Aeropyrum pernix K_1磷脂酶A_2的克隆、表达及其性质  

Cloning,Expression and Characterization of Aeropyrum pernix K_1 phospholipase A_2

作　　者：卢冬梅 吴英松 解桂秋[2] 高仁钧[2] 王智[2] 曹淑桂[2] 冯雁[2] 

机构地区：[1]广州市达瑞抗体工程技术有限公司,广州510665 [2]吉林大学分子酶学工程教育部重点实验室,长春130023

出　　处：《微生物学通报》2006年第5期35-38,共4页Microbiology China

摘　　要：从超嗜热需氧古细菌Aeropyrum pernix K1中抽提出染色体基因组，经PCR扩增得到磷脂酶A2基因，用带有His—tag标记的pET15b作为表达载体，在大肠杆菌BLP中成功地诱导表达。表达产物经过Ni-螯合柱一步得到纯化。SDS—PAGE检测只有一条带，其准确分子量为17，871kD。对纯化后的磷脂酶A2测定其酶活性和生物活性，得出其最适反应温度为90％，最适pH范围为7．8-8．2。至此首次成功地在大肠杆菌中表达了古细菌嗜热磷脂酶A2，这将为以后对该酶的结构和功能以及耐热机制研究打下很好的基础，同时有利于古细菌研究领域的扩展。The sequence of the genome of Aeropyrum pernix K1 was completed and the open reading frame was found. The PCR amplified gene was inserted in pET15b with His-tag mark and expressed in the host E. coli BLP. The recomblned phosphollpase A2 was purified by Ni-chelation chromatography with only a one-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrepho-resis （ SDS-PAGE ） of this enzyme was 17. 871 kD. It showed an optimal activity at 90℃ and a pH of 7.8 ～ 8.2. This is the first successful expression of recombinant PLA2 from archaeon. This study will facilitate the further study of structure-function relationship of PLA2.

关 键 词：AEROPYRUM PERNIX K1 磷脂酶A2 古细菌 热稳定性 

分 类 号：Q78[生物学—分子生物学]