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从含有猫Oct4、Sox2、Klf4及c-Myc的质粒中获取目的基因,将目的基因与携带绿色荧光蛋白(GFP)的慢病毒载体LV5进行定向连接,构建慢病毒表达载体LV-Oct4、LV-Sox2、LV-Klf4、LV-c-Myc,将其连接产物分别转化至细菌感受态细胞中,经筛选阳性克隆、酶切鉴定后测序,通过lipofectamine2000介导转染293T细胞对慢病毒进行包装并测定病毒滴度,探讨猫多能性基因Oct4、Sox2、Klf4及c-Myc慢病毒载体的构建与包装。结果表明:经双酶切鉴定及测序分析,成功构建并包装出LV-Oct4、LV-Sox2、LV-Klf4及LV-c-Myc慢病毒载体,基因测序结果与目标序列完全一致,且病毒滴度均达到107 TU·mL-1以上。说明成功地构建了猫多能性基因慢病毒表达载体,获得稳定产生慢病毒颗粒的包装细胞株。
The target gene was obtained from the plasmid containing Oct4, Sox2, Klf4 and c-Myc, and the target gene was ligated with the lentiviral vector LV5 carrying green fluorescent protein (GFP) to construct the lentiviral expression vector LV-Oct4 and LV- Sox2, LV-Klf4 and LV-c-Myc were transformed into bacterial competent cells respectively. Positive clones were screened and identified by enzyme digestion and sequencing. Lentiviral 293T cells were transfected with lipofectamine 2000 The virus titers were measured to investigate the construction and packaging of the cat multi-energy gene Oct4, Sox2, Klf4 and c-Myc lentiviral vector. The results showed that the lentiviral vectors LV-Oct4, LV-Sox2, LV-Klf4 and LV-c-Myc were successfully constructed and packaged by double enzyme digestion and sequencing analysis. The result of sequencing was in accordance with the target sequence, Degree reached 107 TU · mL-1 above. The successful construction of the lentiviral vector of cat pluripotency gene resulted in a stable cell line producing lentivirus.