Prokaryotic expression of tlyA gene of Leptospira interrogans strain and the significance of the ex-pressed protein in hemolysis and inflammation / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct a prokaryotic expression system for tlyA gene of Leptospira in-terrogans ( L.interrogans) strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages.Methods The fragment of tlyA gene of L.inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR.The PCR product was sequenced after T-A cloning.A prokary-otic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E.coli BL21DE3 strain as the host strain.The expression of rTlyA protein was detected by SDS-PAGE.Ni-NTA af-finity chromatography was performed for purification.The lytic activity of the rTlyA protein on sheep erythro-cytes was determined by plate hemolytic test and spectrophotometry.Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L.interrogans strain Lai.ELISA was per-formed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines ( IL-β, IL-6 and TNF-α) by THP-1 and J774A.1 cells.Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, re-spectively.The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA pro-tein.The rTlyA protein at the concentration of 10μg/ml showed stronger lytic activity on sheep erythrocytes. The transcription levels of tlyA gene (P<0.05) and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L.interrogans strain Lai for 1 to 8 hours.The rTlyA pro-tein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-αby THP-1 and J774A.1 cells (P<0.05).Conclusion The TlyA protein, encoded by the tlyA gene of L.interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α.This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis.