人乳头瘤病毒16型E6和E7细胞毒性T淋巴细胞表位的预测与筛选  

Prediction and screening of cytotoxic T-lymphocyte epitopes targeting human papillomavirus 16E6and E7oncoproteins

作　　者：崔湘杰 苏羽豪 朱兰芳 史磊 史荔 陶玉芬 CUI Xiangjie;SU Yuhao;ZHU Lanfang;SHI Lei;SHI Li;TAO Yufen(Shi Li Laboratory,Institute of Medical Biology,Chinese Academy of Medical Sciences,Kunming,Yunnan 650031,China)

机构地区：[1]中国医学科学院医学生物学研究所史荔实验室,云南昆明650031

出　　处：《中华肿瘤防治杂志》2024年第19期1163-1169,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：云南省重大科技专项基金(202002AA100009);中国医学科学院医学与健康科技创新工程基金(2021-I2M-1-004)。

摘　　要：目的以人乳头瘤病毒16型(HPV16)E6和E7癌蛋白为靶抗原,通过生物信息学方法及小鼠体内实验,筛选出能被人与鼠主要组织相容性复合体(MHC)共呈递的细胞毒性T淋巴细胞(CTL)优势表位。方法在美国国家生物技术信息中心(NCBI)网站上获取HPV16 E6和E7的参考蛋白序列,通过免疫表位数据库(IEDB)网站中的MHC-Ⅰ加工和MHC-Ⅰ结合方法,预测出人类白细胞抗原(HLA)-A*02:01、HLA-A*11:01、HLA-A*24:02限制性和C57BL/6小鼠H-2b限制性的CTL表位,根据评分筛选出两者共同呈递的CTL表位。同时,构建表达HPV16 E6和E7的DNA疫苗(pVAX1-E6E7Com),免疫小鼠后分离脾淋巴细胞,以预测得到的CTL表位作为抗原,与小鼠脾淋巴细胞共培养40 h后,通过酶联免疫斑点法来评估各CTL表位诱导的特异性T细胞免疫应答的能力。所有实验数据均通过单因素方差分析和Dunnett t检验进行组间比较。结果预测筛选获得10条人鼠共呈递的抗原肽。酶联免疫斑点法检测结果表明,实验组的每种表位均能诱导小鼠淋巴细胞产生特异性免疫应答,与阳性参考肽(E7-^(49)RAHYNIVTF^(57))相比,实验组中每种表位差异均无统计学意义(均P>0.05),其中E6-^(38)VYCKQQLL^(45)(175.60±52.88)、E6-^(49)VYDFAFRDL^(57)(202.20±108.50)、E6-^(86)YCYSLYGTTL^(95)(157.00±44.46)和E7-^(77)RTLEDLLMGTL^(87)(162.20±49.35)激发的特异性斑点数比阳性参考肽E7-^(49)RAHYNIVTF^(57)(156.20±97.40)的斑点数量多。结论通过这种生物信息学方法预测和筛选得到的10个CTL表位均具有一定的免疫原性,可作为HPV16型治疗性疫苗的优势候选表位。Objective Using human papillomavirus 16(HPV16)E6and E7oncoproteins as target antigens,to predict and screen cytotoxic T lymphocyte(CTL)-dominant epitopes that can be co-presented by human and murine major histocompatibility complex(MHC)molecules by bioinformatics methods and validated in mice.Methods The oncoproteins sequences of HPV16E6and E7were obtained from the National Center for Biotechnology Information(NCBI)website,and the CTL epitopes of human leukocyte antigen(HLA)-A*02:01,HLA-A*11:01,HLA-A*24:02restriction and C57BL/6mouse H-2brestriction were predicted by the MHC-Ⅰprocessing and MHC-Ⅰbinding methods in the Immune Epitope Database(IEDB)website,and then screened for co-presentation of both based on the scores.A DNA vaccine expressing HPV16E6and E7(pVAX1-E6E7Com)was also constructed,and splenic lymphocytes were isolated after immunisation of mice.The predicted CTL epitopes obtained were used as antigens,and the ability of each CTL epitope to induce specific T cell immune responses was assessed by enzyme linked immunospot assay after co-cultivation with mouse splenic lymphocytes for 40hours.The experimental data were compared between groups by one-way ANOVA and Dunnett t test.Results Ten CTL antigenic epitopes co-presented by human and murine MHC molecules were predicted and screened.The enzyme-linked immunospot assay results showed that each peptide in the experimental group induced specific immune responses in mouse lymphocytes.The difference of each peptide in the experimental group was not statistically significant(P>0.05)compared with the positive reference peptide(E7-^(49) RAHYNIVTF^(57)).Compared with the positive reference peptide(156.20±97.40),E6-^(38) VYCKQQLL^(45)(175.60±52.88),E6-^(49) VYDFAFRDL^(57)(202.20±108.50),E6-^(86) YCYSLYGTTL^(95)(157.00±44.46),and E7-^(77) RTLEDLLMGTL^(87)(162.20±49.35)elicited a higher number of specific spots.Conclusion All 10CTL epitopes predicted and screened by this bioinformatics approach are immunogenic and can be advantageous candidate epitopes for H

关 键 词：人乳头瘤病毒16型 E6 E7 细胞毒性T淋巴细胞表位 

分 类 号：R730.3[医药卫生—肿瘤]