原核表达融合蛋白的亲和层析法纯化

AFFINITY PURIFICATION OF THE TROPIC 1808 GENE FUSION PROTEIN BY PRONUCLEUS EXPRESSION

目的 :以T7·TagAffinityPurificationKit纯化原核表达Tropic 180 8基因编码的融合蛋白 ,并进行免疫印迹检测。方法 :以 0 .4mmol/LIPTG诱导含pET -2 1a -180 8重组质粒的BL2 1(DE3)大肠杆菌 ,T7·TagAffinityPurifica tionKit纯化菌体上清液 ,SDS -PAGE分析 ,将SDS -PAGE蛋白区带转移至NC膜进行免疫印迹检测。结果 :外源性Tropic 180 8基因在大肠杆菌中表达的融合蛋白可经T7·TagAffinityPurificationKit进一步纯化 ,纯化后的蛋白为单一条带 ,分子量约为 32 .0kDa ,其免疫印迹反应结果提示该蛋白为Tropic 180 8基因编码的融合蛋白。结论 :在大肠杆菌中表达的融合蛋白可以免疫亲和法进行纯化和鉴定。

Objective:To purify and detect the fusion proteins in the Escherichia coli with T7·Tag Affinity Purification Kit.Methods:The Escherichia coli BL21(DE3) with pET-21/Tropic 1808 induced by 0.4mmol/L IPTG. Purify the fusion protein with T7·Tag Affinity Purification Kit. After SDS-PAGE, the expression protein was transferred to NC membrane and immunoblotted. The purity of expression protein was examined by the method of immunoblot with T7·Tag Antibody.Results:The fusion protein of pET-21a-1808 in the Escherichia coil can be purified and detected with T7·Tag Antibody. It showed single band with the MW 32.0kDa protein. These results demonstrated that the MW 32.0kDa protein was the Tropic 1808 gene fusion protein.Conclusions:The Tropic 1808 gene fusion protein in the Escherichia coli may be purified and detected by the method of affinity purification.