摘要
目的 建立肾综合征出血热 (HFRS)患者汉滩病毒 (HTNV)核衣壳蛋白 (NP)特异性CTL克隆 ,为HTNVNPT细胞表位鉴定及HFRS患者T细胞免疫功能研究奠定基础。方法 采用Fi coll密度梯度离心法分离HFRS患者外周血单个核细胞 (PBMC) ,用灭活HTNV和IL 2体外刺激 ,有限稀释法建立T细胞克隆 ,流式细胞术鉴定克隆表型。并用EB病毒 (EBV)转化B淋巴细胞 ,建立B淋巴母细胞样细胞系 (B LCL) ,以含HTNVS基因的重组痘苗病毒感染B LCL作靶细胞 ,以CTL克隆作效应细胞进行细胞毒杀伤试验 ,测定T细胞克隆的抗原特异性。结果 T细胞克隆能特异性识别表达NP的B LCL。在 5名患者中 ,3名有较高的杀伤率 ,并自 2名患者建立了 5株HTNV特异性CTL克隆 ,其表型为CD8+ 均大于 6 0 %。结论 成功建立了HFRS患者HTNVNP特异性CTL克隆及其靶细胞。NP是HFRS患者HTNV特异性CTL应答的主要靶抗原之一。
ObjectiveTo establish T cell clone which is specific for nucleocapsid protein (NP) of Hantaan virus (HTNV) in patients with hemorrhagic fever with renal syndrome (HFRS) and to lay a foundation for the study of T cell epitope and T cell immunology. MethodsPeripheral blood mononuclear cells (PBMCs) of patients with HFRS separated by Ficoll-Hypaque density gradient centrifugation were stimulated in vitro with inactivated HTNV and recombinant human IL-2. T cell clones were established with limiting dilution technique and clone phenotype was identified by flow cytometer. PBMCs infected by EB virus (EBV) were used to establish immortalized B lymphoblastoid cell lines (B-LCLs). B-LCLs infected by rVV were used as the target cells of HTNV-specific CTL clones. The antigenic specificity of T cell lines was identified by cytotoxicity assays. ResultsB-LCLs expressed NP were recognized by HTNV-specific T cell clones. Of the 5 individuals tested,three had high specific lysis rate. Five T cell clones were obtained from two patients and CD8 + T cell clone takes more than 60%. ConclusionHTNV NP- specific T cell clones and their target cells have been established. NP is potentially a major target of the CTL response during HTNV infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第8期600-605,共6页
Chinese Journal of Microbiology and Immunology
