Cloning and molecular characterization of outer membrane protein A (ompA) gene from Mycobacterium bovis.
The ompA gene, 981 bp in size and identified in the Mycobacterium bovis genome, was cloned and sequenced. Predicted amino acid sequences showed a protein of molecular weight 33.5 kDa with an isoelectric point of 7.28 and an OmpA conserved domain at the carboxy terminal with signal sequence at the N-terminal. Homology search showed complete homology with the Mycobacterium tuberculosis ompA gene with varying degree of homology with omp genes of other bacteria, so it was predicted that it may act like porin. The ompA gene of M. bovis was expressed in the pProEX HTb expression vector. Recombinant protein OmpA, was purified by using a nickel affinity column. The expressed recombinant OmpA (32 kDa) was confirmed by western blot with Ni-NTA horseradish peroxidase (HRP) conjugate and with specific antiserum raised in rabbits. The effect of low pH (pH 6.0, 5.5 and 5) on the transcription of the ompA gene in M. bovis using real-time PCR showed that there was an increase in transcription of ompA in M. bovis cultured at low pH (maximum at pH 5.5).
Singh
J P N
JP
Division of Bacteriology and Mycology, Indian Veterinary Research Institue, Izatnagar, Uttar Pradesh, India.
Verma
Rishendra
R
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D009163:Mycobacterium bovis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins