Establishment Of ELISA Method Based On A Recombinant Outer Membrane Protein For Detection Of Actinobacillus Pleuropneumoniae

Porcine Pleuropneumonia (PCP) caused by Actinobcillus pleuropneumoniae (APP) is a highly contagious, fibrinous, hemorrhagic, and necrotizing disease that causes important economic losses in the Swine industry around the worldwide. The virulence of APP is multifactorial, but outer membrane protein (OMP) is considered as one of the most important virulence factors in APP. The research aimed at cloning and expressing the OMP gene by molecular biologic technology. That would be laying a foundation for developing new efficient subunit vaccines or specific diagnosis methods of APP.In this study, culture and biological characteristics of APP-1 were analysed. Study on violence and immunogenicity of APP-1 in mice showed that the LD₅₀ were 1.36×10^5.5CFU. Antisera detected by agglutination test against the isolates after two weeks of the first immunity were 4log2; the titer reached 6log2 after the second immunity. 5/6 mice were protected against the corresponding isolates challenge after the first immunity; otherwise, 6/6 mice were protected after the second immunity.According to the sequence of OMP gene in GenBank, a pair of primers was designed with DNAStar software to amplify OMP gene of APP-1 strain by PCR. The product of PCR named APP-1-OMP is approximately 1 319bp in length was amplified from the genomic DNA. The APP-1-OMP gene was cloned into the vector pGEM-T-easy and the recombinant plasmid was named pGEM-T-OMP. The gene comprised a complete open reading frame which is 1 319bp in length and encoding a functional protein consists of 439 Amino acids. Homology analysis indicated that the identity levels of deduced animo acid sequences of OMPs among APP-1 strain, 3 serotypes, 4 serotypes, and 7 serotypes were among 97.9-98.1%. Plasmids containing the right insertion were sequenced to confirm their identity and transformed the combination into E. coli BL21 strain. Bacterial lyses prepared from 1mmol/L IPTG induced cultures were loaded directly onto SDS-PAGE. Upon induction, the recombinant pET15b-OMP produced indeed a new protein with an apparent MW of 48.766KD. The fusion protein was immune response with convalescent sera by Western-blotting analysis.Indirect ELISA was estabished by coating the 96-well plates with purified OMP. Analysis of field sera of pigs collected from different farms revealed that OMP-ELISA was specific and sensitive when compared with other diagnosis methods, which primarily might be used for the diagnosis of porcine contagious pleuropneumonia.