Objective: To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy.
Methods: The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology. Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells (HL60,THP1) were infected by virus.
Results: Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing, and the titers of the packaged viruses were all greater than 1×108 TU/ml. Among them, shRNA-2 lentivirus had the highest interference efficiency, and the expression of c-Cbl gene and CBL protein were decreased about 95% and 60% respectively after leukemia cells were infected with shRNA-2; In addition, the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×109 TU/ml. When leukemia cells were infected with adenovirus, the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively.
Conclusion: Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein. It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.
题目: 靶向人c-Cbl基因重组干扰慢病毒与过表达腺病毒载体的构建、鉴定以及病毒功效研究.
目的: 构建可调控c-Cbl基因表达的重组慢病毒与腺病毒并评估其功效。.
方法: 应用基因重组技术，分别构建靶向人c-Cbl基因的干扰慢病毒和过表达腺病毒。采用定量PCR和免疫印迹法检测病毒感染后白血病细胞（HL60、THP1）c-Cbl基因表达与转录本的变化。.
结果: 3个靶向人c-Cbl基因的重组干扰慢病毒载体经测序验证构建成功，包装的病毒滴度均大于1×108TU/ml，其中shRNA-2号慢病毒干扰效率最高，白血病细胞感染后c-Cbl基因的表达约下调95%，CBL蛋白的表达约下调60%；同时，靶向人c-Cbl基因的重组过表达腺病毒载体也经测序验证构建成功，包装的病毒滴度大于1×109 TU/ml，细胞感染腺病毒后，c-Cbl基因表达可瞬时上调约10倍，CBL蛋白表达约上调1.5倍。.
结论: 重组干扰慢病毒和过表达腺病毒均可高效感染白血病细胞，并能分别下调和上调c-Cbl基因与CBL蛋白的表达，为后续研究肿瘤细胞内c-Cbl基因功能打下前期基础。.
Keywords:
adenovirus vector;