实时荧光定量PCR构建朊病相关蛋白p38 MAPK标准品质粒和标准曲线

Establishment of Standard Curve for p38MAPK Protein Gene Recombinant Plasmid Using Real-time PCR

为进一步揭示朊病的治病机理,根据GenBank中的小鼠p38MAPK的核苷酸序列设计特异性引物,经RT-PCR技术从N2a细胞中扩增和克隆了该蛋白的309 bp核苷酸片段。测序鉴定后,将重组质粒10倍系列稀释后作为标准模板,通过实时荧光定量PCR方法,建立了p38MAPK标准曲线及其直线回归方程。所构建的标准曲线的相关系数r2=0.999,说明成功构建小鼠N2a细胞p38MAPK基因的标准质粒和标准曲线,为研究朊病的发病机理奠定了分子生物学基础。

To investgate the mechanism of the apoptotic effect of PrPSc in prion disease,our study is to establish a tool for the quantification of the protein on RNA level.Primers were designed and based on the p38MAPK nucleotide sequence gene available in GenBank.309 bp fragments were amplified by RT-PCR from the mouse N2a cells,and then the fragments were cloned and sequenced.Recombinant plasmids were diluted by 10-fold serial and used as the real-time PCR standard templates.The constructed standard curve had a relative coefficient r2=0.999.Results indicated that the standard curves were successful.The mRNA level of the p38MAPK can be detected by this real-time PCR assay.