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目的:探讨心肌营养素1(cardiotrophin 1,CT1)/破伤风毒素重链C端片段(tentanus toxin Cfragment,TTC)(CT1/TTC)融合蛋白的构建及其对大鼠嗜铬细胞瘤(pheochromocytoma,PC12)细胞的靶向性。方法:采用聚合酶链式反应(PCR)、T-A克隆等分子生物学方法构建CT1/TTC融合蛋白。体外培养PC12细胞,并将CT1/TTC融合蛋白与PC12细胞共培养,红色荧光免疫组化染色后在激光共聚焦显微镜下观察融合蛋白能否在TTC的靶向作用下进入PC12细胞。结果:成功构建了CT1/TTC融合蛋白,测序显示融合基因序列正确,免疫组化染色结果显示融合蛋白能够进入PC12细胞,并发出红色荧光。结论:采用PCR和T-A克隆等分子生物学方法能成功构建CT1/TTC融合蛋白,并且TTC能够将CT1靶向进入PC12细胞。
OBJECTIVE: To investigate the construction of CT1 / CTT / TTC fusion protein and its effect on the expression of pheochromocytoma (PC12) ) Cell targeting. Methods: CT1 / TTC fusion protein was constructed by molecular biology methods such as polymerase chain reaction (PCR) and T-A cloning. PC12 cells were cultured in vitro and the CT1 / TTC fusion protein was co-cultured with PC12 cells. After red fluorescent immunohistochemical staining, we observed whether the fusion protein could enter into PC12 cells under the TTC targeting effect under confocal laser scanning microscope. Results: The CT1 / TTC fusion protein was successfully constructed. Sequencing showed that the fusion gene sequence was correct. Immunohistochemical staining showed that the fusion protein could enter PC12 cells and emit red fluorescence. CONCLUSION: CT1 / TTC fusion protein can be successfully constructed by molecular biology methods such as PCR and T-A cloning, and TTC can target CT1 into PC12 cells.