发热伴血小板减少综合征病毒核蛋白的原核表达及多克隆抗体制备  

Protein expression and antibody preparation of the nucleoprotein of sever fever with thrombocytopenia syndrome virus

作　　者：陆桦葆 程子文 胡瀛方 王俊竹 张晓茹 孙荟迪 王大为 杨宝玲 Lu Huabao;Cheng Ziwen;Hu Yingfang;Wang Junzhu;Zhang Xiaoru;Sun Huidi;Wang Dawei;Yang Baoling(Jinzhou Medical University,Liaoning Jinzhou 121000)

机构地区：[1]锦州医科大学,辽宁锦州121000

出　　处：《现代畜牧兽医》2025年第3期26-31,共6页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：锦州医科大学大学生创新创业训练计划项目(X202210160050、X202210160022);锦州医科大学-博泽青年科技人才托举项目(JYQT202303、JYBZQT2109);辽宁省教育厅基本科研项目(JYTQN2023433、JYTQN2023435);辽宁省自然科学基金博士科研启动基金计划项目(2022-BS-325、2022-BS-323)。

摘　　要：研究旨在以严重发热伴血小板减少综合征病毒(SFTSV)的核蛋白(NP)为对象,表达SFTSV-NP重组蛋白并免疫小鼠,制备多克隆抗体,为SFTSV诊断试剂的研发奠定基础。试验扩增SFTSV-NP蛋白并连入pET-28a原核表达质粒,通过优化条件进行体外蛋白质的表达与纯化;同时免疫小鼠进行特异性抗体的制备,并对免疫所得抗体的效价进行ELISA检测,利用Western blot和IFA对抗体特异性进行分析。结果显示,试验成功扩增了长度为738 bp的SFTSV-NP蛋白,经菌落PCR和测序验证成功将NP片段连入pET-28a原核表达质粒;通过对表达条件的优化,得出培养温度为28℃、IPTG浓度为0.1 mmol/L时蛋白表达量最大(27 kDa),并进行大量的表达纯化;免疫小鼠所得的特异性抗体效价经ELISA检测符合后续试验要求,且经Western blot和IFA验证后特异性良好。研究表明,试验成功纯化了SFTSV-NP蛋白并制备了多克隆抗体,为后续SFTSV的监测以及功能的深入研究奠定了基础。This study aimed to express recombinant severe fever with thrombocytopenia syndrome virus(SFTSV)nucleoprotein(NP)and immunize mice to prepare polyclonal antibodies,laying the foundation for the development of SFTSV diagnostic reagents.The NP gene of SFTSV was amplified and cloned into the pET-28a prokaryotic expression plasmid.Protein expression and purification were performed under optimized conditions.Mice were immunized to generate specific antibodies,and the antibody titer was evaluated by ELISA.The specificity of the antibodies was further analyzed using Western blot and immunofluorescence assay(IFA).Results showed that the 738 bp SFTSV-NP gene was successfully amplified and ligated into the pET-28a plasmid,as confirmed by colony PCR and sequencing.By optimizing expression conditions,maximum protein yield(27 kDa)was achieved at 28℃with 0.1 mmol/L IPTG induction.Large-scale expression and purification were subsequently performed.ELISA confirmed that the antibody titer met experimental requirements,while Western blot and IFA demonstrated excellent specificity.This study successfully purified SFTSV-NP protein and prepared polyclonal antibodies,providing a basis for further research on SFTSV surveillance and functional studies.

关 键 词：发热伴血小板减少综合征病毒(SFTSV) 核蛋白 原核表达 多克隆抗体 

分 类 号：S855.99[农业科学—临床兽医学]