Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups

Objective:To determine the frequency of mce gene in Leptospira interrogans,and to investigate the gene transcription levels of L.interrogans before and after infecting cells.Methods:The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning.A prokaryotic expression system of mce gene was constructed;the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay.Rabbits were intradermally immunized with rMce to prepare the antiserum,the titer of antiserum was measured by immunodiffusion test.The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR.Results:mce gene was carried in all tested L.interrogans strains,but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc Ⅰ.The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences(GenBank accession No:NP-712236)were 99.02%～100% and 97.91%～100%,respectively.The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins.The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce.After infecting J774A.1 cells,transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated.Conclusion:The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.