关键词: 抗脂多糖因子;大肠杆菌;发酵条件;优化

Abstract:

Anti-lipopolysaccharide factor (ALF), as an important antimicrobial peptide, has strong ability and wide spectrum of antimicrobial activity. An ALF gene isolated from Eriocheir sinensis was cloned in pET32a and transformed into Escherichia coli. In this study, in order to improve the soluble expression of Trx-ALF in Escherichia coli and keep its antimicrobial activity, several fermentation factors were evaluated with one-factor-at-a-time design. The highest level of ALF expression was obtained in the medium containing 2.5 g/L sucrose, 10 g/L yeast extract, 2.4 g/L (NH4)2SO4, 12.8 g/L Na2HPO4, 3 g/L KH2PO4, 10.5 g/L NaCl, 2 mmol/L MgSO4, 0.1 mmol/L CaCl2 with pH 6.5. The optimal culture conditions were as follows: a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 33℃ and a post-induction time of 5 h. The recombinant fusion protein showed an antimicrobial activity against Micrococcus luteus.

Key words: anti-lipopolysaccharide factor, Escherichia coli, ferment condition, optimization