新西兰兔Keap 1基因实时荧光定量PCR检测方法的建立

Development of real-time PCR for detection and quantitation of New Zealand rabbit Keap1 gene

根据GenBank中穴兔Keap1基因序列设计了特异性引物,经PCR扩增,从新西兰兔肝脏中扩增出Keap1基因的C端并连接至T载体上,获得重组质粒pMD-19T-Keap1C。以重组质粒作为质控标准品,通过标准曲线构建、敏感性、特异性和重复性检验,建立了检测兔Keap1基因的实时荧光定量PCR方法,并分析了Keap1基因mRNA在兔各组织中的表达差异。结果显示,Keap1基因检测的Ct值与标准品呈良好的线性关系,扩增效率为103%,R^2>0.99。利用建立的实时荧光定量PCR方法对新西兰兔各组织中Keap1基因mRNA表达水平进行检测,结果显示,Keap1基因在脑、心脏、肝脏、脾脏、肺脏和肾脏中均有表达,且肝脏中表达量最高,脾脏中表达量最低。本试验建立了兔Keap1基因的荧光定量PCR方法并检测了其mRNA在兔各组织中的表达情况,为后续开展兔Keap1-Nrf2-ARE信号通路的研究奠定了基础。

The purpose of this study was to establish a real-time fluorescence quantitative PCR(qPCR)method for detection of rabbit Keap1 gene.Specific pairs of primers were designed based on the nucleotide sequences of Oryctolagus cuniculus Keap1 gene in GenBank.Then C-terminal of Keap1 gene was amplified from liver of New Zealand rabbit.The fragment was cloned into vector and the recombinant plasmid pMD-19T-Keap1C was constructed.The recombinant plasmid was used as standard products to establish standard curve of rabbit Keap1 for detecting sensitivity,specificity and repeatability.The expression level of Keap1 gene in different tissues were detected by qPCR.The results showed that the method of qPCR for rabbit Keap1 gene was successfully established.The amplification efficiency was 103%,and the correlation coefficient R^2>0.99.The expression level of Keap1 gene in different tissues of New Zealand rabbits were detected.The results shaved that Keap1 mRNA existed in all detected tissues(heart,liver,spleen,lung,kidney and brain).The most abundant expression of Keap1 was in liver and the minimal expression was in spleen.Our study lays the foundation for further exploration of rabbit Keap1-Nrf2-ARE signal pathway.