CN1117150C - 编码诊断相关的病毒衣壳抗原的eb病毒dna序列,通过聚合酶链反应产生的表达克隆和该重组抗原在诊断检测中的应用 - Google Patents
编码诊断相关的病毒衣壳抗原的eb病毒dna序列,通过聚合酶链反应产生的表达克隆和该重组抗原在诊断检测中的应用 Download PDFInfo
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Abstract
本申请描述了EB病毒编码基因的鉴定,和迅速构建有效表达该基因或其它基因的质粒的方法。可纯化该EBV基因并用作诊断和治疗EBV相关疾病的抗原。
Description
本发明涉及编码一种EB病毒相关抗原的EB病毒基因组的DNA序列,和定位及分离至少部分个别DNA序列的方法,这一抗原可用于下面所提及的方法和诊断组合物及药物组合物。而且,本发明涉及一种快速、简便、极灵敏和高度专一性的检测针对EB病毒相关抗原的抗体的方法及组合物或试剂盒。在这些检测中,所述EB病毒抗原明显提高了病人血清中特异性抗体种类的检出率。该检出率提供了有关供体感染状态如感染前、初次感染、慢性感染,恢复期和致瘤条件的可信结论。
EB病毒(EBV)感染及结果
EB病毒引起的初始症状为传染性单核细胞增多。这主要涉及儿童或年轻的成人。平均百分之九十多的成年人感染过EB病毒并成为它的终生携带者。这种病毒终生在口咽部产生并通过口的途径传播。而且幼童的感染往往会导致几乎无症状的血清转化现象,成年人的感染则经常引起伴有高烧、白细胞数量显著增加、咽喉炎、肝脏和脾脏肿大的严重疾病。猩红热、弓形体病、白喉和白血病须经诊断手段加以区别。
慢性感染
与急性原发性感染相比,EBV感染很少导致慢性的或慢性波动症状。
毛状白斑
在免疫缺损病人中,EBV能导致舌部的严重病变。
在免疫丧失个体中EBV的重新激活
抑制细胞介导的免疫导致EBV的重新激活。一个结果是抗体滴度升高,而且已描述了对多种可能状况如免疫排斥的有害影响。
X染色体连锁增殖综合症
某些遗传位点与男性同胞感染而导致致命的淋巴网状内皮细胞疾病相关。
Burkitt′s淋巴瘤和EBV
Burkitt′s淋巴瘤的发生与染色体重排有关。并非所有的肿瘤细胞里都含有EBV基因组。但是,至少在高发区,97%的这种肿瘤是EBV相关的,而且控制EBV感染可能会降低发生Buikitt′s淋巴瘤的风险。
鼻咽癌一种与EBV相关的“继发疾病”
另一种与EBV表现出100%相关的疾病为鼻咽癌(NPC)(“TheBiology of Nasopharyngeal Carcinoma”,UICC technical report series,vol.71,M.J.Simons and K.Shanmugaratnam(eds),InternationalUnion Against Cancer,Geneva,p.t(1982)。NPC最常见发生在鼻后腔的Roserrmeller氏窝(咽隐窝)。病人往往只在颈部淋巴结中发生第一个典型转移后才就医。
EBV相关肿瘤的控制
有三种可能的基本策略控制肿瘤:
1.早期检测后进行治疗,
2.将疾病的发生理想地推迟至平均寿命之外,和
3.预防
这些目标也可在多因素疾病中实现,如多种肿瘤。可通过消除一种或多种必需因素(这些因素自身不一定能导致疾病),或者可通过减少那些促进显现肿瘤发生条件的因素来降低疾病的发生率。应用本发明的特异性病毒相关抗原、抗体或遗传物质作为工具,用于病毒相关肿瘤的早期诊断,有利于必需因子的排除。
应用EBV相关基因产物诊断EBV相关的NPC
在不能获得确切组份的重组抗原时,最常规的病毒特异性血清学试验是基于免疫荧光试验,即在免疫荧光和免疫酶试验中使用具有各种特点的EBV基因组阳性细胞和/或预处理成常与细胞相关的抗原来源。表1总合了疾病、抗体和可用于诊断的病毒抗原。
表1:EBV导致的疾病和可通过常规免疫荧光试验来确定的Ig-亚类特异性抗体反应的相关性疾病: VCA IgG IgM IgA EA IgG EBNA IgG MA1IgG正常成人 + - - - +成人急性感染(早期)++ + - + - -慢性感染 + + - +/- +/- +/-?重新激活 + + - + + +XLP2 + - - +/- (+) ?NPC ++ - + +(D) + ++BL ++ - - +(R) + +
VCA:病毒衣壳抗原;EA:早期抗原;EBNA:Epstein-Burr核抗原;1.通过gp240/200免疫沉淀确定;2.XLP作为免疫丧失宿主的一个例子。
因为免疫应答的制约,目前使用的EBV抗原不能被所有的EBV感染的人识别。而且,许多EBV相关抗原在蛋白水平上与其它疱疹病毒具有序列同源性。因此,本发明所解决的技术问题是提供一种更可靠的诊断EBV感染的EBV相关蛋白。上述技术问题可通过所提供的权利要求书中表述的具体实施方案来解决。因此本发明提供一种没有重大遗传变异的EBV编码探针。
本发明EBV特异性抗原的产生
1.所有发现的结果表明,本发明的目的之一是提高检测抗体种类和病毒衣壳抗原种类的特异性抗体试验的灵敏度,改进一允许大量试验和更加标准化的体系。
2.重组DNA技术的应用使确定一对改良EBV抗原性重要的多肽成为可能。用重组DNA分子转化适当的宿主细胞及在适当的培养系统中培养则可达到产生抗原的目的。
3.根据本发明,重组DNA的方法,包括聚合酶链反应的改进引物的选择,被用以在合适的宿主细胞中表达编码23KDa、EBV蛋白(以BLLF2读起)基因或至少部分基因的遗传信息,如在细菌(如大肠杆菌属,沙门氏菌属,假单胞菌属或牙孢杆菌属),酵母(如假丝酵母属或酿酒酵母属),昆虫细胞(如草地夜蛾SF251)及哺乳细胞(如Vero细胞,CHO细胞或淋巴细胞系)中表达。
4.而且,鉴定了编码23KDaEBV蛋白的基因组区,并阐明了它们与诊断目的的相关性。因此,产生这些蛋白或其抗原决定簇的关键信息也在本发明中给予揭示。
该DNA序列可与一任何来源得到的DNA序列杂交,来源包括天然的、合成的或半合成的,其通过突变,包括核苷酸置换、缺失、插入及核苷酸序列的倒位,与编码和EBVB95-8蛋白中读码框架BLRF2编码的23KDa蛋白相关的至少一部分蛋白的DNA序列相关。优选的杂交条件为低于同源双体DNA分子熔点15~27℃的范围内。本发明涉及通过重组DNA技术制备EBV特异性抗体,及其在EBV相关疾病的诊断、预防和治疗中的应用。因此,本发明的一个目的是要鉴定一种新的E-B病毒抗原,其通过免疫学方法与E-B病毒相关疾病如鼻咽癌(NPC),传染性单核细胞增多症和Burkitt′s淋巴瘤(见表1说明)相互关联。本发明的另一个目的是定位和鉴定EBV的基因组区域,例如已从B95-8细胞中克隆得到(American Type Culture Collection,Rockville,MD USA(ATCC)CRL1612)(Skare,J.and Strominger,J.L.Cloning and mapping ofBamHI endonuclease fragments of the DNA from the transformingB95-8 strain of Epstein Barr Virus;Proc.Natl.Acad.Sci.USA 77:3860(1980)),其编码具有诊断学重要性和与医药用途相关的所述抗原。这可通过杂交筛选方法结合与病人血清免疫沉淀来实现(图1)。本发明的又一目的是EBV基因组片段的亚克隆,例如可来自存在的EBV文库,从编码有用抗原p23的至少一部分的B95-8细胞克隆。这可通过聚合酶链式反应(PCR)来实现,PCR可以不要求在读码框架末端有适当限制性内切酶酶切位点而扩增基因组DNA片段,并可通过适当的克隆方法将这些含有引物序列合成末端的扩增基因插入到表达质粒中。很明显,同样的克隆方法可用于从其它与B95-8编码序列不同的病毒分离物中扩增目的基因。针对其目的,可能需要其它引物,其制备和选择在本领域中已阐明。通过选择具有附加序列的引物——序列的5’与编码区互补(5′引物)或序列的3′与编码区互补,使得直接从聚合酶链反应产物建立克隆而不必通过广泛的亚克隆成为可能,因为PCR产物可通过附加的调控信号来优化表达。本发明的另一个目的是通过在适当的宿主细胞中表达各种遗传信息以产生蛋白,如在细菌(例如大肠杆菌属、沙门氏菌属、假单孢菌属或芽孢杆菌属),酵母(如假丝酵母属、酿酒酵母属),动物细胞和人细胞(如Vero细胞;CHO-dhfr-细胞;结合一套适当的选择系统,可选择带有功能性dhfr基因及由适当的调控序列调控的EBV基因的遗传信息的质粒;或淋巴母细胞系)中表达。由这些宿主细胞产生的蛋白含有23KDa相关抗原决定簇(表位),根据表达系统,可合成为融合蛋白或非融合蛋白。为了由细菌产生融合蛋白,编码EBV B95-8的p23的基因组亚片段的表达可导入至已知的质粒pUC8中,并由如异丙基-β-D-硫代半乳吡喃糖苷(IPTG)诱导。不同的表达产物用免疫学方法鉴定。为了产生只含天然存在蛋白或其部分的氨基酸序列的非融合蛋白,可修饰本发明的重组质粒。如果一个寡核苷酸接头被插入到表达载体中细菌蛋白编码区和EBV相关蛋白编码区之间,相应于寡核苷酸接头的氨基酸序列就变成了表达的融合蛋白的一部分。从表达该蛋白的转化子中分离该融合蛋白后,它可通过氨基酸序列特异性蛋白酶在引入的氨基酸接头处进行切割,或者如果氨基酸接头含有对酸切割敏感的肽键,则可通过酸处理(如甲酸)进行裂解。
本发明的又一目的是应用23KDa EBV相关蛋白或其亚区,或者如果适宜的活,EBV相关DNA片段或克隆,以生产诊断组合物(试剂盒),用于临床诊断或科学研究。这些检测是基于已建立的方法,如ELISA、RIA(放免分析)或间接血凝试验。而且,EBV相关蛋白可用于如监视免疫接种进展、分析流行病学问题、病人治疗(药物成份)和生产作为治疗EBV相关疾病如单核细胞增多症、Burkitti′s淋巴瘤和鼻咽癌的预防措施的疫苗。总之,该蛋白可用于预防和治疗EBV相关疾病,因为它能调节患有如NPC、慢性传染性单核细胞增多症或EBV相关的Burkitt′s淋巴瘤的病人的免疫应答。
附图的简要说明
图1:用EBV基因组的BamHI DNA片段选择的杂交RNA体外翻译产物的放射自显影。应用NPC病人的EBV特异性混合血清进行免疫沉淀。通过SDS聚丙烯酰胺凝胶电泳分离免疫沉淀的35S标记蛋白并将X光片对胶曝光。对照,称为“混合液”,含有所有的免疫沉淀EBV特异性蛋白。从放射自显影中可发现针对23KDa的抗体存在于EBV阳性血清中,还显于来自佛波醇酯诱导的P3HR1细胞的RNA可与EBV DNA的BamHI限制性内切酶酶切片段杂交,该片段是在体外洗脱并翻译的。翻译产物用经EBV基因组阴性的BJAB细胞预先吸附的混合EBV反应性血清免疫沉淀,如以前所描述的方法(Seibl,R.and Wolf H.Mapping of Epstein-BarrVirus proteins on the genome by translation of hybrid-selected RNAfrom induced P3HR1 cell and induced Raji cells.Virology 141:1-13(1985))。
图2.与EBV B95-8基因组相关的mRNA 5精细图谱
图的底部给出了EBV B95-8基因组的BamHI酶切位点,并且不同限制性片段用大写和小写字母标注。通过对个个BamHI片段进行杂交选择而定位的蛋白的mRNA用数字和横线来表示。从该精细图谱中可发现针对23KDa的抗体存在于EBV恢复期血清中。
图3.A)在BamHI-L片段中读码框架的定位
23KDa大小的EBV抗原通过杂交选择翻译进行作图。BLLF1编码EBV主要膜抗原,BLLF2、BLRF1和BLRF3编码量论分子量为31.11KDa和12KDa的蛋白。BLLF3也画在附近的BamHI-S片段中,但23KDa抗原编码区只在于BamHI-L中,因此编码23KDa抗原最可能的选择似乎是BLRF2编码的蛋白。
B)BLRF2双链5′和3′端的扩大及应用于PCR的寡聚脱氧核苷酸引物
引物1与编码23KDa蛋白N-末端的DNA序列杂交。互补链在第二个密码子后开始,在翻译起始点附近的区域改成一BspHI位点,用以克隆到ATG-表达载体。这包括将第二个密码子从TCA改为AGC,两者都编码丝氨酸。ATG启始密码子上游的寡脱氧核苷酸序列编码一BamHI限制性内切酶位点和几个额外的碱基,以便于限制性内切酶酶切和克隆。引物2杂交在23KDa的翻译终止位点,并在其5’末端含有一HindIII位点以插入载体中。两个引物的非杂交5′末端在第一个PCR循环时都不合成到互补链上,直到当新产生的PCR产物用作模板时,在后面的循环中才能合成。
图4.扩增DNA的形成和克隆
1(B)所示寡脱氧核苷酸引物在一8700DNA合成仪(Biosearch/MILLIGEN)的1mol柱中合成。因为合成效率很高,所以PCR中使用从柱上分离下来的引物不需进一步纯化。五个含有100μl相同组份的PCR反应按厂家的说明平行进行,其含有引物总产量的0.25%,4ng含EBVBamHI-L片段的质粒(Skare,J.and Stro-minger,J.L.Cloning and Mapping of BamHI endo nuclease fragmentsof the DNA from the fransforming B95-8 strain of Epstein-BarrVirus.Proc.Natl.Acad.Sci.USA 77:3860(1980)),核苷酸,缓冲液和Taq-聚合酶。反应在一Ericomp温度循环仪上进行,50℃退火2分钟,72℃延伸4分钟及94℃变性1分钟,进行50个循环,收集最终DNA,用乙醇沉淀并干燥。重悬于200μl H2O中,然后用BamHI和HindIII进行酶切,该DNA片段经1.5%琼脂糖凝胶电泳进行纯化,分离并与用BamH1和HindIII线性化的pUR289连接。转化至E.coli JM 109中后(Yanish-Perron et al.Improved M13 phageCloning Vectors and host strains nucleotide sequences of M13mp18 andpUC19 vectors,Gene 33:103-119(1985)),通过限制性内切酶消化23KDa抗原编码片段以筛选菌落的质粒。经IPTG诱导阳性克隆、蛋白的PAGE电泳及考马斯亮兰染色或Western印迹显示EBV抗原的产生(图5)。从一生产性克隆pUR23K中,通过BamHI和HindIII的降解重新分离出23KDa编码片段并插入到pDS(pD523K)中。第三步,经BspHI和HindIII剪切而分离到的片段与用Ncol和HindIII线性化的pKK233-2连接(PKK23K)。最后,从PDS23K中得到一370bp的NlaIV/HindIII片段,并连接到因HindII和HindIII线性化的pUC8中,得到pUC8-N234。所有的所述表达克隆方法提供从载体序列到EBV片段的可读框架中的翻译功能。
图5.大肠杆菌中23KDa表达产物的鉴定
IPTG诱导克隆(1mM IPTG 37℃3小时)产生的蛋白经SDS-17.5PAGE和考马斯亮兰染色(左侧系列)或经Western印迹和用高滴度NPC病人混合血清免疫染色来分析。泳道1:pUD289,β-Gal的表达;泳道2:pUR23K;泳道3:pDS23K;泳道4:pKK23K;泳道5:PUCN23K。泳道2中的β-Gal::23KDa融合蛋白比泳道1的β-Gal(116KDa)要大,表明扩增的DNA片段的通读。泳道3的23KDa表达产物大小约为24KDa,产量高。但泳道4中ATG载体的真正23KDa表达产物并未有效产生。泳道5中pUCN23的C-末端23KDa抗原片段似乎是稳定的而且非常有效地产生。所有抗原与NPC混和血清的反应都很强,表明了在病毒感染时这种蛋白的抗原性。
图6.EB病毒读码框架BLRF2的23KDa抗原的DNA和氨基酸序列
下列实施例说明本发明
实施例1:23KDa蛋白编码区的扩增
在EBV的BamHI-L片段上23KDa蛋白的可能编码区(BLRF2)含162个氨基酸。在5’和3’末端没有适当的限制性内切酶位点用以克隆全长的读码框架。因为这一原因,我们合成了两个与编码区5’和3′末端互补的寡脱氧核苷酸,作为引物用于PCR。每个引物在5′末端的附加序列中含有用于克隆扩增DNA的限制性酶切位点(图1)及几个附加核苷酸以便于用这些限制性内切酶剪切。PCR扩增后,扩增的DNA片段用BamHI和HindIII剪切,经琼脂糖凝胶电泳纯化用于用质粒载体克隆。一含有来自EBV B95-8分离株的完整的BamHI-L片段的质粒(Skare,J.and Strominger,J.L.Cloning and mapping of BamHI endonuclease fragments of the DNAfrom the transforming B95-8 Strain of Epstein-Barr Virus,Proc.Natl.Acad.Sci.USA 77:3860(1980))用作模板。
实施例2:作为β-半乳糖苷酶融合蛋白的23KDa VCA在大肠杆菌中的表达。
因为许多重组抗原在大肠杆菌中不能以非融合抗原稳定表达,第一种改进方法是产生β-半乳糖苷酶融合蛋白,其中大细菌蛋白理论上可阻止不稳定蛋白片段的降解。为此,用BamI和HindIII消化的扩增DNA被克隆到βGal表达载体pUR289的lacZ基因的读框3’端(Ruther和Muller-Hill,Easy identification of cDNA clones.EMBO J.2(1983)1791-1794)(图4)。会有这一载体的大肠杆菌克隆用IPTG诱导以便表达βGal::23KDa融合蛋白。图5表明已合成了分子量高于真正βGal的产物。该融合蛋白与含有高EBV抗体滴度的血清持异性反应。这表明已发生了正确的翻译,PCR没有引入重要的错误或终止密码,并且产生了有免疫原性的EBV抗原。
实施例3:23KDa VCA作为一种真正蛋白的表达
从pUR289-23K中用Bam HI和HindIII分离EBV编码区,并克隆到此处重新命名为pDS12/RBSII-2的pDS3中(Bujard,H.etal.The T5 promotor-based trascription translation system for theanalysis in vitro and in vivo.Meth.Enzymol.155:416(1987)),这可提供与23KDa编码区相同的翻译读框(图4)。IPTG诱导后,一23KDa新的蛋白条带在考马斯染色的PAGE上出现。它在Western印迹上可特异性地与EBV血清反应,并约占可从考马斯染色的凝胶上估量的全部大肠杆菌蛋白的约10%(图5)。因为表达产物含有一些来自克隆位点和寡脱氧核苷酸序列的氨基酸,通过用BspHI和HindIII分离该片段并将其插入到ATG载体pKK233-2的NcoI和HindIII位点,以实现真正蛋白的表达(Amann,E.and Brosius,J.`ATG Vectors′for regulated high-level expression of cloned gene inEscherichia coli.Gene 40:183-190(1985))。但是,通过这一载体23KDa抗原的表达产量远低于pDS载体中的量,这可能是由于改变了N-末端序列而导致这种蛋白在大肠杆菌细胞中的低效翻译。
对编码23KDa抗原的C-末端部分的较小片段进行进一步的表达。从编码区中分离NlaIV/HindIII片段并插入到pUC8的HindII/HindIII中(Vieira,B.and Messing,B.The pUC plasmids,a M13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal primers.Gene 19(1982)259-268),以形成PUCN23K。IPTG诱导后,发现非常高效地产生抗原。但因为这一抗原不含真正蛋白的N-末端,所以它不能用于进一步评估。
实施例4:23KDa VCA的反应性和特异性
重组抗原的诊断学价值的初步评估是用从各种血清来源的EBV阳性和阴性血清进行的,例如来自传染性单核细胞增多症病人、NPC病人和健康携带者的血清。
对来自pDS23K的含23KDa抗原的细菌粗裂解物和作为对照的不含外源抗原的裂解物分别进行PAGE。将蛋白转移到硝酸纤维素膜上并将其条带与血清孵育。免疫染色的Western印迹条带的结果总结于表II中。通过与传统的免疫荧光检测比较,来自健康携带者和NPC病人的VCA阳性血清对23KDa抗原显示清楚的阳性。仅有两份在免疫荧光(IF)检测中的反应性血清没有反应。以IF确定的三份来自新感染的人的血清也没有反应,而三份被认为EBV阴性的血清用p23KDa抗原发现为阳性。总之,可以发现IF和Western印迹活性具有广泛的相关性。但需要进一步的测试以检查这种抗原与IF检测相比的相关性和敏感性。
表I:与IF结果的比较,23KDa抗原与各种血清的反应性IF-反应性 EBV状态a 检测血清数 与重组23K抗原的
反应性b
阳性 阴性VCA+EBNA+ 健康携带者 34 32 2VCA+EBNA+ NPC 病人 4 4 0VCA+EBNA- 新感染者 9 6 3VCA-EBNA- EBV 阴性 6 3 3
a.EBV状态通过IF反应性和其它临床指标的常规诊断来确定。
b.23KDa抗原的反应性通过Western印迹来测定。
根据IF检测的血清分类:VCA+,EBNA+=健康携带者;VCA+,EBNA+,临床指标=NPC;VCA+,EBNA-,临床指标=新感染者;VCA-,EBNA-=EBV阴性。
(1)一般信息:
(i)申请人:
(A)姓名:WOLF,hans Joachim
(B)街道:Josef-Jaegerhuber-Strasse 9
(C)城市:Starnberg
(E)国家:德国
(F)邮政编码(ZIP):82319
(A)姓名:REISCHI,Udo
(B)街道:Grosse Leite 7
(C)城市:Grafenau
(E)国家:德国
(F)邮政编码(ZIP):94481
(A)姓名:MOTZ,Manfred
(B)街道:Schachnerstrasse 7
(C)城市:Munich
(E)国家:德国
(F)邮政编码(ZIP):81379
(ii)发明题目:
编码诊断相关的病毒衣壳抗原的EB病毒DNA序列,通过聚合酶链反应产生的表达克隆和该重隆在诊断检测中的应用
(iii)序列数目:2
(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:Patent In Release #1.0,Version #1.25(EPO)
(v)本申请资料
申请号:PCT/EP94/02436
(vi)在先申请的资料:
(A)申请号:EP93 11 1883.0
(B)申请日:1993年7月23日
(2)SEQ ID NO:1的信息:
(i)序列特征:
(A)长度:489碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑型:线性
(ii)分子类型:蛋白质
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1...486
(xi)序列描述:SEQ ID NO:1ATG TCA GCT CCA CGC AAA GTC AGA TTG CCT TCT GTT AAG GCT GTT GAC 48Met Ser Ala Pro Arg Lys Val Arg Leu Pro Ser Val Lys Ala Val Asp1 5 10 15ATG AGC ATG GAA GAC ATG GCC GCC CGC CTG GCT CGC CTG GAG TCT GAG 96Met Ser Met Glu Asp Met Ala Ala Arg Leu Ala Arg Leu Glu Ser Glu
20 25 30AAT AAG GCT CTG AAG CAA CAG GTC CTC AGA GGG GGT GCC TGT GCC TCG 144Asn Lys Ala Leu Lys Gln Gln Val Leu Arg Gly Gly Ala Cys Ala Ser
35 40 45TCT ACC TCT GTT CCT TCT GCT CCA GTG CCT CCG CCT GAG CCG CTT ACA 192Ser Thr Ser Val Pro Ser Ala Pro Val Pro Pro Pro Glu Pro Leu Thr
50 55 60TCT CGA CAG CGA GAG GTA ATG ATT ACG CAG GCC ACG GGC CGT TTG GCG 240Ala Arg Gln Arg Glu Val Met Ile Thr Gln Ala Thr Gly Arg Leu Ala65 70 75 80TCT CAG GCT ATG AAG AAG ATT GAA GAC AAG GTT CGG AAA TCT GTT GAC 288Ser Gln Ala Met Lys Lys Ile Glu Asp Lys Val Arg Lys Ser Val Asp
85 90 95GGT GTA ACT ACC CGC AAT GAA ATG GAA AAT ATA TTG CAA AAT CTG ACC 336Gly Val Thr Thr Arg Asn Glu Met Glu Asn Ile Leu Gln Asn Leu Thr
100 105 110CTC CGC ATT CAA GTA TCT ATG TTG GGT GCA AAA GGC CAA CCC AGC CCT 384Leu Arg Ile Gln Val Ser Met Leu Gly Ala Lys Gly Gln Pro Ser Pro
115 120 125GGT GAG GGA ACA CGA CCA CGA GAA TCA AAC GAC CCC AAC GCC ACC CGA 432Gly Glu Gly Thr Arg Pro Arg Glu Ser Asn Asp Pro Asn Ala Thr Arg
130 135 140CGT GCC CGC TCC CGC TCC CGG GGA CGT GAA GCA AAG AAA GTG CAA ATT 480Arg Ala Arg Ser Arg Ser Arg Gly Arg Glu Ala Lys Lys Val Gln Ile145 150 155 160TCT GAT TAA 489Ser Asp
(2)SEQ ID NO:2的信息
(i)序列特征:
(A)长度:162个氨基酸
(B)类型:氨基酸
(D)拓扑型:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2Met Ser Ala Pro Arg Lys Val Arg Leu Pro Ser Val Lys Ala Val Asp1 5 10 15Met Ser Met Glu Asp Met Ala Ala Arg Leu Ala Arg Leu Glu Ser Glu
20 25 30Asn Lys Ala Leu Lys Gln Gln Val Leu Arg Gly Gly Ala Cys Ala Ser
35 40 45Ser Thr Ser Val Pro Ser Ala Pro Val Pro Pro Pro Glu Pro Leu Thr
50 55 60Ala Arg Gln Arg Glu Val Met Ile Thr Gln Ala Thr Gly Arg Leu Ala65 70 75 80Ser Gln Ala Met Lys Lys Ile Glu Asp Lys Val Arg Lys Ser Val Asp
85 90 95Gly Val Thr Thr Arg Asn Glu Met Glu Asn Ile Leu Gln Asn Leu Thr
100 105 110Leu Arg Ile Gln Val Ser Met Leu Gly Ala Lys Gly Gln Pro Ser Pro
115 120 125Gly Glu Gly Thr Arg Pro Arg Glu Ser Asn Asp Pro Asn Ala Thr Arg
130 135 140Arg Ala Arg Ser Arg Ser Arg Gly Arg Glu Ala Lys Lys Val Gln Ile145 150 155 160Ser Asp
Claims (5)
1.一种检测体外样品中EBV的存在的方法,该方法包括在体外测定获自患者的血清中针对EBV p23kDa抗原的抗体含量,所述EBV p23kDa抗原具有图6所示序列,其中所测定抗体的存在情况和量指示EBV的存在情况。
2.根据权利要求1的方法,该方法包括:
(a)将该获自患者的体外血清样品与其上吸附有所述EBV 23kDa抗原的膜一起孵育;
(b)通过对膜进行免疫染色检测结合的抗体。
3.根据权利要求1或2的方法,其特征在于经免疫测定方法测定EBV的存在。
4.根据权利要求3的方法,其中所述免疫测定方法选自包括免疫荧光测试VCA、ELISA、RIA和间接血细胞凝集试验的组。
5.用于进行权利要求1-4任一项的方法的诊断性组合物,其中包含具有图6所示序列的蛋白质。
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EP93111883A EP0726315B1 (en) | 1993-07-23 | 1993-07-23 | Epstein-Barr virus DNA sequences encoding a diagnostically relevant virus capsid antigen, expression clones derived through polymerase chain reaction and the use of this recombinant antigen in diagnostic tests |
EP93111883.0 | 1993-07-23 |
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US (1) | US5741656A (zh) |
EP (1) | EP0726315B1 (zh) |
JP (1) | JPH09502864A (zh) |
CN (1) | CN1117150C (zh) |
AT (1) | ATE207120T1 (zh) |
AU (1) | AU7495494A (zh) |
CZ (1) | CZ20996A3 (zh) |
DE (1) | DE69330966T2 (zh) |
ES (1) | ES2166757T3 (zh) |
WO (1) | WO1995003415A2 (zh) |
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ATE429517T1 (de) * | 1998-03-04 | 2009-05-15 | Biomerieux Bv | Vervielfältigung und nachweis von ebv barf1 zur diagnose von nasopharyngealen karzinom |
BRPI0709282B8 (pt) | 2006-03-29 | 2021-05-25 | Merial Ltd | vacina contra estreptococos |
CN104139155A (zh) * | 2013-05-06 | 2014-11-12 | 福特汽车公司 | 用于生成模具组件的模的添加制备方法 |
Citations (2)
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EP0173254A1 (en) * | 1984-08-23 | 1986-03-05 | Hans Joachim Wolf | DNA sequences of the EBV genome, recombinant DNA molecules, processes for producing EBV-related antigens, diagnostic compositions and pharmaceutical compositions containing said antigens |
WO1991008224A1 (en) * | 1989-11-24 | 1991-06-13 | The Council Of The Queensland Institute Of Medical Research | Im peptides |
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US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
WO1991002091A1 (en) * | 1989-08-10 | 1991-02-21 | Northwestern University | Method of identifying herpesviruses and oligonucleotides for use therein |
WO1991009127A1 (en) * | 1989-12-20 | 1991-06-27 | Schering Corporation | BCRF1 PROTEINS AS INHIBITORS OF INTERFERON-$g(g) |
DE69332197T2 (de) * | 1992-03-13 | 2003-04-17 | Organon Teknika Bv | Epstein-Barr-Virus verwandte Peptide und Nukleinsäuresegmenten |
-
1993
- 1993-07-23 EP EP93111883A patent/EP0726315B1/en not_active Expired - Lifetime
- 1993-07-23 DE DE69330966T patent/DE69330966T2/de not_active Expired - Lifetime
- 1993-07-23 AT AT93111883T patent/ATE207120T1/de active
- 1993-07-23 ES ES93111883T patent/ES2166757T3/es not_active Expired - Lifetime
-
1994
- 1994-07-22 WO PCT/EP1994/002436 patent/WO1995003415A2/en not_active Application Discontinuation
- 1994-07-22 CN CN94193098A patent/CN1117150C/zh not_active Expired - Lifetime
- 1994-07-22 AU AU74954/94A patent/AU7495494A/en not_active Abandoned
- 1994-07-22 US US08/586,640 patent/US5741656A/en not_active Expired - Lifetime
- 1994-07-22 CZ CZ96209A patent/CZ20996A3/cs unknown
- 1994-07-22 JP JP7504952A patent/JPH09502864A/ja active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0173254A1 (en) * | 1984-08-23 | 1986-03-05 | Hans Joachim Wolf | DNA sequences of the EBV genome, recombinant DNA molecules, processes for producing EBV-related antigens, diagnostic compositions and pharmaceutical compositions containing said antigens |
WO1991008224A1 (en) * | 1989-11-24 | 1991-06-13 | The Council Of The Queensland Institute Of Medical Research | Im peptides |
Non-Patent Citations (2)
Title |
---|
《分子克隆实验手册》1992-01-01 [美]J.萨姆布鲁克等著科学出版社(中国)(第二版) * |
NATURE VOL.310NO,5974,PAGE 207-211 1984-01-01 R.BEAR ET AL,DNA SEQUENCE AND EXPRESSION OF THE B95-8EPSTELN-BARRVIRUS GENOME * |
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Publication number | Publication date |
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EP0726315A1 (en) | 1996-08-14 |
ES2166757T3 (es) | 2002-05-01 |
US5741656A (en) | 1998-04-21 |
EP0726315B1 (en) | 2001-10-17 |
CZ20996A3 (en) | 1996-10-16 |
DE69330966T2 (de) | 2002-06-06 |
AU7495494A (en) | 1995-02-20 |
JPH09502864A (ja) | 1997-03-25 |
WO1995003415A2 (en) | 1995-02-02 |
WO1995003415A3 (en) | 1995-03-16 |
ATE207120T1 (de) | 2001-11-15 |
DE69330966D1 (de) | 2001-11-22 |
CN1129460A (zh) | 1996-08-21 |
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