Article history: Received: 31-12-2017, Accepted: 31-03-2018, Published online: 30-04-2018
Corresponding author: Manju Soman
E-mail: firstname.lastname@example.orgCitation: 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.
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.
1. Evangelista, K.V. and Coburn, J. (2010) Leptospira as an emerging pathogen: A review of its biology, pathogenesis and host immune responses. Future Microbiol., 5: 1413-1425. [Crossref] [PubMed] [PMC]
2. Levett, P.N. (2008) International committee on systematics of prokaryotes. Subcommittee on the taxonomy of leptospiraceae. Int. J. Syst. Evol. Microbiol., 58: 1049-1050. [Crossref]
4. Thibeaux, R., Iraola, G., Ferres, I., Bierque, E., Girault, D., Gilbert, M.E.S., Picardeau, M. and Goarant, C. (2018) Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence, Microb. Genom., 4(1).
6. Sambrook, J. and Russell, J.W. (2001) Molecular Cloning-A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
7. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors (DNA polymerase/nucleotide sequences/bacteriophage 4X174). Proc. Natl. Acad. Sci. U. S. A., 74: 5463-5467. [Crossref] [PubMed] [PMC]
9. McBride, A.J.A., Cerqueira, G.M., Suchard, M.A., Moreira, A.N., Zuerner, R.L., Reis, M.G., Haake, D.A., Ko, A.I. and Dellagostin, O.A. (2009) Genetic diversity of the Leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp., Infect. Genet. Evol., 9: 196-205. [Crossref] [PubMed] [PMC]
10. Palaniappan, R.U.M., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., Mcdonough, S.P., Barr, S.C., Divers, T.J., Simpson, K.W., Mcdonough, P.L. and Mohammed, H.O. (2002) Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans, Infect. Immun, 70: 5924-5930. [Crossref] [PubMed] [PMC]
11. Palaniappan, R.U.M., McDonough, S.P., Divers, T.J., Chen, C.S., Pan, M.J., Matsumoto, M. and Chang, Y.F. (2006) Immunoprotection of recombinant Leptospiral immunoglobulin-like protein against Leptospira interrogans serovar Pomona infection. Infect. Immun., 74: 1745-1750. [Crossref] [PubMed] [PMC]
12. Silva, E.F., Medeirosc, M.A., McBride, A.J.A., Matsunaga, J., Esteves, G.S., Ramos, J.G.R., Santos, C.S., Croda, J., Homma, A., Dellagostin, O.A., Haake, D.A., Reis, M.G. and Ko, A.I. (2007). The terminal portion of Leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine, 14: 6277-6286. [Crossref] [PubMed] [PMC]
13. Hartwig, D.D., Oliveira, T.L., Seixas, F.K., Forster, K.M., Rizzi, C., Hartleben, C.P., McBride, A.J.A. and Dellagostin, O.A. (2010) High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris. Microb. Cell. Fact., 9: 98. [Crossref] [PubMed] [PMC]
14. Sadeghi, M.M., Rabbani, M., Rismani, E., Moazen, F., Khodabakhsh, F., Dormiani, K. and Khazaei, Y. (2011) Optimization of the expression of reteplase in Escherichia coli. Res. Pharm. Sci., 6: 87-92. [PubMed] [PMC]
15. Assaeedi, A.S. and Osman, G.H. (2017) Isolation, cloning, DNA sequencing and bioinformatics analysis of the parasporin-1 gene of Bacillus thuringiensi. J. Proteomics Bioinformatics, 10: 144-151. [Crossref]
16. Dangi, S.K., Yadav, P.K., Tiwari, A. and Nagaleekar, V.K. (2017) Cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of Clostridium chauvoei. Vet World, 10: 1104-1107. [Crossref] [PubMed] [PMC]
17. Thankappan, S., Rana, R., Remesh, A.T., Rekha, V., Nagaleekar, V.K. and Puvvala, B. (2017) Cloning and expression of P67 protein of Mycoplasma leachii. Vet World, 10: 1108-1113. [Crossref] [PubMed] [PMC]
18. Fakruddin, M.D., Mazumdar, R.M., Mannan, K.S.B., Chowdhury, A. and Hossain, M.N. (2013). Critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant E. coli. ISRN Biotech., 2013: 590587. [Crossref] [PubMed] [PMC]19. Mierendorf, R.C., Morris, B.B., Hammer, B. and Novy, R.E. (1998) Expression and purification of recombinant proteins using the pET system. Methods Mol. Med., 13: 257-292. [PubMed]