Genotyping of LipL41 genes from Leptospira interrogans serogroups and immunological identification of the expression products
Objective To determine the existence of LipL41 gene in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogans and 2 international standard strains belonging to 2 serogroups of Leptospira biflexa for understanding physical expression of the gene in the strains, construct prokaryotic expression system of the gene and identify immunity of the expression products. Methods Genomic DNAs from the 17 strains of Leptospira were prepared by routine phenol-chloroform method. Entire LipL41 gene fragments from the strains were amplified by high fidelity PCR. The target amplification products were sequenced for genotyping after T-A cloning and then prokaryotic expression systems of the cloned LipL41 genes were constructed. By using SDS-PAGE, expression of the recombinant proteins (rLipL41) was identified. Western blot assays using rabbit antiserum against genus-specific leptospiral TR/patocⅠ antigen and rabbit anti-rLipL41 sera were applied to determine immunoreactivity and immunogenicity of rLipL41 respectively. Microscopic agglutination test (MAT) was performed to examine across agglutination titers of the rabbit anti-rLipL41 sera and J774A.1 cell adherence model of Leptospira was used to determine blocking effects of anti-rLipL41 sera. Results All strains but 2 of Leptospira biflexa genes, which can be divided into two genotypes LipL41/1 and LipL41/2. Similarities of the nucleotide and putative amino acid sequences of LipL41/1 genotypes from 11 of the strains and LipL41/2 genotypes from 4 of the strains were 88.61%-88.67% and 93.24%-97.18% respectively. Outputs of the rLipL41/1 and rLipL41/2 expressed by the constructed prokaryotic expression systems were 30% and 40% of the total bacterial proteins respectively. Both the rLipL41/1 and rLipL41/2 were able to combine to the TR/patocⅠ antiserum and induce rabbits to produce antibodies. MAT titers of the rabbit anti-rLipL41/1 and anti-rLipL41/2 sera with the 15 strains of Leptospira interrogans were 1∶8-1∶128 and 1∶16-1∶256 dilutions of the antisera could efficiently block adherence of Leptospira to the cells. Conclusion The representatives of 15 Chinese dominant Leptospira interrogans serogroups possessed LipL41/1 or LipL41/2 gene. The prokaryotic expression systems constructed in this study could efficientlyexpressrLipL4 1 1andrLipL4 1 2 ,respectively .rLipL4 1 1andrLipL4 1 2weredemonstratedasthesurfaceprotein antigenswithwellimmunogenicityandantigenicityandwidelyexistedinthedifferentserogroupsofLeptospirainter rogans.