抗菌肽Cecropin D基因在毕赤酵母中的表达与抗菌活性分析  

Expression of antimicrobial peptide Cecropin D in Pichia pastoris

作　　者：郭春和[1] 焦茂兴[2] 何俊[1] 刘德辉[1] 黄毓茂[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]宜春学院,江西宜春336000

出　　处：《中国预防兽医学报》2011年第11期858-861,886,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省现代农业生猪产业技术体系(F10021)

摘　　要：为使抗菌肽Cecropin D在巴斯德毕赤酵母中高效表达,本研究根据Cecropin D全基因序列和毕赤酵母的偏嗜性设计合成4条寡聚西核苷酸,SOEing-PCR技术法合成Cecropin D基因序列,并克隆至酵母表达载体pGAPZαA中,构建重组表达质粒,将其线性化后电击转化毕赤酵母菌株SMD1168,经高浓度博莱霉素筛选,获得高拷贝转化子,菌液PCR鉴定表明Cecropin D基因已整合到酵母基因组中。在GAP强启动子调控下,重组Cecropin D高效分泌表达,产物耐高温耐酸碱,并对部分革兰氏阳性菌和革兰氏阴性菌均有抑菌效果,对E.coliDH5α和S.aureus Cowan I株的MIC值分别为2.17μg/mL和4.55μg/mL。To highly express antimicrobial peptide Cecropin D in Pichia pastoris,Cecropin D gene was amplified by SOEing PCR with 4 synthesized oligonucleotides and cloned into the pGAPZαA vector.The lineared pGAPZαA-CecropinD was transformed into P.pastoris SMD1168 by electroporation,the transformants were screened by Zeocin,it was proved that Cecropin D gene had intergrated into P.pastoris genome detected by PCR.Cecropin D had highly expressed under control of GAP promoter.The production could endure high temperature,acid and base,and it showed antibacterial activity to both Gram-positive and negative bacteria.The minimum inhibitory concentration（MIC） for E.coli DH5α and S.aureus Cowan I were 2.17 μg/mL and 4.55 μg/mL,respectively.

关 键 词：抗菌肽CECROPIN D 毕赤酵母 抗菌活性 

分 类 号：S852.4[农业科学—基础兽医学]