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

摘要：

抗脂多糖因子(anti-lipopolysaccharide factor, ALF)作为一种重要的抗菌肽,具有抗菌性强,抗菌谱广的特点。将中华绒螯蟹(Eriocheir sinensis)抗脂多糖因子基因ALF克隆至pET32a载体获得原核表达载体。为了提高ALF融合蛋白在大肠杆菌中的可溶性表达量,保持抑菌活性,对已构建的重组大肠杆菌的发酵条件进行了优化,通过单因素分析的方法,考察了五种不同的培养基对表达量的影响,并在LB+M9培养基的基础上对培养基各组分进行了选择和优化,确定最佳培养基组分为:2.5 g/L蔗糖,10 g/L酵母膏,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溶液。分析了多种因素对表达量的影响,结果表明,培养基pH调至6.5,在菌体OD600达到0.8时加入诱导剂IPTG至终浓度为0.5 mmol/L,33℃继续诱导5 h,蛋白表达量最高。表达的融合蛋白可以抑制藤黄微球菌(Micrococcus luteus)在平板上的生长。

关键词: 抗脂多糖因子;大肠杆菌;发酵条件;优化