文献类型：期刊文献

中文题名：猪PD-1及其配体PD-L1和PD-L2 SYBR Green Ⅰ Real-time PCR检测方法的建立及应用

英文题名：Establishment and Application of SYBR GreenⅠ Real-time PCR for Detection of Porcine PD-1,PD-L1 and PD-L2 mRNA

作者：岳锋[1,2];周娟娟[1];朱艳平[1,2];李鹏[1,2];孙国鹏[1,2];王选年[1,2]

第一作者：岳锋

机构：[1]新乡学院生命科学技术学院;[2]新乡学院生物技术研究所

第一机构：新乡学院生命科学技术学院

年份：2016

卷号：43

期号：11

起止页码：2892-2899

中文期刊名：中国畜牧兽医

外文期刊名：China Animal Husbandry & Veterinary Medicine

收录：CSTPCD;;北大核心:【北大核心2014】；

基金：国家自然科学基金(31291877);新乡学院科技创新基金重点培育项目(15ZP03);新乡学院博士启动科研项目(1366020053)

语种：中文

中文关键词：Real-time;PCR;PD-1;PD-L1;PD-L2

外文关键词：Real-time PCR; PD-1; PD-L1; PD-L2;

摘要：本试验旨在研究猪PD-1及其配体在疾病状态下的转录水平,建立检测猪PD-1及其配体的Real-time PCR方法。根据GenBank中猪PD-1、PD-L1和PD-L2基因序列,分别设计3对特异性引物,PCR扩增目的基因片段,回收目的基因片段与pMD18-T连接后转化至大肠杆菌DH5α感受态细胞中,提取重组质粒,经PCR及测序鉴定正确,作为标准品绘制标准曲线,并进行特异性和重复性检测。结果显示,Real-time PCR的Ct值与模板浓度对数呈良好的线性反比关系,相关系数R2>0.99,熔解曲线出现狭窄单一峰;Real-time PCR产物经琼脂糖凝胶电泳,呈单一目的条带,无引物二聚体,特异性强;组内和组间重复性检测结果显示,组内和组间的变异系数均小于3%,重复性好;用建立的Real-time PCR方法检测猪自然感染PCV2后PD-1及其配体转录水平,PD-L1和PD-L2转录水平极显著或显著升高(P<0.01;P<0.05),PD-1转录水平无显著变化(P>0.05)。本试验建立了检测猪PD-1、PDL1和PD-L2mRNA的Real-time PCR方法并进行了初步应用,为猪PD-1、PD-L1和PD-L2基因表达模式研究奠定基础。
In order to study the transcriptional level of porcine PD-1and its ligands in the disease state,and establish a Real-time PCR method for detection of porcine PD-1and 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.