乳铁蛋白肽基因工程菌(E.col-ipED-LfcinB BL21)的发酵与诱导表达条件优化

Optimization for the Fermentation Medium and Expression Conditions of the Engineered Bacteria(E. coli-pED-LfcinB BL21) Encoding Lactoferricin

乳铁蛋白肽是乳铁蛋白在酸性条件下经胃蛋白酶裂解,从N端释放的具有两亲性的阳离子多肽,具有广谱抗菌等诸多生理功能。目前,乳铁蛋白肽主要是通过水解乳铁蛋白来获得,成本昂贵,转基因技术是大规模生产乳铁蛋白肽的最佳途径,试验比较了常用碳源和氮源对已构建的基因工程菌的菌体生物量及蛋白表达量的影响,并采用正交试验对工程菌的发酵与诱导表达条件进行优化。结果表明:可溶性淀粉和牛肉膏可以提高菌体生物量,并且具有较高的融合蛋白表达量,所以分别采用淀粉和牛肉膏作为发酵培养基的碳源和氮源。正交试验结果表明可溶性淀粉7 g/L、牛肉膏30 g/L、氯化钠6.7 g/L,发酵温度37℃、诱导时间8h和转速200 r/min时可得到较高的菌体生物量和融合蛋白表达量。

Lactoferricin is an amphipathic,cationic peptide with stronger anti-microbial activity than lactoferrin.It was mainly generated by the pepsin-mediated digestion from N-terminal of lactoferrin in small scale with high cost,Genetic engineering will be the best method to produce lactoferricin in large scale.We compared the effect of commonly used carbon and nitrogen sources on the biomass yield and expression level,and also optimized the fermentation medium and conditions by using orthogonal test.The result showed that the bacteria biomass and protein expression level increased respectively,when used soluble starch as carbon source and beef extract as nitrogen sources.Orthogonal test showed that the maximum of both biomass and expression level can be obtained with soluble starch 7 g/L,beef extract 30 g/L and NaCl 6.7 g/L,respectively,when the bacteria was induced for 8 h with rotating speed 200 r/min at 37 ℃.