靶向人白细胞介素17受体C的特异性shRNA表达载体的构建及鉴定

Construction and identification of human IL-17RC and IL-17RA -specific shRNA eukaryotic expression vector

目的构建同时靶向人白细胞介素17受体C(IL-17RC)的小发夹状干扰RNA(shRNA)真核表达载体。方法设计并合成针对人IL-17RCcDNA的寡聚DNA引物,退火并连接至载体pGenesil-1,将重组质粒转染至293T细胞,提取转染或未转染293T细胞的mRNA,逆转录成cDNA,经荧光定量PCR检测细胞IL-17RC基因表达水平,最后用流式细胞仪检测转染阳性细胞表面IL-17RC的表达。结果1.限制性酶切和测序结果表明成功构建了人IL-17RC的特异性shRNA真核表达载体shRNA-IL-17RC。2.流式细胞仪显示pGenesi1-IL-17RC的转染效率大于40%,并能显著抑制细胞IL-17RC的表达。结论shRNA-IL-17RC的构建为深入研究IL-17在骨关节炎(OA)中的功能以及针对细胞因子受体设计新的治疗OA的药物奠定基础。

Objective To construct eukaryotic expression vector of shRNA specific for human IL-17 receptor C （IL-17RC）.Methods ccording to the published target sequence of IL-17RC, oligonucleotides were designed and synthesized, annealed and inserted into pGenesil-1 subsequently. The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant eukaryotic expression vector was transfected into 293T cells, IL-17RC mRNA levels were detected by flow cytometry.Results The construction of IL-17RC shRNA expression vector were confirmed successfully by the restriction digestion and sequencing. Flow cytometry showed that IL-17RC expression level with IL-17RC-shRNA positve cells was significantly lower than that of wild-type cells.Conclusions The construction of IL-17RC shRNA expression vector lays the foundation for the study of the function of IL-17 in osteoarthritis and the design of new drug for osteoarthritis treatment.