Construction Of A Hla-spaO Fusion Gene Of Salmonella Paratyphi A And Immunoprotection Of Product Expressed By The Fusion Gene

Objective To generate a hla-spaO fusion gene of Salmonella paratyphi A and its prokaryotic expression system, and to determine the immunoprotection of the recombinant expression product rHla-SpaO.Methods A hla-spaO fusion gene was generated as well as amplified from separate hla and spaO genes by PCR using flexible peptide sequence-containing liking primers and then sequenced after T-A cloning. A prokaryotic expression system of the hlaspaO fusion gene was constructed using routine genetic engineered technique and its expressed rHla-SpaO was examined by SDS-PAGE. The antigenicity and immunoreactivity of rHla-SpaO were determined by Western Blot assay and the ability of rHla-SpaO antiserum agglutinating Salmonella paratyphi A was detected by micro-Widal’s test. By using a mouse infection model, the immunoprotection of rHla-SpaO against the lethal dose of S. paratyphi A was determined and the immunoprotective effect of rHla-SpaO was compared with that of equal single recombinant protein Hla and SpaO (rHla and rSpaO).Results The generated hla-spaO fusion gene had100%nucleotide and amino acid sequence identities compared to the single hla or spaO gene. The constructed prokaryotic expression system could express the recombinant protein rHla-SpaO with high efficiency. rHla-SpaO was able combine with rHla or rSpaO antiserum while rHla-SpaO antiserum also could efficiently recognize rHla and rSpaO as well as agglutinate Salmonella paratyphi A through binding with H-antigen. The immunoprotective rate (93.3%) in mice against Salmonella paratyphi A infection due to pre-immunization with100μg rHla-SpaO was significantly higher than that of pre-immunization with equal rHla (60.0%) or rSpaO (53.3%)(p<0.05).Conclusions The recombinant fusion protein antigen rHla-SpaO has more powerful immunoprotective ability than equal single rHla or rSpaO, which can be used as the effective antigen of double-valent paratyphoid A genetic engineered vaccine or typhoid/paratyphoid capsular polysaccharide-protein conjugated vaccine.