独特型TCR Vα1-pIRES-TCR Vβ8表达载体构建及体外表达  

Construction and expression of idiotype TCR Vα1-pIRES-TCR Vβ8 eukaryotic vectors in vitro

作　　者：李闯[1] 吴秀丽[1] 李扬秋[1] 周羽竝[2] 陈少华[1] 杨力建[1] 朱康儿[1] 

机构地区：[1]暨南大学医学院血液病研究所,广东广州510632 [2]暨南大学医学院生物化学教研室,广东广州510632

出　　处：《细胞与分子免疫学杂志》2008年第1期30-33,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02Z114);国家自然科学基金专项基金资助项目(30424003);广东省自然科学基金资助项目(05103293,06025213);国务院侨办重点学科建设基金资助项目(2006年)

摘　　要：目的:构建Jurkat细胞株独特型TCR Vα1-pIRES-TCR Vβ8表达载体,转染后了解其体外表达情况。方法:根据聚合酶链反应-基因扫描(PCR-Genescan)检测Jurkat细胞株TCR Vα及Vβ亚家族表达情况,分别将其所表达的单克隆性的TCR Vα1及TCR Vβ8亚家族基因片段克隆至质粒pIRES的多克隆位点A(MCS A)和多克隆位点B(MCS B)中。通过限制性酶切分析、序列分析、RT-PCR、间接免疫荧光、流式细胞术(FCM)手段鉴定重组表达载体的正确性以及转染A549和Molt4细胞后基因及蛋白的表达情况。结果:构建了2组TCR Vα1-pIRES-TCR Vβ8表达载体,该表达载体转染A549和Molt4细胞后,在mRNA和蛋白水平检测到了TCR Vα1和TCR Vβ8的表达。结论:成功构建了2种独特型Vα1-pIRES-TCR Vβ8真核表达载体,为利用特异性TCR基因修饰T细胞的研究提供方法学依据。AIM： To construct the eukaryotic vectors with idiotype TCR Vα1-pIRES-TCR Vβ8 of Jurkat cell line and investigate their expression in vitro after transferred into eukaryotic cells. METHODS： TCR Vα and Vβ subfamilies of Jurkat cells were analyzed by using RT-PCR and genescan technique. Then the monoclonal TCR Vα1 and Vβ8 genes of Jurkat cells were cloned into multiple clone site （MCS） A and MCS B of the eukaryotic vector plRES respectively. Its sequences were identified by restriction enzyme cutting and sequence analysis. The expression of TCR mRNA and idiotypic protein in transferred A549 and Molt4 cells was tested by RT-PCR, indirect immunophenotyping fluorescein dyeing and flow cytometry, respectively. RESULTS： Two recombinavot eukaryotic vectors with idiotype TCR Vα1-pIRES-TCR Vβ8 were developed successfully and the expression of both idiotypic TCR mRNA and protein was detected in transferred A549 and M549 cells. CONCLUSION： The successful construction of the two eukaryotic vectors with idiotype TCR Vα1-pIRES-TCR Vβ8 will provide a basic method for furthor study of the specific TCR gene modified T cells.

关 键 词：JURKAT细胞株 T细胞受体 转基因 

分 类 号：R733.73[医药卫生—肿瘤] R371.22[医药卫生—临床医学]