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

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.