猪PD-1及其配体PD-L1和PD-L2 SYBR GreenⅠ Real-time PCR检测方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2016.11.013

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (11): 2892-2899.doi: 10.16431/j.cnki.1671-7236.2016.11.013

猪PD-1及其配体PD-L1和PD-L2 SYBR GreenⅠ Real-time PCR检测方法的建立及应用

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

1. 新乡学院生命科学技术学院, 新乡 453003;
2. 新乡学院生物技术研究所, 新乡 453003

收稿日期:2016-04-06 出版日期:2016-11-20 发布日期:2016-11-18
通讯作者: 王选年 E-mail:wangxuannian@163.com
作者简介:岳锋(1983-),男,河南滑县人,博士,讲师,研究方向:动物病理与分子免疫学,E-mail:yuefeng0116@163.com;Tel:0373-3682679
基金资助:
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）

Establishment and Application of SYBR Green Ⅰ Real-time PCR for Detection of Porcine PD-1,PD-L1 and PD-L2 mRNA

YUE Feng^1,2, ZHOU Juan-juan¹, ZHU Yan-ping^1,2, LI Peng^1,2, SUN Guo-peng^1,2, WANG Xuan-nian^1,2

1. College of Life Science and Technology, Xinxiang University, Xinxiang 453003, China;
2. Institute of Biotechnology, Xinxiang University, Xinxiang 453003, China

Received:2016-04-06 Online:2016-11-20 Published:2016-11-18

摘要/Abstract

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、PD-L1和PD-L2 mRNA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。

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

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.

Key words: Real-time PCR; PD-1; PD-L1; PD-L2

中图分类号:

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 GreenⅠ Real-time PCR检测方法的建立及应用[J]. 《中国畜牧兽医》, 2016, 43(11): 2892-2899.

YUE Feng, ZHOU Juan-juan, ZHU Yan-ping, LI Peng, SUN Guo-peng, WANG Xuan-nian. Establishment and Application of SYBR Green Ⅰ Real-time PCR for Detection of Porcine PD-1,PD-L1 and PD-L2 mRNA[J]. , 2016, 43(11): 2892-2899.

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猪PD-1及其配体PD-L1和PD-L2 SYBR GreenⅠ Real-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

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