碳酸酐酶Ⅱ基因克隆及在毕赤酵母中的异源表达

摘要

Carbonic anhydrase Ⅱ (CA Ⅱ ) is an important enzyme involving in various metabolic activities and pathological activity. A gene coding for carbonic anhydraseⅡwas cloned from human placenta and an expression plasmid was constructed with plasmid pPIC9K and introduced into a strain of Pichia. pastoris GS115 by electro-transformation to obtain an engineered strain. The recombinant CA Ⅱ was heterologously overexpressed in the engineered strain GS115 by induction with methanol and purified by anion-exchange chromatography. The purified recombinant CA Ⅱ was verified to have very similar characteristics to a commercial CA Ⅱ through Western blotting,enzyme activity analysis and circular dichroism spectra. This work can lay a foundation for the clinical research and industrial application of CAⅡin the future.%碳酸酐酶Ⅱ（carbonic anhydrase Ⅱ, CA Ⅱ）是参与机体多种代谢活动和病理活动的一种重要催化酶。通过基因重组、电转化等方式，得到能异源高表达人 CA Ⅱ的毕赤酵母（Pichia. pastoris）GS115工程菌。对工程菌进行培养及甲醇诱导后，经离子交换层析柱分离纯化后得到较纯的重组 CA Ⅱ。通过 Western blotting、酶活力及圆二色谱检测发现重组和商品化 CA Ⅱ具有相似的酶活力和构象，为 CA Ⅱ今后进一步在临床的研究和工业应用打下了基础。