Cloning and sequence analysis of a partial CDS of leptospiral gene in pET-32a - DH5α system.

Aim: This study aims at cloning, sequencing, and phylogenetic analysis of a partial CDS of gene in pET-32a - DH5α system, with the objective of identifying the conserved nature of the gene in the genus .

Materials And Methods: A partial CDS (nucleotide 1873 to nucleotide 3363) of the gene was amplified from genomic DNA of serovar Canicola by polymerase chain reaction (PCR). The PCR-amplified DNA was cloned into pET-32a vector and transformed into competent DH5α bacterial cells. The partial gene insert was sequenced and the nucleotide sequences obtained were aligned with the published gene sequences of other serovars, using nucleotide BLAST, NCBI. Phylogenetic analysis of the gene sequence was done by maximum likelihood method using Mega 6.06 software.

Results: The PCR could amplify the 1491 nucleotide sequence spanning from nucleotide 1873 to nucleotide 3363 of the gene and the partial gene could be successfully cloned in DH5α cells. The nucleotide sequence when analyzed for homology with the reported gene sequences of other serovars was found to have 100% homology to the 1910 bp to 3320 bp sequence of gene of strain Kito serogroup Canicola. The predicted protein consisted of 470 aminoacids. Phylogenetic analysis revealed that the gene was conserved in species.

Conclusion: The partial gene could be successfully cloned and sequenced from DH5α cells. The sequence showed 100% homology to the published gene sequences. The phylogenetic analysis revealed the conserved nature of the gene. Further studies on the expression and immunogenicity of the partial LigA protein need to be carried out to determine its competence as a subunit vaccine candidate.