为进行肿瘤细胞免疫应答的研究及制备肿瘤诊断用抗体 ,利用昆虫细胞表达系统表达了肿瘤相关性抗原HCA5 19的重组蛋白 ,并进行了纯化 .所表达的重组蛋白分子量约 10 0kD ,含 75 5个氨基酸残基 ,在HCA5 19cDNA3′端接入FLAG序列 ,则在重组蛋白C端接入 1个 8肽的FLAG尾 .利用其特异性抗体可以有效地纯化重组蛋白 .该FLAGTAG不影响整个重组蛋白的免疫反应性 .纯化的重组蛋白与天然蛋白具有相同或相似的免疫反应性 .它可与识别天然蛋白的血清抗体发生同样的阳性反应 .HCA5 19的足量提供 ,可以免疫动物 ,制备抗体 ;可作为抗原 ,分析其诱导肿瘤病人CTL的特异应答 ,从而评估其作为临床肿瘤疫苗的可能性
Abstract
The recombinant protein of HCA519 cDNA,a tumor\|related antigen encoding gene cloned previously,was expressed in insect cell expression system.The recombinant protein was purified by anti\|FLAG antibody affinity gel.The molecular weight was approximately 100 kD,and the amino acid residues were 755.To the 3′\|end of the HCA519 cDNA,the cDNA fragment encoding FLAG TAG was added,thus an 8 peptide FLAG TAG was added at the C\|terminus of the protein for the purification of the recombinant protein.The FLAG TAG did not change the immunological reactivity of the original recombinant protein.The expressed protein had the same or similar immunological reactivity,compared with the natural HCA519.It reacted with the serum containing antibody which recognized the natural protein.The availability of HCA519 recombinant protein makes it possible to prepare specific antibodies and to study its immunogenecity to induce specific CTL responses,thereby,to evaluate its application potential as candidate protein for vaccination in immunotherapy of cancers.
关键词
HCA519 /
肿瘤相关性抗原 /
重组蛋白 /
免疫反应性 /
昆虫细胞
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Key words
HCA519 /
tumor related antigen /
recombinant protein /
immunoreactivity /
insect cells
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参考文献
1 SahinU ,TureciO ,SchmittH ,CochloviusB ,JohannersT ,SchmitsR ,StennerF ,LuoG ,SchobertI,PfreundschuhM .Humanneoplasmselicitmultiplespecificimmuneresponsesintheautologoushost.ProcNatlAcadSciUSA ,1995,92:11810～11813
2 HeidebrechtHJ,BuckF ,SteinmannJ,SprengerR ,WackerHH ,Par wareschR .p100:anovelproliferation associatednuclearproteinspecifi callyrestrictedtocellcyclephasesS ,G2,andM .Blood,1997,90:226～233
3 vanderBruggenP ,TraversariC ,ChomezP ,LurquinC ,DeplaenE ,VanderEyndeB ,KnuthA ,BoonT .AgeneencodinganantigenrecognizedbycytolyticTlymphocytesonahumanmelanoma.Science,1991,254:1643～1647
4 TureciO .Dissectionoftheantibodyrecognizedcancerimmunome.Can cerImmunosurveillance,LudwigInstituteforCancerResearch,1999,S04
5 LloydJO ,ChenYT .Newpathinhumancancerserology.JExpMed,1998,187:1163～1167
6 KnuthA ,JagerE .PhaseIstudyofimmunizationwithmelanoma associ atedpeptidesderivedfromMelanA ,tyrosinaseandgp100aloneandcombinedwithGS CSFinHLA A2positivepatientswithhigh riskstageⅢormetastaticmelanoma.ClinicalTrialReportofLudwigInstituteforCancerResearch,1999
7 张拥军,余瑞元,徐长法.UtPA抗PAI 1突变体的构建及在sf9细胞中的表达.中国生物化学与分子生物学报(ZhangYong jun,YuRui yuan,XuChang fa.Constructionofamutantchimericalplas minogenactivatorresistingtoinhibitionofPAI 1anditsexpressioninsf9cell.ChinJBiochemMolBiol),2000,16(2):184～187
8 罗寿青,郑德先,刘彦信,李董,饶青,吴克复.SrhM CSFR在昆虫细胞中的表达及其配基结合活性分析.中国生物化学与分子生物学报(LuoShou qing,ZhengDe xian,LiuYan xin,LiDong,RaoQing,WuKe fu.ExpressionofsrhM CSFRininsectcellsandanalysisofbindingactivityforitsligand.ChinJBiochemMolBiol),2000,16(4):443～447
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