Cloning And Expression Of OMLA Of Actinobacillus Pleuropneumoniae And Its Immunogenicity

Actinobacillus pleuropneumoniae is the pathogen of porcine pleuropneumoniae, a disease characterized by hemorrhagic,fibrinous and necrotic lung lesions.To date 15 serotypes have been found,a number of potential factors have been identified associated with the virulence of A.pleuropneumoniae.Studies indicate that virulence is strongly correlated with OMP.OMLA is a part of OMP,so the expressed OMLA may be useful for the studies of molecular biological functions of APP OMLA.In the study,the coding sequences of OMLA genes were amplified by the PCR using the specific primers,then the fragments were inserted into pMD18-T simple vector and sequenced.The result of the sequencing indicated that both of the coding sequences of OMLA genes were 1095bp,encoding 365aa.Compared with other sequences,they shared a nucleic acid identity of 73.0～99.9%,and an amino acid identity 62.2～100.0%.The OMLA mature(mOMLA) gene was amplified by PCR,using the coding sequences of OMLA as template,then inserted into pET-32a(+),constructing the expression plasmid of mature OMLA.The positive recombinant plasmid was transformed into E.coli BL21(DE3),and was induced by IPTG to express the mature OMLA.A fusion protein about 60 kD and its dissolubility was identified by SDS-PAGE,Western-blot.The optimal conditions to express mature OMLA were IPTG 0.4 mmol/L,cultured at 37℃for 4h,the comparative content of recombinant mature OMLA was 47.5%.The fusion protein could react with mice serum against mature OMLA,as confirmed by ELISA.In conclusion,the recombinant pET-mOMLA expression vector was obtained and the mature OMLA protein of Actinobacillus pleuropneumoniae has good immunogenicity.Mice were subcutaneously vaccinated with each recombinant protein to evaluate the immunogenicity.ELISA antibody titre of each protein produced in mice was tested and showed a gradual increase after vaccination.Then immunized mice were challenged with A.pleuropneumoniae serotype 1 at a dose of 5×LD₅₀.the results of ELISA showed that the recombinant proteins of OMLA can induce mice to produce special antibody(＞3200),and the recombinant proteins of OMLA has some immunoprotection effect with the challenging of the SC-A strain of APP.