Cloning, expressing and purification of recombinant C-terminal PMT of Pasteurella multocida
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Abstract
Pasteurella multocida strains produce heat-stable toxin Pasteurella Multocida Toxin (PMT) which is the main virulence factor causing pasteurellosis in pigs. Therefore, PMT protein is considered as a promising candidate to study recombinant vaccine against pasteurellosis in pigs. In this study, we cloned the nucleotide sequence coding C-terminal fragment of PMT (tPMT-780), from acid amine 506 to acid amine 1285, into pRSET-A vector and expressed it on Escherichia coli BL21 bacteria. The SDS-PAGE result showed that tPMT-C780 was well-expressed by IPTG 1mM induction and determined to be insoluble protein, therefore it was purified by denaturation method using Ni-NTA beads. The protein elution samples contained only one protein band similar with the size of tPMT-C780 and positively reacted with PMT-specific antibody. These results suggested that we were successful in expressing and purifying C-terminal protein of PMT toxin, tPMT-C780. This is the primary material for further studies to produce recombinant vaccine against pasteurellosis in pigs.
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References
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