CN1278301A - 新猪环病毒、疫苗及诊断试剂 - Google Patents
新猪环病毒、疫苗及诊断试剂 Download PDFInfo
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Abstract
本发明涉及一种从肺或神经节样本中分离的新型猪环病毒株,所述样本获自患有断乳后多系统性消瘦综合症(PMWS)的牲畜。本发明涉及这些毒株的纯化制品、减毒疫苗或灭活疫苗、重组活体疫苗、质粒疫苗和亚单位疫苗,以及诊断试剂和诊断方法。本发明还涉及可用于在体外表达载体中制备亚单位疫苗的DNA片段,或者整合入病毒或质粒型体内表达载体中的序列。
Description
本发明涉及一种引起PMWS综合症(猪多系统性消瘦综合症,也称为断乳后多系统性消瘦综合症)的新型猪环病毒株(porcine circovirus简称为PCV),涉及检测其的试剂和方法,涉及免疫接种方法及疫苗,和涉及制备这些试剂和疫苗的方法。
最初,在猪肾细胞系PK/15中检测到PCV为非细胞致病性污染物。此病毒与幼禽贫血病毒(简称为CAV)和PBFDV病毒(Pscittacine Beakand feather disease Virus)被分类为环病毒科(Circocuridae)。此病毒为小型无被膜病毒(从15-24nm),共同特征为含有1.76-2.31kb的环状单链DNA形式的基因组。最初认为该基因组编码一种约30kDa的多肽(Todd等人,病毒学文献(Arch Virol),1991,117;129-135)。然而,最近的工作显示其更为复杂的转录(Meehan B.M.等人,1997,78:221-227)。此外,在三种类型已知环病毒之间的核苷酸序列或共同抗原决定簇中无明显同源性。
一直认为来源于PK/15细胞的PCV无致病性。其序列由MeehanB.M.等人,普通病毒学杂志(J.Gen.Virol.),1997,78:221-227可知。直到最近有人意识到PCV株可能为致病性的,与PMWS综合症相关(Gupi P.S.Nayar等人,加拿大兽医杂志(Can.Vet.J.),1997,38:385-387;Clark E.G.,美国猪应用协会进展(Proe.Am.Assoc.SwinePrac.),1997;499-501)。Nayar等人利用PCR技术从患有PMWS综合症的猪中检测到PCV DNA。此前,尚未有野生型PCV毒株被分离和纯化。
在加拿大、美国和法国检出的PMWS综合症临床上的特征为体重逐渐下降,出现诸如呼吸急促、呼吸困难和黄疸的临床表现。从病理学角度而言,这些症状是淋巴细胞或肉芽肿浸润、淋巴结病的临床表现,和更罕见的肝炎、淋巴细胞性或肉芽肿性肾炎(Clark E.G.,美国猪应用协会进展1997;499-501;La Semaine Veterinaire No.26,La SemaineVeterinaire增刊,1996(834);La Semaine Veterinaire1997(857):54;Gupi P.S.Nayar等人,加拿大兽医杂志,1997,38:385-387)。
本申请人从肺或神经节样本中成功分离了五种新PCV毒株,此后称为根据本发明的环病毒,其中这些样本获自位于加拿大、美国(加利福尼亚)和法国(Brittany)的农场。已经从患有PMWS综合症的猪的损伤处检出这些病毒,而在健康猪中未检出。
此外,本申请人测定了这些毒株中的4株之基因组序列,即从加拿大、美国获得的毒株以及从法国获得的两株毒株。在核苷酸水平上这些毒株相互之间显示出强烈的同源性,超过96%;而与PK/15毒株之间同源性较低,约76%。因此,认为新毒株为一种新型猪环病毒的代表,此处称为II型,I型由PK/15代表。
因此,本发明的主题为分离的或纯化制品形式的如上述的II型猪环病毒。
本发明涉及可从患有PMWS综合症病猪中的生理学样本或组织,尤其是损伤组织样本中分离的,尤其是依照实施例所述的方法分离的任何猪环病毒,特别是涉及II型环病毒。
本发明的主题更具体的是五种毒株的纯化制品,其已保藏在ECACC(欧洲动物细胞保藏中心,应用微生物及研究中心,Porton Down,Salisbury,Wiltshire SP40JG,英国):保藏号V97100219(此处称为Imp.1008PCV);保藏号V97100218(此处称为Imp.1010PCV);保藏号V97100217(此处称为Imp.999PCV);于1997年10月2日(星期四)保藏,以及保藏号V98011608(此处称为Imp.1011-48285);保藏号V98011609(此处称为Imp.1011-48121);于1998年1月16日(星期五)保藏。
本发明涉及从患病猪分离的猪环病毒,和/或与本发明毒株具有显著血清学相似性的环病毒,和/或在严格条件下可与本发明的毒株交叉杂交的环病毒,所述严格条件是指例如不能与PCV PK/15毒株杂交。
从患有PMWS综合症的猪中的生理学样本或组织,尤其是损伤组织样本中分离的病毒株可有利地在细胞系中增殖,尤其是猪肾细胞系,特别是无污染物(如PCV以及瘟病毒、猪腺病毒和猪细小病毒)的PK/15细胞中,用于其复制或具体地产生抗原,包括完整抗原(如病毒)和/或抗原亚单位(如多肽)。
非常明显而出人意料的是,已证明这些分离物在PK/15细胞培养物中产量很大,这无疑对病毒或抗原,尤其是对灭活疫苗的产生十分有利。
本发明的主题还涉及环病毒制品,其中在体外培养的细胞特别是细胞系(如:PK/15细胞)传代后分离环病毒,所述细胞或细胞系被至少一种根据本发明的环病毒感染,或者被可从患有PMWS综合症的猪的生理学样本或从组织样本(尤其是损伤组织)中分离的任一种猪环病毒感染。本发明的主题还包括培养物提取物或上清液,任选地,其通过标准技术纯化,以及通常任一种获自体外培养物的抗原性制品。
本发明的主题还涉及免疫活性组分和含有至少一种上述抗原的疫苗。
其可为基于减毒活体全病毒的免疫活性组分,或用这些活性组分制备的疫苗。可根据常规方法进行减毒,例如,通过在细胞上进行传代,优选在猪细胞特别是细胞系(如PK/15细胞)上传代,例如从50至150次,特别是约100次水平的传代。这些疫苗一般包括兽医学可接受的载体或稀释剂,任选地,兽医学可接受的佐剂,以及任选地一种冷冻干燥稳定剂。
这些疫苗优选地包含103-106 TCID50。
其可是免疫活性组分,或基于根据本发明环病毒抗原的灭活状态的疫苗。这些疫苗还包括兽医学可接受的载体或稀释剂,以及任选地兽医学可接受的佐剂。
根据本领域普通技术人员已知的技术,灭活根据本发明的环病毒以及可能存在的组分。灭活优选地通过化学方法进行,如通过使抗原接触一种化学药剂,如甲醛(福尔马林)、多聚甲醛、β-丙醇酸内酯、乙撑亚胺或其衍生物。灭活优选的方法为此处所述的接触化学药剂,特别是接触乙撑亚胺或β-丙醇酸内酯。
优选地,通过本领域技术人员周知的方法,根据本发明的灭活疫苗可添加佐剂有利地以乳液形式提供,例如油包水或水包油型。也可根据佐剂性质将常规佐剂化合物掺入活性组分中。
可用的佐剂中,以举例的方式,包括但不限于氢氧化铝、皂甙(如Quillaja皂甙或Quil A;参见“疫苗设计,亚单位疫苗剂佐剂方法”,1995,Michael F.Powel和Mark J.Newman编,Plennum出版社,New-York及London,210页)、Avridine(疫苗设计,148页)、DDA(溴化二甲基双十八烷基铵,疫苗设计,157页)、聚磷腈(疫苗设计,204页)、或备择的基于矿物油、角鲨烷(例如SPT乳液,疫苗设计,147页)、角鲨烯(例如MF59,疫苗设计,183页)的水包油型乳液,或基于可代谢油(优选根据WO-A-94 20071)的油包水型乳液,以及US-A-5,422,109中所述的乳液。也可选择佐剂的组合,例如Avridine或DDA与一种乳液组合。
这些疫苗优选地包含103-106 TCID50。
活疫苗佐剂可选自那些用于灭活疫苗的佐剂,优选乳液。可在用于灭活疫苗中所示的那些乳液还包括WO-A-9416681中所述的那些乳液。
至于冷冻干燥稳定剂,其以举例方式可为SPGA(Bovarnik等人,细菌学杂志(J.Bacterioligy),59,509,950)、碳水化合物(如山梨醇、甘露醇、淀粉、蔗糖、葡聚糖或葡萄糖)、蛋白质(白蛋白或酪蛋白),或这些化合物的衍生物,或如碱金属磷酸盐的缓冲液。
此外,申请人获得了分离株中4个的基因组,鉴定为SEQ ID NO:1-4和任选地SEQ ID No:6。
因此,本发明的主题是含有这些序列之一的部分或全部的DNA片段。不言而喻,本发明自然涵盖了等同序列,即不改变所述序列的功能或菌株特异性的序列,或由该序列编码的多肽之菌株特异性的序列。当然,由于密码子简并性造成的差异也包括在内。
本发明也涵盖了这样的等同序列,它们在高度严格条件下可与上述序列杂交,和/或与本发明的毒株有高度同源性,且属于上述II型环病毒组。
借助适当载体,这些序列及其片段可有利地用于多肽的体外或体内表达。
特别是,已经鉴定了在II型环病毒基因组序列上的可用于此方面的开放阅读框架,其形成了根据本发明的DNA片段。本发明涉及含有至少一个这些开放阅读框架(相应于氨基酸序列)的任何多肽。优选地,本发明涉及一种基本上由ORF4、ORF7、ORF10或ORF13组成的蛋白质。
为了在体外表达亚单位,作为一种表达方法可优选使用大肠杆菌或杆状病毒(US-A-4,745,051)。将编码序列或其片段整合入杆状病毒基因组(如,杆状病毒苜蓿银纹夜蛾多核型多角体病毒AcNPV),将后者在昆虫细胞中增殖,如草地夜蛾(Spodoptera frugiperda Sf9)(保藏号ATCC CRL 1711)。也可在真核细胞如酵母(如酿酒酵母(Saccharomycescerevisiae))或哺乳动物细胞(如CHO、BHK)中表达亚单位。
本发明的主题也涉及可通过这些表达方法制备,然后任选地根据传统技术纯化的多肽。本发明还涉及一种亚单位疫苗,其包括在兽医学可接受的载体或稀释剂之中的至少一种如此获得的多肽,或其片段,以及任选地兽医学可接受的佐剂。
为了用于制备重组活体疫苗的体内表达,在允许该多肽表达的条件下将编码序列或其片段插入一种合适的表达载体。至于合适的载体,根据本领域技术人员周知的技术可使用活病毒,优选地能在猪中增殖、对猪无致病性(天然无致病性或赋予此性质)。尤其是可使用猪疱疹病毒如假狂犬病病毒、猪腺病毒、疱疹病毒、痘病毒属,特别是牛痘病毒、禽痘病毒、犬痘病毒和猪痘病毒。也可使用质粒DNA作为载体(WO-A-9011092,WO-A-93 19813,WO-A-94 21797,WO-A-95 20660)。
因此,本发明主题也涉及如此制备的载体和重组活体疫苗或质粒疫苗(多核苷酸或DNA疫苗),此外,疫苗包括兽医学可接受的载体或稀释剂。
根据本发明的疫苗(活体减毒疫苗、灭活疫苗、亚单位疫苗、重组疫苗和质粒疫苗)可包括一种或多种(2或3种)根据本发明环病毒之中的一种或多种的一种或多种活性组分(抗原)。
对于每一上述疫苗类型,本发明也提供了针对猪环病毒的免疫接种与针对其它猪疾病(特别是可与PMWS综合征相关的疾病)的免疫接种的联合免疫接种疫苗。因此,根据本发明的疫苗,特别是灭活疫苗可包含相应于其它猪病原体的效价。这些其它猪病原体中,优选地包括PRRS(猪生殖和呼吸综合征)(本领域技术人员可参见WO-A-93/07898,WO-A-94/18311,FR-A-2 709 966;C.chareyre等人,第15届IPVS大会论文集,Birmingham,英国,1998年7月5-9日,139页;此处引入作为参考)和/或猪肺炎支原体(Mycoplasma hyopneumonia)(本领域技术人员可参见EP-A-597 852;EP-A-550 477;EP-A-571 648;O.Martinon等人,157,284和285页以及G.Reynaud等人,150页,均在上述第15届IPVS大会论文集,此处引入作为参考)。其它可能感兴趣的疫苗可包括大叶性肺炎放线杆菌(Actinobacillus pleuropneumoniae)、大肠杆菌、猪萎缩性鼻炎以及伪狂犬病(Aujeszky病)、猪霍乱、猪流感。
本发明的主题还涉及一种方法,从而可在猪中诱导针对本发明环病毒的免疫反应。该方法特别是一种在猪中有效的方法。
该方法可向猪一次或多次施用上述疫苗。也可在同一免疫接种方案中联合上述数种类型的疫苗。
该方法不仅可向成年猪施用,也可向幼猪或怀孕雌猪施用。对后者的免疫接种可对新生猪赋予被动免疫(母本抗体)。
本发明还提供了在猪中诊断根据本发明的环病毒的可能性。因此,本发明的主题还包括利用下述试剂的诊断试验和与其相关的方法。
对不同环病毒序列的了解使得确定共同序列成为可能,这使其可制备能识别所有已知猪环病毒的反应物片段。
为了进行特异性诊断,本领域普通技术人员能选择这样的序列片段,其相应于与对应的PK/15环病毒序列没有或几乎没有同源性的区域。
本领域普通技术人员可通过序列比较选择符合其意愿的反应物片段。
第一种反应物片段存在于此处公开的序列及其片段中,其可特别地在周知的杂交或PCR(聚合酶链反应)技术中用作探针或引物。
第二种反应物片段存在于由来自病毒的这些序列编码的多肽或在载体帮助下表达的多肽(见上),或根据传统用于肽合成的技术通过化学途径合成的多肽。
第三种和第四种反应物片段分别存在于多克隆和单克隆抗体,其可根据传统方法从病毒提取多肽或片段、或由DNA序列编码的多肽或其片段制备。
这第二、三和四种反应物片段可用于本发明主题的诊断方法,其中对获自待测猪的生理学液体样本(血液、血浆、血清等)或组织样本(神经中枢、肝脏、肺、肾等)进行试验,通过搜寻检测抗原自身或针对该抗原的抗体,检验是否存在特异于根据本发明环病毒的抗原。
根据本发明的抗原和抗体可用于任何已知的实验室诊断技术。
然而,优选地在可由兽医、饲养者或牲畜主现场直接使用的技术中使用这些抗原和抗体。本领域技术人员可使用多种实验室和现场技术,因此可有利地改造这些抗原和/或抗体以适于作为诊断试剂的应用。
可在本发明的框架中优选使用的诊断技术为蛋白质印迹、免疫荧光、ELISA和免疫层析。
就免疫层析的使用而言,专业人士可特别参见Robert f.Zurk等人,临床化学(Clin.Chem.),31/7,1144-1150(1985),以及下列专利或专利申请:WO-A-88/08 534、WO-A-91/12528、EP-A-291 176、EP-A-299 428、EP-A-291 194、EP-A-284 232、US-A-5 120 643、US-A-5 030 558、US-A-5 266 497、US-A-4 740 468、US-A-5 266 497、US-A-4 855 240、US-A-5451 504、US-A-5 141 850、US-A-5 232 035和US-A-5 238 652。
因此,优选通过间接试验(通过竞争或通过替换)在样本中检测特异性抗体。为此,使用抗原自身,或保留了抗体识别位点的该抗原片段作为检测试剂。可有利地使用过氧化物酶标记或特定标记进行标记,优选用胶体金。
也期望在特异于该抗原的标记抗体的帮助下检测样本中的抗原本身。标记有利地为如上所述。
对于特异于可特别用于竞争或替换的抗原之抗体,或特异于用来检测抗原自身的抗体,应理解为特异于抗原的多克隆抗体或单克隆抗体、这些抗体的片段,优选为Fab或F(ab)’2片段。
本发明的另一方面是特异于根据本发明的抗原的单克隆或多克隆抗体,这些抗体可特别用作诊断试剂,在生理学液体样本或组织样本中检测抗原,或者甚至在这样的样本或样品中检测抗体。本发明还包括这些抗体的免疫功能性片段,特别是Fab和F(ab)’2片段。
抗体可通过常规技术制备。可特别参见“抗体,实验室手册”,1988,冷泉港实验室,美国,或J.W.Goding。“单克隆抗体:原理与实践”,学院出版社,其内容此处引入作为参考。
也可特别的以自知方法进行小鼠脾细胞与适当的骨髓瘤细胞的融合,其中小鼠用抗原或至少一种该抗原的片段免疫过。
本发明的主题还包括一种特异于抗原的单克隆或多克隆抗体(特别是小鼠或兔抗体)制品,其优选为纯或基本上纯,或甚至为粗品。
本发明还可以确定目标表位,特别是基于此处描述的DNA序列,确定是否存在免疫用的表位或诊断用的表位。从根据本发明的环病毒基因组的DNA序列,本领域技术人员可根据已知方法,例如一种适当的计算机程序或PEPSCAN确定表位。表位是蛋白质的免疫决定区域,是暴露在蛋白质表面的区域。由此,其可通过抗体识别,因而可特别用于诊断领域,用于诊断目的之抗体制备或者用于可用作诊断试剂的相应肽的制备。
一个表位至少是具有8-9个氨基酸的肽。通常优选最少为13-25个氨基酸。
本领域技术人员因此可使用一种或多种这些技术以及其它可能的技术,发现可用于诊断目的的肽或抗体的表位。
本发明的主题还包括诊断试剂盒,其中含有该抗原和/或特异于该抗原的多克隆或单克隆抗体。特别是相应于上述诊断技术的诊断试剂盒。
以下,本发明将以非限制性实施方案结合附图更详细的进行描述,其中:
图1.Imp.1011-48121株的基因组DNA序列。
图2.Imp.1011-48285株的基因组DNA序列。
图3.Imp.999株的基因组DNA序列。
图4.Imp.1010株的基因组DNA序列。
图5.根据图1-4的4个序列与PCVPK/15株序列的比较。
图6.在1997年10月3日法国首次递交的文件中的Imp.999株的基因组DNA序列。
图7.图6序列与PK/15株序列的比较。
序列表SEQ ID
SEQ ID NO:1 Imp.1011-48121株的基因组DNA序列。
SEQ ID NO:2 Imp.1011-48285株的基因组DNA序列。
SEQ ID NO:3 Imp.999株的基因组DNA序列。
SEQ ID NO:4 Imp.1010株的基因组DNA序列。
SEQ ID NO:5 PCV PK/15株基因组的DNA序列。
SEQ ID NO:6在1997年10月3日法国首次递交的文件中的Imp.999株的基因组DNA序列。
实施例实施例1猪环病毒株的培养及分离
从法国、加拿大和美国小猪的肺和淋巴结收集组织样本。这些小猪均显示典型的断乳后多系统消瘦综合症的临床症状。为便于病毒分离,尸检后立即将组织样本于-70℃冷冻。
为了病毒分离,在含有Earle盐(EMEM,BioWhittaker英国有限公司,Wokingham,英国)、青霉素(100IU/ml)和链霉素(100μg/ml)的基本培养基(MEM-SA培养基)中,通过利用无菌马达和杵将组织样本与无菌沙研磨,制备含有约15%组织样本的悬浮液。然后将此研磨制品加入MEM-SA,4℃下于3000g离心30分钟,收获上清液。在接种细胞培养物之前,100μl体积的氯仿加入各个2ml上清液中,室温下连续混合10分钟。然后将此混合物转移入微量离心管中,3000g离心10分钟,收获上清液。将此上清液用作病毒分离实验的接种物。
所有的病毒分离研究均在PK/15细胞培养物上进行,该培养物已知无猪环病毒(PCV)、瘟病毒、猪腺病毒和猪细小病毒污染(Allan G.等人,初乳停断小猪的猪环病毒实验性感染的病原体及猪胎材料的检查,兽医微生物(Vet.Microbiol.),1995,44,49-64)。
根据下列技术进行猪环病毒的分离:
通过从生长至铺满的培养物用胰蛋白酶(胰蛋白酶-依地酸混合物)消化分离PK/15细胞单层,以终浓度约400,000个细胞/ml加入含有15%胎牛血清无瘟病毒污染的MEM-SA培养基(=MEM-G培养基)。然后将此细胞悬浮液的10ml等份与2ml上述接种物等份混合,最终将混合物以6ml等份培养于两个25cm2的Falcon烧瓶中。在含10%CO2的氛围中37℃培养18小时。
培养后,用300mM D-葡糖胺(Cat #G48175,Sigma-Aldich化学有限公司,Poole,英国)处理半铺满单层的培养液(Tischer I.等人,病毒学文献,1987,96:39-57),然后37℃继续培养48-72小时。后一培养结束后,每个接种物的两个Falcon烧瓶之一经过连续3次冻/融循环。所剩Falcon烧瓶之PK/15细胞用胰蛋白酶-依地酸溶液处理,重悬于20mlMEM-G培养基中,以约400,000个细胞/ml浓度接种入75cm2的Falcon中。通过加入经过冻/融循环获得的相应裂解物5ml,“超感染”新鲜接种的烧瓶培养物。实施例2用于通过免疫荧光或原位杂交检测猪环病毒的细胞培养物样本的制备
收集5ml超感染的悬浮液,接种入直径55mm的带有无菌无脂玻璃盖玻片的培养皿。在烧瓶中及玻璃盖玻片上的培养物于37℃培养,如实施例1所述用葡糖胺处理。用葡糖胺处理后24-48小时收集玻璃盖玻片上的培养物,用丙酮室温下固定10分钟,或用10%缓冲的甲醛固定4小时。固定后,所有玻璃盖玻片在硅胶上于-70℃储存,直至用于原位杂交研究和免疫细胞化学标记研究。实施例3通过原位杂交检测PCV的技术
对从患病猪收集的且用甲醛固定的组织进行原位杂交,也对固定于玻璃盖玻片上之为了病毒分离(见实施例2)接种的细胞培养物制品进行原位杂交。
使用相应于PK/15猪环病毒(PCV)以及相应于传染性禽贫血病毒(CAV)的完整基因组探针。使用质粒pPCV1作为PCV的特异性病毒DNA来源,其中含有以单一1.7千碱基对(kbp)插入片段形式克隆的PCV基因组复制形式(Meehan B.等人,猪环病毒DNA基因组序列:与植物环病毒的亲和性,普通病毒学杂志,1997,78:221-227)。使用含有2.3kbp复制形式的禽环病毒病毒CAV之类似质粒pCAA1作为阴性对照。使用两个质粒各自的甘油储藏原种用于根据碱裂解技术制备和纯化质粒(Sambrook J.等人,分子克隆:实验室手册,第二版,冷泉港实验室,纽约,1989),使其可用作模板制备探针。从上述纯化的质粒以及根据供应商说明使用市售非放射性标记试剂盒(DIG DNA标记试剂盒,Boehringer Mannheim,Lewes,英国)从随机六核苷酸引物制备代表PCV和CAV完整基因组的环病毒探针(每个探针1μg)。
在用于原位杂交前将地高辛标记的探针加入体积为50-100μl的无菌水中。
制备包埋在石蜡中用甲醛固定的患猪组织样本以及用甲醛固定的感染细胞培养物制品,用于按如下技术检测PCV核酸:
从包埋于石蜡中的组织切下厚度为5μm的切片,除去石蜡,在浓度递减的连续乙醇溶液中再水合。用甲醛固定的组织切片和细胞培养物在37℃下于0.5%蛋白酶K之0.05M Tris-HCl溶液中(含有5mM EDTA,pH7.6)分别温育15分钟和5分钟。然后将载玻片置于1%甘氨酸的无菌蒸馏水溶液中30秒,用0.01M PBS缓冲液(磷酸缓冲盐液,pH7.2)洗涤两次,最后用无菌蒸馏水洗涤5分钟。将其空气干燥,与探针接触。
每一组织/探针制品加盖洁净无脂玻璃盖玻片,置于90℃炉中10分钟,然后与冰块接触1分钟,最后于37℃培养18小时。将制品短暂浸入2X柠檬酸钠(SSC)缓冲液(pH7.0),以去除保护性玻璃盖玻片,然后于2 X SSC缓冲液中洗涤两次5分钟,最后于PBS缓冲液中洗涤两次5分钟。
经过洗涤后,将制品浸入0.1M马来酸、0.15M NaCl(pH1.5)(马来酸缓冲液)10分钟,然后于37℃在1%封闭试剂(Cat.#1096176,Boehringer Mannheim,英国,Lewis,East Sussex,英国)的马来酸缓冲液溶液中温育20分钟。
然后,制品与稀释于封闭缓冲液中的抗地高辛单克隆抗体1/250溶液(Boehringer Mannheim)37℃下温育1小时,于PBS中洗涤,最后与生物素酰化抗小鼠免疫球蛋白抗体37℃下温育30分钟。制品在PBS中洗涤,通过用0.5%过氧化氢的PBS溶液室温下处理20分钟封闭内源过氧化物酶活性。再次用PBS洗涤制品,用即配即用的3-氨基-9-二乙基咔唑(AEC)底物处理(Cambridge Bioscience,Cambridge,英国)。
最终用自来水洗涤后,将制品用苏木精复染,在自来水中“变蓝”,用封固液(GVA Mount,Cambridge Bioscience,Cambridge,英国)封固于显微镜玻璃盖玻片上。实验对照包括了在获自患病猪和非患病猪的样本上使用非相关阴性探针(CAV)和阳性探针(PCV)。实施例4:通过免疫荧光检测PCV的技术
使用1/100稀释的成体猪血清集合通过间接免疫荧光技术(IIF)进行丙酮固定的所有细胞培养物制品的初次筛选。该血清集合包含来自北爱尔兰的25只成体猪的血清,其中已知含有针对多种猪病毒(包括PCV、猪细小病毒、猪腺病毒和PRRS病毒)的抗体。通过将稀释于PBS中的血清37℃下接触细胞培养物1小时,进行IIF技术,然后在PBS中洗涤两次。再用1/80稀释的与荧光素异硫氰酸结合的兔抗猪免疫球蛋白抗体PBS溶液染色细胞培养物1小时,用PBS洗涤,在紫外光下显微镜观察之前用甘油缓冲液封固。实施例5:对患病猪组织原位杂交的结果
使用由获自法国、加拿大和加利福尼亚小猪的用甲醛固定的组织的PCV基因组探针进行原位杂交,其中小猪患有多系统消瘦综合症,结果显示在数个研究的损伤组织中存在与损伤相关的PCV核酸。当对获自非病患猪的组织使用PCV探针时或者对患病猪使用CAV探针时未观察到信号。在浸润入加利福尼亚小猪肺部损伤中的大量单核细胞之胞浆和细胞核中检出PCV核酸。在肺细胞、支气管和细支气管上皮细胞以及在微动脉、小囊突和淋巴管的内皮细胞中也证实PCV核酸的存在。
在患病法国猪中,在大量滤泡淋巴细胞和淋巴结窦状(小管)内单核细胞的胞浆中也存在PCV核酸。在临时合胞体中也存在PCV核酸。根据这些检测结果,选择加利福尼亚猪肺、法国猪肠系膜淋巴结和加拿大猪器官用于分离新型猪环病毒株。实施例6:新型猪环病毒株细胞培养物及免疫荧光检测结果
在用获自法国小猪(Imp.1008株)、加利福尼亚小猪(Imp.999株)和加拿大小猪(Imp.1010株)接种的细胞培养物中未观察到致细胞病变效应,这些猪显示多系统消瘦的临床症状。然而,获自接种细胞培养物的制品的免疫标记在用丙酮固定及用猪多克隆血清集合染色后,利用加利福尼亚小猪肺(Imp.999株)、法国小猪肠系膜淋巴结(Imp.1008株)、和加拿大小猪器官(Imp.1010株)接种的培养物的大量细胞中显示核荧光。实施例7:猪环病毒基因组DNA的提取
利用感染的PK/15细胞培养物(10只75cm2 Falcon)制备新型猪环病毒毒株的复制形式(见实施例1),该细胞培养物培养72-76小时后收获,用葡糖胺处理,如CAV复制形式克隆时所述(Todd.D等人,利用克隆DNA探针斑点杂交检测禽贫血病毒,临床微生物杂志,1991,29:933-939)。根据改进的Hirt技术提取这些复制形式的双链DNA(Hirt B.从感染细胞培养物中选择性提取多病毒DNA,分子生物学杂志,1967,36:365-369),如Molitor所述(Molitor T.W.等人,猪细小病毒DNA:两种病毒分离物的基因组及复制形式的表征,病毒学,1984,137:241-254)。实施例8:猪环病毒Imp.999株基因组复制形式的限制性图谱
依供应商说明,用S1核酸酶(Amersham)处理根据Hirt技术提取的DNA(1-5μg),然后用多种限制酶(Boehringer Mannheim,英国,Lewis,East Sussex,英国)酶切此DNA,如Todd等人所述,在存在溴乙锭时于1.5%琼脂糖凝胶上电泳分离酶切产物(禽贫血病毒的纯化和生化表征,普通病毒学杂志,1990,71:819-823)。从Imp.999株培养物提取的DNA具有单一EcoRI位点、两个SacI位点,不含有任何PstI位点。因此,此限制性图谱不同于PCV PK/15毒株的限制性图谱,该毒株中含有PstI位点,而不含有任何EcoRI位点。实施例9:猪环病毒Imp.999株基因组的克隆
用限制性酶EcoRI酶切PCV Imp.999毒株双链复制形式产生的约1.8kbp限制性片段,使用Qiagen市售试剂盒(QIAEXII凝胶提取试剂盒,Cat#20021,QIAGEN Ltd.,Crawley,West Sussex,英国)在1.5%琼脂糖凝胶上电泳分离酶切产物(见实施例3)。然后根据标准克隆技术(Sambrook J.等人,分子克隆:实验室手册,第二版,冷泉港实验室,纽约,1989),将此EcoRI-EcoRI限制性片段与已经用相同限制性酶消化并去磷酸化的载体pGEM-7(Promega,医学供应公司,都柏林,爱尔兰)连接。根据标准技术,用获得的质粒转化大肠杆菌JM109宿主菌株(Stratagene,La Jolla,美国)。还将PCV Imp.999毒株EcoRI-EcoRI限制性片段克隆入载体pBlueScript SK+的EcoRI位点(Stratagene,La Jolla,美国)。在每一宿主菌株获得的克隆中,筛选了含有期望片段大小的至少两个克隆。然后培养所得克隆,根据标准质粒制备和纯化技术,以小体积(2ml)或大体积(250ml)纯化含有完整Imp.999毒株基因组的质粒。实施例10:PCV Imp.999毒株基因组DNA(双链复制形式)的序列测定
根据Sanger双脱氧核苷酸技术使用测序试剂盒“AmpliTaq DNA聚合酶FS”(Cat#402070 PE Applied Biosystems,Warrington,英国)以及依制造商说明用Applied Biosystems ABI373A自动测序仪测定两个EcoRI Imp.999克隆(克隆pGEM-7/2和克隆pGEM-7/8)的核苷酸序列。用M13“正向”和“反向”通用引物进行初步测序反应。根据“DNA步行”技术进行如下测序反应。由Life Technologies(Inchinnan BusinessPark,Paisley,英国)合成下列序列测定所需的寡核苷酸。
用MacDNASIS 3.2版软件装配产生的序列并分析(Cat#22020101,Appligene,Durham,英国)。通过由“国家生物工程信息中心”服务器(NCBI,Bethesda,MD,美国)可得的BLAST序列对比工具分析开放阅读框架。
最初从克隆pGEM-7/8获得的完整序列(EcoRI-EcoRI片段)(SEQID NO:6)示于图6。其任意地从EcoRI位点中的G之后开始,从核苷酸角度而言存在几个不确定的地方。
然后优化序列测定,SEQ ID NO:3(图3)给出了此毒株的总序列,其任意地从EcoRI位点开始,即G为第一个核苷酸。
采用类似方法获得其它三种根据本发明的分离物之序列(见SEQ IDNO:1、2和4,以及图1、2和4)。
这四个毒株的基因组大小为:
Imp.1011-48121 1767核苷酸
Imp.1011-48285 1767核苷酸
Imp.999 1768核苷酸
Imp.1010 1768核苷酸实施例11:PCV Imp.999毒株序列分析
当从Imp.999毒株产生的序列用于同GenBank数据库中含有的序列进行同源性测试时,仅检测出与PK/15株(保藏号Y09921和U49186)序列在核酸水平上有约76%的同源性(见图5)。
在氨基酸水平,与数据库的6相序列翻译同源性测试(在NCBI服务器上的BLAST序列对比工具)可证实:与相应于理论上的BBTV病毒之复制酶的开放阅读框架有94%同源性,其类似于由GenBankU49186序列编码的植物环病毒(GenBank鉴定号1841515)。
其它数据库中含有的序列与从PCV Imp.999毒株产生的序列无明显的同源性。
收集自患有多系统消瘦综合症的加利福尼亚小猪损伤组织之培养的Imp.999毒株所产生的序列分析清楚表明,此病毒分离物是一种新型猪环病毒株。实施例12:序列的比较分析
与PCV PK/15株序列进行4株新PCV毒株核苷酸序列的序列比较(图5)。建立考虑了4株新毒株和已有PK/15毒株的同源性矩阵。结果如下:
1:Imp.1011-48121
2:Imp.1011-48285
3:Imp.999
4:Imp.1010
5:PK/15
1 2 3 4 5
1 | 1.0000 | 0.9977 | 0.9615 | 0.9621 | 0.7600 |
2 | 1.0000 | 0.9621 | 0.9632 | 0.7594 | |
3 | 1.0000 | 0.9949 | 0.7560 | ||
4 | 1.0000 | 0.7566 | |||
5 | 1.0000 |
两株法国毒株Imp.1011-48121与Imp.1011-48285的同源性超过99%(0.9977)。
两株北美毒株Imp.999与Imp.1010之间的同源性也大于99%(0.9949)。法国毒株与北美毒株之间的同源性略大于96%。
所有这些毒株与PK/15的同源性在75%-76%之间。
由此可推知,根据本发明的毒株代表了一种不同于由PK/15株代表的类型的新猪环病毒类型。从显示PMWS综合症的猪分离的这一新类型称为II型猪环病毒,PK/15代表I型。虽然它们事实上分离自十分不同的地理区域,属于此II型的毒株之间显示明显的同源性。实施例13:由新PCV毒株基因组编码的蛋白质的分析
认为Imp.1010分离物的核苷酸序列是与多系统消瘦综合症相关的其它环病毒毒株的代表。用BLAST序列对比工具以及MacVecter 6.0软件程序组合(牛津分子组,牛津OX4 4GA,英国),详细分析此序列(Altschul等人,分子生物学杂志,1990,215:403-410)。在此序列(环状基因组)上可检测到体积大于20个氨基酸的13个开放阅读框架(ORF)。这13个ORF为:
名称 | 起始 | 终止 | 链 | ORF大小(核苷酸(nt)) | 蛋白质大小(氨基酸(aa)) |
ORF1 | 103 | 210 | 有义 | 108nt | 35aa |
ORF2 | 1180 | 1317 | 有义 | 138nt | 45aa |
ORF3 | 1363 | 1524 | 有义 | 162nt | 53aa |
ORF4 | 398 | 1342 | 有义 | 945nt | 314aa |
ORF5 | 900 | 1079 | 有义 | 180nt | 59aa |
ORF6 | 1254 | 1334 | 有义 | 81nt | 26aa |
ORF7 | 1018 | 704 | 反义 | 315nt | 104aa |
ORF8 | 439 | 311 | 反义 | 129nt | 42aa |
ORF9 | 190 | 101 | 反义 | 90nt | 29aa |
ORF10 | 912 | 733 | 反义 | 180nt | 59aa |
ORF11 | 645 | 565 | 反义 | 81nt | 26aa |
ORF12 | 1100 | 1035 | 反义 | 66nt | 21aa |
ORF13 | 314 | 1381 | 反义 | 702nt | 213aa |
每个ORF的起始和终止位点参见图4(SEQ ID NO:4)的1010毒株基因组。ORF1-13的界限与999毒株相同。它们1011-48121毒株及1011-48285毒株中ORF的界限也与999毒株中相同,除了ORF3和13:ORF3 1432-1539,有义,108nt,35aaORF13 314-1377,反义 705nt,234aa。
13个ORF中,4个与位于克隆病毒PCV PK/15的基因组之类似ORF具有明显同源性。分析了所有与多系统消瘦综合症有关的环病毒分离物基因组上的每个开放阅读框架。这4个ORF如下:
名称 | 起始 | 终止 | 链 | ORF大小(nt) | 蛋白质大小(aa) | 分子量 |
ORF4 | 398 | 1342 | 有义 | 945nt | 314aa | 37.7 kDa |
ORF7 | 1018 | 704 | 反义 | 315nt | 104aa | 11.8kDa |
ORF10 | 912 | 733 | 反义 | 180nt | 59aa | 6.5 kDa |
ORF13 | 314 | 1381 | 反义 | 702nt | 213aa | 27.8 kDa |
每个ORF的起始和终止位置参见图4(SEQ ID NO:4)的序列。ORF的大小(以核苷酸计=nt)包括终止密码子。
PCV Imp.1010与PCV PK-15分离株的基因组排列之间的比较鉴定了两种病毒基因组中保守的4个ORF。下表为观察到的同源性程度:
ORF Imp.1010/ORF PCV PK-15 | 同源性百分比 |
ORF4/ORF1 | 86% |
ORF13/ORF3 | 66.4% |
ORF7/ORF3 | 61.5%(在重叠水平(104aa)) |
ORF10/ORF4 | 83%(在重叠水平(59aa)) |
在ORF4 Imp.1010与ORF1 PK-15之间观察到最大序列相同性(86%同源性)。由于此蛋白质可能参与了病毒DNA复制,且为病毒复制的关键,预计有这样的同源性(Meehan等人,普通病毒学杂志,1997,78:221-227;Mankertz等人,普通病毒学杂志,1998,79:381-384)。
在ORF13 Imp.1010与ORF2 PK-15之间序列相同性不甚强(66.4%同源性),但这两个ORF各自的确存在高度保守的N-末端基本区域,其与CAV禽环病毒的主要结构蛋白质之N-末端区域相同(Meehan等人,病毒学文献,1992,124:301-319)。此外,在ORF7 Imp.1010与ORF3PK-15之间以及ORF10 Imp.1010与ORF4 PK-15之间观察到许多差异。每个情况中,当与PCV PK-15的ORF3及ORF4比较时,Imp.1010分离株的ORF 7和ORF10的C-末端存在缺失。在ORF7/ORF3以及ORF10/ORF4的N-末端区域水平上观察到最大序列同源性,分别为61.5%同源性(在重叠水平)和83%同源性(在重叠水平)。
由于猪环病毒基因组特别紧密,其基因组排列似乎相当复杂。主要结构蛋白质可能由位于猪环病毒基因组相同链上的数个阅读框架剪接产生。因此,上表中的任一开放阅读框架(ORF1-ORF13)可代表所有或部分由II型猪环病毒编码的抗原性蛋白质,因此可能是一种可用于特异性诊断和/或用于免疫接种的抗原。因此,本发明涉及任一包含至少一种上述ORF的蛋白质。优选地,本发明涉及一种基本上由ORF4、ORF7、ORF10或ORF13组成的蛋白质。实施例14:由新毒株克隆的PCV基因组的传染特性
根据由Meehan等人所述的技术,将含有Imp.999分离株的完整基因组(复制形式)的质粒pGEM-7/8转染入PK/15细胞(用禽贫血病毒感染的细胞之病毒DNA的表征:克隆的复制形式的序列分析及克隆的基因组片段的转染能力,病毒学文献,1992,124:301-309)。对非污染的PK/15细胞上转染后的第一次传代细胞进行免疫荧光分析(见实施例4),显示克隆pGEM7/8的质粒能诱导传染性PCV病毒的产生。含有传染性PCV遗传物质的克隆的获得,允许为制备经修饰的PCV病毒(在猪中减毒的或缺陷型的)对病毒基因组进行任何有用的操作,其中经修饰的PCV病毒可用于制备减毒或重组疫苗,或用于制备用作诊断试剂盒的抗原。实施例15:体外培养制备PCV抗原
根据如实施例1的相同方法进行非污染PK/15细胞的培养和病毒操作。在37℃培养4天后经胰蛋白酶消化并计数后,收集感染细胞。以每ml中400,000个感染的细胞接种下一代细胞。实施例16:病毒抗原的灭活
病毒培养结束后,收集感染的细胞,用超声波(Branson Sonifier)或借助转子-定子型胶体磨(UltraTurrax,IKA)裂解细胞。悬浮液在3700g下离心30分钟。用0.1%乙撑亚胺于37℃下灭活病毒悬液18小时,或用0.5%β-丙醇酸内酯于28℃灭活24小时。如果在灭活前病毒滴度不足,利用300kDa截流分子量的膜通过超滤浓缩病毒悬浮液(MilliporePTMK300)。灭活的病毒悬浮液储存于5℃。实施例17:以基于矿物油的乳液形式制备疫苗
根据如下配方制备疫苗:
灭活猪环病毒悬浮液:250ml
MontanideISA 70(SEPPIC):750ml
分开单独对水相和油相过滤除菌。通过借助Silverson涡轮乳化机混合和匀浆化组分制备乳液。
一只疫苗剂量含有约107.5 TCID50。对皮内施用,一只疫苗剂量体积为0.5ml,对肌内途径给药为2ml。实施例18:可代谢性油基乳液形式的疫苗制备
根据下列配方制备疫苗:
灭活猪环病毒悬浮液:200ml
Dehymuls HRE 7(Henkel):60ml
Radia 7204(Oleofina):740ml
分开单独对水相和油相过滤除菌。借助Silverson涡轮乳化机混合和匀浆化组分制备乳液。
一只疫苗剂量含有约107.5 TCID50。对于肌内途径给药,一只疫苗剂量体积为2ml。实施例19:与美国和法国PCV病毒株有关及与污染有超免疫血清(PCV-T)的PK/15的间接免疫荧光结果,以及从PK/15制备的单克隆抗体F99表和从加拿大株(PCV-C)制备的超免疫血清
*在间接免疫荧光中产生阳性反应的血清或单克隆抗体的最终稀释度之倒数。
病毒 | |||
PK/15 | 美国 | 法国 | |
PCV-T抗血清PCV-C抗血清F99 1H4F994B10F99 2B7F99 2E12F99 1C9F99 2E1F99 1H4 | ≥6400200≥10 000≥10 000≥10 000≥10 000≥10 000≥10 000≥10 000 | 200≥6400<100<100100<100<100<100100 | 800≥6400100<100<100<100100<100<100 |
Claims (29)
1.II型猪环病毒的纯化制品。
2.猪环病毒的纯化制品,其选自保藏于ECACC保藏号为V97100219、V97100218、V97100217、V98011608、V98011609的制品。
3.由细胞产生且从细胞培养物中体外分离的猪环病毒制品,其中所述细胞已用一种猪环病毒感染,该病毒能从患有PMWS综合症的猪的生理学样本或组织样本,特别是损伤组织中分离出。
4.根据权利要求3的猪环病毒制品,其从猪肾细胞系中产生并分离。
5.根据权利要求4的猪环病毒制品,其从未经PCV污染的PK/15细胞中产生并分离。
6.一种培养物提取物或上清液,其收集自用根据权利要求1的环病毒感染的细胞之体外细胞培养物。
7.一种抗原性制品,其收集自用根据权利要求1的环病毒感染的细胞之体外细胞培养物。
8.一种疫苗,其包含根据权利要求7的抗原性制品,或根据权利要求6的培养物上清液或提取物,包含猪环病毒作为抗原。
9.根据权利要求8的疫苗,其特征在于疫苗包括在兽医学可接受的载体或稀释剂中的减毒活完整抗原,以及任选地兽医学可接受的佐剂和任选地冷冻干燥稳定剂。
10.根据权利要求9的疫苗,其特征在于抗原为灭活的,且疫苗还包含兽医学可接受的载体或稀释剂,以及任选地兽医学可接受的佐剂。
11.一种DNA片段,其含有选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:6的序列。
12.一种DNA片段,其含有选自ORF1-13的ORF。
13.根据权利要求12的DNA片段,其特征在于含有选自ORF4、ORF7、ORF10和ORF13的ORF。
14.一种多肽,其由根据权利要求11-13中任一项的DNA片段编码。
15.一种体外表达载体,包括整合入其基因组中的根据权利要求11-13中任一项的DNA序列或片段,以使其可在体外表达。
16.根据权利要求15的表达载体,其特征在于其选自大肠杆菌和杆状病毒。
17.一种由根据权利要求15或16的表达载体产生的,任选地经纯化的多肽。
18.一种亚单位疫苗,包括在兽医学可接受的载体或稀释剂中的至少一种根据权利要求14或17的多肽,以及任选地兽医学可接受的佐剂。
19.一种体内表达载体,包括整合入其基因组中的根据权利要求11-13中任一项的DNA片段,以使其可在体内表达。
20.根据权利要求19的表达载体,其特征在于其选自能在猪中复制而不会使该动物致病的活病毒和质粒。
21.根据权利要求20的表达载体,其特征在于病毒载体选自猪疱疹病毒,如假狂犬病病毒;猪腺病毒;痘病毒属,特别是牛痘病毒,禽痘病毒,犬痘病毒和猪痘病毒。
22.一种活体或质粒疫苗,其特征在于其包括在兽医学可接受的载体或稀释剂中的一种根据权利要求19-21中任一项的表达载体。
23.根据权利要求8-10、18和22中任一项的疫苗,其特征在于其包含如权利要求1-4中定义的数种猪环病毒抗原。
24.根据权利要求8-10、18、22和23中任一项的疫苗,其特征在于还包含至少一种相应于另一种猪病原体的其它效价。
25.根据权利要求24的疫苗,其特征在于包含至少一种下列的其它效价:PRRS、猪肺炎支原体、大叶性肺炎放线杆菌、大肠杆菌、萎缩性鼻炎、伪狂犬病、猪霍乱和猪流感。
26.根据权利要求24的疫苗,其特征在于包含至少一种选自PRRS和猪肺炎支原体的其它效价。
27.一种探针或引物,其包含全部或部分根据权利要求11-13中任一项的序列。
28.一种从根据权利要求1-5中任一项的环病毒、根据权利要求14或17的多肽或其片段制备的多克隆或单克隆抗体。
29.一种检测猪环病毒的方法,其中,在待测猪生理学液体或组织样本中进行检测,通过试图检测抗原本身或针对抗原的抗体进行抗原存在与否的测试。
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FR97/12382 | 1997-10-03 | ||
FR9712382A FR2769321B1 (fr) | 1997-10-03 | 1997-10-03 | Nouveaux circovirus porcins, vaccins et reactifs de diagnostics |
FR9800873A FR2769322B1 (fr) | 1997-10-03 | 1998-01-22 | Nouveaux circovirus porcins, vaccins et reactifs de diagnostic |
FR98/00873 | 1998-01-22 | ||
FR9803707A FR2776294B1 (fr) | 1998-03-20 | 1998-03-20 | Nouveaux circovirus porcins ; vaccins et reactifs de diagnostic |
FR98/03707 | 1998-03-20 |
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CN200910252636.4A Division CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
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CN200910252636.4A Expired - Lifetime CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101688185B (zh) * | 2007-05-11 | 2013-05-22 | 淡马锡生命科学研究院有限公司 | 对2型猪环状构象病毒(pcv2)感染高度容许的均质细胞系的产生 |
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