尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ ， 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ ， 以包含体的形式存在。纯化包含体后， 将产物变性、复性， 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化， 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ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， 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近， 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论

Expression and Biochemical Characterization of Acidic Phospholipase A 2I from Agkistrodon acutus

A cDNA encoding acidic phospholipase A 2I(A.aAPLA 2I)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 acti vity and platelet aggregation inhibiting effect of the expressed A.aAPLA 2I is close to those of denatured refolded native acidic PLA 2 from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured refolded basic phospholipase A 2 from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A 2 are discussed.