Construction Of Tolc Gene Mutant Strain Of Gallibacterium Anatis And Analysis Of Biological Characteristics

Gallibacterium anatis(G.anatis)is a representative species belonging to Gallibacterium and settled in the respiratory and reproductive tract of poultry.It is an important reason of oophoritis,salpingitis,peritonitis,decline in egg production and increased mortality Due to the prevalence of multiple drug resistance and its own antigen diversity,the traditional antibacterial therapy and vaccine prevention effect is not ideal.G.anatis infection causes heavy economic loss and severely affects animal welfare and overall productivity by poultry industries At present,the research on virulence factors and pathogenic mechanism of G.anatis has become a hot topic at home and abroad.TolC protein is a kind of outer membrane channel protein widely existing in Gram-negative bacteria.It has been identified as an important virulence factor in many pathogenic bacteria.Analyzing the biological function of TolC homologous protein in G.anatis is of great significance to the study of its pathogenic mechanism.This study first analyzed the distribution of tolC genes in each strain of G.anatis.We used G.anatis Yu-PDS-RZ-1-SLG(RZ)as the research material to predicte and analyze the localization,structure and B cell antigen epitope of TolC homologous protein,then we analyzed its immunogcnicity.Finally,we constructed the tolC gene deletion strain(RZ?tolC)and explored its biological characteristics The main research contents were as follows1.The amplification of tolC gene and bioinformatic analysis of G.anatisRefering to the genome information of G.anatis UMN179 from GenBank to obtain the tolC gene and a pair of universal primer was designed.Obtain the tolC gene of 28 strains G.anatis isolated from different areas by PCR amplification and sequencing,then its existence state was analysised.The Yu-PDS-RZ-1-SLG was used as the research object to analyze the amino acid sequence,physicochemical properties,signal peptides and domains of TolC homologous protein of G.anatis,and predict the B cell antigen epitope.The results showed that tolC gene is widely existing in G.anatis and has high conservation.The TolC protein of G.anatis was located in the cell membrane by its N terminal signal peptides with a a spiral structure.TolC protein is a long barrel structure formed by three monomers,and its amino acid sequence has only individual amino acid mutations.Each isolate has the same B cell antigen epitope and they may have cross immunogenicity.This study lays the foundation for the establishment of G.anatis detection methods,the development of new vaccines,and the study of biological functions of TolC homologous proteins2.Expression and antigenicity identification of G.anatisThe tolC gene fragment(signal-free peptide)was amplified by PCR and connected to the prokaryotic expression plasmid to construct the prokaryotic expression vector.After transforming BL21(DE3),the recombinant bacteria successfully induced by IPTG and TolC homologous protein expressed efficiently.After purification,we immuned rabbit and obtained polyclonal antiserum against TolC homologous protein,and its immunogenicity was preliminarily identified by western blotting3.The construction and biological characteristics of tolC deletion mutant in G.anatis Yu-PDS-RZ-1-SLGReferring to the genomic information of the UMN179 in the GenBank,the tolC and its upstream and downstream genes were amplified and sequenced,and the upstream and downstream homologous arm primers were designed according to the sequencing results.The upstream homologous arm,downstream homologous arm and kanamycin resistant box were connected into the pMD18-T vector respectively to construct recombinant plasmid pMD18-S-K-X.The specific primers were designed to amplify S-K-X DNA fragments.Then the S-K-X DNA fragments were successfully transferred into the as-prepared competence G.anatis by natural transformation.TolC gene deletion strain RZ?tolC was obtained by kanamycin resistance screening.The the growth characteristics,biofilm formation ability,MIC and adhesion ability of chicken primary oviduct epithelial cells of RZ and RZ?tolC were determined.The results showed that the logarithmic phase was delayed and the growth rate of RZ?tolC was slower than RZ in toto The biofilm formation ability of RZ?tolC was attenuated remarkably compared with that of RZ The MIC value of RZ?tolC for fosfomycin decreased by 32 times,kanamycin increased by 16 times,and neomycin increased by 2 times,doxycycline decreased by 4 times.The construction of G.anatis RZ?tolC and analysis of biological characteristics laid a foundation for exploring TolC function and clarifying pathogenic mechanisms and drug resistance-related mechanism of G anatis.