| Actinobacillus pleuropneumoniae is the pathogen of porcine pleuropneumonia, which causes serious economic losses in pig production worldwide. To date,15 serovars of A. pleuropneumoniae have been identified. The pathogenesis of A. pleuropneumoniae infection is associated with several virulence factors including but not limited to exotoxins, other factors like capsular polysaccharides, lipopolysaccharides, adhesion factors, proteases, outer membrane proteins and transcriptional regulators were also reported to be involved in the pathogenesis.60 putative lipoproteins were predicted by using multiple-selecting standards in the present study. Here, a lipoprotein Lip40, which contains a tandem repeat sequence Q(E/D/P)QPK, was selected for further investigation. The repeat number was different among different strains of A. pleuropneumoniae. Multiple sequence alignments showed that Lip40 was homologous to some known lipoproteins (similarity> 33%). Besides, it exhibited homology with some transferrin-binding proteins (Tbps) from pathogenic bacteria (similarity> 34%). Structure prediction revealed that Lip40 protein consists of an eight-stranded β barrel flanked by a domain made up of four β strands by using SWISS-model method. Superpositions of Lip40 and C lobe/N lobe of TbpB (PDB:3HOL) illustrated that they possess high structural similarity.The lip40 gene without signal peptidase was cloned from A. pleuropneumoniae SLW01, restricted and ligated into pGEX-KG vector. E. coli BL21 (DE3) cells containing pGEX-lip40 was induced by IPTG, so as to obtain recombinant Lip40 (rLip40). Western blotting analysis revealed a hybridization band with a molecular weight of approximate 56 kDa, indicating that antibodies against A. pleuropneumoniae can recognize and bind to the rLip40 protein specifically. Then rabbit hyperimmune serum against rLip40 was prepared. The detection threshold of polyclonal anti-Lip40 antibody was up to a 1:2×105, suggesting that rLip40 could stimulate strong antibody response in rabbit. It also showed that rLip40 is immunoreactive.Though bioinformatics analysis showed that Lip40 is located at the outer membrane, according to the ’+2 rule’, it is needed to be verified by experimental evidence. Subcellular fractions of A. pleuropneumoniae cells were extracted carefully from APP SLW01. A band corresponding to Lip40 (～30 kDa) could be observed localized in the outer membrane fraction in western blotting analysis. Our result confirmed that Lip40 is located in the outer membrane. The present study confirmed that Lip40 was up-regulated both in transcriptional and translational levels by various stress stimulations, suggesting that Lip40 is a muti-stresses responsive factor. Therefore, it was hypothesized that Lip40 might be a potential virulence factor and involved in pathogen-environment and/or pathogen-host interactions. Previous research has reported many lipoproteins and outer membrane proteins have been used in vaccine development. The protective efficacy of Lip40 was then determined using a mouse immunization/infection model. The protection efficacy of Lip40 against virulent challenge of A. pleuropneumoniae was 75%, though a little lower relative to that of ApxIA (100%) and inactivated vaccine (91.7%), it was much more effective than the negative control. Therefore, Lip40 can be used as an effective candidate in A. pleuropneumoniae vaccine research.The lip40 gene deletion mutant of A. pleuropneumoniae strain SLW01, was used as parent strain to generate the complementation strain C△lip40. Biological characterizations of C△lip40 were analyzed. Results revealed that the shuttle vector was stable in C△lip40, and the growth ability of A. pleuropneumoniae was not affected significantly by the shuttle vector. Moreover, it showed SLW01 and △lip40 are highly sensitive to chloramphenicol (Cm) while C△lip40 was more resistant to Cm, compared with SLW01 and C△lip40, indicating that the Cm resistance gene in pJFF224-XN can be stably expressed in the A. pleuropneumoniae. Construction of C△lip40 provides effective material for the analysis of pathogenesis of Lip40. The methods for construction of complementation strain and antibiotic resistance evaluation are established in the present study, which are helpful for further investigation of the biological characteristics and gene function of A. pleuropneumoniae. |
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