基于易错PCR的菌丝霉素的定向进化  

Directed Evolution of Plectasin by Error-prone PCR

作　　者：张琪[1] 胡又佳[1] 陈习平 谢丽萍[1] 

机构地区：[1]中国医药工业研究总院上海医药工业研究院,上海200437

出　　处：《中国医药工业杂志》2014年第6期525-530,共6页Chinese Journal of Pharmaceuticals

基　　金：国家"重大新药创制"科技重大专项(2011ZX09203-001-06)

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。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 amplification 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 identification 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 β-fold,near to the center of active site,which made it easier for the specific binding to lipid Ⅱ.The mutant peptide M-1 was then purificated.The results of antimicrobial activity study showed that the activity of mutant peptide M-1 was 2 times ofplectasin.

关 键 词：菌丝霉素 易错PCR 体外进化 抑菌活性 

分 类 号：Q78[生物学—分子生物学]