人支架蛋白IQGAP1-shRNA慢病毒载体的构建及鉴定

Construction and identification of RNA interference lentiviral vector for IQGAP1 gene

目的制备携带人支架蛋白IQGAP1基因小分子干扰RNA的重组慢病毒。方法分别设计合成针对IQGAP1的4个shRNA慢病毒干扰载体;分别转染293T细胞,利用Western blot验证shRNA的沉默效果,选择其中一个沉默效果较好的shRNA慢病毒干扰载体同源重组产生Lentivirus-IQGAP1-shRNA并测定病毒滴度。结果测序证实,构建携带IQGAP1-shRNA的慢病毒载体具有较好的沉默靶基因的效果,包装慢病毒,病毒滴度为2.0×l010TU/ml。结论成功制备携带IQGAP1-shRNA的重组慢病毒载体并包装出具高效感染力的慢病毒颗粒。

Objective： To construct recombinant lentivirus with IQGAPl-short hairpin RNA （shRNA）. Methods： Four different kinds of lentivirns vectors containing IQGAPI-shRNA were designed and synthesized. 293T ceils were transfected by these IQGAPI-shRNAs, and the method of western blot was applied to verify the silence effects. One of four IQGAPI-shRNAs which had the best silence effect was chosen to recombine lentivirus vector and the titer of purified virus was deter- mined. Results： The titer of virus was 2.0×l010TU/ml. Conclusion： The lentivirus-IQGAPl-shRNA is constructed suc- cessfully.