猪轮状病毒主要非结构蛋白的原核表达、抗体制备及应用OA北大核心CSTPCD

[Background]Rotavirus(RV)is one of the main causes of acute viral gastroenteritis in young children and young animals worldwide,and it is of great significance in public health.Porcine rotavirus disease,caused by Porcine Rotavirus(PoRV),is an acute intestinal infectious disease that often results in gastrointestinal dysfunction in piglets,leading to severe symptoms,such as vomiting,diarrhea,and dehydration.Outbreaks of PoRV can result in significant economic losses in the pig industry.At present,there is no specific drug treatment for PoRV infection,so vaccination is the most economical way to control the infection.However,PoRV genotypes are various and easily mutable,and cross-protection between different genotypes is poor.Therefore,it is urgent to strengthen the epidemiological surveillance and pathogenic mechanism research of PoRV to explore new prevention and control strategies.[Objective]The prokaryotic system of Escherichia coli was employed to express the non-structural proteins(NSP),such as NSP2,NSP4,and NSP5 of PoRV.Subsequently,rabbits were immunized to produce polyclonal antibodies(pAbs)specific to these proteins,which could offer novel insights to the detection and prevention of PoRV.[Method]The NSP2,NSP4,and NSP5 genes of PoRV were codon-optimized and cloned into the pCold-sumo vector.The positive recombinant plasmids,with correct sequencing,were transformed into E.coli BL21(DE3),and the recombinant proteins were obtained with IPTG induction.Protein expression was identified using SDS-PAGE and Western blot assay.The recombinant proteins were then purified and quantified using affinity chromatography.New Zealand white rabbits were immunized with NSP2,NSP4,and NSP5 recombinant proteins by a subcutaneous multi-point injection method to prepare pAbs.The titers of pAbs were determined using indirect ELISA technology.The reactivity of pAbs with PoRV was verified by indirect immunofluorescence(IFA)and Western blot assay,and their applications in PoRV infection were also explored.[Result]SDS-PAGE analysis indicated that the recombinant proteins of NSP2,NSP4,and NSP5 were expressed well in a soluble form.Indirect ELISA analysis showed that the titers of pAbs against the three proteins reached 1﹕81 000,indicating good immunogenicity of the expressed proteins.The results from IFA and Western blot assay demonstrated that the prepared pAbs could specifically react with the dominant prevalent genotypes of PoRV,but had no reaction with other common diarrhea pathogens.Western blot assay results also showed that the pAbs could be used for dynamic expression analysis of NSPs during PoRV infection and validating transfection of the three recombinant eukaryotic plasmids.[Conclusion]Here,the NSP2,NSP4 and NSP5 of PoRV were successfully expressed in E.coli,and their pAbs with high titer and good specificity were obtained,which laid the foundation for the study of PoRV pathogenesis and the development of prevention and control strategies.