Abstract：The aim of this study is to clone a flagellar-associated fliN gene of Leptospira interrogans, and to construct its prokaryotic expression system so as to identify the immunogenicity of the expressed products. The genomic DNA was extracted from Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai strain Lai. By using high fidelity PCR, the entire fliN gene was amplified for sequencing after T-A cloning. A prokaryotic expression system of fliN gene was constructed by using routine method. IPTG was applied to induce the target recombinant protein rFliN expression and SDS-PAGE plus Bio-Rad Agarose Image Analyser was performed to determine the output of rFliN. Then the rFliN was extracted and purified by Ni-NTA affinity chromatography. Western blot assays with rabbit antiserum against the whole cell of L.interrogans serogroup Icterohaemorrhagiae strain Lai was used to identify rFliN and its immunoreactivity. According to the sequencing results, the similarity of both the nucleotide and putative amino acid sequences of the cloned fliN gene was 100% compared to the reported corresponding sequences. The rFliN output expressed by the prokaryotic expression system pET32a-fliN-E.coliBL21DE3 was approximate 30% of the total bacterial proteins. The purified rFliN only showed a single fragment in the gel. The recombinant protein rFliN was able to combine with the antiserum. All these results lead us to have a conclusion that a prokaryotic expression system with high efficiency of L.interrogans fliN gene is successfully constructed in this study and the recombinant protein rFliN shows a fine immunoreactivity, which lay a foundation for further study of pathogenic and immunoprotective effects of fliN.
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