论文部分内容阅读
目的:制备含有融合Flag标签的Runx3基因的复制缺陷型重组腺病毒,感染神经胶质细胞瘤U251,观察外源Runx3在细胞中的表达及亚细胞定位。方法:用PCR的方法扩增Runx3基因,并将Flag标签蛋白的编码基因与RUNX3基因进行融合,构建腺病毒穿梭载体pShuttle-CMV-Runx3,经KpnI/XhoI双酶切鉴定并测序。利用电转化方法将经PmeI线性化的pShuttle-CMV-Runx3穿梭载体导入BJ5183重组细菌,获取重组腺病毒质粒Ad-Runx3,再将经PacI线性化的Ad-Runx3重组病毒骨架质粒转染293A包装细胞,包装并扩增病毒。利用该病毒感染神经胶质细胞瘤U251,用免疫印迹法观察外源Runx3在细胞中的表达,用间接免疫荧光法观察其在细胞内的定位。结果:构建并包装表达Runx3蛋白的重组腺病毒,用重组腺病毒感染U251细胞后,经免疫印迹和间接免疫荧光法检测,可见外源导入的Runx3蛋白在细胞核内的特异性定位。结论:成功制备了含有融合Flag标签的转录因子Runx3基因的重组腺病毒,感染U251细胞,在细胞中观察到该分子表达后定位于细胞核中,为研究Runx3在神经胶质瘤发生中的作用奠定了实验基础。
OBJECTIVE: To prepare replication-deficient recombinant adenovirus containing Flag-tagged Runx3 gene, infect glioma U251 and observe the expression and subcellular localization of exogenous Runx3 in cells. Methods: Runx3 gene was amplified by PCR and fused with RUNX3 gene encoding Flag tagged protein. The shuttle vector pShuttle-CMV-Runx3 was constructed and identified by double digestion with KpnI / XhoI and sequenced. The pShuttle-CMV-Runx3 shuttle vector linearized by PmeI was introduced into BJ5183 recombinant bacterium by electroporation, and the recombinant adenovirus plasmid Ad-Runx3 was obtained. Pac-linear Ad-Runx3 recombinant virus backbone plasmid was transfected into 293A packaging cells , Package and amplify the virus. The virus was used to infect glioma U251, and the expression of exogenous Runx3 in cells was observed by immunoblotting. The intracellular localization was observed by indirect immunofluorescence. Results: The recombinant adenovirus expressing Runx3 protein was constructed and packaged. U251 cells were infected with recombinant adenovirus. After immunoblotting and indirect immunofluorescence assay, we found that Runx3 was specifically localized in the nucleus. CONCLUSION: The recombinant adenovirus containing the Flag-tagged Runx3 gene is successfully infected into U251 cells. The expression of Runx3 in the cell nucleus is observed after the expression of Runx3 in glioma cells. In order to study the role of Runx3 in the development of glioma The experimental basis.