GTP结合蛋白4基因RNAi慢病毒载体的构建与鉴定

发布时间：2018-08-12 14:56

【摘要】：目的:为了建立GTPBP4基因沉默的人结肠癌细胞株,研究GTPBP4基因对人结肠癌细胞株RKO细胞增殖和凋亡的影响,构建并鉴定GTPBP4基因RNA干扰慢病毒载体。方法:针对GTPBP4 mRNA设计siRNA,构建pGCSIL-GFPGTPBP4慢病毒载体,感染293T细胞,包装产生慢病毒,并进行滴度测定。将慢病毒转染人结肠癌细胞株RKO,通过Realtime PCR和Western Blot分析GTPBP4基因敲减效率。结果:结果显示,插入DNA序列与目的基因序列一致,提示插入人GTPBP4基因序列正确。293T细胞中病毒滴度的测定结果 2×108 TU/m L。经siRNA慢病毒感染后,实验组RKO GTPBP4基因mRNA水平表达量受到显著抑制,Western Blot结果显示敲减效率达到72.9%。结论:GTPBP4基因RNAi慢病毒载体构建成功,可用于结肠癌生物行为等各方面的研究。
[Abstract]:Aim: to establish a human colon cancer cell line silenced by GTPBP4 gene, to study the effect of GTPBP4 gene on the proliferation and apoptosis of human colon cancer cell line RKO, and to construct and identify the GTPBP4 gene RNA interfering lentivirus vector. Methods: pGCSIL-GFPGTPBP4 lentivirus vector was constructed according to GTPBP4 mRNA, infected 293T cells, packaged to produce lentivirus, and its titer was determined. Lentivirus was transfected into human colon cancer cell line RKO.The efficiency of GTPBP4 gene knockdown was analyzed by Realtime PCR and Western Blot. Results: the results showed that the inserted DNA sequence was the same as the target gene sequence, indicating that the virus titer of the inserted human GTPBP4 gene was correct. 293T cells showed that the titer of the virus was 2 脳 10 ~ 8 TU/m / L. After infection with siRNA lentivirus, the expression of RKO GTPBP4 gene mRNA in the experimental group was significantly inhibited. Western Blot showed that the knockout efficiency was 72.9%. Conclusion the RNAi lentivirus vector of the RNAi gene of the 10% GTPBP4 gene is successfully constructed and can be used in the study of biological behavior of colon cancer.
【作者单位】：西南医科大学附属医院病理科;
【基金】：四川省科技厅科技计划项目(NO:14JC0091) 泸州市科技局科技计划项目(2013 LZLY-J44)
【分类号】：R735.35

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