摘要
【目的】研究旨在对猫细小病毒(Feline panleukopenia virus,FPV)洛阳分离株VP2蛋白进行原核表达和鼠源多克隆抗体制备。【方法】根据已公布的FPV VP 2基因序列(GenBank登录号:EU018143.1)设计1对特异性引物,以洛阳地区7例疑似FPV感染猫粪便为样本提取DNA,对VP 2基因进行PCR鉴定及测序,利用Mega 11.0软件对VP 2基因进行遗传进化分析;应用在线软件预测分离株VP2蛋白结构特征并找出优势抗原表位,克隆VP2优势抗原(sVP2)并连接pET-32a(+)载体,构建pET-32a-sVP2重组质粒,转化大肠杆菌Rosetta(DE3)感受态细胞后进行诱导表达,对重组蛋白表达条件进行优化,并使用SDS-PAGE与Western blotting方法进行鉴定;将pET-32a-sVP2用镍柱亲和层析蛋白纯化后免疫BABL/c小鼠,制备多克隆抗体,采用ELISA法检测抗体效价。【结果】分离得到1株FPV,命名为LY-1。遗传进化分析发现,该毒株属于FPV-G2亚群,与北京株(GenBank登录号:MT270581)亲缘关系最近。生物信息学分析显示,VP2属于稳定膜内蛋白,无信号肽与跨膜区,在N-端具有丰富的B细胞抗原表位(1―800 bp,sVP2)。试验成功构建pET-32a-sVP2重组质粒并表达,SDS-PAGE显示重组蛋白分子质量约50 ku,主要以包涵体形式存在,在30℃、1 mmol/L IPTG诱导6 h蛋白表达量最佳;Western blotting结果表明,重组蛋白能与鼠抗His单克隆抗体发生特异性反应;ELISA结果表明,该重组蛋白抗体效价为1∶128000,能够较好地识别免疫原sVP2和原核表达VP2蛋白并发生特异性反应。【结论】本试验克隆获得FPV sVP2片段,并制备多克隆抗体,为进一步研究VP2在FPV复制和致病过程中的作用及建立诊断FPV方法提供参考。
【Objective】The aim of this study was to prokaryotically express VP2 protein of Luoyang Feline panleukopenia virus(FPV)isolates and prepare polyclonal antibodies from mice.【Method】A pair of specific primers were designed according to the published FPV VP 2 gene sequence(GenBank accession No.:EU018143.1),DNA was extracted from the feces of 7 cats suspected of FPV infection in Luoyang area,and VP 2 gene was identified by PCR and sequenced.The genetic evolution of VP 2 gene was analyzed by Mega 11.0 software.The online software was used to predict the structure characteristics of VP2 protein and find the dominant epitopes.The VP2 dominant antigen(sVP2)was cloned into pET-32a(+)vector,the recombinant plasmid pET-32a-sVP2 was constructed and transformed into E.coli Rosetta(DE3)competent cell for induction and optimal expression.The recombinant protein was identified by SDS-PAGE and Western blotting.BABL/c mice were immunized with pET-32a-sVP2 purified by nickel column affinity chromatography to prepare polyclonal antibody,and the titer of the antibody was detected by ELISA method.【Result】A FPV strain was isolated and named LY-1.Genetic evolution analysis showed that this strain belonged to FPV-G2 subgroup,which was the closest relative to Beijing strain(GenBank accession No.:MT270581).Bioinformatics analysis showed that VP2 was a stable intramembrane protein with no signal peptide and transmembrane region,and there were abundant B cell epitopes(1-800 bp,sVP2)at the N-terminus.The recombinant plasmid pET-32a-sVP2 was successfully constructed and expressed.SDS-PAGE showed that the molecular mass of the recombinant protein was about 50 ku,mainly in the form of inclusion bodies,the optimal protein expression was induced by 1 mmol/L IPTG at 30℃for 6 h;Western blotting results showed that the recombinant protein could react specifically with mouse anti-His monoclonal antibody.ELISA results showed that the antibody titer of the recombinant protein was 1∶128000.The antibody could recognize the specific reaction between sVP2 and prokaryotic expression of VP2 protein.【Conclusion】FPV sVP2 fragment was cloned and polyclonal antibody was prepared,which provided a reference for further study of the role of VP2 in FPV replication and pathogenesis and establishing a diagnostic method for FPV.
作者
王文婕
茹鹏辉
郁川
杜付熙
陈松彪
尚珂
张春杰
程相朝
WANG Wenjie;RU Penghui;YU Chuan;DU Fuxi;CHEN Songbiao;SHANG Ke;ZHANG Chunjie;CHENG Xiangchao(The Key Laboratory of Animal Disease and Public Health,Henan University of Science and Technology,Luoyang 471003,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第4期1511-1521,共11页
China Animal Husbandry & Veterinary Medicine
基金
河南省自然科学基金项目(232300421263)。