广西眼镜王蛇毒一种新的酸性磷脂酶A_2的基因克隆和性质

Molecular cloning and characterization of a novel acidic phospholipase A_2 from the venom of Guangxi Ophiophagus hannah(king cobra)

通过SephadexG 75 ,CM SepharoseCL 6B和DEAE SephadexA 5 0连续的柱层析 ,从广西眼镜王蛇 (Ophiophagushannah)蛇毒中分离到一种新的酸性磷脂酶A2 (pI 4 .0 ) ,命名为APLA2 2 .它的分子量约为 14 0 0 0 ,具有较高的磷脂酶活力 (4 2 9mol·g- 1·min- 1) .它对小鼠没有致死性 (LD50 >2 0mg·kg- 1) ,但具有抗血小板聚集功能及诱导水肿形成能力 .通过cDNA测序解决了它的完全的一级结构 .由cDNA推导的APLA2 2蛋白质全序列与福建眼镜王蛇和台湾眼镜王蛇蛇毒酸性磷脂酶A2 的同源性分别为73.9% ,64.7% ,它不存在 62～ 66位的“胰腺环” ,因而它可能是一种新的眼镜王蛇毒酸性磷脂酶A2 .APLA2 2基因克隆及其生化药理性质的研究 ,为探讨蛇毒活性蛋白结构与功能关系及蛇伤中毒机理创造了良好的条件 .

An acidic phospholipase A 2(pI 4.0), designated as APLA 2 2, was isolated and purified from the venom of Guangxi Ophiophagus hannah(king cobra), by successive chromatography on Sephadex G 75, CM Sepharose CL 6B and DEAE Sephadex A 50 columns. Its relative molecular mass was 14 000 as determined by SDS PAGE and it had a high phospholipase A 2 activity(429 mol·g -1 ·min -1 ), using egg yolk phospholipids as substrate. It was non lethal to mice (LD 50 >20 mg·kg -1 ), but exhibited anti platelet aggregation activity and edema inducing ability. Its complete primary structure was solved as deduced from the cDNA sequence. Its protein sequence showed 73.9% homology with Fujian Oph. hannah and 64.7% homology with Taiwan Oph. hannah. It possessed no 'pancreatic loop' at residues 62-66, so it might be a novel acidic phospholipase A 2 from the venom of Oph. hannah. The gene cloning and characterization research of APLA 2 2 would provide a good basis for further discussing the relation of structure and function of PLA 2, and the toxic mechansim of PLA 2 from snake venoms in snake bite.