【摘要】 根据无指盘臭蛙(Odorrana grahami)皮肤抗菌肽Odorgrin A的氨基酸序列,合成了以酵母偏爱密码子编码的Odorgrin A基因片段.目的片段从合成质粒上用XhoΙ和EcoRΙ双酶切下后,与经同样限制酶酶切的pPIC9K载体连接而成表达载体pPIC9K-Odo A.PCR扩增、酶切及测序检测的结果表明表达载体构建成功.线性化的pPIC9K-OdoA经电击法转化毕赤酵母(Pichia pastoris)GS115宿主菌,营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测的结果表明,表达载体pPIC9K-Odo A成功地转化并整合入酵母基因组.用甲醇对具遗传霉素G418高抗性的Odorgrin A重组酵母菌进行诱导表达,取酵母发酵液上清经SDS-PAGE电泳检测,结果初步表明,整合进酵母基因组的抗菌肽Odorgrin A基因已获得分泌表达,表达产物相对分子质量为3×103～4×103,与理论值相近.图10参17更多还原

【Abstract】 A DNA fragment based on the amino acid sequence of Odorgrin A,antimicrobial peptide from skin of Odorrana grahami,was designed using yeast biased codons and synthesized chemically.The DNA fragment and vector pPIC9K were firstly digested by Xho Ι and EcoR Ι respectively,then ligated together.It was testified that the ligation product,expression vector named pPIC9K-Odo A,was successfully constructed based on PCR,enzymatic digestion,and DNA sequencing.The linearized pPIC9K-Odo A was transformed into Pichia pastoris GS115 strain by electroporation and homologously integrated with P.pastoris genome,which was proved by auxotrophic screening,geneticin resistant screening,PCR,and DNA sequencing.The positive colony with the highest geneticin resistance was induced by methanol.The SDS-PAGE analysis on the induced product detected a band of 3×103～4×103.It preliminarily indicated that the Odorgrin A had been successfully expressed in P.pastoris.Fig 10,Ref 17更多还原