细胞信号转导抑制因子3特异性shRNA的构建及鉴定

目的构建针对大鼠细胞信号转导抑制因子3(SOCS-3)基因的短发夹RNA(shRNA)表达载体,选取最有效shRNA模板序列。方法设计并合成编码的shRNA的2条寡核苷酸序列,经退火成互补双链,再克隆至pGPU6/GFP/Neo中构建重组表达载体,转化DH5α菌株,进行序列测定;转染至骨骼肌细胞中。Real-time PCR和Western blot检测各组SOCS-3的表达情况。结果测序证实重组质粒构建成功。4对shRNA模板序列对SOCS-3的表达抑制与空白及阴性对照相比差异均有统计学意义(均P<0.05),且以SOCS-3-shRNA a干预效果较好。结论 SOCS-3特异性shRNA重组表达载体构建成功,能有效抑制大鼠骨骼肌细胞SOCS-3的表达,为后续研究及代谢综合征的基因治疗奠定了基础。

Construction and Identification of Short Hairpin RNA Interference Vector of Rat Suppressor of Cytokine Signaling 3 Gene

Objective To construct and identify short hairpin RNA interference vector of rat suppressor of cytokine signaling 3 gene(SOCS-3),for the subsequent study of RNA interference. Methods Two DNA sequences containing a small hairpin structure were designed and synthesized.The complement form was obtained by annealing and cloning into vector pGPU6/GFP/Neo,and the recombinant plasmid was transformed into strain DH5α.The plasmid identified by restriction enzyme analysis was used for sequencing.Skeletal muscle cells were transfected with plasmid vector.The levels of SOCS-3 in skeletal muscle cells were detected by real-time PCR and Western blot. Results The recombinant plasmid was cloned,and the sequence was obtained.Transfection efficiency was above 80%.The mRNA and protein expression of SOCS-3 in SOCS-3-shRNA groups was lower than the blank control group and the negative control group(all P<0.05),and SOCS-3-shRNAa was the most effective one. Conclusion The recombinant vector containing the SOCS-3-shRNA was successfully constructed and could specifically inhibit the SOCS-3 expression,which will provide a gene therapy protocol for the metabolic syndrome.