Distribution Of Salmonella Paratyphi A OmpA Gene And Immunological Identification The Recombinant Expressed Product

Background and Objective In recent years,Paratyphoid fever A,infected by the Salmonella paratyphi,has outbroken in South of China..However,S.paratyphi A has no capsular polysaccharide and it cannot be prevented by Vi polysaccharide Paratyphoid vaccine.So,screening surface antigen of S.paratyphi A to develop vaccine is of vital importance for Paratyphoid fever A prevention and control.On the other hand,outer membrane proteins are major surface antigens of Gram-negative bacteria such as Salmonella.In this study,a prokaryotic expression system of ompA gene from S.paratyphi A was constructed and the antigenicity and immunoreactivity of the recombinant proteins rOmpA were determined.Meanwhile,ompA gene was detected by PCR in 98 strains of S.paratyphi A and the immunoprotective effect of rOmpA against the challenge of S.paratyphi A was understood by a mouse infection model.In view of the above,we look forward to providing experimental evidence for rOmpA that may be chosen as the candidate antigens of S.paratyphi A genetically engineering vaccineMethods The ompA gene was amplified from a clinical S.paratyphi A strain JH01 by using PCR,cloned into T-A cloning vector,and then the prokaryotic expression vector was constructed.The rOmpA expression was determined by SDS-PAGE.Antigenicity and immunoreactivity of rOmpA were determined by immunodiffusion test,Micro-Widal's test and Western-blot assay,ompA gene exiting in 98 S.paratyphi A clinical isolates was detected by PCR.By using immunoprotective effect of rOmpA against the challenge of S.paratyphi A strain 50001 in mice were determinded.Results Both the nucleotide and putative amino acid sequence identities of the cloned ompA gene were 100%compared to the reported corresponding sequences.The expression yield of rOmpA was approximately 65%of the total bacterial proteins. rOmpA was able to induce specific antibody in rabbits,and reacted efficiently with rabbit antiserum or the antiserum against whole cell of S.paratyphi A detected by Western-blot.94.9%(93/98) of the S.paratyphi A isolates had ompA gene.41.7%(5/12) and 58.3%(7/12) of the mice immunized with 100 and 200μg rOmpA were survival after lethal challenge with S.paratyphi A strain 50001.The titer of antibody to the H antigens of Salmonella paratyphi spp in the sera from rOmpA immunized mice and survival mice in the S.paratyphi A challenge detected by Micro-Widal's test was 1:5～1:40.Conclusion ompA gene has an extensive distribution in clinical isolates of Salmonella paratyphi A.rOmpA possesses a immunogenicity and a certain immunoprotective effect,and can be used as the candidate antigen in genetic engineering vaccine.