抗菌肽Magainin基因的克隆及其在Pichia pastoris中的表达

用化学合成法合成了以酵母偏爱密码子编码的抗菌肽magainin基因片段 ,合成片段拼接后 ,与pUC19重组 ,获得magainin基因 .Magainin基因与酵母表达载体pPIC3重组 ,构建胞内表达载体pPIC3 Mag ,电击法转化GS115宿主菌 ,经表型筛选和PCR鉴定证实了目的基因已稳定整合入Pichiapastoris酵母基因组中 .阳性克隆用甲醇诱导表达 ,用免疫印迹法确定了产物的正确性 .利用琼脂孔穴扩散法和液相测定法证明了重组magainin具有抗菌活性

Cloning and Expression of Antibacterial Peptide Magainin in Pichia pastoris

A 97 bp DNA fragment encoding antibacterial peptide magainin was designed based on the amino acid sequence of magainin and the biased codon usage of yeast. Two partially overlapping magainin gene fragments were chemically synthesized, linked and cloned into pUC19 vector. After restriction enzyme analysis and DNA sequencing, the magainin gene was ligated with yeast expression vector pPIC3 and transformed into the GS115 yeast strain by the electroporation.The positive clones,of which the chromosomes were integrated with magainin gene,were identified by the phenotype and PCR.They were induced by methanol and the expression product was examined by dot blot.Results of agarose diffusion assay and liquid phase analysis indicated that the recombinant magainin exhibited the antibacterial activity.