根据Magainin-Ⅱ和Japonicin-Ⅱ的基因序列,用一个铰链结构ser-ser-gly-ser-glyser相连,结合巴斯德毕赤酵母(Pichia pas-toris)偏爱密码子设计出杂合抗菌肽基因MgJ。通过重叠聚合酶链反应(ovedapping PCR)法合成基因片段,再将基因按正确的阅读框架定向克隆至巴斯德毕赤酵母的高效表达载体pPICZα A上。经酶切鉴定及序列分析,所转化的大肠杆菌DH5α菌落中含有插入MgJ基因的重组质粒pPICZα A-MgJ,结果表明,成功构建了杂合抗菌肽MgJ基因真核表达载/ISpPICZαA-MgJ。
According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide MgJ was designed based on the sequence of Magain- in- Ⅱ and Japonicin- Ⅱ, which were synthesized by overlapping PCR and connected by a hinge structure ser-ser-gly-ser-gly-ser. Recombinant gene was cloned into pPICZα A to construct the eukaryotic expression vector pPICZαA-MgJ. By using restriction enzyme and sequence analysis, it can be proved that the pPICZαA-MgJ was transformed into Escherichia coli DH5α. All the tested clones contain MgJ gene, pPICZαA-MgJ was successfully constructed.