The Study Of Immune Protection And Construction Of Mutant Strain Of Outer Membrane Protein A Of Gallibacterium Anatis

Gallibacterium belongs to a new set of Pasteurella families,including Actinobacillus salpingitis(A.salpingitidis)、 Pasteurella anatis and Avian Pasteurella haemolytica-like(Avian P.haemolytica-like),and Gallibacterium anatis(G.anatis)is the type species,blonging to gram-negative bacteria.The study found that G.anatis belonged to the opportunistic pathogens,not only constituted a part of the normal flora of the upper respiratory tract as well as the lower genital tract of chickens,but also had the potential to cause salpingitis and peritonitis in egg-layer,leading to diseases egg production and quality and increased mortality.Because of the existence of multiple drug resistance and antigenic diversity in G.anatis,the prevention and cure effect of antibiotics and vaccines against the disease was becoming more and more unsatisfactory,thus causing huge economic losses to the layer farming.At present,the research on the mechanism and vaccine of G.anatis was very limited,only a few of the predicted virulence factors had been certified,but its pathogenesis had not been elucidated.In this study,part of the property of Gallibacterium anatis outer membrane protein A(Omp A)was researched by methods of PCR and bioinformatics analysis for the first time.Its distribution in G.anatis was studied and possible epitopes were predicted.Polyclonal antibody against purified recombinant Omp A(r Omp A)of G.anatis was produced in rabbit.The immunogenicity was analysed by Western blotting.In order to analysed its immune protection,the mice were immunized with r Omp A.To achieve a better understanding of Omp A gene function,we tried to use the targeted gene manipulation technology to construct gene deletion mutant of G.anatis Omp A.1.Cloning of outer membrane protein A gene of Gallibacterium anatis and analysis of its structure and functionAccording to the nucleotide sequence of omp A of UMN179 published on Gen Bank,a pair of primers were designed to amplify and clone the gene sequence of G.anatis omp A.The amino acid sequence of Omp A was analyzed by bioinformatics software.Physicochemical property,architectural feature and conservative B cell antigen epitopes were predicted by analyzing Omp A amino acid sequence of Yu-PDS-RZ-1-SLG using bioinformatics methods.The result showed that23 strains of G.anatis omp A gene were found,and it was highly conserved;its tertiary structure prediction analysis showed that Omp A was a beta barrel pore structure,a folding piece composed of 8 antiparallel beta;had the same B cell epitopes between different G.anatis,showing that different strains may exist immune cross protection.This study not only laid the foundation for further study of G.anatis omp A gene function,also provided reference for the establishment and development of vaccine G.anatis diagnostic method.2.The identificationof prokaryotic expression,antigenicity and immunogenicity of Gallibacterium anatis Omp AA pair of primers were designed to amplify omp A gene encoding G.anatis Omp A amino acid sequence,and connected to the prokaryotic expression vector p ET-32a(+);successfully constructed the prokaryotic expression vector p ET32a(+)-Omp A and expressed in Escherichia coli BL21;SDS-PAGE analysis showed that the recombinant protein was main in the form of inclusion body;western blotting results showed that the recombinant protein could bind to G.anatis positive serum specificity,and different G.anatis strains Omp A could be combined with rabbit polyclonal antibody of Omp A specificity,which indicated that Omp A had good immunogenicity.3.The study on the immune protection of recombinant outer membrane protein A of Gallibacterium anatisThe purified recombinant protein rOmp A and inactivated bacteria were respectively made into vaccine immunized mice,setting up the control group of PBS at the same time.Totle immunized three times,each immune interval of two weeks.The mice blood samples were obtained after 7days of each immune by docking.The antibody titer were analysed by ELISA.After 14 days of the third immune,all mice were challenged with G.anatis.The result showed that: the antibody level of r Omp A group and inactivated bacteria group were significantly higher than the control group;and their survival rate could reache 50% and 80%,while the PBS group mice all died.These results indicated that Omp A could induce protective antibodies in mice,and lay the foundation for further study on the function of G.anatis Omp A and the prevention and treatment of diseases such as salpingitis and peritonitis.4.The construction mutant strain of Gallibacterium anatis omp A geneAccording to the sequence of UMN179 published on Gen Bank,specific primers were designed to amplify omp A gene upstream and downstream homologous arms,and Kanamycin resistance cassette was selected as a selective marker.Using the genome of G.anatis YU-PDS-RZ-1-SLG as the templet to amplify omp A gene upstream and downstream homologous arms,and cloned into p MD18-T vector to construct the recombinant plasmid p MD18-U-D.The Kanamycin resistance cassette was inserted into the upstream and downstream homologous arms to constuct homologous recombinant plasmid p MD18-U-K-D.The transforming fragment U-K-D obtained by PCR was transformed into G.anatis competent by natural transformation.Breakthrough had been made.