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Avian necrotic enteritis (NE) is a multi-virulence disease caused by the bacterium Clostridium perfringens. Several toxins of this bacteria are components of different candidate vaccines, and have been considered as important factors in pathogenesis of NE. A fusion subunit protein composed of immunodominant segments of NetB, Alpha-toxin and metallopeptidase proteins (NAM) was designed. The high level production of NE subunit vaccine candidate is important for the in vitro and in vivo evaluation and later to decrease the production costs. Therefore, the Response Surface Methodology (RSM) was used for optimizing E. coli culture condition. To this end, induction conditions including cell optical density prior induction (OD600nm), IPTG concentration, post-induction temperature and time were modified. The statistical analysis revealed that all variables except IPTG had significant effects on production and solubility of rNAM. A 7.39 fold increase in production of soluble rNAM was achieved when the post induction temperature, IPTG concentration, the pre-induction OD and time were 19 ºC, 0.55 mM, 0.8 respectively in 8 h after induction. Our study indicated that the RSM method is a simple and superior strategy for protein expression improvement which is considered as a major limitation in production of vaccine candidate and other recombinant proteins using different hosts.
 

Keywords: subunit vaccine, optimization, necrotic enteritis, toxin, RSM method

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