【摘要】 目的构建天蚕素A-死亡素复合基因工程抗菌肽基因CA(1-7)-T(4-19),并在毕赤酵母中表达。方法采用重叠区基因扩增法(SOE)人工合成复合肽基因,同载体pPICZαA连接后转化毕赤酵母受体菌X-33,通过博莱霉素抗性筛选得到的阳性克隆用甲醇诱导表达,并对产物进行抗菌活性检测,建立抗菌谱。结果复合肽基因CA(1-7)-T(4-19)成功克隆到载体pPICZαA上,鉴定结果与预先设计的基因序列一致,在甲醇诱导下复合肽基因得到表达,并得到临床分离的76株革兰阴性和革兰阳性耐药致病菌的最小抑菌浓度,最小抑菌浓度可达到5μg/mL。结论成功获得了具有抑菌活性的复合基因工程抗菌肽,且该复合肽对临床常见多重耐药菌株均有明显的抑杀作用。更多还原

【Abstract】 Objective To construct cecropin A-thanatin combinant gene engineering antimicrobial peptide gene CA(1-7)-T(4-19)for expression in Pichia pastoris.Methods The combinant antimicrobial peptide gene was artificially synthesized via gene splicing by overlap extension(SOE).The gene was cloned into the pPICZαA vector and transformed into Pichia pastoris X-33 by electroporation.The positive clones obtained by the screening of bleomycin resistance were induced by methanol,and the antibacterial activity of the products was detected and the antimicrobial spectrum was established.Results The combinant peptide gene CA(1-7)-T(4-19)was successfully cloned on the carrier pPICZαA.The identification results were consistent with the predesigned gene sequence.The combinant peptide gene was expressed under the induction of methanol,and the minimum inhibitory concentration of 76 strains of Gram-egative and Gram-positive pathogenic bacteria isolated from the clinic was obtained,and the minimum inhibitory concentration was up to 5μg/mL.Conclusion A combinant genetic engineering antimicrobial peptide with antibacterial activity was obtained successfully and it had obvious inhibition effect on clinical common multidrug-resistant strains.更多还原