抗菌肽基因在毕赤酵母中分泌表达及其条件优化  

Expression of the Gene Encoding for the Antibacterial Peptide Cecropin A-melittin Hybrid in Pichia pastoris and Its Optimal Conditions

作　　者：储卫华[1] 李大力[1] 汪信[1] 

机构地区：[1]南京理工大学化工学院生物工程系,南京210094

出　　处：《农业生物技术学报》2006年第6期976-980,共5页Journal of Agricultural Biotechnology

基　　金：江苏省博士后基金(No.2004144);南京理工大学优秀博士基金资助。

摘　　要：用化学合成法合成了以酵母偏爱密码子编码的抗菌肽cecropinA(1-8)-melittin(1-18)基因片段,合成片段与酵母表达载体pPIC9K重组,构建胞外分泌表达载体pPIC9K-came,电击法转化毕赤酵母(Pichiapastoris)SMD1168宿主菌,对CAME抗菌肽重组酵母菌的摇瓶发酵条件进行了优化。结果表明,酵母表达抗菌肽具有抗菌活性且溶血活性显著降低;重组酵母在含2%葡萄糖pH7.0的YPD培养液中,250r/min震荡培养24h后,重组酵母菌的A600为6.0时,经0.5%甲醇在28℃条件下诱导48h能够诱导产生较高抗菌活性的抗菌肽。A 98 bp DNA fragment was designed and synthesized based on the biased codon usage of yeast. Its encoding antibacterial peptide was constructed from residues 1-8 of cecropin A and residues 1-18 of mefittin. The came gene was cloned into plasmids pPIC9K and transformed into Pichia pastoris SMD1168 strain by the electroporation. The positive clones, of which the chromosomes were integrated with came gene were identified by phenotype and antibacterial activity. The results showed that the came gene was expressed in Pichia pastoris successfully, and CAME kept antibacterial activity and decreased the hemolysis activity. The requirements for the flask-shaking culture fermentation of the recombinant Pichia pastoris were optimized. The greater expression of recombinant came could be got when the density of the seed yeast amounted to A600 value 6.0, the pH value of YPD containing 2.0% glucose was 7.0, the yeast was cultured under 28 ℃ for 48 h at the speed of 250 r/min; and induced with 0.5% methanol.

关 键 词：抗菌肽 天蚕素-蜂毒素 摇瓶发酵 抗菌活性 优化 

分 类 号：S188[农业科学—农业基础科学]