猪链球菌2型omp40基因的克隆表达及分布调查

omp40 gene was identified using Suppression Subtractive Hybridization(SSH)in our previous study, whereas the role of omp40 was not clear. Thus, the sequence and distribution of omp40 in 31 different SS were analyzed, which contribute to study the pathogenic mechanism of omp40 in SS2. This experiment analyzed the sequence and distribution of omp40 in 31 different SS , which contribute to study the pathogenic mechanism of omp40 in SS2. Based on predicted protein features sharing same conserved domain with the collagen-binding protein Cna of Staphylococcus aureus, omp40 is likely to function as a direct mediator of collagen adhesion. Using polymerase chain reaction(PCR) detect the distribution of the omp40 in S.suis strains from different sources, serotypes, regions. The results showed that the omp40 can only be found in SS2 which were isolated from diseased pigs, which means this gene is related to the virulence.Using polymerase chain reaction amplified omp40.By digestion with restriction enzymes EcoRⅠand XhoⅠ,the omp40 gene were cloned into pET-28a (+) vector to construct recombinant plasmid.The recombinant plasmid was transformed into E.coil BL21(DE3) and induced with IPTG.The result of SDS-PAGE assay showed that the protein was largely expressed.Western blotting analysis showed that the target protein had a good reactivity with the swine ZY05719 strain rehabilitation serum antibody.Using 96 well plates method determine the difference between the wild strain and the omp40 gene deleted strain in adhesion of collagen, found the omp40 protein and animal collagen type I appearing interaction.