摘要
【目的】探究鸭坦布苏病毒(Duck Tembusu virus,DTMUV)非结构蛋白的亚细胞定位及其在β-干扰素(IFN-β)信号通路中的作用。【方法】通过RT-PCR法扩增DTMUV的7个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)基因,将其克隆至真核表达载体pCAGGS-HA,构建重组真核表达质粒,并分别转染至HEK-293T细胞,通过Western blotting检测蛋白表达;利用间接免疫荧光试验检测非结构蛋白的亚细胞定位情况,通过双荧光素酶报告试验研究DTMUV的非结构蛋白对鸭源IFN-β启动子活性的影响。【结果】试验成功构建DTMUV的7个非结构蛋白带HA标签的真核表达质粒。Western blotting结果显示,真核表达非结构蛋白均正常表达,NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5蛋白分子质量大小分别为38、25、14.4、68、13.9、28和100 ku。间接免疫荧光试验结果显示,7个非结构蛋白在细胞内的表达形态不一,主要定位在细胞浆中。双荧光素酶报告试验结果表明,与对照组相比,过表达NS2B和NS4B蛋白后,鸭源IFN-β启动子活性极显著降低(P<0.01)。【结论】本研究成功构建了DTMUV的7个非结构蛋白的真核表达质粒,非结构蛋白主要表达于细胞浆中,其中NS2B和NS4B蛋白具有颉颃IFN-β活性的功能。试验结果为DTMUV的免疫逃逸研究提供了一定的理论依据,并为深入探究DTMUV在宿主天然免疫信号通路中的作用机制提供了试验材料。
【Objective】This study was aimed to explore subcellular localization of nonstructural proteins of Duck Tembusu virus(DTMUV)and their role in theβ-interferon(IFN-β)signaling pathway.【Method】7 nonstructural protein genes(NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5)of DTMUV were amplified by RT-PCR,cloned into eukaryotic expression vector pCAGGS-HA,constructed recombinant eukaryotic expression plasmid,and transfected into HEK-293T cells,respectively.The proteins expression was detected by Western blotting.The subcellular localization of nonstructural proteins was detected by indirect immunofluorescence assay,and the effect of DTMUV nonstructural proteins on the activity of IFN-βpromoter from ducks was studied by dual luciferase reporting assay.【Result】The experiment successfully constructed 7 eukaryotic expression plasmids of DTMUV with HA tags for nonstructural proteins.Western blotting results showed that the eukaryotic expression of nonstructural proteins was normal,and the molecular weights of NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5 proteins were 38,25,14.4,68,13.9,28 and 100 ku,respectively.The results of indirect immunofluorescence assay showed that the expression patterns of the 7 nonstructural proteins were different in the cell,mainly located in the cytoplasm.The results of dual luciferase reporting assay showed that compared with control group,the activity of IFN-βpromoter from ducks was extremely significantly decreased after overexpression of NS2B and NS4B proteins(P<0.01).【Conclusion】In this study,the eukaryotic expression plasmid of 7 nonstructural proteins of DTMUV was successfully constructed,and the nonstructural proteins were mainly expressed in cytoplasm,among which NS2B and NS4B proteins had the function of opposing IFN-βactivity.The results provided a theoretical basis for the study of immune escape of DTMUV,and provided experimental materials for further exploring the mechanism of action of DTMUV in host natural immune signaling pathway.
作者
张蓉蓉
潘爱銮
吴娟
方兵兵
汪最
卢琴
张腾飞
温国元
罗青平
ZHANG Rongrong;PAN Ailuan;WU Juan;FANG Bingbing;WANG Zui;LU Qin;ZHANG Tengfei;WEN Guoyuan;LUO Qingping(Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology,Key Laboratory of Prevention and Control Agents for Animal Bacteriosis,Ministry of Agriculture and Rural Affairs,Institute of Animal Husbandry and Veterinary,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;Wuhan Elabscience Biotechnology Co.,Ltd.,Wuhan 430074,China;Wuhan Keqian Biology Co.,Ltd.,Wuhan 430070,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第12期5470-5478,共9页
China Animal Husbandry & Veterinary Medicine
基金
现代农业产业技术体系建设专项资金(CARS-41-G13)
湖北省畜禽优质增效与绿色养殖科技创新行动(2018skjcx05)。
