【作者】 陈立；

【导师】 钟国强；

【作者基本信息】 广西医科大学 ， 心血管内科， 2012， 硕士

【摘要】 目的构建缝隙连接蛋白(Cx)43基因shRNA’慢病毒表达载体,并检测其对SD大鼠心肌细胞Cx43基因的沉默效果。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,PCR筛选阳性克隆,测序鉴定。用pHelper1.0和pHelper2.0质粒共转染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表达水平测定病毒滴度,以最适感染复数感染SD大鼠心肌细胞,通过荧光显微镜检测感染效率,应用Real-Time PCR和Western blot检测Cx43mRNA及Cx43蛋白表达,评价其抑制效果。结果经PCR和测序证实,成功构建Cx43慢病毒载体,病毒滴度为8×108TU/mL,慢病毒载体对SD大鼠心肌细胞感染效率为80%,荧光定量PCR检测提示慢病毒载体对心肌细胞Cx43mRNA的抑制率为92.4%,Westernblot检测提示慢病毒载体对心肌细胞Cx43蛋白的抑制率为86.5%。结论成功构建Cx43shRNA慢病毒载体,有效感染SD大鼠心肌细胞后,显著抑制了心肌细胞Cx43的表达。更多还原

【Abstract】 Objective:To construct a lentiviral RNAi vector that is capable of knocking down connexin43(Cx43) gene in rat cardiac myocytes.Methords:Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43gene was designed. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were connect to pGC-LV vectors. Then, The viral particles were generated by cotransfection of293T cells with the pGC-lv-Cx43and two packaging vector(pHelper1.0,pHelper2.0)and the virus titer was determined by counting the percentage of GFP positive cell. After transfection of lentiviral into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43mRNA was determined by RT-PCR, the level of Cx43protein was determined by western blot assay.Results:Target Cx43lentiviral vector was confirmed by PCR, the final titer obtained was8×108TU/mL. the infection rate was80%,the Real-Time PCR show the inhibition rate of C×43mRNA was92.4%and the western blot show the inhibition rate of Cx43protein was86.5%the expression of Cx43in knock down group is significantly lower than in control group.Conclusion:The lentiviral RNAi vector with the capability of knocking down Cx43gene in rat cardiac myocytes has been successfully constructed.更多还原