文献类型：期刊文献

中文题名：IBDV感染鸡后外周血淋巴细胞PD-1及其配体PD-L1/PD-L2转录变化的初步分析

英文题名：An Elementary Analysis for PD-l,PD-L1 and PD-L2 Genes Transcription Level of Peripheral Blood Lymphoctes in Chicken after IBDV Infection

作者：王爱国[1,2];孙国鹏[1];李博文[1,2];岳锋[1];张艳芳[1];朱艳平[1];银梅[2];杨媛[2];郭东光[1];王选年[1,2]

第一作者：王爱国

机构：[1]新乡学院生物技术研究中心生命科学与技术系,新乡453003;[2]河南科技学院动物科学学院,新乡453003

第一机构：新乡学院生命科学技术学院

年份：2014

期号：4

起止页码：654-660

中文期刊名：畜牧兽医学报

外文期刊名：Chinese Journal of Animal and Veterinary Sciences

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD2013_2014】；

基金：国家自然科学基金(31272539)

语种：中文

中文关键词：鸡;传染性法氏囊病病毒;PD-1;PD-L1;PD-L2

外文关键词：chicken;;infectious bursal disease virus;;PD-1;;PD-L1;;PD-L2

摘要：建立鸡PD-1及配体PD-L1/PD-L2SYBR GreenⅠ实时荧光定量RT-PCR检测方法,并用所建立的方法研究和分析雏鸡感染IBDV的不同阶段体内PD-1及配体PD-L1/PD-L2的转录变化。根据GenBank中PD-1、PD-L1和PD-L2的基因序列,分别设计特异引物扩增目的基因,得到各自阳性克隆质粒,以阳性质粒作为标准品建立标准曲线,并进行敏感性、特异性和重复性试验。应用所建立的实时荧光定量RT-PCR方法对4周龄健康雏鸡人工感染IBDV后第3、5、7、10天PBMC中PD-1及配体PD-L1/PD-L2的转录变化进行检测,同时用半定量RT-PCR方法以及IBDV快速检测试纸条对病毒感染后各时间点法氏囊组织中IBDV的载量进行检测。结果表明,建立的方法在1×101~1×109 copies·μL-1模板范围内具有良好的线性关系(R2>0.99),可检测至少101copies·μL-1的阳性标准样品,且具有较好的特异性和重复性。IBDV感染后,病毒在雏鸡体内复制逐渐增加,至第5天病毒载量达到最高。实时荧光定量RT-PCR检测结果表明,PD-1及配体PD-L1/PD-L2在病毒感染后不同阶段转录量均有所升高,其中PD-1在病毒感染后第7天显著(P<0.05)升高,在病毒感染后第3天PD-L1极显著(P<0.01)升高,PD-L2显著(P<0.05)升高。本研究成功建立了PD-1及配体PD-L1/PD-L2实时荧光定量RTPCR检测方法;并应用该方法初步分析了PD-1及配体PD-L1/PD-L2在IBDV感染不同阶段的转录变化,发现IBDV感染后不同阶段PD-1及配体PD-L1/PD-L2的转录均升高,且PD-L1、PD-L2的转录先于PD-1升高,并与病毒载量呈正相关。
An assay of SYBR GreenⅠreal-time fluorescent quantitative RT-PCR was established to investigate the expression pattern of chicken PD-l,PD-L1and PD-L2genes at different stages after IBDV infection.The specific primers were designed according to the sequences of PD-l,PDL1and PD-L2from GenBank to amplify the objective genes for corresponding plasmids construction,and then they were used as quantitative template to construct the standard curve and meltingcurve for detection sensitivity,specificity and repeatability.The established SYBRS GreenⅠrealtime fluorescent quantitative PCR(Rea1time FQ-PCR)assay worked well with a good coefficient correlation(R2>0.99)while the template concentration was from 1×101-1×109 copies·μL-1, the sensitivity was sufficient to detect at least 101 copies of the samples.The relative transcription levels of chicken PD-1,PD-L1and PD-L2mRNA in PBMCs from chicken were detected on the 3rd,5th,7th and 10th day after IBDV infection by the developed Rea1time FQ-PCR assay.At the same time,the semi quantitative RT-PCR and IBDV rapid test strip for detection were carried out to analyze the viral loads in bursal tissue.Real-time RT-PCR analysis show that the expression of PD-1increased significantly(P<0.05)in the 7th day after infection,the expression of PD-L1 and PD-L2increased significantly(P<0.01,P<0.05)in the 3rd day after infection and the IBDV loads in bursal tissue are highest in the 5th days after infection.The established assay was able to detect the expression of the three genes in PBMC.Our results revealing the pattern of chicken PD-l,PD-L1and PD-L2genes at different stages after IBDV infection,which showed that the gene transcription increased positively correlated with IBDV loads,and PD-L1,PD-L2 increasing transcription precede PD-1.