Vet World Vol.11 April-2018 Article-23
Research Article
Veterinary World, 11(4): 557-561
https://doi.org/10.14202/vetworld.2018.557-561
Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a - Escherichia coli DH5α system
2. Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, India.
3. Unidad de Sanidad y Biotecnologia (SaBio), Instituto de Investigacion en Recursos Cinegeticos (IREC), Universidad de Castilla-La Mancha (UCLM), Ronda de Toledo s/n 13071, Ciudad Real, Spain.
4. Department of Animal Husbandry, Veterinary Dispensary Balanthode, Panathady, Kasargod, Kerala, India.
5. ICAR-Central Marine Fisheries Research Institute, Post Box No. 1603, Ernakulam North, Cochin - 682 018, Kerala, India.
Background and Aim: This study aims at cloning, sequencing, and phylogenetic analysis of a partial CDS of ligA gene in pET-32a - Escherichia coli DH5α system, with the objective of identifying the conserved nature of the ligA gene in the genus Leptospira.
Materials and Methods: A partial CDS (nucleotide 1873 to nucleotide 3363) of the ligA gene was amplified from genomic DNA of Leptospira interrogans serovar Canicola by polymerase chain reaction (PCR). The PCR-amplified DNA was cloned into pET-32a vector and transformed into competent E. coli DH5α bacterial cells. The partial ligA gene insert was sequenced and the nucleotide sequences obtained were aligned with the published ligA gene sequences of other Leptospira 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 ligA gene and the partial ligA gene could be successfully cloned in E. coli DH5α cells. The nucleotide sequence when analyzed for homology with the reported gene sequences of other Leptospira serovars was found to have 100% homology to the 1910 bp to 3320 bp sequence of ligA gene of L. interrogans strain Kito serogroup Canicola. The predicted protein consisted of 470 aminoacids. Phylogenetic analysis revealed that the ligA gene was conserved in L. interrogans species.
Conclusion: The partial ligA gene could be successfully cloned and sequenced from E. coli DH5α cells. The sequence showed 100% homology to the published ligA gene sequences. The phylogenetic analysis revealed the conserved nature of the ligA 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. Keywords: cloning, Escherichia coli DH5α, Leptospira, ligA, pET-32a, phylogenetic tree.
Keywords: cloning, Escherichia coli DH5α, Leptospira, ligA, pET-32a, phylogenetic tree.
How to cite this article: Soman M, Mini M, Joseph S, Thomas J, Chacko N, Sumithra TG, Ambily R, Mani BK, Balan R (2018) Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET 32a - Escherichia coli DH5a system, Veterinary World, 11(4): 557-561.
Received: 31-12-2017 Accepted: 31-03-2018 Published online: 30-04-2018
Corresponding author: Manju Soman E-mail: manjuso1993@gmail.com
DOI: 10.14202/vetworld.2018.557-561
Copyright: Soman, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.