Outer Membrane Protein W Gene Identification And Construction Of Mutant Strain Of Gallibacterium Anatis Chinese Isolate

Gallibacterium is a genus in the Gram-negative Pasteurellaceae family,in which Gallibacterium anatis is typical species.The study shows that Gallibacterium anatis is a major cause of lesions in the reproductive tracts of egg layers under certain conditions,causing a drop in egg production and increased mortality.Multiple-drug resistance and a substantial antigenic diversity make it dif?cult to prevent the negative effects of G.anatis using traditional antimicrobial agents and vaccines.Thus,novel prevention and treatment strategies are urgently needed.To date,very little is known about the pathogenesis of G.anatis and only a few putative virulence factors have been identi?ed.These include a new type of RTX toxin Gtx A,a polysaccharide capsule,metalloproteases,and hemagglutinin.In this study,part of the property of Gallibacterium anatis outer membrane protein W(OmpW)was researched,for the first time.By PCR and bioinformatics methods,the distribution of OmpW in Gallibacterium anatis was obtained and possible B cell epitopes was predicted.Polyclonal antibody against purified recombinant Gallibacterium anatis OmpW was produced in rabbit.The immunogenicity was analysised by Western blotting.For better research of OmpW gene function,this study established targeted gene manipulation technique of Gallibacterium anatis interiorly,for the first time,and ?omp W mutant,Gallibacterium anatis OmpW mutant,was developed.1.OmpW gene amplification and bioinformatic analysis of Gallibacterium anatisIn this experiment,a pair of universal primer was designed by reference to the OmpW gene sequence published in GenBank,with witch OmpW gene amplification was accomplished of 32 Gallibacterium anatis isolated from different area in order to analysis the existential state of OmpW.Through analyzing OmpW amino acid sequence of Yu-PDS-RZ-1-SLG by bioinformatics methods,physicochemical property,architectural featurea and conservative B cell antigen epitope were predicted.The results indicated that OmpW was widely existed and more conservativein Gallibacterium anatis genome.Barreled OmpW with four hypervariable regions located at extracellular region had common B cell antigen epitope,so having potential cross immunogenicity among different strains.The results laid the foundation for the diagnosis method establishment of Gallibacterium anatis,the development of efficient vaccine and the function research of OmpW gene.2.Expression and antigenicity identification of Gallibacterium anatis OmpWIn this study,with p ET-28a-OmpW plasmid constructed by the restrict enzyme ligation of prokaryotic expression vector p ET-28 a and OmpW gene segment of Gallibacterium anatis without signal peptide obtained by PCR amplification,transformed into BL21 and inducted with IPTG,recombinant OmpW was successfully expressed.Polyclonal antisera against purified recombinant OmpW was produced in rabbits.Immunogenicity of recombinant OmpW was preliminarily identificated by Western blotting.The results showed that recombinant OmpW possessed the satisfied immunogenicity and polyclonal antisera and different Gallibacterium anatis had immunological reactions,which not only indicate OmpW had the cross-protective potential as vaccine,but also suggested that the establishment of the serological detection method based on recombinant OmpW was possible.3.Construction of a OmpW deletion mutant in Gallibacterium anatis YuPDS-RZ-1-SLGIn this experiment,two specific DNA fragments upstream and downstream of OmpW gene of Gallibacterium anatis PDS-RZ-1-SLG were amplified respectively,and then cloned into p MD-18 T vector to obtain the recominant plasmid p SX.A chloramphenicol resistance cassette was insert into recominant plasmid p SX to generate recominant plasmid p SCX.Linear DNA fragment,SCX,was produced by PCR amplification using the special primer,and transformation with linear DNA.The chloramphenicol resistance cassette replaced a part of the OmpW gene through homologous recombination.Finally,with PCR and Western blotting identification,a OmpW deletion mutant(?OmpW)with chloramphenicol resistance was generated,witch not only laid the foundation for the functional study of Gallibacterium anatis OmpW,but also provided a reliable method for other gene functional study of Gallibacterium anatis.