特异性CDR3区取代型γδTCR转染细胞的构建及生物学功能鉴定

Construction and biological identification of the transfectant cells expressing γδTCR with specific CDR3 sequence

目的:建立特异性CDR3区取代型γδTCR转染细胞平台。方法:利用搭桥PCR的方法将已知的γδT细胞克隆DBS4.3特异性CDR3编码序列嵌入到γ9和δ2cDNA序列中,取代原有的CDR3区序列,获得的全长γ9和δ2链分别插入真核表达载体pREP7和pREP9中,共转染J.RT3-T3.5细胞,双压力抗生素筛选获得表达特异性γδTCR的转染细胞,通过ELISA和Re-altime PCR检测该转染细胞在接受抗原刺激后IL-2的分泌情况。结果:ELISA和Realtime PCR结果显示表达DBS4.3特异性γδTCR的转染细胞在DBS4.3特异性抗原仲丁胺和抗γδTCR抗体刺激作用下,活化分泌IL-2。结论:构建了特异性CDR3区取代型γδTCR转染细胞平台,为研究γδTCR识别抗原的机制奠定了基础。

Objective：To construct of transfectant cells expressing γδTCR with specific CDR3 sequence.Methods：Specific CDR3 region sequence of DBS4.3,a known γδT cell clone,was inserted into γ9 and δ2 chain to substitute original CDR3 sequence using overlapping PCR method.After the full-length γ9 and δ2 chains were ligated with expression vector pREP7and pREP9 respectively,they were co-trasfected into a cell line of human Jurkat cells with TCR β chain gene-defective mutant J.RT3-T3.5.When the transfectant cells expressing specific γδTCR were stimulated by antigen,production of IL-2 was detected by ELISA and Realtime PCR.Results：By ELISA and Realtime PCR,it was exhibited that the transfectant cells expressing DBS4.3 specific γδTCR secreted IL-2 under stimulation of iso-butylamine and anti γδTCR antibody.Conclusion：The transfectant Jurkat cells expressing γδTCR with specific CDR3 sequence are successfully constructed.It provides a platform for the research of recognition mechanism of γδTCR.