实时荧光定量PCR构建朊病相关蛋白P38MAPK标准品质粒和标准曲线

Establishment of standard curve for p38MAPK protein gene recombinant plasmid using Real-time PCR

关键词:  朊病  小鼠神经瘤细胞系N2a  p38有丝裂原激活的蛋白激酶

Key words:  Prion disease  Mouse neuroblastoma cell line N2a  p38MAPK

摘要:

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的309bp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。

Abstract:

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.