鸡β-防御-素8 cDNA在大肠杆菌中的融合表达  

Fusion expression of gallinacin-8 cDNA in Escherichia coli

作　　者：杨涛[1] 钟瑾[2] 涂健[1] 彭开松[1] 祁克宗[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国科学院微生物研究所,北京100101

出　　处：《中国兽医科学》2009年第11期1018-1022,共5页Chinese Veterinary Science

基　　金：国家"十一五"高技术研究发展计划(863)项目(2006AA10Z320);国家自然科学基金项目(30800811;30871851);高等学校省级优秀青年人才基金项目(2009SQRZ036)

摘　　要：为了评价鸡β-防御素的功能与基因工程化生产的可能性,从已构建的重组载体pGEM-T Easy-gal-8中克隆gal-8成熟肽cDNA,将该cDNA插入原核表达载体pGEX-6P-1中,构建表达质粒pGEX-6P-1-gal-8,然后将重组质粒转化到大肠杆菌BL21(DE3)中。挑取阳性克隆菌用IPTG进行诱导表达,SDS-PAGE电泳结果检测显示,融合表达的GST-gal-8蛋白大小约30 ku,经灰度扫描显示该融合蛋白的表达量占细菌总蛋白的119 mg/L。用固定化的谷胱甘肽亲和层析柱进行蛋白纯化,融合蛋白经Prescission蛋白酶酶切并纯化获得了完整的gal-8成熟多肽。抑菌活性试验显示,表达的gal-8成熟肽对黄色微球菌有一定的抑菌活性。To evaluate the function and feasibility of manufacturing chicken β defensin by genetic engineering,the mature cDNA of chicken β defensins gal-8 was amplified from the plasmid pGEM-T Easy-gal-8,and then cloned into prokaryotic expression vector pGEX-6P-1.The recombinant vector pGEX-6P-1-gal-8 was transformed into the competent cell Escherichia coli BL21（DE3）.The positive clones were cultured and induced to express target protein by IPTG.Fusion expression products were approximately 30ku in molecular weight,and examined by SDS-PAGE.Densitometric scanning analysis demonstrated that the fusion protein accounted for about 119mg/L of the total bacterial proteins.The expressed fusion protein was purified with immobilized glutathione affinity chromatography column,and a complete sophisticated gal-8 polypeptide was cut from fusion protein by the Prescission protease.Gal-8 mature peptide possessed the antimicrobial activity to Microccus flavus NCIB 8166 in the assay of drug susceptibility by agar diffusion method.

关 键 词：β-防御素-8 融合表达 Prescission蛋白酶 gal-8成熟肽 

分 类 号：S963.732[农业科学—水产养殖]