人T细胞受体ζ链基因在昆虫细胞中的表达

Expression of Human T Cell Receptor ζ Chain Gene in Insect Cell

目的 :克隆人T细胞受体 (TCR) ζ链基因 ,运用昆虫杆状病毒系统表达该蛋白。方法 :用RT PCR从人外周血单个核细胞 (PBMC)中克隆TCRζ链cDNA ,构建到昆虫杆状病毒系统专用的Transfer载体中 ,并与杆状病毒共转染昆虫sf9细胞。用SDS PAGE电泳和用小鼠抗人ζ链蛋白的单抗在流式细胞仪中鉴定重组蛋白的表达。 结果 :克隆的人TCRζ链cD NA插入昆虫杆状病毒载体 ,并在昆虫sf9细胞中特异地表达了蛋白。表达量约为细胞裂解上清液总蛋白的 11%。经SDS PAGE分析 ,其分子量大小与预期的重组ζ链蛋白一致。用胞内标记流式细胞仪检测证实 ,转染重组杆状病毒的昆虫细胞含有人的 ζ链蛋白。 结论 :从人PBMC中成功克隆了T细胞受体 ζ链的基因 ,并在昆虫细胞中获得高效表达。用生物工程技术获得TCRζ链蛋白有利于深入研究其生物学功能。

Objective: To clone human T cell receptor (TCR) ζ chain gene and express its encoding protein by baculoviral expression system in insect cells. Methods: TCR ζ chain cDNA was cloned from normal human peripheral blood mononuclear cells (PBMC) by RT-PCR and inserted into baculoviral transfer vector. This vector was co-transfected with baculovirus into insect sf 9 cells. The recombinant protein expressed was identified by SDS-PAGE and flow cytometry with mouse anti-human ζ chain monoclonal antibody. Results: Human TCR ζ chain cDNA cloned and inserted into baculoviral vector specifically expressed protein in the insect sf 9 cells, accounting for about 11% of the total protein yield in the supernatant of cell lysate. The molecular weight of the recombinant protein determined by SDS-PAGE was identical to what we anticipated. The insect cells transfected with recombinant baculovirus were demonstrated by intracellular labeling flow cytometry to express ζ chain protein. Conclusion: Human T cell receptor ζ chain gene was successfully cloned from human PBMC, and its encoding protein was highly expressed in insect cells. The TCR ζ chain protein obtained by bioengineering technique is useful for in depth biological function study.