穿膜肽与蛋白微球偶联物的制备及鉴定

Study on Preparation and Determination of Nanospheres Coupled with Cell-penetrating Peptides

目的:制备穿膜肽TAT-EGFP与白蛋白微球(BSA-NP)偶联物,并检测其对膀胱癌EJ细胞的穿膜活性。方法:通过异型双功能交联剂N-羟基琥珀酰亚胺基-3-(2-吡基二硫)-丙酸酯(SPDP)交联的方法制备穿膜肽TAT-EGFP与白蛋白微球偶联物。SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)、电镜、荧光显微镜及共聚焦显微镜检测其共价连接及活性。结果:SDS-PAGE电泳鉴定TAT-EGFP与白蛋白微球是通过共价键连接;电镜显示二联偶联物(TAT-EGFP-BSA-NP)的构象;荧光显微镜及电镜显示了二联偶联物的穿膜活性。结论:成功的制备穿膜肽TAT-EGFP与白蛋白微球偶联物-TAT-EGFP-BSA-NP,且偶联物对膀胱癌细胞有穿膜活性。

Objective：To prepare nanospheres coupled with cell-penetrating peptides（Cpps） and evaluate its ability to cross the plasma membrane of bladder cancer cell（EJ cell）.Methods：The nanosphere coupled with TAT-EGFP was prepared by methods of crosslinking via hetero-bifunctional cross-linker SPDP.Nanospheres and its penetrating activity were tested by SDS-PAGE electrophoresis,scanning electron microscopy and laser scanning confocal microscope（LSCM）.Results：SDS-PAGE electrophoresis revealed the nanospheres linked covalently with TAT-EGFP.The penetrating activity of TAT-EGFP is preserved under scanning electron microscopy and LSCM.Conclusion：The preparation of TAT-EGFP-BSA-NP was succeeded.It was binded by covalent bond and it could penetrate EJ cell.