牛乳铁蛋白功能片段在大肠埃希菌中的表达及其抑菌活性验证  

Lactoferrin functional fragments in Escherichia coli and its activity verification

作　　者：戴珊 赵燕[1] 刘芬 滕慧 张学文[1] DAI Shan;ZHAO Yan;LIU Fen;TENG Hui;ZHANG Xuewen(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha,Hunan 410128,China)

机构地区：[1]湖南农业大学生物科学技术学院,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2024年第5期38-43,共6页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：山东大益生物科技有限公司项目(xczx-2023133)。

摘　　要：以pET-30a和pEGX为出发载体,构建牛乳铁蛋白功能片段(BlfFf)基因工程表达的非融合性表达质粒(pET-BlfFf)和融合性表达质粒(pEGX-BlfFf),并在大肠埃希菌BL21中诱导了蛋白表达。SDS-PAGE分析结果表明,pET-BlfFf表达的目标蛋白主要以包涵体形式存在,而pEGX-BlfFf表达的目标蛋白则可大量溶于细胞破碎上清液中,说明后者更适合表达可溶性目标蛋白。pEGX-BlfFf诱导表达产物经GST标签填料柱亲和层析纯化后,可制备出可溶性融合蛋白GST-BlfFf,其产量约为60 mg/L。该蛋白经凝血酶去除GST标签,获得纯化的目标肽。BlfFf的生物活性试验结果表明,经过胃蛋白酶水解后的多肽对金黄色葡萄球菌和大肠埃希菌均具有抗菌活性。Using pET-30a and pEGX as starting vectors,the non-fusion expression plasmids pET-BlfFf and the fusion expression plasmids pEGX-BlfFf were constructed for preparation of bovine lactoferrin functional fragment(BlfFf)and the protein expression was induced in Escherichia coli BL21.SDS-PAGE analysis showed that the target protein expressed from pET-BlfFf is mainly in the form of inclusion bodies,while the target protein expressed with pEGX-BlfFf could be dissolved in a large number of cell-shredding supernatants.This indicates that the pEGX-BlfFf is more applicable for soluble BlfFf production.The pEGX-BlfFf expression product was purified by GST label affinity column chromatography,and the soluble protein GST-BlfFf was purified.The yield was about 60 mg/L.The target protein BlfFf was finally purified after remove its GST by treated with thrombin.The bioactivity analysis of BlfFf showed that the peptides from BlfFf hydrolyzed by pepsin shows antibacterial activity against both Staphylococus aureus and Escherichia coli.

关 键 词：牛乳铁蛋白功能片段 大肠埃希菌表达 蛋白纯化 抑菌活性 

分 类 号：Q786[生物学—分子生物学]