尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A2 (A .aBPLA2 )基因克隆至温敏表达载体 pBLMVL2 ,在大肠杆菌RR1中成功诱导表达 .表达产物A .aBPLA2 约占细菌蛋白质总量的 2 0 % ,并以包涵体的形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用FPLCSuperoseTM12纯化 ,产物经过SDS 聚丙烯酰胺凝胶电泳检测只有单一条带 .对纯化后的表达A .aBPLA2 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 ,表达A .aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2 酶活性相近 ,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2 的溶血活性 ,没有抑制血小板聚集活性 .最后对磷脂酶A2 的结构与这些活性的关系进行了讨论

Expression and Biochemical Characterization of A Basic Phospholipase A2 from Agkistrodon acutus

A cDNA encoding a basic phospholipase A 2 ( A.a BPLA 2) from Agkistrodon acutus was inserted into a bacterial expression vector pBLMVL2 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.a BPLA 2 is close to those of denatured refolded native acidic phospholipase A 2 from Agkistrodon halys Pallas, A.a BPLA 2 has the same hemolytic activity as denatured refolded basic phospholipase A 2 from Agkistrodon halys Pallas, but its inhibiting effect on platelet aggregation is negligible. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A 2 are discussed.