Influence Of Different Culture Medium On Expression Of Riemerella Anatipestifer Omps And Cloning And Expression Of OmpA Gene Fragment

The infection of Riemerella anatipestifer (RA) is one of the most important disease in duck. It is report that RA has 21 serotypes in the world, and little cross reaction in different serotypes. Lots of research had been done in RA, but little on virulence and protective antigen.Outer membrane proteins (Omps) are the whole proteins in the Outer membrane, one of specific structure in Gram-negative bacteria. Studies showed that Omps were associated with the virulence and immunogenicity of Gram-negative bacteria.RA1 was cultured in TSB (Trypicase Soy Broth) medium containing EDTA and its Omps profile was analyzed by SDS-PAGE. The results showed that more protein components from 34kD to 100kD occurred when EDTA was added to the medium. Especially 39kD, 60kD and 110kD proteins comprised most of the protein content of Omps . The western-blot analysis revealed that 39kD protein band could be distinguished in EDTA group, but not in control group. It is suggested that EDTA has a significant effect on expression and immunogenicity of Omps from RA1.Using bioinformatics software DNAStar, Outer membrane protein A (OmpA) gene sequences from two RA strains, ATCC 11845 and CVL 110/89, a pair of primers were designed using primer premier 5.0. A 498bp DNA fragment was PCR amplified from all the chromosomes of RA reference train and separated strains .By digestion with EcoR I and sequence analysis for the specific fragment, the separated strains were identified as RA. PCR was confirmed to be an ideal method for specially and rapidly identifying RA Analysing Outer membrane protein A (OmpA) gene sequences from two RA strains ,ATCC 11845 and CVL 110/89, a mutual sequence,which might exist in all RA strains, was obtained ,A pair of primers were desighed using primer premier 5.0.A 501bp DNA fragment was PCR amplified from the chromosomes of RA2 reference strain and cloned into Pet-32a(+). The recombinant plasmid was transformed into its host E.coli strain BLai and a protein band with MW of 27kD was expressed. The western-blot analysis revealedthat the protein band could be distinguished by both RA1 and RA2 antisera. It is suggested that the protein is one of protein that on the surface of the bacteria.