长白山白眉蝮蛇毒类凝血酶Gussurobin基因的克隆、表达与纯化(英文)  

Cloning, Expression and Purification of Gussurobin, A Thrombin-like Enzyme from the Snake Venom of Gloydius ussuriensis

作　　者：杨青[1] 胡学军[1] 许小明[1] 安利佳[1] 袁晓东 苏志国[3] JANSON Jan-Christer 

机构地区：[1]大连理工大学化工学院生物工程研究所,大连116012 [2]宝生物大连公司,大连116600 [3]中国科学院化工冶金研究所生物化学工程国家重点实验室,北京100080 [4]Biosurface Science Center

出　　处：《生物化学与生物物理学报》2002年第1期6-10,共5页

基　　金：辽宁省科学技术基金项目 (No .9930 5 0 0 1);瑞典政府NUTEK基金资助~~

摘　　要：根据同源性 ,在高度保守的上游信号肽区域设计引物 ,通过RT PCR反应 ,从长白山白眉蝮蛇 (Gloydiusussurensis)毒腺总RNA中克隆得到类凝血酶 gussurobincDNA ,双向测序得到 gussurobin基因的全序列并由此推测出相应的氨基酸序列。与其他已知的类凝血酶不同 ,gussurobin只含有一个可能的糖基化位点 ,即Asn12 4 Ser12 5 Thr12 6。将gussurobin基因克隆到表达载体 pPIC9K中 ,电极转化至毕氏酵母菌株GS115中 ,经G418抗性筛选和营养缺陷型筛选获得重组子。经摇瓶培养 ,获得表达。经过柱层析分离 ,获得SDS PAGE电泳纯的重组gussurobin。Total RNAs were extracted from the venom gland of the snake Gloydius ussuriensis. The cDNA of gussurobin, a thrombin-like enzyme from Gloydius ussuriensis, was cloned and amplified by RT-PCR. Assay of the nucleotide sequence of the cDNA allowed postulation of the complete amino acid sequence for Gloydius ussuriensis, Chinese Viperdae. Its amino acid sequence exhibits significant homology with that of other snake thrombin-like enzymes. The cDNA of gussurobin was inserted into the vector pPIC 9K and expressed successfully in Pichia pastoris, strain GS115. The recombinant gussurobin was separated and purified from 500 ml culture and showed one band on SDS-PAGE.

关 键 词：类凝血酶 gussurobin CDNA序列 表达 纯化 长白山白眉蝮蛇 蛇毒 

分 类 号：Q78[生物学—分子生物学]