PRELPshRNA稳定转染Saos-2细胞系的构建及鉴定

Establishment and identification of Saos-2 sublines with stable transfection of PRELP shRNA

目的构建PRELP（proline/arginine-rich end leucine-rich repeat protein）基因sh RNA慢病毒表达载体,并对其在Saos-2细胞中沉默效果进行鉴定。方法针对PRELP m RNA设计2对干扰序列,化学合成后插入至慢病毒载体,将慢病毒载体以不同MOI值分别转染Saos-2细胞,筛选细胞内GFP阳性率最高MOI值。将合成的慢病毒载体以筛选出的MOI值分别转染细胞,嘌呤霉素筛选稳定转染细胞株,Western blot法验证两株稳定转染细胞株PRELP下调效率。结果成功构建PRELP sh RNA慢病毒载体,当MOI为50-70时,细胞内荧光蛋白表达阳性率最高;嘌呤霉素筛选到稳定转染的细胞株;构建的稳转细胞系均特异性抑制PRELP蛋白的表达。结论成功构建了PRELP sh RNA慢病毒载体,并建立稳定的Saos-2细胞PRELP下调表达细胞模型,为研究PRELP在骨关节炎中的作用奠定了基础。

Objective To construct the expression vector for proline/arginine-rich end leucine-rich repeat protein（PRELP） sh RNA and identify its silencing effect in Saos-2 cells. Methods Two pairs of interfering sequences were designed according to the m RMA sequences of PRELP, synthesized chemically and inserted into lentivirus vector, with which Saos-2 cells were transfected at various MOIs, and the MOI at which the positive rate of GFP in cells was the highest was screened. Saos-2 cells were transfected with the constructed lentivirus vector at the screened MOI, from which the stably transfected strains were screened with puromycin. The down-regulation efficacy of PRELP in two stably transfected cell strains were verified by Western blot. Results The lentivirus vector for PRELP sh RNA was constructed successfully. When the MOI was 50, the positive rate of GFP in cells reached the maximum. Stably transfected cell strains were obtained by screening with puromycin, in which the expression of PRELP was significantly inhibited. Conclusion The lentiviral expression vector for PRELP sh RNA was constructed successfully, and the Saos-2 cell model with downregulated expression of PRELP was established, which laid a foundation of study on role of PRELP in osteoarthritis.