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Optimization of Expression Conditions of Hybrid Antimicrobial Peptide LPCB in Pichia pastoris
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- Author:
- No author available
- Journal Title:
- Chinese Journal of Applied & Environmental Biology
- Issue:
- 1
- DOI:
- 10.3724/SP.J.1145.2012.00065
- Key Word:
- 杂合抗菌肽;Lfcin-Pro-CecropinB;毕赤酵母;表达条件优化
Abstract： 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-Lfin-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,ψ)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.
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