嵌合γδ TCR结构域的αβ TCR分子的同源建模及真核表达  

Homology modeling and eukaryotic expression of a modified αβ TCR harboring the immunoglobulin-like domain of γδ TCR

作　　者：陶嫦立 邵红伟[1] 沈晗[1] 黄树林[1] 

机构地区：[1]广东药科大学生命科学与生物制药学院,广东省生物技术候选药物研究重点实验室,广东广州510006

出　　处：《细胞与分子免疫学杂志》2016年第8期1026-1030,1035,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31300737)

摘　　要：目的设计并构建嵌合γδTCR类免疫球蛋白(Ig)结构域的αβTCR分子,并在Jurkat T细胞中观察其表达。方法通过生物信息学分析确定TCR△Ig分子的融合位点;应用同源建模模拟嵌合TCRα和TCRβ蛋白的三维结构,并应用组合扩展方法(CE)对TCR△Ig分子与野生型TCR分子的蛋白骨架以及抗原识别活性部位进行空间结构比对和分析。采用重叠PCR法分别在TCRα△Ig和TCRβ△Ig的末端融合增强型青色荧光蛋白(ECFP)和增强型黄色荧光蛋白(EYFP),并分别克隆入质粒p IRES2-EGFP内部核糖体进入位点(IRES)的上下游。最后将重组表达质粒p IRES-TCRβ△Ig-EYFP/TCRα△Ig-ECFP转染Jurkat T细胞,共聚焦显微镜扫描分析该嵌合TCR分子的表达。结果 TCRα△Ig和TCRβ△Ig的同源建模及三维结构比对结果显示,相比于野生型TCR,该嵌合TCR的可变区(V)结构无改变并且抗原识别活性中心结构保持稳定。重组表达质粒经PCR鉴定及测序分析证明载体构建成功;并且共聚焦显微镜扫描结果显示嵌合TCR分子能定位于T细胞膜表面并正常表达。结论该嵌合TCR分子设计合理,能在Jurkat T细胞表面正常表达。Objective To design, construct and express a chimeric αβ TCR harboring the immunoglobulin-like （Ig） domain of γδ TCR in Jurkat T cells. Methods The fusion sites of TCR △Ig were determined by bioinformatics analysis. Then the protein structures of TCR a lg and TCR β /klg were predicted by homology modeling. Furthermore, the structures of TCR a Ig and TCR β △Ig were compared with the wild type （wt） TCR a and TCR β respectively by combinatorial extension （CE）. After that, the TCR a △Ig and TCR 15 △Ig were fused to fluorescent protein ECFP and EYFP respectively via the overlap PCR, and then the fusion genes （TCR a △Ig-ECFP and TCR β △Ig-EYFP） were cloned into plRES2-EGFP vector and respectively located at the upstream and downstream of an internal ribosome entry site （IRES）. The recombinant prokaryotic expression vector plRES-TCR β A Ig-EYFP/TCR a △Ig-ECFP was transferred into Jurkat T cells. Finally, the expression of TCR alg in Jurkat T cells was monitored by confccal laser scanning microscopy （CLSM）. Results The variable region structure of the TCR A Ig did not change and the antigen recognition active regions remained stable compared to the wtTCR. The recombinant expression plasmid was successfully constructed as confirmed by PCR identification and sequencing analysis, CLSM showed that TCR AIg was expressed and located at the plasma membrane of Jurkat T cells. Conclusion The design of TCR △Ig was reasonable and the TCR A Ig could be expressed on Jurkat T cell surface.

关 键 词：T细胞受体 蛋白结构预测 同源建模 融合基因 

分 类 号：R392.11[医药卫生—免疫学]