| 摘要: |
| 目的：应用T2细胞结合实验以及亲和力稳定性实验鉴定由网络表位预测服务和通用原则预测得到的乙肝病毒X蛋白（HBx）来源的HLAA0201限制性CTL表位。方法：选择中国人群最常见的B、C基因型中的adw、adr血清型的乙肝病毒X基因序列，通过网络在线表位筛选服务，分析得到共同并且得分较高的表位，并根据超基序、延展基序及量化基序方案等通用表位筛选原则进行进一步的筛选，得到4条较为理想的九肽(HBx1,HBx2,HBx3,HBx4)作为候选表位。随后借助于流式细胞技术，通过T2细胞实验分析各候选肽以及阳性、阴性、空白对照的荧光系数，从而对所筛选的表位进行体外鉴定。结果：获得的4个候选表位 (HBx1:VLCLRPVGA,HBx2:CLFKDWEEL,HBx3:VLHKRTLGL,HBx4:HLSLRGLPV) 中HBx2的亲和力较高，HBx2和HBx4的稳定性较好。结论：CLFKDWEEL是一种乙肝病毒X蛋白潜在的HLAA0201限制性CTL表位，下一步可通过体内试验对其免疫原性作一步的验证。 |
| 关键词: 肝炎病毒，乙型 HLA-A0201 HBx 表位 筛选 鉴定 T淋巴细胞，细胞毒性 流式细胞术 |
| DOI：10.3724/SP.J.1008.2007.01169 |
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| 基金项目:国家自然科学基金重大研究计划项目(30530660). |
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| Screening and identification of HLA-A0201 restricted CTL epitope from hepatitis B virus X protein in vitro |
| ZHU Yan，LI Ka，GUO Ying jun，SUN Shu-han* |
| (Department of Medical Genetics，College of Basic Medical Sciences，Second Military Medical University，Shanghai 200433，China) |
| Abstract: |
| Objective：To identify the HLAA0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro. Methods: HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr,with the highest frequencies in Chinese,were computed and analyzed by screening service offered by Internet combined with peptide supermotif,extended motif and quantitative motif prediction. Four most ideal ninepeptides (HBx1,HBx2,HBx3,and HBx4) were selected as candidate peptides. Using flow cytometry,the fluorescence index of both control and experimental groups were detected and the 4 ninepeptides were evaluated with T2 binding assay and DC50 assay. Results: The ninepeptides VLCLRPVGA (HBx1),CLFKDWEEL (HBx2),VLHKRTLGL (HBx3) and HLSLRGLPV (HBx4) were selected as candidate targets. Among the 4 candidate peptides,HBx2 showed higher HLAA0201 affinity and HBx2,HBx4 showed better stability.Conclusion: Our study indicates that CLFKDWEEL might be a potential HLAA0201 restricted CTL epitope from hepatitis B virus X protein; further study is needed for verification of its immunity in vivo． |
| Key words: hepatitis B virus HLA-A0201 HBx epitope screening identification T lymphocytes,cytotoxic flow cytometry |
