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

Manju Soman,Mangattuparambil Mini,R Ambily,Nirmal Chacko,Binu K Mani,Jobin Thomas,Rinsha Balan,Siju Joseph,T G Sumithra

doi:10.14202/vetworld.2018.557-561

Abstract

AimThis 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 MethodsA 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.ResultsThe 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.ConclusionThe 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.

Highlights

Epidemics of leptospirosis continue to occur in tropical developing countries, due to poor standards of sanitation and hygiene that leave people and animal at risk to the disease [1]
The polymerase chain reaction (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
This study aims at cloning of a highly immunodominant region of the ligA gene of Leptospira in the pET32 vector - E. coli DH5α system followed by sequencing and phylogenetic analysis of the partial ligA gene

Summary

Introduction

Epidemics of leptospirosis continue to occur in tropical developing countries, due to poor standards of sanitation and hygiene that leave people and animal at risk to the disease [1]. Leptospirosis is caused by a Spirochaete of the genus, Leptospira, which comprises about 22 genomospecies, further divided into about 300 antigenically different serovars [2,3]. 12 new species of Leptospira have been identified [4]. Available whole-cell inactivated vaccines provide only serovar-specific, short-term. 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

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