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目的:探讨靶向多肽与标签蛋白(绿色荧光蛋白)的位置关系是否会影响融合蛋白与细胞之间的结合能力。方法:将获得的GE11和LyP1两种靶向多肽分别与增强型绿色荧光蛋白在不同位置融合表达,通过原核系统表达纯化,将纯化的蛋白加入血清饥饿的SMMC-7721肝癌细胞株培养液中,处理3h,通过荧光显微镜观察细胞中绿色荧光蛋白的情况检查融合多肽与细胞的结合情况。结果:绿色荧光蛋白的羧基端和GE11、LyP1多肽分别融合表达,处理细胞后,融合蛋白显示与细胞有很强的结合能力;当GE11、LyP1在绿色荧光蛋白氨基端融合时,融合蛋白几乎不能与细胞结合。在此基础上,检测了多种靶向肽对多种细胞的靶向效应。结论:不合适的融合策略会降低,甚至消除靶向多肽的结合能力;融合大分子量蛋白也会改变靶向肽的靶向效应。因此,当使用靶向多肽携带基因进行研究时,其在融合蛋白中的位置应该非常谨慎。
Objective: To investigate whether the positional relationship between a target polypeptide and a tagged protein (green fluorescent protein) can affect the binding ability between the fusion protein and the cell. Methods: The two targeting polypeptides of GE11 and LyP1 were fused with enhanced green fluorescent protein (EGFP) at different positions, respectively. The expressed protein was purified by prokaryotic expression system. The purified protein was added to the serum-starved SMMC-7721 hepatoma cell line. After 3 h of treatment, the binding of the fusion polypeptide to the cells was examined by observing the green fluorescent protein in the cells by a fluorescence microscope. Results: The fusion protein of GFP and GE11 and LyP1 were fused respectively. After the cells were treated, the fusion protein showed strong binding ability with the cells. When the fusion protein of GE11 and LyP1 was fused with the amino-terminal of GFP, Binds to cells. On this basis, the targeting effects of various targeting peptides on various cells were examined. CONCLUSIONS: Inappropriate fusion strategies reduce or even eliminate the binding ability of targeted peptides; fusion of large molecular weight proteins also alters the targeting effect of the targeted peptide. Therefore, when using the targeted polypeptide to carry the gene for study, its location in the fusion protein should be very cautious.