Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用

Construction of a lentiviral vector for RNA interference targeting rat cardiac myocytes connexin 43 gene and its effect

目的构建缝隙连接蛋白(Cx)43基因shRNA慢病毒载体,并检测其对大鼠心肌细胞Cx43基因的作用。方法针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,挑选阳性克隆行PCR鉴定及测序。用pHelper1.0和pHelper2.0质粒转染293T细胞,包装产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的细胞数量计算病毒滴度;以最适感染复数感染大鼠心肌细胞,通过荧光显微镜观察感染效率;应用real-time PCR和Western blot法检测大鼠心肌细胞Cx43 mRNA及Cx43蛋白表达,并评价其抑制效果。结果经PCR鉴定和测序证实,Cx43慢病毒载体构建正确,其病毒滴度为8×108TU/mL、转染效率为82.59%。荧光定量real-time PCR法检测Cx43 mRNA的抑制率为96.10%,Western blot法检测Cx43蛋白的抑制率为77.16%。结论成功构建了Cx43基因shRNA慢病毒载体,其能显著抑制大鼠心肌细胞Cx43基因的表达。

Objective To construct a lentiviral RNAi vector and to detect its effect on connexin 43 （Cx43） gene in rat cardiac myocytes. Methods short hairpin RNA （shRNA） sequence targeting rat cardiac myocytes Cx43 gene was de- signed. After synthesis and annealing; the double-stranded oligonucleotides （ds oligo） were connecting to pGC-LV vectors. The positive clones were selected and conducted by PCR identification and sequencing. Then, The viral particles were gen- erated by cotransfection of 293T cells with the pGC-lv-Cx43 and two packaging vector （pHelperl. 0, pHelper2.0）, and the virus titer was determined by counting the number of GFP positive cells. After transfection of lentiviral vector into rat cardi- ac myocytes with multiplicity of infection （MOI）, the level of Cx43 mRNA in rat cardiac myocytes was determined by real- time PCR and the level of Cx43 protein was determined by Western blot assay. The inhibitory effect was evaluated. Results The construction of Cx43 lentiviral vector was confirmed by PCR identification and sequencing. The final titer obtained was 8 × 10^8 TU/mL. The transfection efficiency was 82.59% , the real-time PCR showed the inhibition rate of Cx43 mRNA was 96.10% and the Western blot showed the inhibition rate of Cx43 protein was 77.16%. Conclusion The lentiviral RNAi vector successfully constructed in rat cardiac myocytes has the capability of inhibiting Cx43 gene.