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目的:构建携带报告基因eGFP及人内皮他丁en-dostatin-K5的嵌合型腺病毒载体Ad5/11。方法:采用重叠PCR及在细菌E.coli.BJ5183中同源重组的方法,获得表达eGFP报告基因的嵌合腺病毒骨架载体,然后以经PacI线性化的嵌合腺病毒载体骨架与PacI线性化的表达endostatin-K5的腺病毒E1穿梭载体共转染真核细胞HEK293细胞中获得携带报告基因eGFP及人endostatin-K5的嵌合型腺病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP。以获得的嵌合型腺病毒载体感染U87MG细胞,在荧光显微镜观察eGFP的表达,采用RT-PCR检测endostatin-K5的表达;将所构建的嵌合病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP与未经修饰的腺病毒载体Ad5-E1-CMV-eGFP在体外感染人胶质瘤细胞系A172及乳腺癌细胞系MDA-MB-231;通过荧光蛋白eG-FP的表达,比较它们对以上肿瘤细胞的感染效率。结果:嵌合病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP可成功地表达eGFP及endostatin-K5。以经过修饰的嵌合型腺病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP感染人脑胶质瘤细胞系A172及人乳腺癌细胞MDA-MB-231的感染效率,明显高于对照未经修饰的腺病毒载体Ad5-E1-CMV-eGFP。结论:携带eGFP及人endostatin-K5的嵌合型腺病毒载体Ad5/11,可明显提高对人脑胶质瘤细胞系A172及人乳腺癌细胞系MDA-MB-231的感染效率。
OBJECTIVE: To construct chimeric adenovirus Ad5 / 11 harboring reporter gene eGFP and human endostatin en-dostatin-K5. Methods: Chimeric adenovirus backbone vectors expressing eGFP reporter gene were obtained by overlapping PCR and homologous recombination in E. coli strain BJ5183. The recombinant adenovirus vector was linearized with PacI Of adenovirus E1 shuttle vector expressing endostatin-K5 was co-transfected into HEK293 cells to obtain the chimeric adenoviral vector Ad5 / 11-E1-CMV-endo-K5 / E3 carrying reporter gene eGFP and human endostatin-K5 -CVV-eGFP. The chimeric adenovirus vector was used to infect U87MG cells. The expression of eGFP was observed by fluorescence microscopy. The expression of endostatin-K5 was detected by RT-PCR. The constructed chimeric viral vector Ad5 / 11-E1-CMV-endo- In vitro, human glioma cell line A172 and breast cancer cell line MDA-MB-231 were infected with K5 / E3-CMV-eGFP and the unmodified adenoviral vector Ad5-E1-CMV- Expression, compare their efficiency of infection of the above tumor cells. Results: The chimeric viral vector Ad5 / 11-E1-CMV-endo-K5 / E3-CMV-eGFP could successfully express eGFP and endostatin-K5. Infection rate of human glioma cell line A172 and human breast cancer cell line MDA-MB-231 infected with the modified chimeric adenoviral vector Ad5 / 11-E1-CMV-endo-K5 / E3- Significantly higher than the control unmodified adenovirus vector Ad5-E1-CMV-eGFP. Conclusion: Ad5 / 11, a chimeric adenovirus carrying eGFP and human endostatin-K5, can significantly improve the efficiency of human glioma cell line A172 and human breast cancer cell line MDA-MB-231.