α-珠蛋白基因的RNAi慢病毒载体的构建

Construction of a lentiviral vector for α-globin gene RNA interference

目的构建α-珠蛋白基因RNAi重组慢病毒载体。方法合成靶序列Oligo DNA,退火形成双链DNA,克隆到慢病毒p LKO载体,重组载体进行酶切和测序鉴定。在脂质体的介导下将ps PAX2、pMD2.G及慢病毒载体三质粒共转染293T细胞,48h后收集细胞培养上清液。采用终点稀释法测定病毒滴度。结果酶切和测序证实重组载体构建成功。病毒的滴度为3.9×106TU/ml。结论成功构建α-珠蛋白基因RNAi慢病毒载体,为采用RNAi技术治疗β-地贫的研究创造了条件。

Objective：To construct a lentiviral vector for α-globin gene RNA interference（RNAi）. Methods：An interfering sequence targeting α-globin gene and a negative sequence were designed,synthesized and inserted into p LKO lentiviral vector.The recombined vector was confirmed by sequencing and restriction enzyme analysis. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293 T cells. The virus titer was tested by end-point dilution method. Results：The recombinant plasmids were confirmed by sequencing and restriction enzyme analysis. The titer of virus was over 3.9 × 106TU/m L. Conclusion：The lentiviral RNAi vector targeting α globin gene was constructed successfully,which provides possibility for further investigations of Thalassemia.