Cloning and sequencing of protein L-isoaspartyl O-methyl transferase of Salmonella Typhimurium isolated from poultry

17. Cloning and sequencing of protein L-isoaspartyl O-methyl transferase of Salmonella Typhimurium isolated from poultry - S. K. Dixit, D. P. Hota, M. Kumawat, T. K. Goswami and M. Mahawar

Veterinary World, 7(9): 712-716

   doi: 10.14202/vetworld.2014.712-716

S. K. Dixit: Immunology Section, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

sunildixit1987@gmail.com

D. P. Hota: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India; durgaprasad.hota04@gmail.com

M. Kumawat: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India; 0711mworld@gmail.com

T. K. Goswami: Immunology Section, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India; goswami.tapas@gmail.com

M. Mahawar: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India; manishbiochemistry@gmail.com

Received: 16-05-2014, Revised: 28-07-2014, Accepted: 04-08-2014, Published online: 25-09-2014

Corresponding author: S. K. Dixit, e-mail: sunildixit1987@gmail.com

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Abstract

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Aim: To clone the Salmonella Typhimurium protein L-isoaspartyl O-methyl transferase (PIMT) enzyme and to analyze the sequence withPIMT gene of other pathogenic serovars of Salmonella.

Materials and Methods: Salmonella Typhimurium strain E-2375 was procured from the National Salmonella Center, IVRI. The genomic DNA was isolated from Salmonella Typhimurium. Polymerase chain reaction (PCR) was carried out to amplify PIMT gene using the designed primers. The PCR product was cloned into pET28c plasmid vector and transformed into Escherichia coli DH5α cells. The plasmid was isolated from E. coli and was sequenced. The sequence was analyzed and submitted in Genbank.

Results: The PCR product revealed a distinct amplicon of 627 bp. The clone was confirmed by PCR. Sequencing data revealed 100% homology between PIMT sequences from Salmonella Typhimurium strain E-2375 (used in the current study) and PIMT sequences of standard reported strain (Salmonella Typhimurium str. LT2) in NCBI data base. This submitted sequence in Genbank having accession no. KJ575536.

Conclusions: PIMT gene of Salmonella is highly conserved in most of the pathogenic Salmonella serovars. The PIMT clone can be used to isolate PIMT protein. This PIMT protein will be helpful to identify isoaspartate containing proteins thus can help in study Salmonella virulence.

Keywords: cloning, sequencing, Salmonella Typhimurium protein L-isoaspartyl O-methyl transferase, virulence.