猪TNF-α竞争定量RT-PCR标准曲线的建立.pdfVIP

第33卷第6期 湖南农业大学学报(自然科学版) 、b1．33 No．6 2007年 12月 Journal of Hunan Agricultural University(Natural Sciences) Dec．2O0r7 文章编号：1007—1032(2007)06—0730—04 猪TNF一0【竞争定量RT—PCR标准曲线的建立 司兴奎，张济培，陈建红，顾万军 (佛山科学技术学院 动物医学系，广东 佛山 528231) 摘 要：根据GenBank中猪TNF-~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-(I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PeR方法(qc—PeR)，建立猪TNF-~ 的标准竞争曲线和直线回归方程． 关 键 词：猪；TNF 竞争定量RT-PCR 中图分类号：$828；$852．4+3 文献标识码：A Establishment of a quantitative competitive RT-PCR assay for porcine TNF．n mRNA detection S1Xing-kui．ZHANGJi-pei·CHENJiamlmng．GUWan-jun (Department of Veterinary Medicine，Foshan Science and Technology University，Foshan，Guangdong 52823 1，China) Abstract：An 402 bp fragment of 11NF—a 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 1’NF-a and，couldbeamplifiedbythe san-lepairofprimerforthe402bpfragment。was constructedby reversePCRbasedon the premititive plasmid．By the quantitative competitive PCR between the logarithm of ratios of am plified copies of primitive plasmid(which will be replaced by cDNA of F-amRNA)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-amRNA detection was verified．It will be convenient for porcine TNF-amRNA detection with the competitive plasmid as competiti