[目的]对新型阿卡斑病毒(Akabane virus,AKAV)非结构蛋白(non-structura,NSs)进行生物信息学分析,进而制备抗多克隆抗体,为新型AKAV检测及功能研究提供必要材料.[方法]使用蛋白生物信息学在线分析网站,对新型AKAV NSs蛋白进行系统分析.克隆NSs全长基因,利用大肠杆菌表达系统表达NSs重组蛋白,优化蛋白诱导条件并分析重组蛋白的可溶性;收获破碎菌体,经8 mol/L尿素变性、Ni2+-NTA亲和层析纯化、透析及浓缩后进行SDS-PAGE、Western blotting鉴定;将纯化的重组NSs蛋白免疫新西兰大白兔,制备多克隆抗体;应用间接ELISA、Western blotting和间接免疫荧光试验(IFA)测定多克隆抗体效价及特异反应性.[结果]生物信息学分析表明,NSs蛋白由91个氨基酸残基构成,等电点为12.10,属于碱性蛋白,不稳定指数约为79.04,平均亲水性为指数为-0.059,为不稳定的亲水蛋白;无跨膜区和信号肽,属于非分泌蛋白.试验成功构建了 NSs蛋白原核表达载体pET-32a-NSs,经SDS-PAGE和Western blotting鉴定显示,成功表达重组NSs蛋白,分子质量为29.3 ku,主要以包涵体形式存在,与生物信息学分析结果一致;制备的多克隆抗体效价可达1:16 384 000,Western blotting和IFA结果表明所制备抗体能够与NSs蛋白发生特异性反应,具有较好的特异性.[结论]试验成功获得纯化的新型AKAV NSs蛋白,该蛋白具有较好的免疫原性;制备获得效价高、特异性好的多克隆抗体,为后续新型AKAV疫苗开发、检测及NSs蛋白功能研究提供了基础.
[Objective]This study was aimed to perform the bioinformatics analysis of non-structura(NSs)protein of novel Akabane virus(AKAV)and prepare anti-polyclonal antibody,so as to provide necessary materials for detection and functional study of novel AKAV.[Method]The novel AKAV NSs protein was systematically analyzed using some online analysis website for protein bioinformatics.The full-length NSs gene was cloned and the recombinant NSs protein was expressed by the expression system of Escherichia coli.The induction conditions of the recombinant protein were optimized and the solubility of the recombinant protein was analyzed.The crushed Escherichia coli expressing NSs protein was collected,denatured by 8 mol/L urea,and purified by Ni2+-NTA affinity chromatography.After dialysis and concentration,the purified NSs protein was identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared by immunizing New Zealand White rabbits with purified recombinant NSs protein,then the titer and specific reactivity of the polyclonal antibody were determined by indirect ELISA,Western blotting and indirect immunofluorescence assay(IFA).[Result]Bioinformatics analysis results showed that the NSs protein was composed of 91 amino acid residues,with an isoelectric point of 12.10,belonging to basic protein.Its instability index was about 79.04,and the average hydrophilicity index was-0.059,indicating that it was an unstable hydrophilic protein.There was no transmembrane region and signal peptide,the NSs protein was a non-secreted protein.The NSs protein prokaryotic expression vector pET-32a-NSs was successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant NSs protein was successfully expressed,with a molecular weight of 29.3 ku and mainly existed in the form of inclusion body,which was consistent with the results of bioinformatic analysis.The titer of the prepared polyclonal antibody reached 1:16 384 000.The results of Western blotting and IFA showed that the prepared antibody could react specifically with NSs protein,having good specificity.[Conclusion]The novel AKAV NSs protein was successfully purified and showed good immunogenicity.The polyclonal antibody with high titer and good specificity was prepared,which laid a foundation for the development of vaccine,disease detection and the function study of NSs protein.
蔡畅;刘兴友;马玲;吴健敏;陈赫威;宋瑞鹏;覃绍敏;刘金凤;秦树英;孙倩;陈童锦悦;欧长波
广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,南宁 530001||河南科技学院动物科技学院,新乡 453003||农业农村部(广西)-东盟跨境动物疫病防控重点实验室,南宁 530001新乡学院生命科学与基础医学学院,新乡 453003广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,南宁 530001||农业农村部(广西)-东盟跨境动物疫病防控重点实验室,南宁 530001广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,南宁 530001||农业农村部(广西)-东盟跨境动物疫病防控重点实验室,南宁 530001||广西大学动物科学技术学院,南宁 530004河南科技学院动物科技学院,新乡 453003||广西大学动物科学技术学院,南宁 530004
畜牧业
新型阿卡斑病毒(AKAV);NSs蛋白;生物信息学;原核表达;多克隆抗体
novel Akabane virus(AKAV);NSs protein;bioinformatics;prokaryotic expression;polyclonal antibody
《中国畜牧兽医》 2024 (005)
2015-2026 / 12
国家自然科学基金(32160830);广西兽医生物技术重点实验室开放基金项目(20-065-23-B-2);广西兽医研究所基本科研业务费专项(桂科专项20-4、桂科专项21-7)
10.16431/j.cnki.1671-7236.2024.05.023
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