重组人胰岛新生相关蛋白的表达、纯化及免疫活性研究

摘要 目的：对胰岛新生相关蛋白（Islet neogenesis associated protein ,INGAP）进行表达、纯化，并检测其免疫活性。方法： INGAP基因片段插入表达载体pET22b(+)，在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解，Heparin Agrose亲合柱层析为第一步纯化，Superdex75凝胶过滤层析作为第二步精细纯化，HPLC测定INGAP蛋白的浓度，将纯化的INGAP蛋白经注射途径免疫家兔，制备兔抗INGAP血清，采用免疫双扩、ELISA及Western Blot分析INGAP的免疫活性。结果INGAP以包涵体形式表达，表达产量高达总菌体蛋白的40%左右,经Heparin Agrose亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白，经HPLC测定目的蛋白的最终纯度为98.81%，表达及纯化的INGAP具有良好的免疫活性。

The expression with purification and the immunological activity study of recombine islet neogenesis associated protein in human

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.