化学合成的α-降钙素基因相关肽基因在大肠杆菌中的融合表达及产物分离纯化

Expression and Purification of a Biosynthetic α-CGRP Gene in E. coli

化学法合成的α－降钙素基因相关肽基因从pXZ101表达质粒转移到pUC18质粒中测序鉴定后，克隆到pGEX表达载体上，在大肠杆菌C600中表达．融合蛋白经谷胱甘肽亲和层析初步纯化，用凝血酶切去融合蛋白，经SephadexG－50柱纯化后，用溴化氰裂解，再经SephadexG－25柱纯化后得到纯品CGRP．经ELISA反应及放射免疫鉴定具有生物活性；经末端氨基酸测定与设计的一致．动物活体实验证明，大肠杆菌中表达的蛋白有降压活性．

A biochemically synthesized α-calcitonin gene related peptide (CGRP) gene was cut down from plasmid pXZ101, and ligated to pUC18 to be sequenced. The CGRP gene was then cloned into an expression vector, pGEX-4T-2, and expressed in an E. coli strain C600.The fusion protein was seperated by glutathione affinity chromatography, then cut off most of it with thrombin, and finally purified by Sephadex G-50 chromatography. The pre-CGRP was lysed with BrCN and re-purified by Sephadex G-25 chromatography. Mature CGRP was then acquired. Its biological activity was detected by ELISA and immunoradioactive reactions. Its N-terminal amino acid was identical to the authentic CGRP as would be expected.The animal assay demonstrated that it had the same biological activity as the authentic CGRP.