慢病毒载体的构建及其介导的绿色荧光蛋白在胰岛β细胞中的表达

Construction of Lentiviral Vector Containing Green Fluorescent Protein Gene and Its Expression in Pancreatic Islet β Cells

目的获得能够在大鼠胰岛β细胞中高效表达的慢病毒载体。方法以PCR的方法扩增绿色荧光蛋白(green fluorescent protein,GFP)片段,并将其通过穿梭质粒装入pLenti6/V5表达质粒,然后用脂质体转染试剂将plenti6/V5GFP、pLP1、pLP2以及pLP/VSVG转染入293FT细胞,获得的病毒用人纤维瘤细胞系的HT1080细胞进行滴定。然后用一定滴度的慢病毒转导大鼠胰岛β细胞系INS-1,观察转导效率。结果通过限制性内切酶和琼脂糖凝胶电泳方法,观察到所克隆入pLenti6/V5表达质粒的GFP片段大小正好与PCR扩增出的片段一致。经测序验证,序列与NCBI网站上GFP序列完全一致。转染结果显示,经293FT细胞所产生的慢病毒,转导效率达到80%以上。而在1×106/ml病毒颗粒的情况下,AAV-GFP病毒几乎不能转导β细胞。结论与同一滴度的AAV-GFP病毒相比,胰岛β细胞的转导效率有非常显著的差异。即慢病毒载体表达系统在对胰岛β细胞转导的能力上,明显优于重组腺辅病毒表达系统。表明慢病毒载体在糖尿病基因治疗中,特别是对胰岛β细胞的转基因工作中,有着良好的应用前景。

Objective To obtain a lentiviral vector which can transduce the pancreatic islet β cells efficiently. Method GFP fragment was amplified by PCR, and introduced into pLenti6/V5 by use of GatewayTM pENTRTM Vector system. Then, plenti6/V5-GFP, pLP1, pLP2 and pLP/VSVG were transfected into 293FT cells. The lentivirus was titrated in human fibroma cells HT1080. The lentivirus was used to infect islet β cells INS-1 and the transduction efficiency was detected. Result The cloning accuracy of the PCR-amplified GFP fragment into pLenti6/V5 plasmid was checked by the restriction enzyme method and agarose gel electrophoresis. The fragment was sequenced and the GFP sequence was completely consistent with that provided by NCBI website. When 1 × 10^6/ml of virus was used, AAV-GFP was hardly able to infect the islet β cells, but the transduction efficiency of lentivirus could reach 80%. Conclusion The transduction efficiency of lentivirus produced by 293 cells is obviously higher that of AAV-GFP at the same titer. The lentiviral vector expression system may be more valuable than rAAV system in the transduction of the pancreatic islet β cells.