福建农业学报2026，Vol.41Issue(6)：653-661,9.DOI:10.19303/j.issn.1008-0384.2026.06.002
犬细小病毒非结构蛋白NS1的原核表达及多克隆抗体制备
Prokaryotic Expression and Polyclonal Antibody of Canine Parvovirus NS1 Protein
摘要
Abstract
[Objective]Polyclonal antibodies against the key pathogenic and viral life cycle-related NS1 of canine parvovirus(CPV)were prepared for studying the molecular mechanism of the viral infection on dogs.[Method]PCR primers were designed to amplify the non-structural NS1 gene to be inserted into the pET28a(+)vector for construction of prokaryotic expression plasmid pET28a(+)-His-NS1.Using an E.coli expression system,NS1 was expressed with the conditions optimized and protein purified by nickel ion affinity chromatography.The purified protein was then analyzed by circular dichroism spectroscopy prior to immunization on BALB/c mice.Subsequently,orbital venous serum sample of the mice was analyzed by ELISA,western blot,and confocal microscopy to determine the polyclonal antibody titer and specificity.[Result]A double enzyme digestion verified the successful construction of pET28a(+)-His-NS1 expression vector.The expressed and purified NS1 recombinant protein had a molecular weight of approximately 76 kDa.It was optimally expressed by induction with 0.1 mmol·L-1 IPTG at 25 oC for 12 h.The secondary structure of NS1 as indicated by circular dichroism spectroscopy was primarily composed of random coils and consistent with structural predictions.The polyclonal antibody in the mouse serum had a high immunogenicity with a titer exceeding 1:409 600.It was confirmed by western blot and confocal microscopy analyses to specifically recognize NS1 in the CPV-infected cells.[Conclusion]The recombinant NS1 of CPV obtained using a prokaryotic expression system in this study successfully prepared the murine polyclonal antibody that would become the essential tool to pursue previously lacked research relating to the functions of the protein and pathogenicity of CPV.关键词
犬细小病毒/NS1蛋白/原核表达/多克隆抗体Key words
canine parvovirus/NS1 protein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
王春林,马晓霞,刘金超,洪菊霜,李英奇,张胜卫,刘俊林..犬细小病毒非结构蛋白NS1的原核表达及多克隆抗体制备[J].福建农业学报,2026,41(6):653-661,9.基金项目
甘肃省自然科学基金项目(23JRRA715) （23JRRA715）
