Transcription and expression characteristics of Leptospira virulence-associated protein InvA / 中华微生物学和免疫学杂志

ABSTRACT

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 116.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.