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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.
The chemically synthesized α-calcitonin gene-related peptide gene was transferred from the pXZ101 expression plasmid to the pUC18 plasmid and sequenced and identified. The gene was cloned into the pGEX expression vector and expressed in E. coli C600. The fusion protein was purified by glutathione affinity chromatography. The fusion protein was excised by thrombin. After purified by SephadexG-50 column, the fusion protein was cleaved with cyanogen bromide and purified by SephadexG-25 column to obtain pure CGRP. The biological activity was confirmed by ELISA and radioimmunoassay; the amino acid determination by end was consistent with the design. Animal living experiments show that the expression of E. coli protein antihypertensive activity.