siCASK重组载体构建及效应检测

目的　构建针对人CASK基因的siRNA质粒 ,建立CASK下调表达的细胞模型 ,为CASK相关的细胞信号研究奠定基础。方法　选择人CASK基因不同靶点 ,体外合成DNA模板 ,经退火后插入到pSilencer3 .1hygro载体的多克隆位点。阳性重组质粒转染ECV3 0 4细胞 ,蛋白印迹。观察siCASK对目的蛋白表达的抑制效率。结果　酶切、测序表明以pSi lencer3 .1hygro为载体的重组质粒siCASK构建成功。siCASK转染细胞后 ,能够明显抑制目的蛋白的表达。结论　成功构建了siRNA重组质粒 ,为下一步CASK功能研究奠定基础。

Construction of siRNA plasmid targeting CASK and its roles in CASK expression

Objective To construct small interference RNA (siRNA) plasmid targeting human calcium, calmodulin-associated serine/threonine kinase (CASK) gene and to establish CASK-downregulation ECV304 cell line in order to lay a foundation for the study of CASK related signal. Methods Two potential target sequences in CASK open reading frame were selected for the synthesis of DNA templates in vitro. The two DNA strands were annealed into dsDNA, and then the dsDNA was cloned into pSilencer3.1hygro vector. ECV304 cells were transfected with positive siRNA plasmids and the expression of CASK was observed by Western blotting. Results Restriction endonuclease digestion and DNA sequencing showed that siRNA plasmids based on pSilencer3.1 hygro were constructed successfully. Western blotting revealed that the CASK protein was obviously suppressed by siCASK transfection. Conclusion The two siRNA plasmids targeting human CASK gene, successfully constructed by us, could suppress the human CASK gene expression.