Cloning and Sequencing of Gene Encoding Outer Membrane Protein LipL32 of Leptospira interrogans Vaccinal Serovar Canicola

Background and objective Leptospirosis is one of the most important zoonoses with worldwide distribution. LipL32 is the major leptospiral outer membrane lipoprotein expressed during leptospiral infections. Antigenic characterization of the members of the species Leptospira interrogans is a necessary step towards to understanding the interactions between leptospires and the immune system. The aim of this study was Cloning and Sequencing of Gene Encoding Outer Membrane Protein LipL32 of Leptospira interrogans vaccinal serovar Canicola Leptospira interrogans serovar Canicola (LC-RTCC2805) was used in this study which obtained from the Leptospira Reference Laboratory, Razi Vaccine and Serum Research Institute, Karaj, Iran. The bacteria were subcultured into the selective culture medium EMJH. The genomic DNA was extracted by standard Phenol-Chlorophorm method. The specific primers for proliferation of lipl32 gene were designed. The lipl32 gene was amplified and cloned into a cloning vector plasmid and transformed in competent E. coli Top10 cells. Recombinant plasmid was isolated from cells by kit. PCR amplification of the lipl32 gene using the designed primers resulted in an 835bp lipl32 gene product. The amplified gene was cloned in pJET1.2/ blunt vector and transformed into E.coli (Top10) cells. The confirmation of the recombinants was made by picking the white colonies and carrying out colony PCR amplification of the gene. The sequence was deposited in the GenBank database.The percentage identity and divergence among different leptospiral serovars was deduced using the Blast programme.DNA sequence analysis revealed that serovar Canicola (LCRTC 2805) was most closely related to Leptospira interrogans, serovar Canicola (accession No: AB094434 and DQ092412) in GenBank with (99.6% idenditity). The serovars Canicola (accession No: AY763509) was more distantly related (99.4% idenditity). The results showed that the lipl32 gene was highly conserved among pathogenic Leptospira serovars (>90% idenditity). In conclusion, the protein expressed by lipl32 gene may be used in diagnostic methods like ELISA and also can be a good candidate for recombinant vaccine against leptospirosis.