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

Abstract

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

CLC Number:

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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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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