重组抗菌肽基因克隆及其在Pichia pastoris中的表达及鉴定

Cloning and Expression of Antibaterial Peptide Gene in Pichia pastoris and Identifying of Activity

根据毕赤酵母对遗传密码的偏爱性,在不改动氨基酸序列的前提下,用化学法合成抗菌肽麻蝇素A的基因片段,合成片段拼接后,与α因子重组,重组基因再构建到表达载体pPIC9K上,得到受乙醇氧化酶1基因(AOX1)的启动子与转录终止区控制的酵母表达质粒,经限制性酶切鉴定及核苷酸序列分析,阳性重组子转化pastoris GS115宿主菌,经表型筛选,阳性克隆用甲醇诱导表达.表达产物经PAGE凝胶电泳分析、纯化及抗菌活性检测,结果表明,重组抗菌肽基因在酵母中获得高效表达,表达产物经α因子信号肽分泌到胞外,具有较强的抗菌活性。

A 120bp DNA fragment encoding antibacterial peptide of sarcophaga peregrina A was designed based on the amino acid sequence of antibacterial peptide and the biased codon usage of pastoris.Eight oligonucleotides were chemically synthesized,linked and then recombinant with a-factor gene,recombinanted gene was cloned into yeast expression vector pPIC9K.After restriction enzyme analysis and DNA sequencing,the recombinanted gene of antibacterial peptide was txansfected the Pichia pastoris GS115 strain.The positive clones screened by the phenotype were induced by methanol,the expression product was tested by SDS-PAGE and inhibit zone using E.coli SD and E.coli C3000 as tested strain.The results showed that antibacterial peptide gene was expressed in Pichia pastoris successfully.The expression product was secreted outside with the lead of α-factor signal and had strong antibacterial activity.