Identification And Application Study Of Riemerella Anatipestifer Antigenic Genes Induced In Vivo

Riemerella anatipestifer(RA),the etiological agent of septicemia anserum exsudativa,may develop as an acute or chronic septicemia,leading to fibrinous pericarditis,perihepatitis,airsacculitis,caseous salpingitis and meningitis.This disease has spread worldwide and accounts for major economic losses in duck producing industry. However,little was known about virulence factors that mediate the pathogenesis of RA infection.Notably,pathogenic immunogens expressed in vivo specifically induced by the pathogen infection have been thought to be important for the pathogenicity of the pathogen.To characterize potential virulent factors expressed by RA during the RA infection,the genomic DNA isolated from RA serotypeⅠwas digested by restriction endonuclease Sau3AI.DNA fragments with 1-4Kb were ligated into the plasmid pET28a/b/c digested with BamHI,the recombinant plasmids were transformed into E.coli BL21 to construct the RA genomic library with the capability about 3×10~4.To screen the IVI antigen,anti-RA sera from RA-infected ducks were pre-absorbed with BL21 and RA grown in vitro.Using in vivo-induced antigen technology(IVIAT) and the pre-absorbed anti-RA sera,we screened genomic DNA library of RA and obtained 28 positive clones, these new genes may be induced in vivo during the RA infection,and encoding important proteins associated with the RA infection in ducks.Bioinformatic analysis of these genes and deducted proteins revealed that they were classified into different categories, involving metabolism and synthesis of virulent factors,Signaling transduction pathway, secretory pathway,virulence plasmid,and so on.Therefore,our findings provide a new framework for studying the pathogenesis and the development of diagnostic reagents for RA infection in duck.The specific primers of three genes ive-1,ive-2,ive-6 were designed and these ORFs were amplified successfully,which size was respectively 537bp,1062bp or 933bp.After sequence analysis,deduction of amino acid sequence,antigenicity and secondary structure analysis,they were cloned into expression vector pGEX-KG,and all recombinant plasmids were transformed into E.coli BL21,and high effective expression of antigens induced successfully by IPTG.the moleculer weight of recombinant protein were respectively 46KDa,66KDa and 60KDa,then their antigenicities were characterized by Western blot assays using pre-absorbed sera against RA grown in vitro.The result indicated that the expression of these genes were inducible and these proteins were antigenic.Importantly,they are likely to be newly expressed in vivo during the RA infection and be important to study the mechanisms of RA infection.Using the purified proteins Ive-2 and Ive-6 expressed in the E.coli BL21 as coating antigen,two indirect ELISA method was developed,the results suggested that the two ELISA have high specificity,repeatability and sensibility,the result of ELISA using the sera of earlier period infected ducks and serun from inactive vaccine immunized ducks as primary antibody indicated that,Ive-2 was specificly induced in vivo during the RA infection,the two proteins are likely to be RA earlier period infection-related antigens and the protein Ive-2 may be used as antigen for the differential diagnosis of RA infection in ducks.