CLONING, SEQUENCING AND EXPRESSION OF IMMUNODOMINANT OUTER MEMBRANE PROTEIN OMP31 FROM BRUCELLA SPP
Loading...
Files
Date
2011
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
GADVASU, Ludhiana
Abstract
Brucellosis is a re-emerging zoonosis with serious implications for both humans and animals. It is caused by members of genus Brucella. In the present study, for rapid detection of Brucella from clinical samples, PCR was carried out by using published primers based on immune-dominant outer membrane protein gene omp31. A new set of primers based on omp31 gene were designed and by using this primer set, it was possible to differentiate Brucella abortus and Brucella melitensis. The amplicons of 513bp and 477bp were amplified by PCR in case of B. abortus and B. melitensis respectively. Subsequently omp31 gene was amplified from a clinical isolate of Brucella abortus and cloned in to TA cloning vector. The cloned product was sequenced and the sequence data obtained (Accession No.JF734338) was analysed. For expression of omp31 gene, pPROExHT b prokaryotic expression vector (Invitrogen, USA) was used. His-tagged recombinant Omp31 protein was successfully expressed which showed a specific band of 34kDa in SDS-PAGE analysis. Expressed recombinant Omp31 protein was successfully purified to homogeneity by Ni-NTA affinity chromatography under denaturing conditions and the purified recombinant protein was confirmed by DOT and Western blotting. The present study is a first step in providing rapid diagnosis and differentiation of Brucella species as well as in development of recombinant antigen based indigenous ELISA