重组鸡抗菌肽Gallinacin-9的原核表达及其抗菌活性的鉴定  

Expression of Recombinant Gallinacin-9 from Chicken in E.coli and Determination of Its Antimicrobial Activity

作　　者：韩宗玺[1] 马得莹[2] 刘胜旺[1] 李一经[3] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所生物技术国家重点实验室禽传染病研究室,哈尔滨150001 [2]东北农业大学动物营养研究所,哈尔滨150030 [3]东北农业大学动物医学院,哈尔滨150030

出　　处：《畜牧兽医学报》2008年第10期1426-1431,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30600435);NOVUS国际科研基金;黑龙江省教育厅重点项目(1153LZ05)

摘　　要：采用RT-PCR方法,从鸡舌组织中扩增到Gallinacin-9(Gal-9)基因,测序表明Gal-9为201 bp,其成熟肽由67个氨基酸残基组成。进一步将克隆的Gal-9基因亚克隆到大肠杆菌原核表达载体pGEX-6p-1的EcoRⅠ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-Gal-9,经序列分析鉴定确证目的基因克隆入载体的预期位点。将重组质粒转化大肠杆菌BL21,于37℃诱导培养不同时间,SDS-PAGE电泳表明该基因在大肠杆菌中高水平表达,表达的重组鸡Gal-9融合蛋白相对分子质量约为32 ku。重组蛋白占菌体总蛋白的40%。表达的重组鸡Gal-9融合蛋白以包涵体的形式存在。重组蛋白经纯化后,分别以对数生长中期的大肠杆菌BL21(DE3-)株与致病性链球菌CAB株为检测菌,利用薄层平皿琼脂糖孔穴扩散法测定了重组Gal-9蛋白的抗菌活性,结果表明,重组Gal-9对这2种细菌都具有抗菌活性。Gallinacin(Gal)-9 was cloned from tongue of chicks by RT-PCR and sequenced.The full length cDNA of Gal-9 consists of 201 bp,encoding 67 amino acids.The cDNA of chicken Gal-9 gene was sub-cloned into EcoR Ⅰ and Sal Ⅰ sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-Gal-9.The recombinant plasmid was transfected into E.coli BL21 and the transformed bacteria was induced with IPTG.It was demonstrated by SDS-PAGE that a 32 kDa protein which was equal to chicken Gal-9 protein in molecular weight was expressed in E.coli BL21.The recombinant fusion protein was purified.The recombinant Gal-9 was expressed as inclusion bodies.The recombinant Gal-9 exhibit expected anti-E.coli and anti-Pathogenic streptococcus activity,as measured by inhibition zone assaying.

关 键 词：Gallinacins-9 鸡 融合蛋白 抗菌活性 

分 类 号：S852.42[农业科学—基础兽医学] Q78[农业科学—兽医学]