天蚕素多肽分子对大肠埃希菌的抑制作用

Inhibition of Escherichia coli growth by cecropin A

目的体外重组表达家蝇天蚕素成熟肽分子,观察其对临床分离的多重耐药大肠埃希菌(E.coli)体外抑制作用。方法 PCR克隆天蚕素成熟肽(Mature Cecropin,MC)基因,在MC基因前加入色氨酸密码子(TGG)连接于pET32a质粒的硫氧还蛋白基因后,构建重组质粒pET32a-MC。转化E.coli/BL21(DE3)中。IPTG诱导重组融合蛋白质Thioredoxin(硫氧还蛋白,Trx)-MC表达。融合蛋白质纯化后,经胃蛋白酶37℃水解。融合蛋白质中的天蚕素氨基酸序列具有抗胃蛋白酶能力不被水解,融合蛋白质其余部分被胃蛋白酶水解成小于2kD的碎片,使用透析袋分离、纯化、浓缩,获得天蚕素成熟肽分子。水解产物经Tricine-SDS-PAGE验证,获得一条与天蚕素分子大小相近的条带。采用液态生长抑制试验检验天蚕素分子对E.coli抑制作用,观察其对标准菌株ATCC 25922及临床分离多重耐药菌株的抑制活性。结果 MC成熟肽分子体外对E.coli标准菌株的抑制活性较强,标准菌株较多重耐药菌株受抑制得更快,但两者均能被天蚕素分子抑制。结论天蚕素多肽分子对E.coli多重耐药菌株具有抑制作用。

The aim of this study is to express cecropin A from the Musca domestica and identify its activity in vitro. Housefly cecropin mature peptide gene was cloned to construct pET32a mc plasmid. The plasmid pET32a-mc was then trans- formed into BL21 （DE3） to express the recombinant protein after the induction of IPTG. The TrxMC fusion protein was pu rifled and the TrxMC fusion protein was digested by pepsin. After that the antibacterial activity of MC was detected by liquid growth inhibition. Results showed that the MC molecule had the same activity in vitro on standard strains and multiple drug resistant strains with E. coli, which concluded that MC molecules had inhibitory on multiple drug-resistant （MDR） E. coil strain and could be as an alternative to traditional antibiotics.