将尖吻蝮蛇毒酸性磷酯酶 A2 ( A.a APLA2 )基因克隆至温敏表达载体 p BLMVL2 ,在大肠杆菌 RR1中成功诱导表达 .表达产物 A.a APLA2 约占细菌蛋白质总量 2 5 % ,并以包涵体形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用 FPLC Superose TM1 2纯化 ,产物经过 SDS- PAGE检测只有单一条带 .对纯化后的表达 A.a APLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,A.a APLA2 的酶活性处于变性后复性江浙蝮蛇酸性磷脂酶 A2 和碱性磷脂酶A2 的酶活性之间 ,没有抑制血小板聚集活性 ,只有微弱溶血活性 .最后对 PLA2 的结构与这些活性的关系进行了讨论
Abstract
A cDNA encoding an acidic phospholipase A 2Ⅱ (A.aAPLA 2Ⅱ) from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies.After partial purification by washing the inclusion bodies with Triton X 100,denaturing and refolding,the renatured recombinant protein was purified by FPLC column superose TM 12. The enzymatic activity of the expressed A.aAPLA 2Ⅱ was between those of denatured refolded native acidic phospholipase A 2 and basic phospholipase A 2 from Agkistrodon halys pallas.A.aAPLA 2Ⅱ had the rarely hemolytic activity,and lacked inhibiting effect on platelet aggregation.The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A 2 are discussed.
关键词
尖吻蝮蛇酸性磷脂酶A_2Ⅱ /
表达 /
活性测定 /
结构 /
功能
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Key words
A.aAPLA 2Ⅱ /
Expression /
Activity assay /
Structure
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参考文献
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