BTG2基因干扰重组慢病毒的制备及在A549细胞中的表达鉴定

Construction of BTG2-siRNA Lentiviral and Identification the Lentiviral Activity in A549 cell

目的用已构建好的BTG2-siRNA重组的慢病毒表达载体包装成具有感染性的慢病毒颗粒,获得稳定BTG2-siRNA表达的A549细胞。方法构建的BTG2-siRNA慢病毒表达载体,经测序后证实与标准序列一致。将BTG2-siRNA慢病毒表达载体与其他两种慢病毒包装载体质粒共转染293T细胞,获得BTG2基因siRNA重组慢病毒。感染A549细胞后,用25μg/m L的嘌呤霉素筛选稳定干扰BTG2表达的A549细胞。CCK8法检测感染BTG2干扰慢病毒的A549细胞的增殖能力变化。结果 Western blot检测提示感染BTG2-siRNA慢病毒的A549细胞总蛋白中BTG2表达要明显低于正常的A549细胞。CCK8实验显示,感染BTG2干扰慢病毒的A549细胞的增殖能力增加。结论成功制备了BTG2基因siRNA重组慢病毒颗粒,感染A549细胞后可检测到BTG2蛋白的表达明显降低。为进一步研究BTG2基因在恶性肿瘤中的生物学功能与相关机制的研究奠定了基础。

Objective BTG2-siRNA reconstructed lentiviral expression vector was used to pack infectious lentiviral parti-cles in order to obtain BTG2-siRNAstably expressing A549 cells. Methods BTG2-siRNAlentiviral expression vector was recon-structed in the sigma company and the expressed sequence was verified by direct sequencing to be consistent with standard se-quence. 293 cells were co-transfected with BTG2-siRNAlentiviral expression vector and two other lentiviral expression vectors yielding BTG2 siRNA reconstructed lentiviral. After infected, A549 cells that stably interrupt expression of BTG2 was identified through 25 ug/ml puromycin. CCK8 was utilized to evaluate proliferation after knocking down BTG2. Results Expression level of BTG2 was significantly decreased in A549 cells transfected with BTG2-siRNAlentiviral expression vector compared with normal cells by western blot evaluation of total protein preparation. Proliferation of A549 cells was increased after transfected with BTG2 shRNA by CCK8. Conclusion BTG2-siRNAlentiviral expression vector was successfully constructed and BGT2 protein in A549 was significantly decreased after infected. This research made foundation for further investigation of biologic function of BTG2 in malignancy.