PINCH-1基因RNAi慢病毒载体的构建及鉴定

Construction and identification of RNAi lentiviral vector targeting for PINCH-1

目的构建人PINCH-1基因的RNA干扰(RNAi)慢病毒载体,并转染人骨髓基质细胞初鉴定转染效果。方法利用RNAi设计软件,设计合成针对人PINCH-1基因的特异性siRNA序列,利用酶切反应合成包含特异性shRNA序列的pGC-SIL-GFP慢病毒载体GC-shBC047440,通过GC-shBC047440与pHelper 1.0载体和pHelper 2.0载体共转染包装293FT细胞产毒,并利用GFP蛋白表达水平进行滴度测定。获取的病毒上清筛选最适感染复数(MOI值),并感染骨髓基质细胞,RT-PCR和Western blot检测重组慢病毒对骨髓基质细胞PINCH-1蛋白和RNA表达的影响。结果酶切鉴定证实成功构建针对人PINCH-1基因的RNAi慢病毒干扰载体GC-shBC047440,载体感染人骨髓基质细胞后PINCH-1RNA表达较未感染组和空载体对照组显著下降。结论成功构建针对人PINCH-1基因的RNAi慢病毒载体,该载体能有效沉默PINCH-1基因的表达。

Objective To construct RNAi lentiviral vector targeting for PINCH-1. Methods SiRNA sequence targeting for PINCH-1 was designed and synthesized,and the specific shRNA targeting for PINCH-1 was inserted into the pGCSIL-GFP vector so called GC-shBC047440. 293FT ceils mediated by lipofectamin 2000 were transfected and the packaging plasmids （Helper 1.0 and pHelper 2.0plasmids） so as to produce the lentivirus. The titer of lentivirus was detected by GFP expression level. Results Enzyme digestion confirmed that the RNAi lentiviral vector targeting for PINCH 1 was successfully constructed,and it can effectively infec- ted bone marrow stromal cells and decreased the RNA level of PINCH-1. Conclusion The RNAi lentiviral vector targeting for PINCH-1 was successfully constructed,and it could effectively silence the expression of PINCH-1.