猪TNF-α竞争定量RT-PCR标准曲线的建立

Establishment of a quantitative competitive RT-PCR assay for porcine TNF-α mRNA detection

根据GenBank中猪TNF-α的序列设计引物,采用RT-PCR技术扩增402 bp的部分基因片段,并克隆到pGEM-T载体中,构建成含TNF-α部分基因片段的重组质粒.利用反向PCR技术构建与扩增402 bp片断共用同一对引物,但缺失137 bp的竞争重组子.通过重组质粒与竞争重组子竞争定量PCR方法(qc-PCR),建立猪TNF-α的标准竞争曲线和直线回归方程.

An 402 bp fragment of TNF-α was amplified from the total RNA of porcine peripheral blood mononuclear cells （PBMC） by RT-PCR method, and one recombinant （primitive plasmid） was obtained by ligation of the 402 bp framgment and pGEM-T vector. The other recombinant （competitive plasmid）, contained 265 bp fragment of TNF-α and, could be amplified by the same pair of primer for the 402 bp fragment, was constructed by reverse PCR based on the premititive plasmid. By the quantitative competitive PCR between the logarithm of ratios of amplified copies of primitive plasmid （which will be replaced by cDNA of TNF-α mRNA） to the amplified copies of series 2-fold dilution competitive plasmid copies in comparison with the molecules of series 2-fold dilution competitive plasmid, the linear regression equation of the standard competitive curve for TNF-α mRNA detection was verified. It will be convenient for porcine TNF-α mRNA detection with the competitive plasmid as competitive template in the future.