皖南尖吻蝮蛇毒中类去整合蛋白/富含半胱氨酸两结构域cDNA的克隆和表达  

Cloning,Expression and Characteristics of Disintegrin-like/cysteine-rich Domains cDNA from Agkistrodon acutus Venom

作　　者：刘庆都[1] 张家琳 承新[1] 刘爱平[1] 刘兢[1] 

机构地区：[1]中国科学技术大学生命科学学院,合肥230027

出　　处：《中国生物化学与分子生物学报》2000年第4期462-467,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金! ( 3 9670 176)资助项目

摘　　要：为了探讨出血毒金属蛋白酶结构功能关系 ,通过 RT- PCR方法 ,从皖南尖吻蝮蛇( Agkistrodon acutus)毒腺总 RNA中扩增得到编码 P- 型出血毒金属蛋白酶的完整类去整合蛋白和富含半胱氨酸两个结构域 c DNA( AA/DC) .它全长 964bp的 c DNA,开放阅读框架编码 2 1 6个氨基酸残基 ,序列比较分析表明它同来自 Bothrops jararaca的 jararhagin- C、来自 Crotalus atrox的 catrocollastatin- C有很高的同源性 .在类去整合蛋白结构域中 ,Ser- Glu- Cys- Asp( SECD)代替了去整合蛋白中相应部位的 Arg- Gly- Asp( RGD)三肽序列 .将编码区基因克隆入 p GEX- 2 T载体中 ,转化大肠杆菌 TG- 1 ,用 IPTG诱导表达 ,表达产物具有抑制胶原诱导的血小板凝集活性 ,但不抑制ADP诱导的血小板凝集 .该研究为进一步阐述蛇毒金属蛋白酶结构功能关系和药物开发奠定了基础 .By means of RT PCR, a cDNA gene was obtained from the total RNA of Agkistrodon acutus venom.The clone was 964 bp in length,which encoded a complete open reading frame of 216 amino acid residues.The cDNA deduced amino acid sequence was rich in cysteines and highly similar to jararhagin C and catrocollastatin C.The Arg Gly Asp(RGD) tripeptide sequence found in disintegrin was replaced by Ser Glu Cys Asp (SECD) in the disintegrin like domain.The recombinant disintegrin like/cysteine rich domains were expressed as a fusion protein with glutathione S transferase in E.coli and the recombinant protein was an inhibitor of the collagen induced but not ADP induced platelet aggregation.

关 键 词：蛇出血毒素 类去整合蛋白 半胱氨酸 基因克隆 

分 类 号：R996.3[医药卫生—毒理学] Q78[医药卫生—药学]