表达结核特异性Vγ9Vδ2 TCR双链单体的果蝇 S2恒定细胞系的构建和鉴定  

Construction and identification of a drosophlia S2 cell line with stoble expr ession of Mycobacterium tuberculosis-specific Vγ9Vδ2 T cell receptor monomers

作　　者：涂晓欣 张洁[1] 孟繁荣[1] 方毅敏[2] 杨芳芳[1] 赖小敏[1] 

机构地区：[1]中山大学中山医学院微生物学教研室,热带病防治研究教育部重点实验室,海洋微生物功能分子广东省高校重点实验室,广东省重大传染病预防和控制技术中心,广州510080 [2]广州市胸科医院

出　　处：《中华微生物学和免疫学杂志》2015年第1期23-26,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金面上项目（81271779）;十二·五传染病重大专项分任务（2012ZX10004903-004-002）

摘　　要：目的：转染及筛选鉴定能够稳定表达和分泌Vγ9Vδ2 T细胞受体（TCR）双链单体的果蝇S2恒定细胞系。方法利用氯化钙转染法将TCR γ9-FB-pMT／V5-His B质粒、TCR δ2-JB-pMT／V5-His A质粒、pMT／Bip-BirA质粒和pCoHygro潮霉素质粒转染进S2细胞中，用潮霉素B筛选恒定细胞系后表达生物素化的结核特异性Vγ9Vδ2 TCR双链单体，并加以鉴定。结果斑点印迹试验、SDS-PAGE和Western blot表明细胞培养上清中有Vγ9Vδ2 TCR单体，且已被成功生物素化。结论成功筛选出能稳定分泌表达Vγ9Vδ2 TCR的果蝇S2恒定细胞系，为研究γδ T细胞的免疫识别提供实验资料。Objective To construct and identify a drosophila S2 cell line that could stably express Mycobacterium tubercuolsis-specific Vγ9Vδ2 T cell receptor （TCR） monomers.Methods Four vectors in-cluding the TCR γ9-FB-pMT/V5-His B expression vector , TCR δ2-JB-pMT/V5-His A expression vector , pMT/Bip-BirA expression vector and pCoHygro plasmid were transfected into S2 cells by using calcium phos-phate transfection.The S2 cells with stable expression of the Vγ9Vδ2 TCR monomers were screened out with Hygromycin-B.CuSO4 and biotin were used together to induce the expression of biotinylated Vγ9Vδ2 TCR monomers in S2 cells after 4 weeks of culturing .The target proteins in the supernatants of cell culture were identified with dot blot , SDS-PAGE and Western blot assays .Results The results of dot blot , SDS-PAGE and Western blot assays showed that the Vγ9Vδ2 TCR monomers could be stably expressed by the screened S2 cell line.Moreover , the expressed Vγ9Vδ2 TCR monomers were successfully biotinylated .Concl usion The drosophila S2 cell line that could stably express Vγ9Vδ2 TCR monomers was successfully established . This study has paved the way for further investigation on the antigen recognition of γδT cells and its possible mechanism.

关 键 词：γδT细胞受体 双链单体 S2细胞 

分 类 号：R392[医药卫生—免疫学]