Cloning, Phylogenetic Analysis and Expression of Recombinant LipL41, Loa22 and LipL21 Proteins from Leptospira interrogans

摘要

Leptospirosis, a worldwide anthropozoonotic infection with multisystemic involvement, is emerging in North India. These days, focus is to develop recombinant outer membrane protein based diagnostic tests. In this study, three genes lipL41 (1088 bp), loa22 (608bp) and lipL21 (581bp) of Leptospira interrogans were cloned and sequenced. Multiple sequence alignment and phylogenetic analysis revealed that loa22 and HpL21gene sequences of L. interrogans serovars Grippotyphosa and Canicola respectively wereconserved in nature but lipL41 gene sequence of L. interrogans serovars Grippotyphosa showed variation in nucleotide sequence which contributes to serovar evolution within species. For protein expression truncated lipL41 (1028 bp), loa22 (548bp) and lipL21 (472bp) genes were amplified, cloned and expressed in prokaryotic expression system and His-tagged ~45kDa (HpL41 gene), ~28kDa (loa22 gene) and ~17kDa (HpL21 gene) proteins were purified by nickle-nitriloacetic acid (Ni-NTA) affinity chromatography.Purified proteins were confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. For immunological characterization, total four doses of recombinant proteins were injected subcutaneously into Swiss-albino mice at 50mug quantity along with Freund's adjuvant and after 21 days immunogenicity of expressed proteins was tested by blotting using mice raised serum.