hsa-miR-381过表达慢病毒载体的构建、转染和表达

Construction,transfection and expression of hsa-miR-381 over-expressed lentivirus vector

目的构建hsa-miR-381过表达慢病毒载体病毒载体,并对其在食管鳞癌TE10细胞中miR-381表达调节效果进行鉴定。方法利用PCR法扩增miR-381基因序列,将目的基因miR-381克隆到携带Green&Puro的慢病毒载体LV3中,经双酶切及测序鉴定后大量抽提;利用脂质体将含目的基因的重组质粒和包装质粒p Gag/Pol、pRev、p VSV-G共转染293T细胞;用得到的慢病毒转染TE10细胞,通过荧光显微镜观察转染状况,实时荧光定量PCR分析转染前后miR-381的表达。结果酶切与测序结果证明成功构建重组质粒,并成功包装成慢病毒,实验组病毒滴度为2×108TU/ml,空白阴性对照组病毒滴度为2×108TU/ml。TE10细胞转染过表达慢病毒后miR-381的表达升高,TE10-381组has-miR-381的表达丰度是TE10组的456.05倍。结论 LV3-hsa-miR-381慢病毒载体构建成功,并能明显增加TE10细胞中miR-381的表达量。

Objective To construct hsa-miR-381-over-expressed lentivirus vector,and to investigate the regulation effect of miR-381 in esophageal squamous cell carcinoma TE10 cells after transfection. Methods The miR-381 sequence was obtained by PCR amplification,and then inserted into the lentiviral vector LV3 with GreenPuro. The miR-381 sequence was confirmed by double-enzyme cleavage and DNA sequencing and extracted. The recombinant lentivirus plasmid and packaging plasmid p Gag / Pol,pRev,and p VSV-G were co-transfected into 293 T cells by liposomes. The viral yielded by 293 T cell was transfected into TE10 cells. The status of transfection was observed by fluorescence microscope,and the expression of miR-381 was detected by real-time quantitative PCR before and after transfection. Results Restriction enzyme digestion and sequencing results showed that recombinant plasmid was successfully constructed and packaged to lentivirus. The miR-381 sequence was confirmed by restriction enzyme digestion and DNA sequencing,and successfully inserted into the vector. The viral titer was 2 × 10^8 TU / ml. After transfection,the expression of miR-381 was significantly up-regulated in TE10 cells,and the expression abundance of hsa-miR-381 in TE10- 381 increased by 456. 05 times. Conclusion The LV3-has-miR-381 lentiviral vector is successfully constructed,and it could significantly increase the miR-381 expression in TE10 cell.