Molecular cloning of genes flhA and flhB_2 for flagellar biosynthesis of Leptospira interrogans and functional prediction of the prokaryotic expressing products

摘要

To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemorrhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type Ⅲ secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L.interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.