重组人胰岛新生相关蛋白的高水平表达与纯化  

High Level Expression and Purification of Human Islet Neogenesis Associated Protein

作　　者：沙建平[1] 薛耀明[1] 陈炫[2] 朱永红[3] 李时升[4] 王晓勤[3] 卓凤婷[1] 曾展军[1] 

机构地区：[1]南方医科大学附属南方医院内分泌科,广州510515 [2]暨南大学第一附属医院临床实验中心,广州510630 [3]重庆市生物制药工程技术中心第三军医大学临床微生物学及免疫学教研室,重庆400038 [4]第三军医大学附属西南医院麻醉科,重庆400038

出　　处：《中国生物工程杂志》2007年第11期1-5,共5页China Biotechnology

基　　金：国家"十五""863"计划资助项目(2001AA215161)

摘　　要：通过RT-PCR体外扩增目的基因胰岛新生相关蛋白(isletneogenesis associated protein,INGAP),并将其克隆入原核表达载体pET22b(+),在大肠杆菌BL21(DE3)中诱导表达。表达的目标蛋白主要以包涵体形式存在,洗涤杂蛋白后用尿素溶解,经Heparin Agrose亲合柱层析分离后,再用Superdex75凝胶过滤层析进一步纯化。纯化后的INGAP皮下注射免疫家兔,制备兔抗INGAP血清,采用免疫双扩、ELISA评价INGAP的免疫活性。结果显示INGAP表达量高达菌体总蛋白的40%左右,经HPLC测定,分离纯化后的目标蛋白纯度达到98.81%,且具有良好的免疫原性。The gene of human Islet neogenesis associated protein （INGAP）was amplified with RT-PCR and cloned into prokaryotic expression vector pET22b （ ＋ ）. INGAP was expressed in the E. coli BL21 （DE3） and purified by affinity chromatography and gel filtration chromatography. The inclusion bodies of the expressed protein were extracted and dissolved in 8mol/L. The heparin Agrose affinity chromatography was used to separated the desired protein,and the further purified protein was obtained by the Superdex75 the gel filtration chromatography. The purified INGAP protein immune the rabbits by injection, and the polyclonal antibody against INGAP protein was prepared. The immunological activity of expressed and purified LexA protein was detected by ELISA , and Western blot. The result showed that the INGAP was accounted for about 40 % of the total bacteria protein. The final purity of the INGAP was 98. 81%, which was determined by the HPLC. The expressed and purified LexA protein had satisfactory immunological activity,which was confirmed by immunodiffusion and ELISA.

关 键 词：胰岛新生相关蛋白 表达 纯化 免疫原性 

分 类 号：Q786[生物学—分子生物学]