重组牛乳铁素融合表达质粒的构建及其抑菌活性分析

为建立生物合成牛乳铁素的方法，根据牛乳铁素氨基酸序列确定其编码序列，利用PCR 技术获得基因扩增产物，接着将该产物与编码鱼腥蓝细菌Anabaena sp. PCC 7120 的DNA 聚合酶基因dnaENI 中断裂的内含肽基因Asp dnaEIn 的PCR 产物进行重组PCR，将获得的重组融合基因再克隆到表达载体pET-28a 构建重组融合表达质粒。结果显示，融合表达质粒转化大肠杆菌后经诱导能在大肠杆菌表达宿主Escherichia.coli BL21(DE3) 中大量表达，且细胞裂解液与纯化的Anabaena sp. PCC 7120中含断裂内含肽Asp DnaEIc的DNA聚合酶蛋白DnaECI混合反应后对金黄色葡萄球菌具有抑制作用。

Prokaryotic Expression and Antibiotic Activity of Recombinant Bovine Lactoferricin Fusion Protein

In order to establish a bovine lactoferricin biosynthesis method, the bovine lactoferricin-encoding gene was determined on the basis of amino acid sequence. A recombinant fusion gene was obtained by means of overlapping PCR following the purification of the separate PCR products of the bovine lactoferricin-encoding sequence and the split intein Asp dnaEIn gene from the DNA polymerase gene dnaENI of Anabaena sp. PCC 7120, and the obtained fusion gene was subsequently cloned into the expression plasmid pET-28a to construct a recombinant fusion expression plasmid. The results showed that recombinant fusion proteins were over-expressed after the expression host Escherichia coli BL21 (DE3) containing the recombinant bovine lactoferricin fusion expression plasmid was induced and that reaction products of over-expressed recombinant bovine lactoferricin lysates and the purified Anabaena sp. PCC 7120 DnaECI, which contained split intein Asp DnaEIc, could inhibit Staphylococcus aureus.