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[摘要]
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TCR ζ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sf 9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TCR ζ链cDNA插入昆虫杆状病毒载体,并在昆虫sf 9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TCR ζ链蛋白有利于深入研究其生物学功能
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[Abstract]
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.
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