犬细小病毒非结构蛋白NS1的原核表达及多克隆抗体制备OA

[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.