人乳头状瘤病毒11型E7抗原HLA-A＊0201限制性细胞毒性T淋巴细胞表位的功能鉴定  

Identification of predicted epitopes of HLA-A＊ 0201-restricted cytotoxic T lymphocytes derived from human papillomavirus type 11 E7 antigen

作　　者：徐妍[1] 程浩[1] 朱可建[1] 赵可佳[1] 陈贤帧[1] 卢忠明[1] 

机构地区：[1]浙江大学医学院附属邵逸夫医院皮肤科,杭州310016

出　　处：《中华微生物学和免疫学杂志》2008年第3期239-243,共5页Chinese Journal of Microbiology and Immunology

基　　金：教育部科技重点项目（G20050139）

摘　　要：目的筛选和鉴定人乳头状瘤病毒11型E7抗原（HPV11E7）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HPV11E7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HPV11E7 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HPV11ET表位多肽均能诱导DC的成熟分化；E77-15、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；E77-15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HPV11E7／293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HPV11E7HL-A＊0201限制性CTL表位E77-15（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。Objective To screen and identify the predicted epitopes of synthesized HLA-A＊ 0201 restricted CTL derived from HPV11E7 antigen. Methods Five HPV11E7 CTL epitope peptides and tetramers consisting of HLA-A＊ 0201 were selected by way of computer and synthesized by Sanquin company, including HPV11E7 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 HPV11E7 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 cells 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 HPV11E7 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 infections.

关 键 词：人乳头状瘤病毒11型 E7抗原 细胞毒性T淋巴细胞 四聚体 

分 类 号：R686[医药卫生—骨科学]