蛇毒类凝血酶Calobin cDNA的设计合成与克隆  

The design,chemical synthesis and cloning of cDNA of Agkistrodon caliginosus thrombin-like enzyme Calobin

作　　者：袁盛凌[1] 段海清[1] 张兆山[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2003年第5期357-360,共4页Letters in Biotechnology

摘　　要：人工合成了朝鲜蝮蛇(Agkistrodoncaliginosus)类凝血酶Calobin的编码序列。在设计引物时选择大肠杆菌和酵母菌的偏爱密码子,通过相互搭桥的方式,将全长为789bp的编码序列分成14个片段,每个片段长度为78个碱基左右,采用“核心模板法”并加以改进,通过4次PCR延伸反应,得到了全长基因。经扩增后回收目的片段,双酶切后连接到克隆载体,将得到的转化子酶切鉴定后,任意选择6个克隆进行双向测序,得到了全序列正确的3个克隆。本工作为类凝血酶在大肠杆菌和酵母系统中的高效表达奠定了基础,也为长度为700～900bp片段的合成提供了帮助。By selecting the high-usage codons both in Escherichia coli and in yeast,the cDNA of snake venom thrombin-like enzyme Calobin,consist of789bp,was designed by dividing it into14oligonucleotides fragments,with78bases length and21bases crossover.The synthetic fragments were assembled by4steps PCR occording to the idea of″core templete″.PCR product was inserted into pGEM-3zf(+),positive clones were selected and six of them were sequenced from two directions.The result showed three of recombinant clones were correct and corresponded to the gene designed.This work provides effective method of assembly gene which length is700～1000bp.

关 键 词：蛇毒 类凝血酶 基因合成 聚合酶链式反应 序列分析 

分 类 号：R996.3[医药卫生—毒理学] Q781[医药卫生—药学]