南方农业学报2025，Vol.56Issue(3)：962-972,11.DOI:10.3969/j.issn.2095-1191.2025.03.026
坦布苏病毒NS1蛋白的原核表达、生物信息学分析及鼠源多克隆抗体制备
Prokaryotic expression and bioinformatics analysis of NS1 pro-tein of Tembusu virus and preparation of mouse-derived polyclonal antibody
摘要
Abstract
[Objective]Bioinformatics analysis was performed on the NS1 protein of Tembusu virus(TMUV),the NS1 protein was expressed in prokaryotes,and mouse-derived NS1 polyclonal antibody was prepared,providing theoreti-cal basis for TMUV detection and research on the function of NS1 protein.[Method]ProtParam,ProtScale,TMHMM-2.0,SignalP-5.0,NetPhos 3.1,NetNGlyc-1.0,YinOYang-1.2,SOPMA and AlphaFold2 bioinformatics softwares were used to analyze the amino acid composition,theoretical isoelectric point,hydrophilicity/hydrophobicity,transmembrane domain,signal peptide,phosphorylation site,glycosylation site,secondary structure and tertiary structure of NS1 pro-tein.The NS1 gene was amplified,the recombinant plasmid was constructed,NS1 recombinant protein was expressed in Escherichia coli BL21(DE3)receptor cells.NS1 protein was purified and concentrated by Ni2+-NTA affinity chromato-graphy after urea treatment,and then identified by SDS-PAGE and Western blotting.Mouse-derived NS1 polyclonal anti-body was prepared,and an indirect ELISA method was established to detect the titer and specificity of mouse-derived NS1 polyclonal antibody.[Result]The bioinformatics analysis showed that the NS1 protein was a stable hydrophilic pro-tein,comprised by 320 amino acid residues,with a molecular weight of approximately 36.1 kD and a theoretical isoelec-tric point of 8.24.The NS1 protein lacked transmembrane domain and signal peptide,contained 42 phosphorylation sites(22 serine,17 threonine and 3 tyrosine residues)as well as 13 glycosylation sites(3 N-glycosylation sites and 10 O-glycosylation sites).In the secondary structure of the NS1 protein,α-helix constituted 21.88%,extended strands consti-tuted 25.00%,β-turns constituted 11.87%,and random coils constituted 41.25%,and the tertiary structure consisted of 2 domains.The prokaryotic expression vector pET-28a-NS1 was successfully constructed,and NS1 protein was efficiently purified.The prepared mouse-derived anti-NS1 polyclonal antibody exhibited a high titer of 1∶3276800 as determined by indirect ELISA,it could also be used for the specific detection of TMUV and indirect immunofluorescence analysis.[Conclusion]The NS1 protein lacks signal peptides and transmembrane domains,and is a hydrophilic protein suitable for expression in prokaryotic system.The pET-28a-NS1 is successfully constructed using a prokaryotic expression system.Fol-lowing urea denaturation,the NS1 protein is purified by Ni²⁺-NTA affinity chromatography,yielding a protein with high purity and biological activity.A mouse-derived anti-NS1 polyclonal antibody with high titer and strong specificity is suc-cessfully prepared,enabling its use for the specific detection of TMUV.关键词
坦布苏病毒/NS1蛋白/原核表达/多克隆抗体Key words
Tembusu virus/NS1 protein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
李娜,林翠,吴诚诚,张帆帆,谭佳,黄江南,李海琴..坦布苏病毒NS1蛋白的原核表达、生物信息学分析及鼠源多克隆抗体制备[J].南方农业学报,2025,56(3):962-972,11.基金项目
国家自然科学基金项目(32360875) （32360875）
江西省农业科学院基础研究与人才培养项目(JXSNKYJCRC202334) （JXSNKYJCRC202334）
江西省水禽产业技术体系项目(JXARS-09) （JXARS-09）
江西省重点研发计划项目(20224BBF61031) National Natural Science Foundation of China(32360875) （20224BBF61031）
Basic Research and Talent Training Project of Jiangxi Academy of Agricultural Sciences(JXSNKYJCRC202334) （JXSNKYJCRC202334）
Jiangxi Agriculture Research System—Wa-terfowl(JXARS-09) （JXARS-09）
Jiangxi Key Research and Development Plan Project(20224BBF61031) （20224BBF61031）
