尖吻蝮蛇毒磷脂酶A_2基因的克隆与序列分析  

Cloning and Sequencing of Genes Encoding Phospholipase A 2 from Agkistrodon acutus

作　　者：刘小龙[1] 潘华[1] 杨冠珍[1] 吴祥甫[1] 周元聪[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《生物化学与生物物理学报》1999年第1期41-45,共5页

摘　　要：从尖吻蝮蛇毒腺中抽提总ＲＮＡ，经ＲＴＰＣＲ扩增磷脂酶Ａ２的基因，以江浙蝮蛇的酸性磷脂酶Ａ２基因为探针杂交筛选克隆，分离得到４种磷脂酶Ａ２基因。经双向测序测定了这些磷脂酶Ａ２同功酶基因的全序列，并由此推导出编码的氨基酸序列。运用计算机软件推算了它们的等电点，按照等电点和结构特征将它们分别命名为尖吻蝮蛇毒酸性磷脂酶Ａ２Ｉ（Ａ．ａＡＰＬＡ２Ｉ）、尖吻蝮蛇毒酸性磷脂酶Ａ２ＩＩ（Ａ．ａＡＰＬＡ２ＩＩ）、尖吻蝮蛇毒碱性磷脂酶Ａ２（Ａ．ａＢＰＬＡ２）和尖吻蝮蛇毒Ｌｙｓ４９磷脂酶Ａ２（Ａ．ａＬｙｓ４９ＰＬＡ２）。其中Ａ．ａＡＰＬＡ２Ｉ的１～１０位氨基酸残基序列同以前分离得到的尖吻蝮蛇酸性磷脂酶Ａ２已测定的１～１０位氨基酸残基序列完全一致。Ａ．ａＬｙｓ４９ＰＬＡ２基因则由于其推导出的４９位氨基酸残基由Ｌｙｓ代替了Ａｓｐ而区别于以前克隆到的磷脂酶Ａ２基因。最后运用计算机软件比较了它们的同源性。这一组磷脂酶Ａ２基因的克隆，将为进一步研究磷脂酶Ａ２的结构与功能的关系提供更多的信息。Synthetic oligonucleotides were used to amplify phospholipase A 2 (PLA 2) gene by RT PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA 2 gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA 2 isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA 2I、A.aAPLA 2II、A.aBPLA 2 and A.aLys 49 PLA 2 according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1-10 residues of A.aAPLA 2I deduced from the cDNA is identical to that of acidic PLA 2 which had been isolated from Agkistrodon acutus . A.aLys 49 PLA 2 is unique because of the usual Asp 49 is replaced by Lys 49 , which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure function relationship of PLA 2 family.

关 键 词：尖吻蝮蛇毒 蛇毒 磷脂酶A2 基因克隆 序列分析 

分 类 号：Q556.1[生物学—生物化学] Q959.62