摘要
根据GenBank中猪IL-12p40的序列设计引物,采用RT-PCR技术扩增377 bp的部分基因片段,并克隆到pGEM-T Easy载体中,构建成含IL-12p40部分基因片段的重组质粒.利用反向PCR技术构建与扩增377 bp片段共用同一对引物但扩增255 bp片段的竞争重组子.通过重组质粒与竞争重组子竞争定量PCR方法(qc-PCR),建立了猪IL-12p40的标准竞争曲线和直线回归方程.
A 377 bp fragment of IL-12p40 was amplified from the total RNA of porcine peripheral blood mononuclear ceils （PBMC） by RT-PCR, and one recombinant （primitive plasmid） was obtained by ligation of the 377 bp fragment and pGEM-T Easy vector. The other recombinant （competitive） plasmid containing 255 bp fragment of lL-12p40 was amplified by the same pair of primers for the 377 bp fragment, was constructed by reverse PCR based on the primitive plasmid. By the quantitative competitive PCR between the logarithm of ratios of amplified copies of primitive plasmid （ which will be replaced by eDNA of IL-12p40 mRNA） to the amplified copies of series 2-fold dilution competitive plasmid copies in comparison with the logarithm of molecules of series 2-fold dilution competitive plasmid, the linear regression equation of the standard competitive curve for IL-12p40 mRNA detection was verified.
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2008年第4期91-94,共4页
Journal of South China Agricultural University
基金
广东省自然科学基金博士科研启动基金(06300063)
