CAJ | 학술논문

目的筛选和鉴定人乳头状瘤病毒11型E7抗原(HPVllE7)HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位.方法预测HPVllE7抗原HLA-A*0201限制性CTL表位并合成相对应的表位多肽和四聚体(tetramer),即HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)和82-90(LLLGTLNIV).从健康HLA-A*0201成人外周血单一核细胞诱导树突状细胞(DC)并负载上述表位多肽,流式细胞技术检测DC成熟分化标记及ELISA法检测DC分泌的IL-12;成熟DC负载各组多肽后观察DC激活T淋巴细胞的效应,ELISA法检测T细胞分泌的IFN-γ;四聚体检测抗原特异性CD8+ T细胞及乳酸脱氢酶(LDH)释放法评价DC诱导的CTL对靶细胞的特异性体外杀伤效应.结果预测的5条HPVllE7表位多肽均能诱导DC的成熟分化;E7 7-15、82-90和15-23多肽负载的DC能激活T淋巴细胞分泌高水平IFN-γ;E7 7-15多肽负载的DC能刺激特异性tetramer+CD8+细胞增殖且其诱导的CTL对HPVllE7/293细胞产生高效率的特异性杀伤作用(P<0.05).结论筛选并鉴定出1条HPVllE7HLA-A*0201限制性CTL表位E7 7-15(TLKDIVLDL),负载该表位肽的DC体外可诱导高效、特异性的CTL效应,抗原性较强,有可能作为HPV感染治疗用肽疫苗的候选表位.
목적 사선화감정인유두상류병독11형E7항원(HPVllE7)HLA-A*0201한제성세포독성T림파세포(cytotoxic T lymphocyte,CTL)표위.방법 예측HPVllE7항원HLA-A*0201한제성CTL표위병합성상대응적표위다태화사취체(tetramer),즉HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)화82-90(LLLGTLNIV).종건강HLA-A*0201성인외주혈단일핵세포유도수돌상세포(DC)병부재상술표위다태,류식세포기술검측DC성숙분화표기급ELISA법검측DC분비적IL-12;성숙DC부재각조다태후관찰DC격활T림파세포적효응,ELISA법검측T세포분비적IFN-γ;사취체검측항원특이성CD8+ T세포급유산탈경매(LDH)석방법평개DC유도적CTL대파세포적특이성체외살상효응.결과 예측적5조HPVllE7표위다태균능유도DC적성숙분화;E7 7-15、82-90화15-23다태부재적DC능격활T림파세포분비고수평IFN-γ;E7 7-15다태부재적DC능자격특이성tetramer+CD8+세포증식차기유도적CTL대HPVllE7/293세포산생고효솔적특이성살상작용(P<0.05).결론 사선병감정출1조HPVllE7HLA-A*0201한제성CTL표위E7 7-15(TLKDIVLDL),부재해표위태적DC체외가유도고효、특이성적CTL효응,항원성교강,유가능작위HPV감염치료용태역묘적후선표위.
Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.