人类免疫缺陷病毒-1型负向调节因子多肽特异性CD4＋T淋巴细胞克隆的建立和筛选  

Construction and screening of human immunodeficiency virus-1 negative regulation factor peptide-specific CD4＋T lymphocyte clone

作　　者：宋培新[1] 李彦磊[2] 郑楠[2] 

机构地区：[1]南京大学医学院附属鼓楼医院感染科,210008 [2] 南京大学医学院公共健康医学研究中心

出　　处：《中华传染病杂志》2015年第4期215-219,共5页Chinese Journal of Infectious Diseases

基　　金：江苏省自然科学基金资助项目（BK20130591）;教育部博士点基金资助项目（20130091120022）

摘　　要：目的 建立和筛选慢性HIV感染者体内HIV-1型负向调节因子（Nef）多肽特异性CD4＋T淋巴细胞克隆.方法 采集5例无症状期HIV感染者PBMC,Bulk培养,用Nef蛋白C末端的Nef末端多肽刺激,双荧光流式细胞术分析细胞表面CD4分子和细胞内γ干扰素表达.有限稀释法进行Nef末端多肽特异性CD4＋T淋巴细胞克隆,酶联免疫斑点法（ELISPOT）筛选特异性克隆,挑选生长最好的细胞扩大培养,完成克隆.结果 1例H1V感染者的PBMC中存在特异性CD4＋T淋巴细胞,成功建立T淋巴细胞克隆.经ELISPOT法确认为Nef末端多肽特异性CD4＋T淋巴细胞克隆.人白细胞抗原（HLA）-DRB1分型检测,提示该多肽表位可能为HLA-DRB1＊ 0406限制性.结论 成功建立Nef末端多肽特异性CD4＋T淋巴细胞克隆,发现并鉴定了Nef末端表位及其HLA限制性.Objective To construct and screen the human immunodeficiency virus-1 （HIV-1） negative regulation factor （Nef） peptide-specific CD4＋ T lymphocyte clone.Methods Peripheral blood mononuclear cells （PBMC） from five asymptomatic HIV-1 infected patients were collected and Bulkcultured with Nef end peptides.The CD4 molecule and intracellular interferon （IFN）-gamma of cultured cells were detected by two-color flow cytometry.The Nef end peptide-specific T cell clone was then constructed by limited dilution and confirmed through enzyme linked immunospot assay （ELISPOT）.The best grown cells were selected and cultured as the final clone.Results The Nef end peptide-specific-T lymphocyte clone was successfully constructed from PBMC of one HIV-infected patient and confirmed by ELISPOT.The detection of human leukocyte antigen （HLA）-DRB1 type showed that the epitope of this peptide was probably HLA-DRB1 ＊ 0406.Conclusion The Nef end specific-T cell clone is successfully constructed,and a new epitope in the C-terminus of Nef protein and its HLA restriction are identified.

关 键 词：HIV-1 负向调节因子多肽 CD4+T淋巴细胞克隆 

分 类 号：R512.91[医药卫生—内科学]