少一个ORF编码的蛋白质或者相同或不同ORF蛋白质的组合、最佳地由至少一个ORF-2核酸序列编码的蛋白质的方法,该方法包括:
(a)在适合产生蛋白质的条件下,培养转化的或转染的宿主有机体,该宿主有机体含有能够指导宿主有机体产生某蛋白质的核酸序列,该蛋白质与从HEV分离出的,具有SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3或其组合的氨基酸序列的天然HEV蛋白质基本同源。
在一个例子中,如下对HEV株SAR-55的病毒基因组的RNA序列进行分离并克隆cDNA。从用SAR-55感染的猕猴收集生物样品,然后从中抽提出病毒RNA,再对病毒RNA进行反转录,并用互补于HEV面甸株(Tam等人(1991))基因组或SAR-55基因组正链或负链的引物,通过聚合酶链反应加以扩增。将PCR片段亚克隆入pBR322或pGEM-32,然后测序双链PCR片段。
可用于本发明的载体包括任何载体,只要在该载体中可以插入所述的核酸序列,以及任何优选的或所需的可操作元件,并且该载体可以随后被转入宿主有机体并在该有机体中复制。优选的载体是,那些限制性位点已记载清楚而且含有转录核酸序列所需的或优选的可操作元件的载体。
如此处所用,“可操作元件”包括至少一个启动子、至少一个操纵基因、至少一个前导序列、至少一个终止密码子和其他任何对于载体核酸序列的合适转录和随后翻译所必需或优选的DNA序列。特别地,在构思之中的是这样的载体,它含有至少一个被宿主有机体识别的复制起始点和至少一个供选择的标记以及至少一个能够引发核酸序列转录的启动子序列。
在构建本发明的克隆载体过程中,还应注意,可在每个载体中插入多拷贝的核酸序列及其可操作元件。在这样的例子中,对于每个载体而言,每个宿主有机体会产生更多的所需HEV蛋白质。可以插入载体的多拷贝DNA序列的数目仅取决于形成的载体的能力,这些能力受其大小、转入以及在合适的宿主微生物中复制和转录等因素的影响。
在另一例子中,将含有HEV蛋白编码序列的限制性消化片段插入合适的、会在原核或真核细胞中起作用的表达载体。“合适的”指载体能够携带和表达为HEV蛋白(较佳地,至少一个完整的ORF蛋白)编码的完整核酸序列。在ORF-2的情况下,表达的蛋白质会形成病毒样颗粒。优选的表达载体是那些能在真核细胞中起作用的种类。这类载体的例子包括但并不限于:用于在酵母中表达的载体(如pPIC9载体-Invitrogen)、痘苗病毒载体、腺病毒或疱疹病毒,尤其是杆状病毒转化载体、pBlueBac。优选的载体是p63-2(它含有完整的ORF-2基因)和P59-4(它含有完整的ORF-3和ORF-2基因)。这些载体于1992年9月10日保藏在美国典型培养物保藏中心(ATCC,12301 Parklawn Drive,Rockville,MD20852,USA),保藏号为75299(P63-2)和75300(P59-4)。实施例1讲述了将ORF-2基因克隆入pBlueBac以产生p63-2的过程。该方法包括:用限制酶NruI和BglII消化HEV株SAR-55的基因组,将含有BlnI和BglII位点的多接头插入载体上唯一的NheI位点,然后用衔接头将NruI-BglII ORF-2片段插入BlnI-BglIIpBlueBac。
在另一例子中,选定的重组表达载体可接着被转染入合适的真核细胞系统,以便表达重组蛋白。这种真核细胞系统包括但并不限于:酵母和诸如HeLa、MRC-5或Cv-1之类的细胞系。一种优选的真核细胞系统是SF9昆虫细胞。一种优选的方法涉及使用pBlueBac表达载体,其中通过钙沉淀法用重组pBlueBac和AcMNPV杆状病毒共转染昆虫细胞SF9。
表达的重组蛋白可用本领域中已知的方法进行检测,其中包括考马斯蓝染色法和Western印迹法(使用含有抗HEV抗体的血清),如实施例2中所示。另一方法是用免疫电子显微镜检测病毒样颗粒,如实施例3中所示。
在另一例子中,由SF9细胞表达的重组蛋白可以粗裂解液形式获得,或者用本领域中已知的标准蛋白质纯化程序加以纯化。纯化方法包括:差示沉淀、分子筛色谱、离子交换、等电聚焦、凝胶电泳、亲和色谱和免疫亲和色谱等。在免疫亲和色谱中,重组蛋白可通过含有树脂的柱而纯化,其中在树脂上结合有对ORF蛋白特异的抗体。在实施例10中提供了纯化重组表达的HEV ORF蛋白的方案的一个例子。
在另一例子中,表达的本发明的重组蛋白被用于免疫分析,以诊断或预后哺乳动物中的戊型肝炎。其中哺乳动物包括但并不限于:人、黑猩猩、旧大陆猴、新大陆猴、其他灵长目动物。在一个优选例子中,免疫分析被用于诊断人的戊型肝炎感染。使用HEV蛋白,尤其是ORF蛋白,特别是ORF-2蛋白而进行免疫分析,提供了高特异性的、灵敏的、可重复的、诊断HEV感染的方法,这与采用不完整的ORF蛋白而进行的免疫分析形成了鲜明的对比。本发明的免疫分析可以是放射免疫分析、Western印迹分析、免疫荧光分析、酶免疫分析、化学发光分析、免疫组织化学分析等。本领域中已知的ELISA标准技术在
“免疫诊断方法”(Methods in Immunodiagnosis),第2版,Rose和Bigazzi编辑,John Wiley andSons,1980和Campbell等人,
“免疫学方法”(Methods of Immunology),W.A.Benjamin,Inc.,1964中有描述,这两篇文献都在此引用作为参考。这种分析可以是本领域中所述的直接的、间接的、竞争性的或非竞争性的免疫测定。(Oellerich,M.1984.
“临床化学临床生物化学杂志”(J.Clin.Chem.Clin.Biochem.)22:895-904)。适用于这种检测分析的生物样品包括但并不限于:组织解剖抽提物、全血、血浆、血清、脑脊液、胸膜液、尿液等。
在一个例子中,测试血清与一固相试剂进行反应,该固相试剂表面结合有作为抗原的重组HEV蛋白,较佳地为ORF蛋白或不同ORF蛋白的组合形式如ORF-2和ORF-3、ORF-1和ORF-3等。较佳地,HEV蛋白是会形成病毒样颗粒的ORF-2蛋白。可以用已知的将蛋白质结合于固相载体材料的技术制备固相表面试剂。这些结合方法包括:蛋白质与载体的非特异吸附或蛋白质与载体上反应基团的共价结合。在抗原与抗HEV抗本反应之后,通过洗涤而除去未结合的血清组份,然后抗原-抗体复合物再与次级抗体(如标记的抗-人抗体)反应。标记物可以是在合适荧光试剂或生色试剂的存在下孵育固相载体而被检测出的酶。其他的合适标记物也可使用,如放射性标记物或胶体金等。
在一个优选的例子中,由含有完整的SAR-55 ORF-2序列的重组载体pBlueBac所表达的蛋白被用作特异性结合剂,以检测抗HEV抗体,尤其是IgG或IgM抗体。实施例4和5显示了ELISA的结果,其中固相试剂具有重组的ORF-2作为表面抗原。这个由完整的ORF-2核酸序列所编码的蛋白质优于不完整的ORF-2蛋白,因为与ORF-2部分抗原相比,它可与更多的、来自被HEV感染的不同灵长目的抗血清反应。本发明的蛋白质还能检测出针对不同HEV株而产生的抗体,但是不会检测出针对甲型、乙型、丙型或丁型肝炎而产生的抗体。
HEV蛋白和类似物可以单独地或与其他试剂如次级抗体一起被制成试剂盒形式用于免疫测定。
本发明的重组HEV蛋白,较佳地为ORF蛋白或ORF蛋白的重组蛋白,更佳地为ORF-2蛋白和基本同源的蛋白质及类似物,可以被用作疫苗以保护哺乳动物免受戊型肝炎的侵袭。作为免疫原的疫苗,可以是细胞、用重组表达载体转染过的细胞的细胞裂解液或含有表达蛋白的培育上清液。或者免疫原是部分或基本纯化的重组蛋白。尽管可以将免疫原以纯的或基本纯的形式进行施用,但是最好还是以药物组合物、药剂或制剂形式使用。
本发明的药剂可用作兽药或用于人类,它含有上述的免疫原和一种或多种药学上可接受的载体和任选的其他治疗成分。载体必须是“可接受的”,意味着与药剂的其他成分相容而且对接受者无害。药剂可以方便地作为单位剂量形式,并且可用药物领域中公知的方法制备。
所有的方法包括将活性成分与作为一种或多种附属成分的载体混合起来。一般,药剂的制备是通过:将活性成分与液态载体或细分固相载体,或与两者一起均匀地和紧密地混合在一起,然后如果需要将产品制成所需的药剂。
适用于静脉内、肌内、皮下或腹膜内给药的药剂,可以是活性成分与其他溶液(最好与接受者的血液是等渗的溶液)形成的无菌水溶液。这种药剂可以方便地将固体的活性成分溶解在含有生理相容物质如氯化钠(如0.1-2.0M)、甘油等并且具有与生理条件相容的缓冲pH的水中,以产生水溶液,然后使该溶液无菌。它们可以装在单剂量或多剂量容器中如密封的安瓿或小瓶。
本发明的药剂可以加入稳定剂。代表性的稳定剂是聚乙二醇、蛋白质、糖类、氨基酸、无机酸和有机酸,它们可以单独使用或混合使用。这些稳定剂较佳的加入量相对每份重量的免疫原而言为0.11-10,000重量份数。如果使用两种或多种稳定剂,那么它们的重量最好在上述范围之内。这些稳定剂被用于合适浓度和pH下的水溶液中。这种水溶液的比渗透压一般为0.1-3.0渗透压摩尔,较佳地为0.8-1.2渗透压摩尔。水溶液的pH被调节至范围5.0-9.0,较佳地为6-8之内。在配制本发明的免疫原时,可使用抗吸附剂。
还可采用额外的药物学方法来控制作用的持续时间。通过使用聚合物来复合或吸附蛋白质或其衍生物,可以获得控释剂。还可以通过选择合适的大分子(如聚酯、聚氨基酸、乙烯类聚合物、吡咯烷酮、乙烯乙酸乙烯酯、甲基纤维素、羧甲基纤维素或硫酸鱼精蛋白)和大分子浓度以及控释的掺入方法,来实施受控的给药。另一种通过控释制剂而控制作用持续时间的可行方法是,将蛋白质、蛋白质类似物或其功能衍生物掺入聚合物材料如聚酯、聚氨基酸、水凝胶、聚(乳酸)或乙烯乙酸乙烯酯共聚物。或者,不将这些试剂掺入聚合物颗粒,而是将这些颗粒截留在例如通过团聚技术或界面聚合反应制备的微囊中,如羟甲基生物素或明胶微囊和聚(甲基丙烯酸甲酯)微囊,或者截留在胶体药物给药系统中如脂质体、白蛋白微球、微乳、纳米级颗粒和纳米级胶囊,或者截留在大乳状液(macroemulsion)中。
当需要口服制剂时,组合物可以与典型的载体混合,其中有如乳糖、蔗糖、淀粉、滑石硬脂酸镁、结晶纤维素、甲基纤维素、羟甲基纤维素、甘油、藻酸钠或阿拉伯胶。
本发明的蛋白质可以单独作为试剂盒形式提供,也可以上述的药物组合物的形式提供。
接种疫苗可以用常规方法进行。例如,可以使用在适当稀释剂如盐水或水,或完全或不完全的佐剂中的免疫原。此外,为了使蛋白质有免疫原性,免疫原可以结合或不结合于载体。载体分子的来自包括但并不限于:牛血清白蛋白(BAS)、匙孔血蓝蛋白(KLH)、破伤风类毒素等。免疫原可以通过任何适合抗体产品的途径进行给药,如静脉内、腹膜内、肌内、皮下等给药。免疫原可以给药一次或者按固定间隔给药,直至产生大量的抗HEV抗体。抗体可用免疫测定在血清中检测出。
在另一例子中,免疫原是能指导宿主有机体合成HEV ORF蛋白的核酸序列。这种核酸序列可以用本领域技术人员已知的方法插入合适的表达载体。适合在体内产生高效基因转化的表达载体包括但并不限于:反转录病毒、腺病毒和痘苗病毒载体。这些表达载体的可操作元件在本说明书之前已经公开并且为本领域技术人员所知晓。这些表达载体可以静脉内、肌内、皮下、腹膜内或口服施用。
在另一例子中,通过肌内注射例如基于质粒的真核表达载体(该载体含有能指导宿主有机体合成HEV ORF蛋白的核酸序列)而实现直接基因转化。这种方法以前被用过,以便在体内产生乙型肝炎表面抗原并导致了针对表面抗原的抗体应答(Davis,H.L.等人(1993)“人类分子遗传学”(Human Molecular Genetics),2:1847-1851;还参见Davis等人(1993)“人类基因治疗”(Human Gene Therapy),4:151-159和733-740)。
当免疫原是部分纯化或基本纯化的重组HEV ORF蛋白时,引发保护性的、针对HEV的抗体应答的有效剂量为约2微克-100微克。更佳的范围为约5微克-70微克,最佳的范围为约10微克-60微克。
对于编码HEV ORF蛋白的核酸序列而言,引发保护性的、针对HEV的抗体应答的有效剂量为约1微克-5000微克。更佳的范围为约300微克-1000微克。
含有能指导宿主有机体合成HEV ORF蛋白的核酸序列的表达载体,可以单独作为试剂盒形式提供,也可以以上述的药物组合物的形式提供。
可以出于预防或治疗目的而施用本发明的免疫原。当预防性地施用时,免疫原是在暴露于HEV之前或在出现HEV感染症状之前施用。预防性施用免疫原起到在哺乳动物体中防止或减轻随后的HEV感染的作用。当治疗性地施用时,免疫原在感染发生时(或发生后不久),或者在出现HEV感染或疾病的症状时施用。治疗性施用免疫原起到减轻感染或疾病的作用。
一个优选例子是用重组ORF-2蛋白制备的疫苗。该重组ORF-2蛋白由HEV株SAR-55的ORF-2序列或其等价序列所表达。因为已表明重组ORF-2蛋白可与多种HEV阳性血清反应,所以表明可用于防止多种HEV株。
除了用作疫苗之外,可用组合物制备针对HEV病毒样颗粒的抗体。这些抗体可直接用作抗病毒剂。为了制备抗体,用病毒颗粒免疫宿主动物,或者合适的话,将产生在病毒颗粒上的非颗粒抗原结合在载体上,如上对疫苗所述的那样。按适当的时间间隔收集宿主的血清或血浆,从而提供含有与病毒颗粒反应的抗体的组合物。例如通过使用饱和硫酸铵或DEAE Sephadex,或者本领域技术人员知晓的其他技术,可以获得γ球蛋白或IgG抗体。这些抗体基本上没有那些伴随其他抗病毒剂如药物的不良副作用。
通过减弱潜在的不良的免疫系统应答,可以使抗体组合物与宿主系统更相容。这是通过除去外来物种抗体的全部或部分Fc区,或者使用与宿主动物同物种的抗体(如使用来自人/人杂交瘤的抗体)而实现的。通过用相应的但是无免疫原性的部分来替换抗体的免疫原性部分(即嵌合抗体),可以产生人源化抗体(即在人体中无免疫原性)。这种嵌合抗体可含有一个物种抗体的活性或抗原结合部分和另一个物种抗体的Fc区(无免疫原性)。这种嵌合抗体的例子包括但并不限于:非人哺乳动物-人嵌合体、啮齿动物-人嵌合体、鼠科动物-人嵌合体和大鼠-人嵌合体(Robinson等人,国际专利申请184,187;Taniguchi M.,欧洲专利申请171,496;Morrison等人,欧洲专利申请173,494;Neuberger等人,PCT申请WO86/01533;Cabilly等人,1987“美国科学院院报”84:3439;Nishimura等人,1987“癌症研究”(Canc.Res.)47:999;Wood等人,1985“自然”(Nature)314:446;Shaw等人,1988,“国家癌症研究院杂志”(″J.Natl.CancerInst.″)80:15553,这些文献都在此引用作为参考)。
“人源化”嵌合抗体的总回顾由Morrison S.,1985“科学”229:1202和Oi等人,1986“生物技术”(″BioTechniques″)4:214中提供。
合适的“人源化”抗体也可以用CDR或CEA取代法而制备(Jones等人,1986“自然”321:552;Verhoeyan等人,1988“科学”239:1534;Biedler等人,1988,“免疫学杂志”(J.Immunol.)141:4053,这些文献都在此引用作为参考)。
还可通过遗传工程来产生抗体或抗原结合片段。在大肠杆菌中表达重链和轻链基因的技术是PCT专利申请出版号WO901443。WO901443和WO9014424,以及Huse等人,1989“科学”246:1275-1281中的主题。
抗体还可用作增加免疫应答的手段。可以按与其他治疗性地施用抗体时相类似的剂量来施用抗体。例如在其他病毒疾病如狂犬病、麻疹和乙型肝炎的潜伏早期,按0.02-0.1毫升/磅体重施用混合的γ球蛋白以干扰病毒进入细胞。因此,与HEV病毒颗粒反应的抗体可被动地单独或与其他抗病毒剂一起给受HEV感染的宿主施用,以增加抗病毒药物的功效。
或者,通过将抗独特型抗体作为免疫原施用,可以诱导出抗HEV抗体。方便地,可以用上述制备的、纯化的抗HEV抗体制剂在宿主动物中诱导抗独特型抗体。将在合适稀释剂中的组合物施用给宿主动物。在施用(通常是重复施用)后,宿主会产生抗独特型抗体。为了消除对Fc区的免疫应答,可使用与宿主动物相同物种产生的抗体,或者将所用抗体的Fc区去除。在宿主动物中诱导抗独特型抗体之后,取出血清或血浆,从而提供抗体组合物。组合物可用上述抗HEV抗体的纯化方法进行纯化,或者使用结合于亲和基质的抗HEV抗体通过亲和色谱而纯化。产生的抗独特型抗体在构象上与真的HEV抗原相似,它可在不使用HEV颗粒抗原的情况下用于制备HEV疫苗。
当用作在动物中诱导抗HEV病毒抗体的手段时,可以用与疫苗一样的方式即肌内、腹膜内、皮下等注射有效浓度的、在生理上合适的稀释剂中的抗体,其中可以有或没有佐剂。进行一次或多次的加强注射是有利的。
本发明的HEV衍生蛋白也可用于产生暴露前或暴露后预防用的抗血清。其中,HEV蛋白或蛋白混合物与合适的佐剂一起配制,然后根据已知方法通过注射给志愿者而进行给药,以便产生人抗血清。在免疫后数周期间,通过定期采集血清样本,用此处所述的免疫测定检测抗HEV血清抗体的存在与否从而监测对注入蛋白质的抗体应答情况。
来自免疫个体的抗血清可作为受接触感染威胁的个体的暴露前预防手段。抗血清还可用于治疗暴露后的个体,这类似于使用高效价的针对乙型肝炎病毒的抗血清进行暴露后预防。当然,本领域的技术人员会轻易地理解,用标准技术从免疫个体的抗血清中纯化出的免疫球蛋白(HEV免疫球蛋白),可用作暴露前预防措施或用于治疗暴露后的个体。
对于体内使用针对HEV病毒样颗粒和蛋白的抗体和抗独特型抗体以及诊断用途,优选使用单克隆抗体。单克隆抗病毒颗粒的抗体或抗独特型抗体可如下产生。用本领域技术人员知晓的方法,从免疫动物中取出脾或淋巴细胞,并进行使之无限增殖化,或者用于制备杂交瘤(Goding,J.W.1983.“单克隆抗体:原理和实践:(″Monoclonal Antibodies:Principles and Practice″),Pladermic Press,Inc.,NY,NY,pp.56-97)。为了产生人-人杂交瘤,可选择人淋巴细胞供体。已知可被HEV感染的供体(其中通过例如在血液中存在抗病毒抗体或通过病毒培育而表明发生了感染)可作为合适的淋巴细胞供体。淋巴细胞可从外周血样品中分离出,或者如果供体进行脾切除术,那么可使用脾细胞。可用Epstein-Barr病毒(EB病毒)使人淋巴细胞无限增殖化,或者使用人融合配偶物(partner)来产生人-人杂交瘤。用肽进行的原发性体外免疫也可用于产生人单克隆抗体。
可筛选由无限增殖化细胞分泌的抗体,以确定分泌具有所需特异性的抗体的克隆。对于单克隆的抗病毒颗粒抗体,抗体必须结合HEV病毒颗粒。对于单克隆抗独特型抗体,抗体必须结合抗病毒颗粒抗体。选择出产生具有所需特异性的抗体的细胞。
上述的抗体和抗原结合片段可以单独地以试剂盒形式提供,或者作为药物组合物提供,供体内使用。如本文所述,抗体可用于治疗用途、免疫测定中的诊断用途或作为纯化ORF蛋白的免疫亲和剂。
材料
实施例中使用的材料如下:
灵长动物:黑猩猩(Chimp)(Pan troglodyes)。旧大陆猴:猕猴(Cyno)(Macaca fascicularis)、恒河猴(Rhesus)(M. mulatta)、豚尾猴(PT)(M.nemestrina)、和非洲绿猴(AGM)(Cercopithecus aethiops)。新大陆猴:须绢毛猴(mustached tamarins)(Tam)(Saguinus mystax)、松鼠猴(SQM)(Saimirisciureus)和夜猴(OWL)(Aotus trivigatus)。灵长动物在生命危险受控制的条件下单独饲养。动物的居住、维护和照顾达到或超过灵长动物饲养业的所有标准。
大多数动物用0.5毫升稀释在胎牛血清中粪便悬浮液中所含的HEV株SAR-55进行静脉内接种,如Tsarey,S.A.等人(1992),
美国科学院院报,89:559-563;和Tsarev,S.A.等人(1993),
“感染疾病杂志”(″J.Infect.Dis.″)(167:1302-1306)中所述。Chimp-1313和1310用从7个巴基斯坦戊型肝炎病人中收集而来的粪便混合物进行接种。
在接种之前和之后每周约两次收集血清样品。用市售的测试品(Medpath Inc.,Rockville,MD)分析肝酶血清中丙氨酸氨基转移酶(ALT)、异柠檬酸脱氢酶(ICD)和γ谷氨酰转移酶(GGT)的水平。血清学测试如上所述进行。
实施例1
鉴别HEV株SAR-55基因组的DNA序列
制备用于PCR的病毒RNA模板。混合HEV感染的猕猴的胆汁(10微升)、20(重量/体积)SDS(至终浓度为1%)、蛋白酶K(10mg/ml;至终浓度为1mg/ml)、1微升tRNA(10mg/ml)和3微升0.5M EDTA,最终体积为250微升,然后在55℃温育30分钟。在65℃,用苯酚/氯仿(1∶1,v/v)从胆汁中抽提总核酸二次,然后用氯仿抽提一次,再用乙醇沉淀,用95%乙醇洗涤,然后用于RT-PCR。当RNA被更全面地加以纯化时,通过RT-PCR扩增来自粪中,尤其来自血清中的HEV RNA会更有效。同上用蛋白酶K处理血清(100微升)或10%粪便悬浮液(200微升)。在温育30分钟后,加入300微升CHAOS缓冲液(4.2M硫氰酸胍、0.5N月桂酰基肌氨酸/0.025M Tris-HCl, pH8.0)。在65℃,用苯酚/氯仿萃取核酸两次,然后在室温下用氯仿萃取。再将7.5M乙酸铵(225微升)加入上相,并用0.68毫升2-丙醇沉淀核酸。沉淀溶解在300微升CHAOS缓冲液中,然后加入100微升水。重复氯仿萃取和2-丙醇沉淀步骤。将核酸溶解在水中,用乙醇沉淀,用95%乙醇洗涤,再用于RT-PCR。
引物。用Applied Biosystems 391型DNA合成仪合成94个引物,它们的长度为21-40核苷酸(nt)并且互补于缅甸HEV株(BUR-121)基因组或SAR-55基因组的正链或负链(Tam,A.W.等人(1991),“病毒学”(″Virology″),185:120-131)。
这94个引物的序列示于下面,从SEQ ID NO.5一直到SEQ ID NO.98。
HEV引物表:引物
ORF
序列
区域D3042B 1 ACATTTGAAT TCACAGACAT TGTGC SEQ ID NO.5R3044B 1 ACACAGATCT GAGCTACATT CGTGAG SEQ ID NO.6D3044B 1 AAAGGGATCC ATGGTGTTTG AGAATG SEQ ID NO.7R3045B 1 ACTCACTGCA GAGCACTATC GAATC SEQ ID NO.8R261S 1 CGGTAAACTG GTACTGCACA AC SEQ ID NO.9D260S 1 AAGTCCCGCT CTATTACCCA AG SEQ ID NO.10D259S 1 ACCCACGGGT GTTGGTTTTT G SEQ ID NO.11R255S 1 TTCTTGGGGC AGGTAGAGAA G SEQ ID NO.12R254S 2 TTATTGAATT CATGTCAACG GACGTC SEQ ID NO.13D242S 1 AATAATTCAT GCCGTCGCTC C SEQ ID NO.14R241S 1 AAGCTCAGGA AGGTACAACT C SEQ ID NO.15R231S 1 AAATCGATGG CTGGGATCTG ATTC SEQ ID NO.16R230S 1 GAGGCATTGT AGAGCTTTGT G SEQ ID NO.17D229S 1 GATGTTGCAC GGACAGCAAA TC SEQ ID NO.18D228S 1 ATCTCCGATG CAATCGTTAA TAAC SEQ ID NO.19D227B 1 TAATCCATTC TGTGGCGAGA G SEQ ID NO.20R218B 2 AAGTGTGACC TTGGTCCAGT C SEQ ID NO.21D217B 2 TTGCTCGTGC CACAATTCGC TAC SEQ ID NO.22D211B 1 CATTTCACTG AGTCAGTGAA G SEQ ID NO.23D202B 2 TAATTATAAC ACCACTGCTA G SEQ ID NO.24R201B 2 GATTGCAATA CCCTTATCCT G SEQ ID NO.25R200S 1 ATTAAACCTG TATAGGGCAG AAC SEQ ID NO.26R199S 1 AAGTTCGATA GCCAGATTTG C SEQ ID NO.27R198S 2 TCATGTTGGT TGTCATAATC C SEQ ID NO.28R193B 1 GATGACGCAC TTCTCAGTGT G SEQ ID NO.29R192B 1 AGAACAACGA ACGGAGAAC SEQ ID NO.30R191B 1 AGATCCCAGC CATCGACTTT G SEQ ID NO.31R190S 2 TAGTAGTGTA GGTGGAAATA G SEQ ID NO.32D189B 2 GTGTGGTTAT TCAGGATTAT G SEQ ID NO.33D188B 2 ACTCTGTGAC CTTGGTTAAT G SEQ ID NO.34R187S 2 AACTCAAGTT CGAGGGCAAA G SEQ ID NO.35D186S 2 CGCTTACCCT GTTTAACCTT G SEQ ID NO.36D185B 2,3 ATCCCCTATA TTCATCCAAC CAAC SEQ ID NO.37D184S 2,3 CTCCTCATGT TTCTGCCTAT G SEQ ID NO.38R181S 2 GCCAGAACGA AATGGAGATA GC SEQ ID NO.39R180B 1 CTCAGACATA AAACCTAAGT C SEQ ID NO.40D179S 1 TGCCCTATAC AGGTTTAATC G SEQ ID NO.41D178B 1 ACCGGCATAT ACCAGGTGC SEQ ID NO.42D177B 2 ACATGGCTCA CTCGTAAATT C SEQ ID NO.43R174B 1 AACATTAGAC GCGTTAACGA G SEQ ID NO.44D173S 1 CTCTTTTGAT GCCAGTCAGA G SEQ ID NO.45D172B 1 ACCTACCCGG ATGGCTCTAA GG SEQ ID NO.46R166B 2 TATGGGAATT CGTGCCGTCC TGAAG(EcoR I) SEQ ID NO.47R143B 1 AGTGGGAGCA GTATACCAGC G SEQ ID NO.48D141B 1 CTGCTATTGA GCAGGCTGCT C SEQ ID NO.49R142S 1 GGGCCATTAG TCTCTAAAAC C SEQ ID NO.50D135B 1 GAGGTTTTCT GGAATCATC SEQ ID NO.51R134B 1 GCATAGGTGA GACTG SEQ ID NO.52R133B 1 AGTTACAGCC AGAAAACC SEQ ID NO.53D132S 2,3 CCATGGATCC TCGGCCTATT TTGCTGTTGC SEQ ID NO.54
TCC(BamHI)D131B 5′NC AGGCAGACCA CATATGTG SEQ ID NO.55R119B 1 GGTGCACTCC TGACCAAGCC SEQ ID NO.56D118B 1 ATTGGCTGCC ACTTTGTTC SEQ ID NO.57R117B 1 ACCCTCATAC GTCACCACAA C SEQ ID NO.58R116B 1 GCGGTGGACC ACATTAGGAT TATC SEQ ID NO.59D115B 1 CATGATATGT CACCATCTG SEQ ID NO.60D114B 1 GTCATCCATA ACGAGCTGG SEQ ID NO.61R112B 2 AGCGGAATTC GAGGGGCGGC ATAAAGAACC AGG SEQ ID NO.62
(EcoR I)R111B 2 GCGCTGAATT CGGATCACAA GCTCAGAGGC SEQ ID NO.63
TATGCC(EcoR I)D110B 2 GTATAACGGA TCCACATCTC CCCTTACCTC(BamHI) SEQ ID NO.64D109B 2 TAACCTGGAT CCTTATGCCG CCCCTCTTAG(BamHI) SEQ ID NO.65D108B 1 AAATTGGATC CTGTGTCGGG TGGAATGAAT SEQ ID NO.66
AACATGTC(BamHI)R107B 1 ATCGGCAGAT CTGATAGAGC GGGGACTTGC CGGATCC SEQ ID NO.67D101B 2 TACCCTGCCC GCGCCCATAC TTTTGATG SEQ ID NO.68R100B 1 GGCTGAGATC TGGTTCGGGT CGCCAAGAAG GTG SEQ ID NO.69
(Bgl II)R99B 2 TACAGATCTA TACAACTTAA CAGTCGG(Bgl II) SEQ ID NO.70R98B 2 GCGGCAGATC TCACCGACAC CATTAGTAC(Bgl II) SEQ ID NO.71D97S 1 CCGTCGGATC CCAGGGGCTG CTGTCCTG(BamHI) SEQ ID NO.72R96B 2 AAAGGAATTC AAGACCAGAG GTAGCCTCCT C SEQ ID NO.73
(EcoR I)D95B 2 GTTGATATGA ATTCAATAAC CTCGACGG SEQ ID NO.74R94B 3′NC TTTGGATCCT CAGGGAGCGC GGAACGCAGA SEQ ID NO.75
AATGAG(BamHI)D90B 2 TCACTCGTGA ATTCCTATAC TAATAC(EcoR I) SEQ ID NO.76R89B 3′NC TTTGGATCCT CAGGGAGCGC GGAACGCAGA SEQ ID NO.77
AATG(BamHI)R88B 1 TGATAGAGCG GGACTTGCCG GATCC(BamHI) SEQ ID NO.78R87B 1 TTGCATTAGG TTAATGAGGA TCTC SEQ ID NO.79D86B 1 ACCTGCTTCC TTCAGCCTGC AGAAG SEQ ID NO.80R81B 1 GCGGTGGATC CGCTCCCAGG CGTCAAAAC(BamHI) SEQ ID NO.81D80B 1 GGGCGGATCG AATTCGAGAC CCTTCTTGG(EcoR I) SEQ ID NO.82R79B 1 AGGATGGATC CATAAGTTAC CGATCAG(BamHI) SEQ ID NO.83D78B 1 GGCTGGAATT CCTCTGAGGA CGCCCTCAC(EcoR I) SEQ ID NO.84R77B 1 GCCGAAGATC TATCGGACAT AGACCTC(Bgl II) SEQ ID NO.85R76B 2 CAGACGACGG ATCCCCTTGG ATATAGCCTG(BamHI) SEQ ID NO.86D75B 5′NC GGCCGAATTC AGGCAGACCA CATATGTGGT SEQ ID NO.87
CGATGCCATG(EcoR I)D72B 1 GCAGGTGTGC CTGGATCCGG CAAGT(BamHI) SEQ ID NO.88R71B 1 GTTAGAATTC CGGCCCAGCT GTGGTAGGTC(EcoR I) SEQ ID NO.89D63B 1 CCGTCCGATT GGTCTGTATG CAGG SEQ ID NO.90D61B 1 TACCAGTTTA CTGCAGGTGT GC SEQ ID NO.91D60B 1 CAAGCCGATG TGGACGTTGT CG SEQ ID NO.92R59B 2,3 GGCGCTGGGC CTGGTCACGC CAAG SEQ ID NO.93D50B 1 GCAGAAACTA GTGTTGACCC AG SEQ ID NO.94R49B 2 TAGGTCTACG ACGTGAGGCA AC SEQ ID NO.95R48B 1 TACAATCTTT CAGGAAGAAG G SEQ ID NO.96R47B 1 CCCACACTCC TCCATAATAG C SEQ ID NO.97D46B 1 GATAGTGCTT TGCAGTGAGT ACCG SEQ ID NO.98
在序列左边的缩写含义如下:R和D分别指反向或正向引物;B和S分别指序列衍生自戊型肝炎缅甸-121株和戊型肝炎SAR-55株;5′NC和3′NC分别指HEV基因组5’和3’非编码区域;1,2和3分别指来自开放阅读框1、2或3的序列。在某些所示序列右边的符号()中给出了插入这些序列中的人工限制性位点。
为了克隆PCR片段,在引物的5′端添加了位于3-7个核苷酸之后的EcoRI、BamHI或BglII限制性位点。
RT-PCR。通常100微升RT-PCR混合物含有:模板、10mM Tris-HCl(pH8.4)、50mM KCl、2.5mM MgCl2、所有4种dNTP(各0.2mM)、50pmol正向引物、50pmol反向引物、40单位RNasin(Promega)、16单位鸟成髓细胞过多症病毒反转录酶(Promega)、4单位AmpliTaq(Cetus),在其上方为100微升轻矿物油。混合物在42℃温育1小时,然后扩增35个PCR循环:94℃1分钟,45℃1分钟和72℃1分钟。PCR产物在1%琼脂糖凝胶上分析。
克隆PCR片段。在末端含有限制性位点的PCR片段用EcoRI和BamHI,或用EcoRI和BglII限制性酶进行消化,然后克隆入EcoRI/BamHI-消化过的pBR322或pGEM3Z(Promega)。或者,用TA克隆试剂盒(Invitrogen)将PCR片段克隆入pCR1000(Invitrogen)。
PCR片段和质粒的测序。从1%琼脂糖凝胶中切取PCR片段,用Geneclean(Bio 101,La Jolla,CA)纯化。按Winship,P.R.(1984),Nucleic Acid Rev,17:1266中所述,用Sequenase(United States Biochemical)测序双链PCR片段。
用Sequenase试剂盒(United States Biochemical)测序经氯化铯梯度纯化的双链质粒。
序列的计算机分析。用7.5版本的Genetics Computer Group(Madison,WI)软件包(Devereaux,J.等人(1984),“核酸研究回顾”,12:387-395),在VAX8650计算机(国立癌症研究院,Frederick,MD)上比较HEV株的核酸序列。
实施例2
构建重组表达载体P63-2
含有HEV株SAR-55基因组的完整ORF-2的质粒(Tsarev,S.A.等人(1992),美国科学院院报,89:559-563)被用于获得限制性片段NruI-BglII。NruI在ORF-2的ATG起始密码子上游5个核苷酸处切割HEV cDNA。刚好在polyA序列之前的HEV基因组3′端以前已有一个人工BglII位点(Tsarev,S.A.等人(1992)。美国科学院院报,89:559-563)。为了将该片段插入pBlueBac-Transfer载体(Invitrogen),在载体唯一的NheI位点处引入一个合成的多接头。该多接头含有HEV cDNA和pBlueBac序列中都缺乏的BlnI和BglII位点。用衔接头将NruI-BglII ORF-2片段插入BlnI-BglII pBlueBac,如图1所示。
实施例3
P63-2在SF9昆虫细胞中的表达
按照Invitrogen的方案,用钙沉淀法将p63-2和AcMNPV杆状病毒(Invitrogen)共转染入SF9细胞(Invitrogen)。按照这个方案,AcMNPV杆状病毒DNA可产生活的、完整的杆状病毒,它会将p63-2包装起来形成重组杆状病毒。通过噬菌斑纯化该重组杆状病毒4次。得到的重组杆状病毒63-2-IV-2被用于感染SF9细胞。
SDS-PAGE和Western印迹法。将昆虫细胞再悬浮于加样缓冲液中(50mMTris-HCl,pH6.8,100mM DTT,2%SDS,0.1%溴酚蓝和10%甘油),然后按Laemmli,U.K.(1970),
″自然″,227:680所述的方法进行SDS-聚丙烯酰胺凝胶电泳。凝胶用考马斯蓝染色,或者将蛋白质电印迹于BA-85硝酸纤维素滤膜(Schleicher&Schuell)。在转化后,硝酸纤维素滤膜在含10%胎牛血清和0.5%明胶的PBS中封闭。将1∶1000稀释的黑猩猩-1313的超免疫血清用作第一抗体。将1∶2000稀释的、磷酸酶标记的、经亲和纯化的、针对人IgG的羊抗体(Kirkegaard & Perry Laboratories,Inc.)用作第二抗体。滤膜在Western蓝稳定化的底物中对碱性磷酸酶进行显色(Promega)。所有的温育都在封闭缓冲液中进行,洗涤是用含0.05%Tween-20(Sigma)的PBS进行。
HEV ORF-2的表达。在用重组杆状病毒63-2-IV-2感染的SF9细胞中合成的主要蛋白是表观分子量为74KD的蛋白质(图2A,泳道3)。该大小比完整ORF-2的预计分子量(71KD)稍大。大小差异可以是由蛋白质的糖基化造成的,因为在N-末端部分有至少一个潜在的糖基化位点(Asn-Leu-Ser)。没有在未感染细胞(图2A,泳道1)或用野生型非重组杆状病毒感染的细胞(图2A,泳道2)中检测到这个蛋白质。在后一种情况下,检测到的主要蛋白质是多角体蛋白。用黑猩猩-1313(已用HEV超免疫过)的血清,通过Western印迹分析同样的裂解液时(图2B),只有重组细胞裂解液中的蛋白发生反应(泳道3),而且主条带仍是74KD的蛋白质(图2B)。在考马斯蓝染色的凝胶中约25、29、35、40-45和55-70千道尔顿的次条带(图2A,泳道3)也在Western印迹中与血清反应(图2B,泳道3)。某些分子量大于74KD的条带是不同程度的糖基化所造成的,而分子量较小的条带可能反映了加工和/或降解。用Western印迹法显示,在用HEV接种之前从黑猩猩-1313中取出的血清不与任何蛋白质反应。
实施例4
重组感染的SF9细胞的免疫电镜显微观察
5×106个重组感染的SF9细胞,在含10mM Tris-HCl,pH7.4,0.3%十二烷基肌氨酸钠的氯化铯(1.30克/毫升)溶液中进行超声波处理,然后在40,000rpm(SW60Ti)离心68小时。将50微升ELISA应答最高的、浮力密度为1.30克/毫升的组份稀释于1毫升PBS中,然后加入5微升黑猩猩-1313的超免疫血清。超免疫血清是通过用戊型肝炎的第二种株(墨西哥HEV)再侵袭已感染过的黑猩猩而制备的。样品在室温下温育1小时,然后在4℃温育过夜。用SW60Ti离心机转头在30,000rpm,4℃下离心2小时而沉淀出免疫复合物。将沉淀再悬浮于蒸馏水中,用3%PTA负染色,置于碳载网上,在电子显微镜EM-10(Carl Zeiss,Oberkochen,德国)上,于40,000放大倍数下检查。
VLP的检测。用野生型杆状病毒或重组杆状病毒63-2-IV-2感染的昆虫细胞的细胞裂解液,通过氯化铯密度离心进行分级。当来自重组感染昆虫细胞的氯化铯梯度组份与黑猩猩-1313超免疫血清混合时,在浮力密度为1.30克/毫升的组份中观察到两种覆盖着抗体的病毒样颗粒(virus-like particle,VLP):第一种(图3A-3A),是覆盖有抗体的各颗粒,其大小(30纳米)和形态结构暗示它是HEV,第二种(图3B),是被抗体包裹的、由小于HEV(约20纳米)但其他方面很象HEV的颗粒构成的聚集体。直接的电子显微镜观察显示,存在非常不均一的物体群,其中包括某些直径分别为30纳米和20纳米的物体,它们看上去象病毒颗粒,但是在没有结合的抗体的情况下,不能被证实为HEV。大量免疫电镜显微试验揭示,至少部分从HEV基因组ORF-2区域合成出的蛋白质已被装配成颗粒结构。观察到,感染晚期的昆虫细胞,此时较小的蛋白质的比例更高,一律在ELISA中给出更佳的结果。因此,来自感染晚期的重组昆虫细胞的未分级裂解液,在随后的试验中被用作ELISA的抗原。
实施例5
在用不同株的HEV感染后,根据来自表达完整的抗HEV ORE-2的昆虫细胞
的抗原而进行ELISA检测
将5×106个用63-2-IV-2病毒感染过的SF9细胞悬浮在1毫升的10mMTris-HCl,pH7.5,0.15NaCl中,然后冻融3次。将10微升这种悬浮液溶解在10毫升碳酸盐缓冲液(pH9.6)中,然后用于覆盖一个柔性的微效价分析板(Falcon)。以1∶20、1∶400和1∶8000,或者以1∶100、1∶1000和1∶10000的比例稀释血清样品。在ELISA中使用Western印迹法中所述的同样的封闭剂和洗涤溶液。作为次级抗体,使用针对人IgG的偶连过氧化酶的羊IgG片段或偶连辣根过氧化物酶的羊抗-旧大陆猴或抗-新大陆猴的免疫球蛋白。结果是在405纳米处通过测量光密度(O.D.)而确定。
为了确定代表HEV巴基斯坦株的、来自昆虫细胞的抗原是否能检测出用HEV墨西哥株感染的猕猴中的抗-HEV抗体,检查了3只猴子(图4)。用含有墨西哥’86 HEV株(Ticehurst,J.等人,(1992),传染病杂志,165:835-845)的粪便感染2只猴子cyno-80A82和cyno-9A97。而第3只猴子用相同株的第二代进行感染。作为对照,在同一试验中测试来自cyno-374(被巴基斯坦HEV株SAR-55感染的)的血清样品。所有3只用墨西哥株感染后的猴子血清转变为抗HEV。用第一代感染的动物在15周时血清转变,而用第二代感染的动物在5周时血清转变。令人感兴趣的是,在4只动物中,最高的抗HEV效价是在cyno-83中发现的,而cyno-83是用墨西哥株的第二代接种的。用第一代墨西哥株接种的猕猴产生最低的效价,而用第一代巴基斯坦株接种的猕猴产生介于其中的效价。
实施例6
根据来自表达完整ORF-2的昆虫细胞的抗原,进行抗-HEV特异性的ELISA
分析
为了估计此处所述的ELISA分析是否排斥其他类型的肝炎相关抗体而特异性地检查抗HEV,分析一组4只用其他已知肝炎病毒(Garci,P.等人(1992),
传染 病杂志,165:1006-1011;Farci,P.等人(1992),
科学(出版中);Ponzetto,A.等人(1987)、
传染病杂志,155:72-77;Rizzetto,M.等人(1981)
肝炎学,1:567-574;有关黑猩猩-1413、1373、1442、1551(HAV)和有关黑猩猩-982、1442、1420、1410(HBV)的参考资料是来自Purcell等人的未发表资料。)感染的黑猩猩的血清样品(表1)。在接种前、接种后5周和15周时的血清样品在1∶100、1∶1000和1∶10000的血清稀释比下进行HEV ELISA分析。用HAV、HBV、HCV和HDV感染的动物中,没有一种血清在ELISA分析中呈现HEV抗体反应,但是
用HEV接种的所有4只黑猩猩都产生了IgM和IgG级的抗HEV抗体。
表1
血清学分析用不同的肝炎病毒(甲型、乙型、丙型、丁型、戊型)
感染的黑猩猩中的抗HEV抗体
黑猩猩 | 接种的病毒 | 对接种病毒发生血清转变的周数 | 前血清 | 接种后的周数 | ||||||
5 | 15 | 20/25 | ||||||||
IgG | IgM | IgG | IgM | IgG | IgM | IgG | IgM | |||
黑猩猩-1413 | HAV | 5 | - | - | - | - | - | - | ||
黑猩猩-1373 | HAV | 7 | - | - | - | - | - | - | ||
黑猩猩-1442 | HAV | 5 | - | - | - | - | - | - | ||
黑猩猩-1451 | HAV | 5 | - | - | - | - | - | - | ||
黑猩猩-982 | HBV | 3 | - | - | - | - | - | - | ||
黑猩猩-1442 | HBV | 7 | - | - | - | - | - | - | - | - |
黑猩猩-11420 | HBV | 9 | - | - | - | - | - | - | ||
黑猩猩-1410 | HBV | 5 | - | - | - | - | - | - | ||
黑猩猩-51 | HCV | 10 | - | - | - | - | - | - | ||
黑猩猩-502 | HCV | 12 | - | - | - | - | - | - | ||
黑猩猩-105 | HCV | 28 | - | - | - | - | - | - | ||
黑猩猩-793 | HCV | 13 | - | - | - | - | - | - | ||
黑猩猩-904 | HDV | 8 | - | - | - | - | - | - | ||
黑猩猩-814 | HDV | 7 | - | - | - | - | - | - | ||
黑猩猩-800 | HDV | 10 | - | - | - | - | - | - | ||
黑猩猩-29 | HDV | 10 | - | - | - | - | - | - | - | - |
黑猩猩-1310 | HEV | 5 | - | - | 1∶10,000 | 1∶100 | 1∶10,000 | - | ||
黑猩猩-1374 | HEV | 3 | - | - | 1∶8000 | -* | 1∶8000 | - | ||
黑猩猩-1375 | HEV | 3 | - | - | 1∶8000 | 1∶400 | 1∶400 | - | ||
黑猩猩-1313 | HEV第一次** | 5 | - | - | 1∶10,000 | 1∶100 | 1∶1000 | - | ||
黑猩猩-1313 | HEV第二次** | 0.5 | 1∶100 | - | 1∶10,000 | - | 1∶10,000 | - |
*黑猩猩-1374在接种后3、4周左右对抗HEV IgM呈阳性(见图5)。
**黑猩猩-1313用HEV接种2次。第一次用7个巴基斯坦病人的混合样品接种,第二次是45个月后用HEV墨西哥株接种。
实施例7
确定HEV SAR-55株在除人之外的灵长目动物中的宿主范围
用HEV的标准粪便悬浮液通过静脉内接种不同的灵长目动物,然后收集一系列的血清样品以监测感染情况。确定血清ALT水平作为有肝炎的标志,而血清转变被定义为抗HEV的增加。将这些结果与在猕猴和黑猩猩中获得的结果进行比较。
用HEV接种的两只恒河猴(表2)都表现出非常明显的丙氨酸氨基转移酶活性和强抗-HEV应答。丙氨酸氨基转移酶活性于第3 5天时在2只猴子中观察到,而血清转变发生于21天。抗HEV的最大效价在29天时达到。在该研究中所用的2只非洲绿猴(表2)都表现出增加的丙氨酸氨基转移酶活性和抗-HEV。尽管非洲绿猴230在接种后7周时死亡,但是在死亡之前已获得了感染证据。对于猴子74而言,丙氨酸氨基转移酶活性的峰值超过接种前血清的平均值约3倍;对于猴子230而言,超过约5倍。在猴子74和230中,丙氨酸氨基转移酶活性和血清转变的峰值分别在28天和21天同时出现。
表2在8种灵长目物种中HEV感染的生物化学和血清学情况
动 物 | 丙氨酸氨基转移酶(单位/升) | 抗-HEV IgG | |||
接种前,平均值(SD) | 天 | 数值 | 第一次检查的日期(效价) | 最大效价 | |
黑猩猩1374 | 51(12) | 27 | 114 | 27(1∶400) | 1∶8000 |
1375 | 41(14) | 27 | 89 | 27(1∶400) | 1∶8000 |
猕猴374* | 46(20) | 26 | 608 | 19(1∶400) | 1∶8000 |
381* | 94(19) | 35 | 180 | 28(1∶20) | 1∶8000 |
恒河猴726 | 43(6) | 35 | 428 | 21(1∶20) | 1∶800 |
938 | 29(10) | 35 | 189 | 21(1∶20) | 1∶800 |
非洲绿猴74 | 72(21) | 28 | 141 | 28(1∶400) | 1∶800 |
230 | 102(45) | 21 | 334 | 21(1∶8000) | 1∶800 |
豚尾猴98 | 37(8) | 21 | 47 | 21(1∶400) | 1∶800 |
99 | 41(6) | 28 | 59 | 21(1∶400) | 1∶800 |
绢毛猴616 | 28(7) | 70 | 41 | - | |
636 | 19(4) | 7,56 | 30 | - | |
松鼠猴868 | 90(35) | 40 | 355 | 41(1∶20) | 1∶20 |
869 | 127(63) | 42 | 679 | 35(1∶20) | 1∶20 |
夜猴924 | 41(7) | 97 | 21(1∶20) | 1∶800 | |
925 | 59(6) | 49,91 | 78,199+ | 21(1∶20) | 1∶800 |
注:-,没有检测到抗-HEV。
*,以前曾经用在细菌中表达的HEV蛋白片段作为抗原进行过研究[18]
+,丙氨酸氨基转移酶的生物式(biomodal)升高。
SD=标准差
豚尾猴99显示,其丙氨酸氨基转移酶活性比接种前的血清平均值的增加>3SD,而豚尾猴98不显示。然而2只猴子都在第21天发生血清转变,而且抗-HEV效价与黑猩猩和旧大陆猴的效价相当。因为在豚尾猴中的丙氨酸氨基转移酶峰值低,所以不能完全排除是免疫而不是感染的可能性。然而,出于多种原因,不太可能是免疫。首先,在2只绢毛猴中都没有观察到免疫。这些绢毛猴只有豚尾猴的1/4大小但接受了相同量的接种物。其次,众所周知在粪便中排泄出的HEV数量一般是非常少的,因而在本研究中所用的0.5毫升10%粪便悬浮液不太可能含有数量足以免疫动物的抗原,尤其是通过静脉内接种时。
在本研究中接种的绢毛猴,其丙氨酸氨基转移酶活性都没有显著上升,也没有出现抗-HEV(表2)。因此,这些动物似乎不被感染。松鼠猴产生抗-HEV,但是水平明显低于黑猩猩和旧大陆猴(表2)。此外,在其他动物中血清转变出现得更晚。松鼠猴868在41天发生血清转变,而869在35天时发生。在大于3个月的监测期中,任何时候的抗-HEV都不大于1∶20,很明显在47-54天达到峰值之后2只动物中的抗-HEV变弱。但是,在2只动物中丙氨酸氨基转移酶活性的增加相当显著,并且与血清转变的时间暂时相关。
夜猴对HEV感染的应答与旧大陆猴物种相似(表2)。2只夜猴在21天血清转变,并且在28天之前抗-HEV效价达到1∶8000的数值。夜猴924的丙氨酸氨基转移酶活性于35天达到峰值,而猴925在49天才达到峰值。
实施例8
在黑猩猩中检测IgM和抗-HEVIgG
在2只黑猩猩中,在接种后约4周其血清ALT水平增加(表2,图5)。2只黑猩猩在ALT酶上升时或更早的时候发生血清转变(图5A,5C)。还测定了黑猩猩抗-HEV IgM的水平。在黑猩猩-1374中,抗-HEV IgM的效价(图5B)没有IgG效价(图5A)那么高,而且在2周后下降。尽管IgG和IgM抗体在20天时首先在该动物中检测到,但是在那一天抗-HEV IgM的效价最高而IgG的效价在那一天最低,但其随后上升并在3个月多的时间停留在几乎相同的水平上。在黑猩猩-1375中,在20天仅检测到抗-HEV IgG(图5D)。该效价比黑猩猩1374中的高,而抗-HEV IgM在整个监测期间都检测到。在该动物中,抗-HEV IgG先在27天被观察到(图5D),而且它在整个试验中基本维持相同水平。
实施例9
基于昆虫细胞中表达的完整ORF-2蛋白的ELISA和基于大肠杆菌中表达的结
构蛋白片段的ELISA之间的比较
为了估计在真核细胞中表达HEV基因组的完整ORF区域是否比在大肠杆菌中表达结构蛋白片段更有利,我们用以前的ELISA中的抗原来重新测试早先已分析过的猕猴血清(Tsarev,S.A.等人(1992),
美国科学院院报,89:559-563;和Tsarev,S.A.等人(1993)
传染病杂志,167:1302-1306),其中使用在细菌中表达的抗原片段(表3)。
表3.基于来自表达完整ORF-2蛋白的昆虫细胞的抗原的ELISA和基于来自
表达结构蛋白片段的大肠杆菌的抗原的ELISA之间的比较
猕猴编号 来自细菌细胞的抗原
来自昆虫细胞的抗原(完整的ORF-2)
(ORF-2片段)* 抗-HEV
第一次检测到抗-HEV 第一次检测到的 效价 最大效价
的日期 日期
猕猴-376 28 21 1∶400 1∶8000
猕猴-369 54 40 1∶100 1∶8000
猕猴-374 19 19 1∶400 1∶8000
猕猴-375 26 26 1∶400 1∶8000
猕猴-379 21 19 1∶100 1∶8000
猕猴-381 28 28 1∶400 1∶8000
*血清还用更不灵敏的ORF-3抗原进行测试[..]。
(Tsarev,S.A.等人(1993)
传染病杂志,167:1302-1306)
对于用ELISA检查的6只猴子中的3只,在昆虫细胞中表达的抗原可比在大肠杆菌中表达的抗原更早地检测出血清转变。使用来自昆虫细胞的抗原,我们能在所有6只猴子的血清中在测试的最高稀释度(1∶8000)下检测到抗-HEV抗体。用来自大肠杆菌的抗原(缅甸株)时,没有获得抗-HEV效价的信息,因为所有的血清仅在1∶100的稀释度下进行测试(Tsarev,S.A.等人(1992),
美国科学院 院报,89:559-563;和Tsarev,S.A.等人(1993)
传染病杂志,167:1302-1306)。
在另一研究中,戊型肝炎病毒SAR-55株以10倍递增进行连续稀释,将10-1至10-5稀释物接种于各对猕猴以测定病毒的效价。测量血清的ALT水平以确定肝炎,而针对HEV的血清抗体用本发明的ELISA法进行测定(数据在图中)或通过Genelab的ELISA法进行测定(三个ELISA,每个ELISA基于Yarbrough等人(
病毒学杂志,(1991)65:5790-5797)的、名称为4-2、3-2和612的抗原中的一种。(数据在图6a-g的底部,阳性为(+),阴性为(-))。所有的样品都按标准进行测定。
在所有接种过的猕猴和所有稀释度的病毒中,本发明的ELISA法都检测到了向抗-HEV IgG的血清转变。
相反,Genelab′s法的结果变化很大,如下面所总结的那样。
表4
病毒的稀释度 Genelab′s ELISA 本发明的ELISA
10-1 未测试 阳性
10-2 对2只动物呈阳性,有限期限内 阳性
10-3 对2只动物呈阴性 阳性
10-4 猕猴389:对IgM和IgG为阳性 阳性
猕猴383:阴性 阳性
10-5 猕猴386:阴性 阳性
猕猴385:阳性 阳性
因为猕猴385(10-5)在用Genelabs和本发明的ELISA测试时都为阳性,所以10-4(接种的病毒大10倍)和10-3(接种的病毒大100倍)也应是阳性的。本发明ELISA将其定为阳性。与之相反,Genelab′s ELISA在10-3时漏了2个阳性,而在10-4时漏了一个阳性,尽管猕猴383的ALT水平暗示为阳性肝炎。因此,这些数据表明,本发明的ELISA法优于已有技术中检测针对HEV抗体的方法。
实施例10
基于重组ORF-2抗原的ELISA比较,其中该抗原或者由从HEV巴基斯坦
SAR-55株的完整ORF-2区域表达出的55kDa蛋白质,或者由较短的作为融合
蛋白在细菌中表达的ORF-2区域的构成
如实施例3中所述和如图2A和2B中所示,在用含完整ORF-2的重组杆状病毒感染的昆虫细胞中表达出许多种不同分子量的蛋白质。如下,在接种后7天时从5×108个SF-9细胞中部分纯化出分子量约为55kDa的蛋白质:离心感染的细胞,再悬浮于10毫升10mM Tris-HCl(pH8.0)、50mM NaCl、含40微克/毫升苯基甲基磺酰氯(Sigma,St Louis,Missouri)的溶液中,超声波破细胞,然后裂解液在4℃于90,000×g离心30分钟。将上清液上样于用10mM Tris-Hcl(pH8.0)、50mM NaCl平衡的DEAE-琼脂糖凝胶CL-6B(PHarmacia,Uppsala,Sweden)柱。用上样缓冲液洗涤柱,然后用10mM Tris-HCl(pH8.0)、250mM NaCl洗脱55kDa蛋白。合并含55kDa蛋白的组分,通过向10毫升蛋白质溶液中加入3克(NH4)2SO4而沉淀出蛋白质。将蛋白质沉淀溶解在10mMTris-HCl(pH8.0)、50mM NaCl中。再将55kDa蛋白用作ELISA分析中的昆虫细胞表达的HEV抗原,与基于细菌表达的两种HEV抗原中任何一种(3-2(墨西哥)(Goldsmith等人,(1992)
柳叶刀,339:328-331)或SG3(缅甸)(Yarbough等人,(1993):Assay Development of diagnostic tests for Hepatitis E在″International Symposium on Viral Hepatitis and Liver Disease.Scientific Programand Abstract Volume.″中,东京:VHFL p.87))而进行的ELSA进行比较试验。这些细菌抗原是26kDa谷胱甘肽S-转移酶(GST)与3-2(M)抗原序列(该序列由位于ORF-2C末端上游6个氨基酸处的42个氨基酸构成)(Yarbough等人,(1991)病毒学杂志,65:5790-5797),或者与ORF-2的327个C末端氨基酸形成的融合蛋白(Yarbough等人,(1993))。ELISA如下进行。
将处于碳酸盐缓冲液(pH9.6)中的60ng/孔55kDa蛋白或200ng/孔融合抗原在聚苯乙烯微量滴定板(Dynateck,S.Windham,ME)于37℃温育2小时。板用含10%胎牛血清和0.5%明胶的PBS进行封闭。来自用含10-1-10-8(如表5和图7A-7J和8A-8D中所示)SAR-55株HEV的粪便稀释液静脉内接种的猕猴的血清样品(注意:猕猴389和392是口服接种的),在封闭液中以1∶100稀释。将1∶1000或1∶2000稀释的偶连过氧化物酶的羊抗-人IgM(Zymed,San Francisco,CA),或者按1∶1000稀释的过氧化物酶标记的羊抗-人免疫球蛋白用作检测抗体。
在除了针对2只口服接种猴子(猕猴-387和猕猴-392)之外的所有的ELISA测试中,在同一实验室中同时测试55kDa抗原和融合抗原,这样仅有的可变因素便是所有的抗原。评分与55kDa抗原的抗HEV ELISA中阳性反应的标准是,光密度值≥0.2并且大于同一动物的接种前血清样品光密度值的2倍。此外,因为在细菌中表达的2种抗原都是与GST的融合蛋白,所以与这些抗原测试的样品的光密度值必须比用非融合的GST获得的数值大3倍才能被认为是阳性的(Goldsmith等人,1992)。
结果
用10-1稀释度的标准HEV粪便悬浮液接种的2只猕猴(377,378),其ALT活性在接种4-5周后有明显增加,这表示有肝炎(表5,图7A和7B)。
表5 用含10-1-10-8稀释度的SAR-55HEV接种物标准液接种后,
在猕猴中发生的生物化学和血清学事件的总结
猕猴 | 病毒接种物原液的稀释度 | ALT | 55kDa抗原试验中检测到抗-HEV峰时的接种后周数 | 融合抗原试验中检测到抗-HEV时的接种后周数 | ||||||
接种前平均值(SD)1 | 周 | 峰值(U/L) | IgG | IgM | IgGSG3 3-2(M) | IgMSG3 3-2(M) | ||||
377 | 10-1 | 76(39) | 5 | 264 | 4-15+ | 3-7 | 4-10 | 4-5 | 3-4 | 3-5 |
378 | 10-1 | 50(9) | 4 | 285 | 4-15 | - | - | - | - | - |
394 | 10-2 | 62(14) | 4 | 89 | 3-15 | 3-10 | - | 4-6 | - | - |
395 | 10-2 | 121(21) | 15 | 314 | 5-15 | - | - | - | - | - |
380 | 10-3 | 89(20) | 1 | 135 | 5-15* | - | 6-15 | - | - | - |
383 | 10-3 | 29(8) | 4 | 77 | 5-15 | 5-13 | - | - | - | - |
389 | 10-4 | 60(7) | 15 | 114 | 6-15 | 6-8 | - | - | - | - |
393 | 10-4 | 41(4) | 5 | 87 | 6-15 | - | - | - | - | - |
385 | 10-5 | 59(32) | 7 | 56 | 11-15 | - | - | 7-15 | - | - |
386 | 10-5 | 31(4) | 4 | 34 | 8-15 | 8-13 | - | - | - | - |
397 | 10-6 | 60(4) | 8 | 94 | - | - | - | - | - | - |
398 | 10-6 | 36(3) | 2 | 55 | - | - | - | - | - | - |
399 | 10-7 | 102(16) | 2 | 93 | - | - | - | - | - | - |
400 | 10-7 | 57(4) | 9 | 188 | - | - | - | - | - | - |
403 | 10-8 | 33(3) | 2-3 | 49 | - | - | - | - | - | - |
406 | 10-8 | 56(4) | 2 | 73 | - | - | - | - | - | - |
387 | 10-1(口服)§ | 32(4) | 4 | 38 | - | - | ND | - | ND | - |
392 | 10-1(口服)§ | 49(6) | 3 | 70 | - | - | ND | - | ND | - |
1接种前血清的ALT平均值和标准差(SD)数值。
+该试验在15周后结束。
*猕猴-380的接种前血清的OD值,当用55kDa抗原进行ELISA进行测试时,为其他猕猴接种前血清平均值的2倍高。
§除了猕猴-387和猕猴-392之外的所有ELISA测试都在相同试验中进行。
-未检测。ND-未进行。
所有3种测试的抗原都在从猕猴377中于接种后3周取出的样品中检测到抗-HEV IgM(表5,图8A),但是不能在来自第二只猴子猕猴-378的所有样品中检测到抗-HEV IgM。在基于55kDa的ELISA中,在2只猴子中都检测到抗-HEVIgG,但是在基于融合抗原的ELISA中仅在猕猴-377中被检测到。抗-HEV IgG的OD值明显高于抗-HEV IgM的OD值。用55kDa抗原获得的ELISA值也明显高于用2种融合抗原中任一种所获得的ELISA值(图7A和7B)。在2种来源的抗原的ELISA中所观察到的OD值的模式也明显不同。在基于融合抗原的ELISA中,阳性信号在血清转变后不久便到达最大值,然后在15周的试验中下降。在基于55kDa抗原的ELISA中,阳性信号在血清转变后不久便到达最大值,并且试验中(15周)基本维持一样高的水平。
在2只用10-2稀释度标准HEV粪便悬浮液接种的猴子(394和395)中ALT活性的升高情况,在预期的肝炎时间明显低于用高10倍剂量接种的动物(表5,图7C和7D)。在接种前以及在接种后15周时,猕猴-395确实有更高的ALT活性。后者可能与HEV感染无关。弱阳性的抗-HEV IgM仅在猕猴-394中(图8B)和基于55kDa抗原的试验中检测到。然而,2只动物都是被感染的,因为在2只动物中都观察到抗-HEV血清转变。在猕猴-394中,在第3周首先由55kDa抗原以及在晚一周之后用3-2(M)检测到抗HEV IgG。SG(3)抗原不能在猕猴-395中检测出血清转变,而且抗-HEVIgG仅被55kDa抗原检测出。在该动物中,抗HEV的效价倾向于随时间而下降。
猕猴-380和猕猴383是用10-3稀释度标准HEV粪便悬浮液接种的(表5,图7E、7F和8C)。猕猴-380在接种之前和之后有波动的ALT活性;因此,在该动物中ALT水平不能用于记录戊型肝炎。在猕猴-383中,观察到ALT活性的轻微上升(图7F),这与血清转变同时发生,因此这可能是中度的戊型肝炎造成的。在猕猴-380的血清中,用3种抗原中的任何一种都不能检测出抗-HEV IgM。当用SG3(B)测试时,猕猴-380发生了向抗-HEV IgG的血清转变,但是用3-2(M)抗原测试时不显示。虽然用55kDa抗原测试时,该动物有已有的抗-HEVIgG,但是从5周时开始抗-HEV IgG有大大的增加(图7E)。早先已报道过在来自另一猕猴的血清中鉴别出早已存在的抗体(Ticehurst,J.等人,(1992),
传染 病杂志,165:835-845;Tsarev等人,(1993)
传染病杂志,168:369-378)。在预计时间发生了血清转变,但是在来自猕猴-383的样品中抗-HEV IgG水平仍低,仅能用55kDa抗原检测出。
猕猴-389和猕猴-393是用10-4稀释度标准HEV粪便悬浮液接种(图7G、7H、8D和表5)。没有一只动物的ALT活性明显上升,尽管在5周时在猕猴-393的血清中确定了一个小的但明显的ALT峰(图7H),这暗示界线型肝炎。基于SG(B)或3-2(M)的ELISA将2只动物都评为HEV感染阴性。与之相反,55kDa抗原于接种后6-8周在猕猴-389的血清中检测到抗-HEV IgM(图8D)而且在6周-15周时在2只动物中都检测出抗-HEV IgG。
没有1只用10-5稀释度标准HEV粪便悬浮液接种的动物产生ALT活性的明显上升(表5),但是在猕猴-386中,在8-13周和8-15周用55kDa抗原分别检测出抗-HEV的IgM和IgG(图7J,8E)。猕猴-385的抗-HEV IgG可用55kDa和3-2(M)抗原检测出但不能用SG3(B)抗原检测出。与前面模式相反,用融合蛋白检测出抗-HEV IgG的时间比用55kDa抗原检测出早4周,而且检测出的抗-HEV IgG水平也更高。
没有1只用10-6至10-8稀释度标准HEV粪便悬浮液接种的动物产生对3种抗原的任一种的抗体,而且在那些动物中没有观察到ALT活性的上升,除了在猕猴-400中于第9周时有一个相当显著的ALT活性峰(表5)。然而,在该动物中没有血清转变的情况表明,这个峰可能与HEV感染无关。
至于用10-1稀释度的10%粪便悬浮液口服接种2只猕猴(387和392)中,没有一只猴子被感染,因为ALT水平没有上升而且用3-2(M)和55kDa抗原进行的ELISA没有检测出对HEV的血清转变(表5)。
最后,在用10-1至10-5稀释度的粪便悬浮液接种的所有动物中都发现了HEV感染的血清学证据;在接受更高稀释度的动物中没有这种证据。用ALT升高而确定的明显肝炎,仅在用10-1稀释度显示的2只猴子中观察到。在某些用更高稀释度粪便悬浮液接种的动物中观察到ALT活性低得多的升高,而在另一些动物中则没有发现升高。单独考虑时,这些低幅度的ALT上升不能诊断肝炎。但是血清转变和这些ALT峰出现的同时发生暗示,在这些动物中存在中度的肝炎。在用10-1至10-5稀释度的粪便悬浮液接种的所有动物中都检测到抗-HEV IgG血清转变。在用更高稀释度原液接种的动物中,观察到抗-HEV IgG的水平趋于下降而且血清转变的时间趋晚。
总之,55kDa巴基斯坦ORF-2抗原比3-2(M)或SG3(B)抗原更有效地检测被HEV巴基斯坦株感染的猕猴中的抗-HEV IgM和IgG。例如对于除猕猴-385之外的所有动物血清,用55kDa抗原都比3-2(M)或SG3(B)抗原更有效地检测出抗-HEV IgM和IgG。在所有10只用10-5或更低稀释度粪便接种的动物中检测抗-HEV IgG时,用55KDa抗原进行的ELISA产生了内在的一致的和可重复的结果。ELISA信号的大小也随着接种物的被稀释而下降。融合抗原在各对动物之间不产生一致的结果。在用10-1、10-2、10-3或10-5稀释度接种的各对动物中仅有1只显示出向抗-HEV IgG的血清转变,而且用这些抗原仅检测出一个向抗-HEV IgM的血清转变。没有1只用10-4稀释度的原接种物接种的动物发生针对两种融合抗原中任何一种的血清转变,尽管1只动物(猕猴-393)用55KDa抗原分析时有持续高水平的抗-HEV IgG。如上所述,尽管对抗-HEV IgM的ELISA的灵敏度明显低于对猕猴抗-HEV IgG的ELISA的灵敏度,但是与3-2(M)或SG3(B)抗原相比55KDa抗原能够在更多的动物中检测出抗-HEV IgM。总之,有关该研究中所用的巴基斯坦病毒接种物感染效价的确定性的结论,不能根据基于3-2(M)和SG3(B)的ELISA的合并数据而得出,但是可仅根据55KDa巴基斯坦株的ELISA获得的数据而得出。
至于猕猴-385,在2个测试结果中的抗-HEV IgG的差别是4周。这些数据暗示,在3-2(M)抗原上存在区别性的、在较大的55kDa和SG3(B)抗原上不存在的、被该动物识别的表位。当含有完整ORF-2(75kDa)和55kDa蛋白的全昆虫细胞裂解液被用作抗原重新测试这些样品时,结果与仅用55kDa时的结果一样。这个发现暗示,55kDa蛋白可能不缺少3-2表位氨基酸,而是在该研究中所用的3种抗原中的3-2表位序列的构象有差别。最后,感兴趣地注意到,尽管抗原SG3(B)含有更长的ORF-2部分而且包含表位3-2的全部序列,但是它不能比3-2(M)抗原检测出更多的阳性血清。
实施例11
用RT-PCR确定HEV SAR-55病毒原液的感染效价
有关接种物感染效价的知识对于解释在动物模型的试验感染中许多获得的数据是至关重要的。然而,直到目前为止,还没有报道过HEV病毒原种的感染效价。抽提10倍稀释的、含有HEV SAR-55株的粪便悬浮液,然后如下进行RT-PCR扩增,以确定能检测出HEV基因组的最高稀释度。将200微升悬浮液与0.4毫升1.5M NaCl加15%聚乙二醇(PEG)8000混合,在4℃保温过夜。在微离心机(Beckman,Palo Alto,CA)上于16,000g离心3分钟而收集沉淀,然后将沉淀溶于475微升溶液(含4.2M异硫氰酸胍,0.5%N-月桂酰基肌氨酸、0.25MTris-HCl(pH8.0),0.15M二硫苏糖醇(DTT)和1.0微克tRNA)中。接着加入50微升1M Tris-HCL(pH8.0)、100mM EDTA和10%SDS。RNA用苯酚-氯仿(1∶1)抽提2次,然后在室温下用氯仿抽提一次。向上相中加入250微升7.5M乙酸铵,用0.6毫升2-丙醇沉淀出核酸,用75%乙醇洗涤,用100%乙醇洗涤,然后用于逆转录(RT)PCR。
为了检测HEV基因组,使用2套代表SAR-55基因组的3′区域(ORF-2)序列的嵌套引物。用于逆转录和第一次PCR的引物分别为SEQ IDNO.:99:GTATAACGGA TCCACATCTC CCCTTACCTC和SEQ ID NO.:100:TACAGATCTATACAACTTAA CAGTCGG。而用于第二次PCR的引物分别是SEQ IDNO.:101:GCGGCAGATC TCACCGACAC CATTAGTAC和SEQ ID NO.:102:TAACCTGGATCCTTATGCCG CCCCTCTTAG。将RNA沉淀溶解在20微升0.05M Tris-HCl(pH7.6)、0.06M KCl、0.01M MgCl2、0.001M DTT、40单位的RNasin(Promega Biotec,Madison,WI)、16单位禽成髓细胞瘤病毒逆转录酶(Promega Biotec),和10pmol逆转录引物,然后在42℃温育1小时。向20微升逆转录酶混合物中加入100微升0.01M Tris-HCl(pH8.4)、0.05M KCl、0.0025M MgCl2、0.0002 M各种dNTP,50pmol直接引物,50pmol逆转录引物和4单位AmpliTaq(Perkin-Elmer Cetus,Norwalk,CT),再覆盖100微升轻矿物油。HEV cDNA扩增35个循环:94℃1分钟,55℃1分钟和72℃1分钟。PCR产物在1%琼脂糖凝胶上分析。然后将5微升这种混合物用于第二轮的相同条件下的扩增反应,不同点是延伸时间增加到3分钟。
用10-1至10-5的各种稀释度的标准HEV粪便产生的RT-PCR产物(图9),在2%琼脂糖凝胶上分离,然后通过溴化乙锭(EB)对凝胶染色而进行检测。观察到在更高的稀释度下特异PCR产物数量的下降,而检测出HEV基因组的10%粪便悬浮液的最高稀释度为10-5。因此,考虑到稀释因素,HEV基因组效价约为106.7/克粪便。
此外,仅那些用RT-PCR显示含有HEV基因组的稀释度是对猕猴有感染的。因此,标准粪便悬浮液的感染效价及其用RT-PCR检测的基因组效价基本上相同。在RT-PCR和感染力效价之间的类似相关关系在另一丙型肝炎株中发现过(Cristiano等人,(1991),
肝炎学,14:51-55;Farci等人(1991)
新英格兰医学杂 志,25:98-104;Bukh等人,(1992)
美国科学院院报,89:187-191)。
实施例12
将ORF-2蛋白用作疫苗进行主动免疫
和用抗-HEV阳性恢复血清进行被动免疫
在基于55KDa ORF-2蛋白的ELISA中为HEV抗体阴性(<1∶10)的猕猴(Macaca fascicularis)在BL-2生命危险防范下被单独居住,然后将含巴基斯坦HEV株SAR-55的、已稀释至含有10,000或1000 CID50的粪便悬浮液(于胎牛血清中)用于静脉内接种动物。
对于主动免疫研究,从5×108个在感染后7天收获的SF-9细胞中纯化出杆状病毒重组表达的55kDa ORF-蛋白,如实施例10中所述。将3毫克纯化的55kDa蛋白用明矾沉淀,然后用0.5毫升含有50微克明矾沉淀出的55kDa蛋白的疫苗,通过肌内注射而免疫8只猕猴。4只猴子接受一次剂量,而另4只猴子接受二次剂量,其中间隔4周。在最后一次免疫后4周,用1,000-10,000CID50的HEV静脉内侵袭这些灵长目动物。
4只猕猴在主动免疫研究中作为对照。猕猴-412和413接受一次空白对照剂(0.5毫升磷酸盐缓冲液),而猕猴-397和849接受了2次空白对照剂。对照动物用1,000-10,000CID50的HEV进行侵袭。
对于被动免疫研究,用0.5毫升10%混合的、含有两种中国HEV分离株KS1-1987和KS2-1987的粪便悬浮液感染猕猴-384,然后在恢复期从动物体重复收集血浆(Yin等人,(1993)
医学病毒学杂志,41:230-241)。约1%猕猴-396和猕猴-399的血液以及10%猕猴-401和猕猴-402的血液用来自猕猴-384的、HEV抗体效价为1∶10,000的恢复期血浆加以替换。在注入血浆后2天,用1000CID50HEV侵袭动物。作为对照,在用HEV感染之前,10%猕猴-405的血液用从猕猴-384获得的抗-HEV阴性血浆加以替换。然后用1000CID50 HEV侵袭猕猴-405。
对于被动和主动免疫研究,在接种前和接种后15周,对肝进行皮肤针刺活组织检查和收集血清和粪便样品。用市售的测试手段(Metpath Inc.,Rockville,MD)测试血清的丙氨酸转移酶(ALT)水平,并将患肝炎的生物化学证据定义为ALT增加2倍或更多。肝活组织检查按标准进行,而采用的抗-HEV ELISA描述于实施例10中。RNA抽提和RT-PCR的进行如实施例11中所述,不同点在于:按制造商的方案,用TRIzol试剂(Gibco BRL,Gaithersburg,Maryland)抽提来自100微升血清或来自100微升粪便悬浮液的RNA。对于定量,合并来自各动物的PCR阳性系列血清或粪便,然后在胎牛血清中按10倍进行连续的稀释。将100微升各种稀释液如该实施例前面所述的那样,用于RNA抽提和RT-PCR。用于该研究的PCR方案检测水平可低至10 CID50 HEV/毫升血清和低至100 CID50/毫升粪便。
对于接种之前5周和接种之后15周的每周血清样品的ALT峰值,对各个动物以比例(之后/之前)的形式表示。用Simes测试法,将对照组动物的该比例的几何平均值与被动或主动免疫的动物的数值进行比较(Simes,R.J.(1986)Biometrika,73:751-754)。
用Wilcoxon测试法(Noether,G.(1967)
非参数统计学要素(Elements of nonparametric statsitics)(John Wiley & Sons Inc.,New York),pp.31-36),将对照组动物的病毒血症和散布在粪便中病毒的持续时间以及HEV基因组效价与被动或主动免疫的动物进行比较。用相同的测试比较被动免疫和主动免疫动物之间的上述各参数。
对于统计分析,在1毫升血清中HEV基因组<10的血清样品被指定为效价为1∶1,而在1克粪便中HEV基因组<100的粪便样品被指定为效价为1∶10。
结果
在非免疫动物中戊型肝炎的感染进程。
在5只用HEV侵袭的非免疫动物中的3只,记录到了肝炎的生物化学证据,因为血清ALT值增加至少2倍。在2只动物中,没有发现ALT活性的明显增加。但是,组织病理学数据记录了所有5只动物中的肝炎,如表6所示。表6在接种过的动物和对照动物用HEV侵袭后的组织
病理学、生物化学、血清学和病毒学情况
*在肝中的坏死.炎症变化分成1+、2+、3+和4+,然后将每周的评分加起来。
+免疫预防
;在猕猴-396中在2周内检测到1+等级的坏死一炎症变化,但是它们与仅持续一周的病毒性肝炎相符。
§猕猴013在侵袭后9周死亡。
坏死-炎症变化一般在1+至4+的尺度下为1+和2+之间,而且在具有这种升高中的动物中暂时性地与ALT活性的升高相关。
所有的对照动物在侵袭后3-5周发生血清转变,并产生为1∶1,000至1∶32,000最大HEV抗体效价。在感染即肝炎的严重程度和抗-HEV应答水平之间有良好的相关性。猕猴-405具有最高的累积肝炎评分,它也具有最长时间的病毒血症和病毒排泄以及最高水平的抗-HEV(表6)。散布在粪便中的病毒的持续时间与病毒血症的持续时间一样长或更长。对于所有的对照动物,在血清中的HEV基因组效价(10-3至10-4.7)比在粪便中效价(10-5.7至10-7)低。在所有这些5只动物中,病毒血症和散布在粪便中的病毒在4-11周内被检测到,即平均为在血清转变后4.2周内被检测到(从2-9周)。
被动免疫。对于猕猴-396和399,约1%的它们血液用抗-HEV阳性恢复期血浆加以替换,结果在转输(侵袭的时刻)后2天测定时它们的HEV抗体血浆为1∶40(表6)。在转输后1周时在2只动物中观察到HEV抗体效价的2倍下降,而且在转输后2周内HEV抗体下降至可检测水平(<1∶10)之下。在猕猴-396中在侵袭后5周而在猕猴-399中在侵袭后4周,同样检测到抗-HEV,这表示发生了HEV感染。在侵袭后9-10周达到最大的HEV抗体效价(1∶8,000)。二只猕猴中没有一只在侵袭后表现出ALT活性的明显上升。在猕猴-396中检测到肝炎的组织学证据,而且在2只动物的血清和粪便中都检测到HEV基因组(表6)。
猕猴-401和402的约10%血液用恢复期血浆加以替换。在转输(侵袭的时刻)后2天,在2只猕猴中的HEV抗体效价为1∶200(表7)。
表7在对照的和免疫的猕猴中的HEV抗体情况
对照动物 | HEV抗体 | 被动免疫动物 | HEV抗体 | 主动免疫动物 | HEV抗体 | ||||
效价(第一次检测到的周) | 最大效价(周) | 侵袭时的效价 | 最大效价(侵袭后的周) | 最大效价(第一次检测到的周) | 最大效价(第二次免疫后的周) | 最大效价(侵袭后的周) | |||
猕猴-405 | 1∶80(3) | 1∶32,000(9) | 猕猴-396 | 1∶40 | 1∶8000(10) | 猕猴-003 | 1∶10,000(3) | 1∶10,000(5) | |
猕猴-412 | 1∶100(5) | 1∶10,000(7) | 猕猴-399 | 1∶40 | 1∶8000(9) | 猕猴-009 | 1∶10,000(3) | 1∶10,000(1) | |
猕猴-413 | 1∶100(5) | 1∶10,000(7) | 猕猴-401 | 1∶200 | 1∶4000(6) | 猕猴-013 | 1∶100(2) | 1∶10,000(3) | |
猕猴-849 | 1∶100(3) | 1∶10,000(5) | 猕猴-402 | 1∶200 | 1∶80(12) | 猕猴-414 | 1∶1,000(3) | 1∶1,000(0) | |
猕猴-397 | 1∶100(3) | 1∶10,000(7) | 猕猴-398 | 1∶1,000(3) | 1∶10,000(5) | 1∶10,000(0) | |||
猕猴-407 | 1∶1,000(4) | 1∶10,000(5) | 1∶10,000(0) |
抗-HEV在两只动物中于侵袭后15周连续地进行检测,在猕猴-401中达到最大效价1∶4,000但在猕猴-402中最大效价仅为1∶80。在猕猴-401中达到最大效价1∶4,000但在猕猴-402中最大效价仅为1∶80。生物化学和组织学分析没有揭示在2只动物中有肝炎。然而,在2只动物中,观察到病毒血症和散布在粪便中的病毒,这表明发生了感染(表6)。因此,达到较高抗体效价的被动性免疫预防可以保护猕猴抵抗HEV侵袭后的肝炎。
主动免疫。用50微克55kDa蛋白免疫的4只灵长目动物产生了针对重组蛋白的、效价为1∶100-1∶10,000(表7)的抗体。一头动物(猕猴013)在侵袭后9周死于一次麻醉事故,并包含在分析中(表6)。接受双剂量抗原的4头动物产生效价为1∶10,000的HEV抗体。4头动物中的2头在HEV静脉内侵袭后死亡。这也可能是麻醉事故的结果,但是没有确定确切的病原。这2头动物从进一步的分析中删去。6只余下的动物中没有一只在侵袭后产生异常的ALT水平或肝炎的组织学证据(表6)。用1或2剂量的55kDa蛋白免疫的猕猴没有得病毒血症。然而,4只接受1个剂量的免疫原的动物中有3只在粪便中排泄病毒。与之相反,在2只接受2个剂量疫苗的受侵袭动物中都没有观察到排泄病毒。
大多数主动免疫动物比被动免疫动物产生更高的HEV抗体。然而,与用抗-HEV血浆被动免疫的2只动物中1∶200的效价相比,猕猴-013在侵袭时HEV抗体效价为1∶100。但是,猕猴-013显示出比被动免疫动物更大的抗HEV感染的保护能力。猕猴-009(它在侵袭时的HEV抗体的效价为1∶1000)是完全抗肝炎和HEV感染的(表6)。与之相反,尽管在侵袭时的HEV抗体效价为1∶10,000,但是猕猴-003被感染而且在粪便中有散落的HEV。但是在该动物以及接受1个剂量免疫原且HEV抗体效价为10,000或更高的猕猴中,没有检测到肝炎或病毒血症。
对照的和免疫的动物中HEV感染进程的比较。
如用组织病理学手段检测的那样,所有免疫的动物,除了一只被动免疫的猴子,都在用HEV静脉内侵袭后有抗肝炎的保护。将对照组与被动免疫和主动免疫动物之间的肝炎严重程度和病毒复制水平的平均值的比较揭示,一般地,感染严重程度与侵袭时HEV抗体效价反相关,而且以下列次序递减:未免疫的>被动免疫(1%)>被动免疫(10%)>主动免疫(1剂量)>主动剂量(2剂量)(表6,8)。然而,在被动和主动免疫动物的2个亚组中的每一组中的动物数目都不足以进行统计分析。因此,统计分析是将合并后的被动免疫组和合并后的主动免疫组分别与合并的对照组进行的。该分析的结果列于表8。表8在对照的和免疫的动物中HEV感染的平均值总结
*几何平均值
+被动和主动免疫预防
α-P<0.01
β-P<0.05
γ-不显著
它们显示,在对照组中的组织病理学评分和组织变化的持续时间与被动或主动免疫的动物在统计学上是不同的。当与被动或主动免疫的动物相比时,在对照组中ALT峰值的更高的接种前/接种后比例在统计上是显著的,这表明,在2组免疫动物中部有针对肝炎生物化学显示的保护。病毒血症的持续时间和粪便中HEV的效价在2组免疫动物中明显低于对照组。然而,病毒散落持续时间和血清中HEV效价的差异在对照组和被动免疫组之间无统计学差异,尽管当对照组与主动免疫组进行比较时这些参数是显著不同的。对于病毒血症和粪便散落以及HEV效价,还在被动免疫组和主动免疫组的动物之间发现了显著差异。
总之,在表6-8中所示的数据显示,被动地或主动地获得的HEV抗体两者都保护猕猴在用毒HEV侵袭后抵抗肝炎。尽管在用1,00-10,000 CID50的SAR-55侵袭后,所有的5只未免疫的猕猴都产生了肝炎的组织学证据,但是被动性地获得1∶200抗体效价的2只动物没有患肝炎,而且抗体效价低至1∶40的2只动物中的1只也没有患肝炎。
然而,应注意,主动免疫动物表现出完全的抗肝炎保护而且能比被动免疫动物更有效地抗HEV感染。例如,与被动免疫动物中获得的结果不同,在用HEV侵袭后没有在主动免疫动物中检测到病毒血症。在用重组55kDa蛋白免疫一次或两次后,可在猕猴中达到高至1∶10,000的HEV抗体效价。尽管1只猴子(013)在主动免疫后产生1∶100的效价,但是这个水平仍然防止了肝炎和病毒血症。
主动免疫研究还表明,尽管单剂量疫苗可防止HEV病毒血症,但是仍可检测到在粪便中散落的病毒。但是,观察到2剂量的疫苗可防止肝炎和HEV感染的所有症状。因此,这些暗示,例如给个体在外出旅行前施用单剂量的免疫可以保护它们在高危环境中不得戊型肝炎。
最后,注意到给出的结果与以前报道的、对非人类的灵长动物进行被动和主动免疫预防以防甲型肝炎而获得的结果非常类似:被动性免疫预防可防止肝炎但是不能防止感染,而接种不仅可防止肝炎而且可防止HAV感染(Purcell,R.H.等人(1992)
疫苗,10:5148-5149)。此处所给出的有关HEV免疫预防的研究与以前的针对HAV的免疫预防的研究,在确定保护的抗体效价(<1∶100)和在用毒病毒静脉内侵袭后的结果这2方面是相一致的,这是人们感兴趣的。因为其他研究已经表明了在人中被动性和主动性获得相当效价的抗-HEV的有效性,而且已经证实了在肝炎研究中灵长动物研究的预测性价值(Stapleton,J.,等人(1985)胃肠学,89:637-642;Innis,B.L.,等人,疫苗,10:S159),所以很有可能用这些猕猴中的结果可预期在人中可起保护作用。
实施例13
在酵母中完整ORF-2蛋白和更小分子量片段的直接表达
4种cDNA ORE-2片段编码:
1.完整的ORF-2蛋白(氨基酸1-660,分子量70979),片段1778-1703。(其中片段号码指下面给出的引物号码)
2.从第34个氨基酸开始的ORF-2蛋白(氨基酸34-660,分子量67206),片段1779-1703
3.从第96个氨基酸开始的ORF-2蛋白(氨基酸96-660,分子量60782),片段1780-1703
4.从第124个氨基酸开始的ORF-2蛋白(氨基酸124-660,分子量58050),片段1781-1703
它们是通过PCR而获得的,其中将质粒P63-2用作模板并使用下列合成的寡核苷酸:
SEQ ID NO.:103(反向引物#1073):GCACAACCTA GGTTACTATA ACTCCCGAGTTTTACC,
SEQ ID NO.:104(正向引物#1778):GGGTTCCCTA GGATGCGCCC TCGGCCTATTTTG,
SEQ ID NO.:105(正向引物#1779):CGTGGGCCTA GGAGCGGCGG TTCCGGCGGTGGT
SEQ ID NO.:106(正向引物#1780):GCTTGGCCTA GGCAGGCCCA GCGCCCCGCCGCT
和SEQ ID NO.:107(正向引物#1781):CCGCCACCTA GGGATGTTGA CTCCCGCGGCGCC。
在SEQ ID NO.:103-107中所示的所有序列都在其5′端的4个核苷酸之后含有人工序列CCTAGG。人工序列是Avr II(Bln I)限制酶的识别位点。用Bln I切割合成的PCR片段,然后将其连入pPIC9载体(图10)(Invitrogen)的Avr II位点。通过限制性分析而确定方向正确的片段,其中使用存在于ORF-2序列和载体上的不对称EcoR I位点。按照Invitrogen的方案,使用纯化的重组质粒pPIC9-1778(含有片段1778-1703)、pPIC9-1779(含有片段1779-1703)、pPIC9-1780(含有片段1780-1703)和pPIC9-1781(含有片段1781-1703)转化酵母原生质球(Picha株)。用相同的方案进行重组克隆的筛选和表达的分析。如本申请中所述,这些表达的蛋白质可用作疫苗中的免疫原和用作免疫分析中的抗原。最后,本领域的技术人员会意识到,用于所述实施例的载体和酵母菌株可以用其他载体(如pHIL-F1;Invitrogen)或酵母菌株(如酿酒酵母)。
实施例14
在用重组杆状病毒63-2-IV-2感染的SF-9昆虫细胞中合成的HEV ORF-2基因
产物的纯化和氨基端序列分析
如实施例10中所述,用重组杆状病毒63-2-IV-2感染SF-9细胞并在接种后7天收获。昆虫细胞裂解液在SDS-PAGE上的主蛋白条带的分子量约55kDa。该55kDa条带的进一步纯化是使用DEAE-琼脂糖凝胶和150-450mM NaCl梯度,通过离子交换柱色谱而实现的。通过SDS-PAGE然后进行考马斯蓝染色而分析DEAE组分是否存在55kDa条带。峰组分在无SDS的聚丙烯酰胺凝胶电泳中被分离成55kDa、61kDa和中等分子量的3条条带。通过氨基端微蛋白测序对来自聚丙烯酰胺凝胶的各蛋白质条带进行的分析揭示,55和61kDa蛋白共有SEQ IDNO.:2 Ala-112的单一N-末端。据信,2种ORF-2切割产物的大小差别可能反映了不同的糖基化方式或者更大产物的羧基端切割。
在聚丙烯酰胺凝胶上的第三种中等的蛋白质是杆状病毒的壳多糖酶蛋白。通过对DEAE峰组分进行反向HPLC(使用微孔系统和NaCl及乙腈溶剂),将55和61kDa ORF-2蛋白分成不含壳多糖酶的单一的对称峰组分。
实施例15
55KDa和61kDa切割产物的直接表达
通过PCR,用质粒p63-2作为模板而获得编码从第112个氨基酸开始的ORF-2蛋白(ORF-2的氨基酸112-660)的cDNA ORF-2片段。然后将cDNA片段插入pBlueBac-3Transfer载体中的BamHI-PstI位点。用该载体产生的重组杆状病毒感染SF9昆虫细胞,然后通过考马斯蓝染色聚丙烯酰胺凝胶而分析昆虫裂解液中是否存在55和61kDa ORF-2蛋白。如此处所述,直接表达的蛋白质可用作疫苗中的免疫原和免疫测定中的抗原。
Claims (19)
1.一种核酸分子,其特征在于,它具有一序列,该序列基本上由编码戊型肝炎病毒开放阅读框2蛋白的氨基酸112-660的核苷酸构成。
2.如权利要求1所述的核酸分子,其特征在于,该分子由编码SEQ ID NO.:2的氨基酸112-660的核苷酸构成。
3.一种表达载体,其特征在于,它含有权利要求1或2所述的核酸分子。
4.一种用权利要求3所述的表达载体转化或转染的宿主细胞。
5.一种戊型肝炎病毒蛋白,其特征在于,其分子量约55千道尔顿,而且该蛋白具有戊型肝炎病毒的开放阅读框2蛋白的氨基酸112作为其氨基端。
6.一种产生戊型肝炎病毒开放阅读框2蛋白的方法,其特征在于,它包括:
在适合的条件下,培养含有权利要求3所述表达载体的宿主生物体,从而表达该蛋白质。
7.如权利要求6所述的方法,其特征在于,该蛋白质的分子量约为55千道尔顿。
8.一种在生物样品中检测针对戊型肝炎病毒的抗体的方法,其特征在于,该方法包括:
将样品与权利要求5所述的戊型肝炎病毒蛋白接触,从而与抗体形成免疫复合物。
9.如权利要求8所述的方法,其特征在于,该生物样品选自下组:全血、血浆、血清、脑脊液、组织、尿和胸膜液。
10.如权利要求8所述的方法,其特征在于,检测IgM或IgG抗体。
11.如权利要求8所述的方法,其特征在于,该蛋白被连于固相载体。
12.如权利要求8所述的方法,其特征在于,用标记的抗体来检测免疫复合物。
13.一种试剂盒,其特征在于,它含有权利要求5所述的戊型肝炎病毒蛋白。
14.一种药物组合物,其特征在于,它含有权利要求5所述的蛋白质和适当的赋形剂、稀释剂或载体。
15.一种使哺乳动物对戊型肝炎感染产生免疫的疫苗,其特征在于,该疫苗含有权利要求5所述的蛋白质。
16.一种权利要求5所述的蛋白质的用途,其特征在于,它用于制造在哺乳动物中预防戊型肝炎的方法中所用的药物,该方法包括将有效量的该药物施用于哺乳动物,以刺激产生保护性的抗体。
17.一种体外在生物样品中检测戊型肝炎病毒存在与否的方法,其特征在于,该方法包括:
将生物样品与对权利要求5所述的戊型肝炎病毒蛋白特异的抗体进行接触,其中形成该抗体与该戊型肝炎病毒蛋白构成的免疫复合物就表示该生物样品中存在戊型肝炎病毒。
18.一种抗体,其特征在于,它对权利要求5所述的戊型肝炎病毒蛋白有特异性的结合亲和性,并且能够起抵抗戊型肝炎的保护作用。
19.一种权利要求18所述的抗体的用途,其特征在于,它用于制造在哺乳动物中预防戊型肝炎的方法中所用的药物,该方法包括将预防有效量的该药物施用于哺乳动物。
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US08/316,765 US6706873B1 (en) | 1992-09-18 | 1994-10-03 | Recombinant proteins of a pakistani strain of hepatitis E and their use in diagnostic methods and vaccines |
US08/316,765 | 1994-10-03 |
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AU8405798A (en) | 1997-07-18 | 1999-02-10 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | A swine hepatitis e virus and uses thereof |
US6458562B1 (en) | 1998-04-09 | 2002-10-01 | The United States Of America As Represented By The Secretary Of Health And Human Services | Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines |
EP1169341A1 (en) * | 2000-02-07 | 2002-01-09 | Department of Science and Technology | Infectious cdna clone of hepatitis e virus (hev) genome, its production and uses |
CN1345775A (zh) * | 2000-09-30 | 2002-04-24 | 养生堂有限公司 | 用于预防、诊断及治疗戊型肝炎病毒的多肽,及它们作为诊断试剂和疫苗 |
EP1400588A4 (en) * | 2001-06-25 | 2006-07-19 | Toshiba Kk | POLYNUCLEOTIDE PROBE AND PRIMER ORIGINATING IN JAPANESE HEPATITIS E VIRUS, FISH WITH SUCH PROBE, KITS HAVING THE SAME, AND METHOD FOR DETECTING HEPATITIS E VIRUS USING THE SAME |
CN100446813C (zh) * | 2005-06-24 | 2008-12-31 | 东南大学 | 甲型肝炎-戊型肝炎联合疫苗及其制备方法 |
CN112225783B (zh) * | 2020-09-16 | 2021-08-31 | 东莞市朋志生物科技有限公司 | Hcv重组抗原及其突变体 |
CN114957411B (zh) * | 2022-05-25 | 2023-08-18 | 徐州医科大学 | 一种正戊型肝病毒属基因c1型病毒orf2重组蛋白及其制备方法和应用 |
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NO326399B1 (no) | 2008-11-24 |
AU712901B2 (en) | 1999-11-18 |
KR100359526B1 (ko) | 2003-02-25 |
WO1996010580A3 (en) | 1996-05-23 |
JPH11500001A (ja) | 1999-01-06 |
NO971529D0 (no) | 1997-04-03 |
AU3830995A (en) | 1996-04-26 |
JP3889808B2 (ja) | 2007-03-07 |
WO1996010580A2 (en) | 1996-04-11 |
NO971529L (no) | 1997-05-26 |
CN1168698A (zh) | 1997-12-24 |
EP0784631A1 (en) | 1997-07-23 |
ATE518879T1 (de) | 2011-08-15 |
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NZ295056A (en) | 1999-06-29 |
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