鸡TET2及截短体真核表达载体的构建及其对先天免疫反应的影响  

Construction of Chicken(Gallus gallus)TET2 and Truncated Eukaryotic Expression Vector and Its Effects on Innate Immune Response

作　　者：马克姣 蔡清清 王佳兴 王强州 白皓 陈世豪 常国斌 MAKe-Jiao;CAI Qing-Qing;WANG Jia-Xing;WANG Qiang-Zhou;BAI Hao;CHEN Shi-Hao;CHANG Guo-Bin(Institute of Epigenetics and Epigenomics,Yangzhou University,Yangzhou 225009,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学表观遗传学与表观基因组学研究所,扬州225009 [2]扬州大学兽医学院,扬州225009

出　　处：《农业生物技术学报》2024年第10期2371-2380,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32002271);国家肉鸡产业技术体系(CARS-41)。

摘　　要：甲基胞嘧啶双加氧酶2(ten-eleven translocation 2,TET2)通过甲基化依赖和不依赖的两种方式调控哺乳动物的天然免疫反应,而鸡(Gallus gallus)TET2的先天免疫调控作用尚未阐明。本研究旨在克隆鸡TET2基因并构建TET2及截短体的真核表达载体,初步探讨TET2及其功能域对鸡先天免疫反应的影响。根据鸡TET2基因组(GenBank No.NM_001277794.1)信息,克隆其全长CDS序列。将TET2全长分为N端N1126和C端CD两个截短体,扩增其基因片段,将其同源重组连接至pCAGGS-Myc真核表达载体。将重组质粒pCAGGS-Myc-TET2及截短体分别转染至鸡胚成纤维细胞DF-1中,通过Western blot和免疫荧光检测鸡TET2及截短体的蛋白表达和细胞定位。通过qRT-PCR检测过表达pCAGGS-Myc-TET2及截短体基因对ploy(I:C)诱导的先天免疫反应相关基因表达的影响。PCR结果显示,成功克隆鸡TET2全长序列及其截短体N1126和CD。生物信息学分析表明,与鸡TET2亲缘关系最为相似的是鸿雁(Anser cygnoides)和绿头鸭(Anas platyrhynchos)。Western blot检测表明,Myc标签融合的TET2全长和截短体均能在DF-1细胞正常表达。免疫荧光检测表明,鸡TET2定位于细胞核。qRT-PCR结果表明,过表达鸡TET2全长及截短体均能显著促进poly(I:C)诱导的黑色素瘤分化相关基因5(melanoma differentiation associated gene 5,MDA5)和三基序蛋白25(tripartide motif containing 25,TRIM25)的表达(P<0.05),而对干扰素调节因子7(interferon regulatory factor 7,IRF7)的表达无显著影响(P>0.05);与过表达截短体N1126相比,过表达TET2全长和CD功能域极显著促进poly(I:C)诱导的β干扰素(interferon-β,IFN-β)表达(P<0.05)。本研究初步探索了TET2及其截短体对鸡先天免疫反应的影响,为鸡TET2调控先天免疫反应的分子机制提供了基础信息。Ten-eleven translocation 2(TET2)regulates the innate immune response of mammals in 2 ways:Methylation-dependent and methylation-independent.However,the innate immune regulation of chicken(Gallus gallus)TET2 has not been elucidated.The aim of this study was to clone chicken TET2 gene and construct the eukaryotic expression vector of TET2 and its truncated form,and preliminarily explore the effect of TET2 and its functional domain on chicken innate immune response.According to the chicken TET2 genome(GenBank No.NM_001277794.1)information,primers were designed to clone the full-length CDS sequence.The full length of TET2 was divided into N-terminal N1126 and C-terminal CD 2 truncated fragments,and then the truncated gene fragment was amplified.It was connected to the pCAGGS-Myc eukaryotic expression vector by homologous recombination.The recombinant plasmid pCAGGS-Myc-TET2 and its truncated form were transfected into chicken embryo fibroblasts DF-1,respectively.The protein expression and cellular localization of chicken TET2 and its truncated form were detected by Western blot and immunofluorescence.The effects of overexpression of pCAGGS-Myc-TET2 and truncated vectors on the expression of genes related to the innate immune response induced by ploy(I:C)were detected by qRT-PCR.PCR results showed that the full-length sequence of chicken TET2 and its truncated N1126 and CD were successfully cloned.Bioinformatics analysis showed that Anser cygnoides and Anas platyrhynchos were most closely related to G.gallus TET2.The results of Western blot showed that the full-length and truncated TET2 fused with Myc tag could be expressed normally in DF-1 cells.The results of immunofluorescence showed that the TET2 were localized in the nucleus.The results of qRT-PCR showed that overexpression of full-length and truncated chicken TET2 could significantly promote the expression of melanoma differentiation associated gene 5(MDA5)and tripartide motif containing 25(TRIM25)induced by poly(I:C)(P<0.05).However,there was no significant eff

关 键 词：鸡 甲基胞嘧啶双加氧酶2(TET2) 功能域 真核表达载体 先天免疫 

分 类 号：S381.2[农业科学—农艺学]