Cloning and Expression of Brucella Cyclic ?-1, 2 Glucan Transporter Gene (cgt)

Abstract

Background: Brucellosis is an important cosmopolitan infection disease caused by organisms belonging to the genus Brucella. The cgt gene (cyclic ?-1, 2 glucan transporter gene) is a virulent factor in Brucella genus. The present study was conducted with the aim of cloning and expression of Brucella cgt gene. Materials and methods: Brucella melitensis cgt gene was amplified from extracted chromosomal DNA by PCR, then : PCR product was cloned into pTZ57R and subcloned into pGEMEX-1 expression vector, then expressed in JM109 E.coli strain. Recombinant protein was confirmed by western blot analysis using patient's serum. Results: The PCR product was cloned in pTZ57R plasmid via T/A cloning method. Recombinant plasmid was digested by BamHI and SacI restriction enzymes, the released band was purified and subcloned into pGEMEX-1 expression vector. Then, sample cells were lysed using lyses buffer and sonicated, then electrophoresed on SDS-PAGE. Protein bands were transferred on nitrocellulose membrane and reacted by patient's serum and detected by HRP conjugated anti : human antibody. Conclusion: We cloned and expressed Brucella abortus cyclic -1, 2-glucan transporter gene (cgt) which is an important agent in brucellosis. Using cgt gene mutant may be an effective way for inhibiting or decreasing the pathogenicity of bacteria