竹叶青蛇毒丝氨酸蛋白酶的分子克隆和序列比较  

Molecular Cloning and Sequence Comparison of Serine Proteases from the Venom of Trimeresurus stejnegeri

作　　者：李文辉[1] 高荣[1] 张云[1] 

机构地区：[1]中国科学院昆明动物研究所,云南昆明650223

出　　处：《Zoological Research》2003年第3期180-185,共6页动物学研究（英文）

基　　金：云南省自然科学基金资助项目 ( 99C0 0 85M);中国科学院西部之光项目;中国科学院"十五"计划预研项目

摘　　要：利用逆转录酶与聚合酶链反应相结合的RT PCR法 ,扩增出 5个竹叶青 (Trimeresurusstejnegeri)蛇毒丝氨酸蛋白酶的cDNAs ;将扩增的cDNA片段克隆入pGEM T载体中 ,筛选得到它们的基因 ,分别命名为TSSP 1、TSSP 2、TSSP 3、TSSP 4和TSSP 5。经末端终止法测定核苷酸序列 ,推导出 5个丝氨酸蛋白酶的全序列 ;结合纯化的蛋白酶N -末端序列测定结果 ,推导TSSP 2、 3和 4分别编码凝血酶样酶stejnobin、纤溶酶stejnefibrase 1和 2。 5个丝氨酸蛋白酶分别含有 1～ 6个N -型糖基结合位点 ,表明它们的计算分子量与纯化蛋白表观分子量之间的差异是由糖含量的不同造成 ,而其氨基酸序列相似度在 6 0 %～ 90 %。TSSP 1和 2编码的成熟蛋白酶由 2 36个氨基酸残基组成 ,TSSP 3、 4和 5的则由 2 34个氨基酸残基组成。TSSP 1编码的蛋白酶在组成丝氨酸蛋白酶三联体催化活性中心产生了His41 Arg41的天然突变 ,这与其他自然界已发现的丝氨酸蛋白酶明显不同。Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to amplify cDNAs constructed from Trimeresurus stejnegeri venom glands poly(A)+ RNA to facilitate the cloning and sequencing of serine protease genes.The PCR products were then subcloned into pGEM-T vector and transformed into E.coli strain JM109.Five serine protease cDNAs (named TSSP-1,-2,-3,-4 and -5,respectively) were cloned and sequenced.The protease encoded by TSSP-1 is characterized by a His 41-Arg 41 mutation in the catalytic triad.Combined the results of N-terminal sequence determinations of the purified proteins,it is concluded that TSSP-2,-3 and -4 encode stejnobin (thrombin-like enzyme),and stejnefibrase 1 and 2 (fibrinolytic enzymes),respectively.Mature proteases encoded by TSSP-1 and -2 are composed of 236 amino acid residues,and those proteases encoded by TSSP-3,-4 and -5 are all composed of 234 amino acid residues.The cloned 5 mature proteases contain 1 to 6 N-type glycosylation sites,indicating that the differences among calculated molecular weights and apparent molecular weights determined by SDS-PAGE are caused by carbohydrate content variations.Sequence identities among the cloned proteases are around 60%-90%.The differences in their structures resulted in their various substrate specificities.

关 键 词：蛇毒 丝氨酸蛋白酶 序列 底物专一性 竹叶青 分子克隆 

分 类 号：Q55[生物学—生物化学]