人β防御素3融合蛋白在大肠杆菌中的表达、纯化与活性分析

The Cloning,High Level Expression in Escherichia coli of Human Beta-Defensin 3 and Its Antimicrobial Activity Analysis

近年来由于抗生素的不规范使用等多种原因 ,细菌耐药问题日益严重 ,人们正努力从各个方面来解决 ,其中机体天然产生的肽抗生素 (peptideantibiotics)由于其对耐药菌的强大抗菌作用而受到人们的关注。肽抗生素是一种阳离子小分子多肽 ,在天然免疫和获得性免疫中都发挥着重要作用。防御素 (defensin)是肽抗生素中较为重要的一种 ,主要来源于皮肤、呼吸道等的上皮组织 ,是正常机体抵抗外界病原微生物入侵的重要防线。人 β 防御素 3(humanbeta defensin 3,hβD 3)参与人体免疫屏障 ,并因具有广谱抗菌和抗菌活性不被盐离子浓度抑制等特点而具特别的研究开发价值。提取中国人扁桃体组织总RNA ,以RT PCR技术扩增编码hβD 3成熟肽的cDNA并构建于原核表达载体pQE 80L ,IPTG诱导表达后利用SDS PAGE、免疫印记等方法对重组蛋白进行分析。重组蛋白表达量达到细菌表达总量的 4 0 %。重组蛋白自表达菌包涵体中提取后 ,经亲和层析法纯化目的蛋白达到电泳纯 ,经多步透析法复性 ,在体外抗菌实验中表现出了对金黄色葡萄球菌、多重耐药金黄色葡萄球菌等的抗菌活性 。

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons,. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human β defensin 3, was isolated and demonstrated a salt insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hβD 3 specific cDNA sequence was amplified with RT PCR . After sequenced, the target DNA fragment was cloned into pQE 80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E.coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS PAGE and Western blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hβD 3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.