中国畜牧兽医2016，Vol.43Issue(11)：2892-2899,8.DOI:10.16431/j.cnki.1671-7236.2016.11.013
猪PD-1及其配体PD-L1和PD-L2 SYBR GreenⅠReal-time PCR检测方法的建立及应用
Establishment and Application of SYBR Green Ⅰ Real-time PCR for Detection of Porcine PD-1,PD-L1 and PD-L2 mRNA
摘要
Abstract
In order to study the transcriptional level of porcine PD-1 and its ligands in the disease state,and establish a Real-time PCR method for detection of porcine PD-1 and its ligands, three pairs of specific primers were designed according to porcine gene sequences of PD-1, PD-L1 and PD-L2,respectively.Fragments of target genes were amplified by PCR.The target genes were cloned into the multiple cloning site of pMD18-T vector.After being transfected into DH5α,recombinant plasmids were identified by PCR and genetic sequencing,as being standard samples for Real-time PCR standard curve.Specificity and repeatability were conducted.The results showed that it had a good linear inverse relationship between the Real-time PCR values of Ct and logarithm of template concentration.R2 were all more than 0.99.Melt curves were the narrow single peak.The amplification products of Real-time PCR were the single band and no primer dimers.The coefficients of variation were less than 3% within and between groups of repeated test.It showed good specificity and repeatability.The mRNA levels of PD-L1 (P<0.01) and PD-L2 (P<0.05) were remarkably increased in the PBMCs of diseased pigs compared to healthy pigs,whereas no change was observed for PD-1(P>0.05).In this study,the methods of Real-time PCR for porcine PD-1,PD-L2 and PD-L1 mRNA were established,which laid a foundation for the study of the expression pattern of PD-1,PD-L1 and PD-L2 genes in pigs.关键词
Real-timePCR/PD-1/PD-L1/PD-L2Key words
Real-time PCR/PD-1/PD-L1/PD-L2分类
生物科学引用本文复制引用
岳锋,周娟娟,朱艳平,李鹏,孙国鹏,王选年..猪PD-1及其配体PD-L1和PD-L2 SYBR GreenⅠReal-time PCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(11):2892-2899,8.基金项目
国家自然科学基金(31291877) （31291877）
新乡学院科技创新基金重点培育项目(15ZP03) （15ZP03）
新乡学院博士启动科研项目(1366020053) （1366020053）
