杂合抗菌肽LPCB在毕赤酵母中的表达条件优化

为获取能表达具有天然活性的杂合抗菌肽的重组酵母菌,确定其最佳表达条件,将构建好的重组酵母表达载体pPICZα-A-CecropinB(1～10)-Magainin2(1～12)(简称pPICZα-A-CBMA)和pPICZα-A-Lfcin-Pro-CecropinB(简称pPICZα-A-LPCB)经SacⅠ线性化处理,分别电转入巴斯德毕赤酵母Pichia pastoris X-33,利用抗生素Zeocin筛选阳性克隆,PCR扩增验证,甲醇诱导表达.采用抑菌圈法初步测定产物活性,优化杂合肽LPCB诱导时间、诱导剂浓度、诱导温度、培养基pH等表达条件,传代检测其质粒稳定性.结果显示,基因工程菌P.pastoris X-33/pPICZα-A-CBMA和P.pastoris X-33/pPICZα-A-LPCB成功构建.表达产物前者抑菌活性微弱,后者抑菌活性良好.杂合肽LPCB的最佳表达条件为:25℃,pH中性培养,每24 h添加0.5%(V/V,φ)甲醇,诱导表达72 h.重组酵母菌pPICZα-A-LPCB在培养超过100代后,未发生质粒丢失.

Optimization of Expression Conditions of Hybrid Antimicrobial Peptide LPCB in Pichia pastoris

To construct recombinant yeast strains which can produce hybrid antimicrobial peptides with natural activity and determine the optimum conditions for expression,the SacⅠ-linearized recombinant expression vectors pPICZα-A-CecropinB(1~10)-Magainin2(1~12)(pPICZα-A-CBMA) and pPICZα-A-Lfcin-Pro-CecropinB(pPICZα-A-LPCB) were transformed into Pichia pastoris X-33 by electroporation,respectively.The positive clone strains were screened with Zeocin and identified by PCR.The protein expression was induced by methanol and the antibacterial activity was tested through antibacterial experiments.Furthermore,the expression conditions of hybrid peptide LPCB were optimized and the stability of plasmid was tested by culture.The results showed that the genetic engineering strains P.pastoris X-33/pPICZα-A-CBMA and P.pastoris X-33/pPICZα-A-LPCB were successfully constructed.The expression product of the former exhibited little antimicrobial activity,while the latter exhibited relatively stronger activity.And the optimum expression conditions of LPCB were as follows: Temperature 25 ?C,pH neutral,adding 0.5%(V/V,φ) methanol to culture every 24 h,induced expression time 72 h.P.pastoris X-33/pPICZα-A-LPCB was cultured for 100 generations and no plasmid was lost.