Targeting knock out of Leptospira interrogans flagellum-associated fliN gene and pathogenic function alteration of the mutant / 中华微生物学和免疫学杂志

plasmid can be used to study the pathogenic mechanism of target gene products of L.interrogans.

Prokaryotic expression of Leptospira interrogans FliH/Ⅰ/Y/N genes and determination of the protein locations in leptospiral envelope / 中华微生物学和免疫学杂志

Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.