Cloning, Expression And Experimental Immunity Research Of Porin Gene Of Riemerella Anatipestifer Outer Membrane

The infection of Riemerella anatipestifer was a contagious disease of domestic ducks, geese, turkeys, other poultry and wild birds caused by Riemerella anatipestifer and can lead to high incidence, death rate and slow growth of2-3weeks old ducklings. It has become one of the most serious bacterial infectious diseases for duck industry and caused serious economic losses to the duck industry for its distribution in most countries. The most effective control method of RA infection was injection of inactivated vaccine with strains isolated from endemic area. But as many as21kinds of serotypes and lack of cross-protection between serotypes, application of inactivated vaccine was limited. This problem can be solved by finding a useful subunit component of cross protection for each serotype RA strains. According to study data show subunit ingredients have been reported such as OmpA, pN45, Camp and Vap etc with development value and no ideal protective effect. So screening subunit composition with protective effect for each serotype is still the focus of study. Porins were generally major outer membrane protein composition of Gram-negative bacteria and existing research shows it highly conserved among strains can be used for detection and bacterial antigenic components can provide cross-protection for each type strain.Study for outer membrane porin of RA was carried out for the first time. Porin gene of1222bp was successfully amplified by specific primers designed in serotype1,2,4,6,10,11,13,14and17of RA strains and sequence alignment analysis showed homology between8serotypes RA and RA-JL-1、RA-JL-2and RA-CH-2(CP004020.1)、RA-GD (CP002562.1)、DSM15868(CP002346.1)×ATCC11845(CP003388.1) reported in Genbank were100%. Only some base sequence of RA-CH-1(CP003787.1) exist difference and homology was93.8%. The porin homology of RA with other bacteria of Flavobacterium was lower than70%by blast analysis. Result indicated that the gene has a very high conservatisim and homology can be used for the identification of the RA strains better than16S rDNA and OmpA. Specific primers according to a highly conserved357bp sequence of porin gene were designed and PCR method for detection was established. The PCR method was proven to have good specificity, sensitivity and100%coincidence with bacteriological identification. The PCR method can be used for rapid detection RA infection. Secondary structure, signal peptide, transmembrane region and tertiary structure model of Porin were predicted by biological analysis software. Result showed Porin was a barrel protein mainly constituted by β-pleated sheet and the transmembrane region was formed by integration of (3-pleated sheet with cellular membrane. Only one a-helix was the signal peptide of Porin. The antigenic analysis by DNASTAR software showed14potential B cell antigen epitops and15potential T cell antigen recognition sites in Porin.Prokaryotic expression plasmids pET28a-porin were constructed and induced for expression. Result showed that molecular size of expressed Porin was approximately48.5ku and soluble analysis showed the protein was expressed with form of insoluble inclusion bodies. Expressed products were purified and analysed by Western blot. The analysis showed specific immune response occued between expressed protein and positive serum of serotype1,2,10,11and17RA strains whole cell. This indicated Porin has a good immunological cross reaction. Eukaryotic pVAX1-porin recombinant expression plasmid was constructed on this basis and then transfected into Marc-145cells with liposomes method. Porin expression in eukaryotic cells were identified by RT-PCR and indirect immunofluorescence method from gene transcription and protein level.BALB/c mice were immunized with eukaryotic expression plasmid pVAXl-porin and the results showed pVAX1-porin eukaryotic expression plasmid can induce specific antibody of mice and was significantly higher than pVAXl empty vector and saline control group (P<0.05). This indicated that Porin of RA was able to induce specific humoral immune response in mice. The MTS assay results showed significantly proliferation of mice spleen lymphocytes immunized with pVAX1-porin eukaryotic expression plasmid stimulated by RA antigen and simultaneous detection of IL-2and IL-4levels in serum were significantly elevated, compared with pVAXl empty vector and saline group(P<0.05). Result indicated that Porin of RA can induce cellular immune responses of mice.Preliminary study on porin of RA confirmed that the gene existed in the RA strains of each serotype with a high degree of homology and conservation and can be used for the identification of RA strains. Porin eukaryotic expression plasmid induced humoral and cellular immune responses of mice and indicated the Porin has a good immunogenicity. The findings laid theoretical foundation for the development of subsequent RA genetic engineering vaccine.