fa Vaccine development, efficacy and safety evaluation Vaccine development, efficacy and safety evaluation پژوهشي Research Avian necrotic enteritis (NE) is a multi-virulence disease caused by the bacterium <em>Clostridium perfringens</em>. 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 <em>in vitro</em> and <em>in vivo</em> evaluation and later to decrease the production costs. Therefore, the Response Surface Methodology (RSM) was used for optimizing <em>E. coli</em> 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 &ordm;C, 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.<br> &nbsp; subunit vaccine, optimization, necrotic enteritis, toxin, RSM method 5 12 http://vacres.pasteur.ac.ir/browse.php?a_code=A-10-125-2&slc_lang=fa&sid=1 Camellia Katalani Katalanik.bio@gmail.com 0000-0001-5033-2263 No Agriculture Science and Natural Resource University (SANRU), Faculty of Agricultural science Ghorbanali Nematzadeh gh.nematzadeh@sanru.ac.ir 0000-0002-1371-0293 Yes Sari Agriculture Science and Natural Resource University (SANRU), Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT) Gholamreza Ahmadian gholamrezaahmadian@yahoo.com 0000-0001-8273-0813 No Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB) Jafar Amani jafar.amani@gmail.com 0000-0002-5155-4738 No Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences5 Ghafar Kiani ghkiani@gmail.com 0000-0001-6547-8142 No Agriculture Science and Natural Resource University (SANRU), Faculty of Agricultural science Parastoo Ehsani p_ehsani@yahoo.com 0000-0003-3592-2008 No Department of Molecular Biology, Pasteur Institute of Iran