体外研究汉滩病毒(Hantaan virus, HTNV) S基因及其5’端、3’端在BLCL（EBV-transformed B lymphoblastoid cell line, BLCL）内稳定表达的意义, 为核蛋白T细胞表位的研究奠定基础。本研究从肾综合征出血热(HFRS)患者的外周血单个核细胞(PBMC)建立与HTNV特异性CTL同源的患者自身的EB病毒转化的B淋巴母细胞系，将含有不同HTNV S基因片段的重组真核表达载体转染入BLCL，获得长期稳定表达，作为靶细胞系，为下一步进行CTL杀伤试验提供靶细胞系。设计4条引物，用PCR方法从PBV220-S22原核质粒中扩增出S基因全读码框(37-1326bp)及S基因5’端(37-501bp)，S基因3’端（502－1326bp），用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中，成功构建pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体，并通过脂质体转染至BLCL细胞中，进行了稳定表达。间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C在BLCL细胞中的表达。pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体有较高的转染效率，目的基因能在宿主细胞中长期稳定表达，有利于研究HTNV-S基因在T细胞表位研究中的意义。建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应，平均杀伤率分别为50.2%、39.0%和25.4%。HTNV-NP优势T细胞表位可能主要位于病毒核蛋白的羧基端。

In order to provide some data for the study on T cell epitopes of Hantaan virus nucleocapsid protein, we constructed EBV-transformed B lymphoblastoid cell line (BLCL) from HFRS patients’ PBMC. We inserted HTNV S and 5’ and 3’terminals of S gene segment into a eukaryotic expression vector pcDNA 3.1/V5-His TOPO. The recombinant expression plasmids pcDNA3.1-S and pcDNA 3.1-S-N pcDNA3.1-S-C were constructed. BLCL were transfected in vitro with them, selected by G418 and used as target cells for CTL cytotoxicity assay. The expression of HTNV S and 5’ and 3’terminals of S gene segment in BLCL was detected by indirect immunofluorescence assay (IFA). These recombinant plasmids can be expressed efficiently and steadily in BLCL. We can provide a tool for the study on T cell eptitopes of Hantaan virus nucleocapsid protein. The CTL clone recognized and killed the target cells with specificities of HTNV-NP. Cytotoxicities(%) were 50.2%, 39.0% and 25.4%, respectively. HTNV T cell antigenic epitopes were mainly localized on the C-terminal of the virus nucleocapsid protein.