【摘要】 以菌丝霉素成熟肽经大肠杆菌密码子优化后的基因为基础,采用易错PCR技术,使用低保真的Taq酶,调整Mg2+浓度、添加Mn2+,改变PCR扩增程序,获得易错PCR产物。经克隆、转化后,挑取200株突变体进行测序鉴定,并进行抑菌活性筛选,筛得突变体M-1。基因比对结果显示,突变体M-1中有1个碱基发生突变,使得31位氨基酸由丙氨酸(Ala)变成精氨酸(Arg)。蛋白质分子空间结构模拟显示,突变位点位于蛋白质的?折叠上,靠近活性中心,氨基酸残基由疏水性变成极性,推测更有利于与lipidⅡ的特异性结合,增强抑菌活性。本研究还对突变体小肽M-1进行了分离纯化。抑菌活性试验表明,突变体小肽M-1的抑菌活性是菌丝霉素的2倍。更多还原

【Abstract】 The gene coding plectasin mature peptide was optimized according to the E.coli codon usage bias. Error-prone PCR was applied to evolve this peptide by usage of EX Taq DNA polymerase and adding Mn2+ and different concentrations of Mg2+. Meanwhile, the amplifi cation program was changed to improve the mutation rate. The PCR product was cloned and transformed into E.coli and 200 mutants were screened. After sequence identifi cation and antimicrobial activity screening, a mutant M-1 was obtained. It was revealed that there was one amino acid substitution. The 31-alanine was substituted by arginine. According to the simulated protein of mutant M-1 peptide, the mutation site was located at ?-fold, near to the center of active site, which made it easier for the specifi c binding to lipidⅡ. The mutant peptide M-1 was then purifi cated. The results of antimicrobial activity study showed that the activity of mutant peptide M-1 was 2 times of plectasin.更多还原