猴痘病毒主要抗原蛋白的原核表达、纯化和反应原性评价OA

Monkeypox virus(also called Mpox virus,MPXV)is a zoonotic Orthopoxvirus that primarily transmitted in humans through direct contact with infected skin or bodily fluids.Addressing the ongoing public health challenge of MPXV spread,this study aims to produce key six structural proteins of MPXV(A35R,M1R,B6R,A29L,H3L,and E8L)by using a prokaryotic expression system and further evaluate their immunoreactivity,providing potential targets for the development of diagnostic tests,therapeutic drugs,and novel vaccines.Firstly,bioinformatics analyses were performed on the target proteins to predict their physicochemical properties,structures,and antigenic epitopes.Subsequently,the sequences encoding the extracellular regions of the proteins were codon-optimized,synthesized,and cloned into the pET-28a-SUMO vector.The recombinant plasmids were transformed into E.coli BL21(DE3)competent cells,and protein expression was optimized by adjusting IPTG concentration,induction temperature and expression time.The expressed proteins were purified using nickel-affinity chromatography,and their immunoreactivity was assessed via Western blot using specific monoclonal antibodies and MPXV-positive serum as primary antibodies.The results demonstrated that all six recombinant proteins were successfully expressed and purified.Among them,A35R,B6R,and A29L were mainly expressed in soluble form,while M1R,H3L and E8L were predominantly present as inclusion bodies.Furthermore,five recombinant proteins(A35R,M1R,B6R,A29L and H3L)specifically reacted with their corresponding monoclonal antibodies and MPXV-positive serum.However,the E8L recombinant protein was only recognized by its specific monoclonal antibody and showed weak reactivity with positive serum.This study successfully obtained five MPXV recombinant antigen proteins(A35R,M1R,B6R,A29L and H3L)with good immunoreactivity,which served as potential candidates for novel serological detection as well as vaccine and drug development.The research can provide significant theoretical and practical support for the prevention and control of Mpox and the development of related products.