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

doi:10.11843/j.issn.0366-6964.2014.04.021

摘要/Abstract

摘要：

建立鸡PD-1及配体PD-L1/PD-L2 SYBR Green Ⅰ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1×10¹～1×10⁹copies·μL^－1模板范围内具有良好的线性关系（R²>0.99），可检测至少10¹ copies·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RT-PCR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。

Abstract:

An assay of SYBR Green Ⅰ real-time fluorescent quantitative RT-PCR was established to investigate the expression pattern of chicken PD-l，PD-L1 and PD-L2 genes at different stages after IBDV infection.The specific primers were designed according to the sequences of PD-l，PD-L1 and PD-L2 from GenBank to amplify the objective genes for corresponding plasmids construction，and then they were used as quantitative template to construct the standard curve and melting curve for detection sensitivity，specificity and repeatability.The established SYBRS Green Ⅰ real-time fluorescent quantitative PCR (Rea1 time 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-l，PD-L1 and PD-L2 mRNA in PBMCs from chicken were detected on the 3rd，5th，7th and 10th day after IBDV infection by the developed Rea1 time 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-1 increased significantly (P<0.05) in the 7th day after infection，the expression of PD-L1 and PD-L2 increased 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-L1 and PD-L2 genes 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.

中图分类号:

S858.315.3

王爱国，孙国鹏，李博文，岳锋，张艳芳，朱艳平，银梅，杨媛，郭东光，王选年. 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[J]. 畜牧兽医学报, 2014, 45(4): 654-660.

WANG Ai-guo，SUN Guo-peng，LI Bo-wen，YUE Feng，ZHANG Yan-fang，ZHU Yan-ping，YIN Mei，YANG Yuan，GUO Dong-guang，WANG Xuan-nian. An Elementary Analysis for PD-l，PD-L1 and PD-L2 Genes Transcription Level of Peripheral Blood Lymphoctes in Chicken after IBDV Infection[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 654-660.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.04.021

https://www.xmsyxb.com/CN/Y2014/V45/I4/654

参考文献

［1］van den BERG T P.Acute infectious bursal disease in poultry：a review［J］.Avian Pathol，2000，29(3)：175-194.
［2］LUO J，ZHANG H，TENG M，et al.Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus ［J］.Avian Pathol，2010，39(5)：359-365.
［3］SHARMA J M，KIM I J，RAUTENSCHLEIN S，et al.Infectious bursal disease virus of chickens：pathogenesis and immunosuppression［J］.Dev Comp Immunol，2000，24(2-3)：223-235.
［4］RAUW F，LAMBRECHT B，van den BERG T P.Pivotal role of ChIFNgamma in the pathogenesis and immunosuppression of infectious bursal disease ［J］.Avian Pathol，2007，36(5)：367-374.
［5］RODRÍGUEZ-LECOMPTE J C，NIÑO-FONG R，LOPEZ A，et al.Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells［J］.Comp Immunol Microbiol Infect Dis，2005，28(4)：321-337.
［6］VELU V，TITANJI K，ZHU B，et al.Enhancing SIV-specific immunity in vivo by PD-1 blockade［J］.Nature，2009，458(7235)：206-210.
［7］BARBER D L，WHERRY E J，MASOPUST D，et al.Restoring function in exhausted CD8 T cells during chronic viral infection［J］.Nature，2006，439(7077)：682-687.
［8］YAMAZAKI T，AKIBA H，IWAI H，et al.Expression of programmed death 1 ligands by murine T cells and APC［J］.J Immunol，2002，169(10):5538-5545.
［9］KEIR M E，BUTTE M J，FREEMAN G J，et al.PD-1 and its ligands in tolerance and immunity［J］.Annu Rev Immunol，2008，26：677-704.
［10］FRANCISCO L M，SAGA P T，SHARPE A H.The PD-1 pathway in tolerance and autoimmunity［J］.Immunol Rev，2010，236：219-242.
［11］FREEMAN G J，LONG A J，IWAI Y，et al.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation［J］.J Exp Med，2000，192(7)：1027-1034.
［12］LATCHMAN Y，WOOD C R，CHERNOVA T，et al.PD-L2 is a second ligand for PD-1 and inhibits T cell activation［J］.Nat Immunol，2001，2(3):261-268.
［13］LIANG S C，LATCHMAN Y E，BUHLMANN J E，et al.Regulation of PD-1，PD-L1，and PD-L2 expression during normal and autoimmune responses［J］. Eur J Immunol，2007，33(10)：2706-2716.
［14］OKAZAKI T，HONJO T.PD-1 and PD-1 ligands：from discovery to clinical application［J］.Int Immunol, 2007，19(7)：813-824.
［15］ISHIDA M，IWAI Y，TANAKA Y，et al.Differential expression of PD-L1 and PD-L2，ligands for an inhibitory receptor PD-1，in the cells of lymphohematopoietic tissues［J］.Immunol Lett，2002，84(1):57-62.
［16］SALAMA A D，CHITNIS T，IMITOLA J，et al.Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis［J］.J Exp Med，2003，198(1)：71-78.
［17］DAY C L，KAUFMANN D E，KIEPIELA P，et al.PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression［J］.Nature，2006，443(7109)：350-354.
［18］MUENST S，HOELLER S，WILLI N，et al.Diagnostic and prognostic utility of PD-1 in B cell lymphomas［J］.Dis Markers，2010，29:47-53.
［19］BLACKURN S D，SHIN H，FREEMAN G G，et al.Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade［J］.Proc Natl Acad Sci U S A，2008，105(39):15016-15021.
［20］TRAUTMANN L，JANBAZIAN L，CHOMONT N，et a1．Upregulation of PD-1 expression on HIV-specific CD8⁺T cells leads to reversible immune dysfunction［J］．Nat Med，2006，12(10)：1198-1202.
［21］BONI C，FISICARO P，VALDATTA C，et al. Characterization of hepatitis B virus(HBV)-specific T-cell dysfunction in chronic HBV infection［J］．J Virol，2007，81(8)：4215-4225.
［22］PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR［J］.Nucleic Acids Res，2001，29(9)：e45.
［23］HAMANISHI J，MANDAI M，IWASAKI M，et al.Programmed cell death 1 ligand 1 and tumor infiltrating CD8⁺ T lymphocytes are prognostic factors of human ovarian cancer［J］.Proc Natl Acad Sci U S A, 2007，104(9)：3360-3365.
［24］IKEBUCHI R，KONNAI S，SHIRAI T，et al.Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade［J］.Vet Res，2011，42(1)：103-117.
［25］PARVIZI P，ANDRZEJEWSKI K，READ L R，et al.Expression profiling of genes associated withregulatory functions of T-cell subsets in Marek′s disease virus-infected chickens［J］.Avian Pathol，2010，39(5)：367-373.
［26］MATSUYAMA K A，MURATA S，ISEZAKI M, et al.Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek′s disease virus［J］.Viro J，2012，9(1)：94.
［27］BALAMURUGAN V，KATARIA J M.Economically important non-oncogenic immunosuppressive viral diseases of chicken-current status［J］.Vet Res Commun，2006，30(5)：541-566.

[1]	陈璐, 常新宇, 沈嘉忱, 沈瑞廷, 赵振华, 侯晓林. 鸡传染性支气管炎病毒感染HD11细胞的转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4860-4865.
[2]	王建琳, 江之瑶, 栾庆东, 尹燕博. 我国主要流行的4种血清型禽腺病毒-I群感染SPF鸡的致病性比较研究[J]. 畜牧兽医学报, 2022, 53(5): 1598-1607.
[3]	李丽, 唐国毅, 冯贺龙, 薛玉涵, 任助, 王国康, 贾妙妙, 商雨, 罗青平, 邵华斌, 温国元. 基于马赛克HA序列的H9亚型禽流感灭活疫苗的免疫效力分析[J]. 畜牧兽医学报, 2021, 52(12): 3569-3577.
[4]	徐磊, 钟佳莲, 余勋信, 刘毅发, 黄瑜, 刘小龙, 赖隆永, 闫丽萍, 许秀梅, 宋素泉, 张渊魁. 猪脾转移因子提高LaSota株鸡新城疫弱毒疫苗的免疫保护率[J]. 畜牧兽医学报, 2021, 52(12): 3578-3587.
[5]	余祖华, 丁轲, 贾艳艳, 郁川, 何雷, 廖成水, 李静, 张春杰, 李银聚, 吴庭才, 程相朝, 门凯凯, 张瑾, 余祖玲, 周子誉, 罗俊. 鸡TGFβ1对MDCC-MSB1细胞增殖、凋亡、迁移与侵袭的影响[J]. 畜牧兽医学报, 2020, 51(10): 2567-2575.
[6]	刘丹华, 郑世民, 刘晓静, 吕晓萍, 高雪丽, 刘超男. 禽网状内皮组织增生病病毒感染对SPF雏鸡血液和局部淋巴组织CD4⁺/CD8⁺细胞及相关细胞因子表达的影响[J]. 畜牧兽医学报, 2020, 51(6): 1447-1454.
[7]	段世豪, 路建彪, 李旭勇, 刘成, 张树金, 殷国政, 司振书, 李玉保. 鸡马立克病肝组织肿瘤病变与热休克蛋白转录、表达的相关性研究[J]. 畜牧兽医学报, 2019, 50(2): 415-421.
[8]	向华, 杨国淋, 曹三杰, 黄小波, 伍锐, 赵勤, 文心田, 文翼平. 同时检测禽类六种病毒的金标银染可视化基因芯片的构建及初步应用[J]. 畜牧兽医学报, 2018, 49(12): 2698-2706.
[9]	周忠文, 林桂芳, 王好好, 苏帅, 崔治中, 孙淑红. 表达禽网状内皮组织增生病病毒env基因的重组马立克病毒的生物学特性[J]. 畜牧兽医学报, 2018, 49(7): 1482-1491.
[10]	付礼胜, 侯宁, 王晓燕, 高雪丽, 刘超男, 吕晓萍, 郑世民. SPF雏鸡感染REV后中枢免疫器官细胞凋亡及相关因子的变化[J]. 畜牧兽医学报, 2017, 48(10): 1983-1989.
[11]	曾婷婷，谢芝勋，谢丽基，邓显文，谢志勤，罗思思，黄莉，黄娇玲. 应用多重GeXP-PCR同时检测6种鸡免疫抑制病病毒的研究[J]. 畜牧兽医学报, 2016, 47(6): 1198-1208.
[12]	周业飞，周梅仙. 面筋蛋白外啡肽B5对传染性支气管炎灭活疫苗免疫效果的影响[J]. 畜牧兽医学报, 2016, 47(3): 566-583.
[13]	宿靖伟，滕蔓，罗俊，迟佳琦，余祖华，禹乐乐，胡博，张改平. 马立克病病毒与禽网状内皮增生病病毒的混合感染及在传代过程中的重组[J]. 畜牧兽医学报, 2016, 47(1): 128-134.
[14]	王巧，李庆贺，刘冉冉，郑麦青，文杰，赵桂苹. 禽流感病毒PA-N182与鸡COPD蛋白互作的鉴定[J]. 畜牧兽医学报, 2015, 46(10): 1899-1904.
[15]	裴兰英，冯秀娟，李娟，李玉保，王守荣，朱育伟，吴伟胜. 马立克病病毒感染鸡心组织肿瘤病变与热休克蛋白表达的相关性分析[J]. 畜牧兽医学报, 2014, 45(12): 2013-2018.