CAJ | 학술논문

目的采用T细胞表位预测软件结合体外实验鉴定丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位.方法采用T表位预测软件Rankpep预测HCV特异性CTL表位,选择候选CTL表位加以合成;用候选CTL表位肽分别刺激HCV感染者以及健康志愿者的外周血单个核细胞(PBMC),采用酶联免疫斑点试验(ELISPOT)检测PBMC中肽特异性分泌IFN-γ的斑点形成细胞(spots forming cells,SFC)的水平,采用细胞内细胞因子染色(intracellular cytokine staining,ICS)检测PBMC中肽特异性IFN-γ+CD8+T细胞的水平.结果用5条候选CTL表位肽[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)和NS5a 367(TVSSALAEL)]分别刺激10个HCV感染者和2个健康者的PBMC后,健康者的PBMC不产生IFN-γ而7个HCV感染者的PBMC产生IFN-γ;HCV感染者的PBMC中肽特异性分泌IFN-γ的细胞的频率为(5-36)SFC/105 PBMC,肽特异性IFN-γ+CD8+T细胞占总CD8+T细胞的百分比为0.02%～0.25%.结论 ELISPOT结果和ICS结果证实5条肽NS3 450、NS3 594、NS4b 78、NSSa 416和NS5a 367为全新的HCV特异性CTL表位.
목적 채용T세포표위예측연건결합체외실험감정병형간염병독(HCV)특이성세포독성T세포(CTL)표위.방법 채용T표위예측연건Rankpep예측HCV특이성CTL표위,선택후선CTL표위가이합성;용후선CTL표위태분별자격HCV감염자이급건강지원자적외주혈단개핵세포(PBMC),채용매련면역반점시험(ELISPOT)검측PBMC중태특이성분비IFN-γ적반점형성세포(spots forming cells,SFC)적수평,채용세포내세포인자염색(intracellular cytokine staining,ICS)검측PBMC중태특이성IFN-γ+CD8+T세포적수평.결과 용5조후선CTL표위태[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)화NS5a 367(TVSSALAEL)]분별자격10개HCV감염자화2개건강자적PBMC후,건강자적PBMC불산생IFN-γ이7개HCV감염자적PBMC산생IFN-γ;HCV감염자적PBMC중태특이성분비IFN-γ적세포적빈솔위(5-36)SFC/105 PBMC,태특이성IFN-γ+CD8+T세포점총CD8+T세포적백분비위0.02%～0.25%.결론 ELISPOT결과화ICS결과증실5조태NS3 450、NS3 594、NS4b 78、NSSa 416화NS5a 367위전신적HCV특이성CTL표위.
Objective To identify hepatitis C virus(HCV)-specific cytotoxicity T lymphocytes (CTL)epitopes by the combination of T epitopes prediction software and in vitro assays.Methods HCVspecific CTL epitopes were predicted by T epitope prediction software Rankpep and then candidate HCV-specific CTL epitopes were selected.Candidate HCV-specific CTL epitopes were used to stimulate PBMC of HCV-infected patients and healthy volunteers.and then enzyme-linked immunospot(ELISPOT)and intracellular cytokine staining(ICS)were used to measure the frequencies of IFN-γ-producing cells in total PBMC and the percentages of IFN-γ+CD8+T cells in total CD8+T cells,respectively.Results Five candidate CTL epitopes[NS3 450(TVPQDAVSR),NS3 594(GPTLLYRL),Ns4b 78(sMMAFSAAL),NS5a 416(SEENVSVVF)and NS5a 367(TVSSALAEL)]were used to stimulate PBMC of ten HCV-infected patients and two healthy volunteers.PBMC of seven HCV-infected patients secreted IFN-γ while PBMC of healthy volunteers did not produce IFN-γ.The frequencies of peptide-specific IFN-γ-producing cells ranged from 5 to 36 SFC/105 PBMC and the percentages of peptide-specific IFN-γ+CD8+T cells ranged from 0.02%-0.25%.Conclusion Resuhs of ELISPOT assay and ICS assay confirm that these five peptides NS3 450,NS3 594,NS4b 78,NS5a 416 and NS5a 367 are identified as Hovel HCV-specific CTL epitopes.