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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。
According to the preference of Pichia pastoris for the genetic code, the gene fragment of antibacterial peptide idein A was chemically synthesized without changing the amino acid sequence. After the synthetic fragments were spliced, the gene was recombined with the α factor and the recombinant gene was then constructed into the expression vector pPIC9K, we got the yeast expression plasmid controlled by the promoter and transcription termination region of the alcohol oxidase 1 gene (AOX1). After restriction enzyme digestion and nucleotide sequence analysis, the positive recombinant was transformed into the host strain of pastoris GS115, And the positive clones were induced by methanol.The expression products were analyzed by PAGE gel, purified and tested for antibacterial activity. The results showed that the recombinant antimicrobial peptide gene was highly expressed in yeast and the expressed product was secreted by the alpha factor signal peptide , With strong antibacterial activity.