鸡β-防御素-1基因在大肠杆菌中的融合表达及其初步纯化与抗菌活性测定  

Fusion Expression of Chicken Gallinacin-1(Gal-1) in Escherichia coli and its Purification, Detection of Antibacterial Activity

作　　者：吴静[1,2] 史玉颖[1] 李玉峰[1] 马秀丽[1] 黄兵[1] 宋敏训[1] 

机构地区：[1]山东省农业科学院家禽研究所,济南250023 [2]山东大学生命科学学院,济南250100

出　　处：《家禽科学》2013年第1期6-11,共6页Poultry Science

基　　金：山东省农业科学院高技术自主创新基金(No.2006YCX026);山东省自然科学基金(No.ZR2009DQ018)

摘　　要：防御素是广泛存在于动、植物体内的一大类阳离子抗菌肽,它们在宿主—家禽天然免疫中发挥着重要作用,是机体抵御外界病原微生物入侵的一道重要防线,在鸡体内只存在Gallinacins类β-防御素。鸡β-防御素-1(Gal-1)对多种致病菌都具有杀菌作用,具有很好的药用开发前景。通过PCR技术从重组质粒pMD-Gal1中克隆出Gal-1基因成熟肽编码区,将该基因克隆至原核表达载体pET-30a(+)中,构建重组质粒pET30a-Gal1,将该质粒转化大肠杆菌Rosetta(DE3)菌株。筛选阳性克隆菌株,用浓度为1.0mmol/L的IPTG进行诱导表达。SDS-PAGE分析表明,Gal-1基因在大肠杆菌中以融合形式成功表达,表达产物表观分子量约为12kDa。对融合蛋白表达条件进行优化,提高可溶组分所占比例。表达上清用Ni2+-NTA Sepharose 4B进行亲和层析纯化,在洗脱液中显示单一条带。琼脂糖孔穴扩散法检测显示,表达产物对多种革兰氏阴性菌和阳性菌均具有明显的抑制活性。Gal-1融合蛋白成功表达为进一步研究鸡β-防御素的功能奠定基础。Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimierobial activity against pathogens. Gallinacins（Gal 1-14） are the only subfamily of 13-defensins found in chicken. These peptides exhibit a wide range of antimicrobial activity against both gram-positive and gram-negative bacteria, they have shown potential for development into therapeutic drugs and are worthwhile being further investigated. In this study, the mature peptide coding fragment of Gal-1 gene was amplified from the plasmid of pMD-Gall, and then inserted into the EcoR I and Not I enzyme-cutting sites of pET-3Oa（＋） plasmid. The recombinant vectors named pET3Oa-Gall was transformed into the competent cell of E.coli Rosetta （DE3） strain. The positive clones were cultured and induced to express target protein by addition of 1.0mmol/L IPTG in LB. Expression products with molecular weight of approximately 12kDa were examined by SDS-PAGE and Western Blot, which showed that Gal-I gene was expressed in the form of fusion protein. The expressed fusion proteins were purified by with Ni2 ＋-NTA Sepharose 4B affinity chromatography column. Highly purified proteins were achieved in the eluate. The recombinant Gall was able to kill both Gram negative bacteria and Gram positive bacteria by the means of agar well diffusion assay （AWDA）. Further study should will be continued based on the results of this research.

关 键 词：抗菌肽 β-防御素-1(Gal-1) 融合表达 分离纯化 亲和层析 抑菌活性 

分 类 号：S852.4[农业科学—基础兽医学]