利用套叠PCR技术合成人胰岛素原基因的临床研究  

The study of molecular design and cloning of human proinsulin gene by SOE-PCR

作　　者：马建忠[1] 周贤婧[1] 王永刚[1] 

机构地区：[1]兰州理工大学生命科学与工程学院,730050

出　　处：《中国现代医药杂志》2010年第2期8-11,共4页Modern Medicine Journal of China

基　　金：甘肃省自然科学基金资助项目;项目编号(3ZS062-B25-O23)

摘　　要：目的设计并分子克隆适宜于大肠杆菌表达系统的人胰岛素原基因。方法以美国国家基因库(GenBank)发布的人胰岛素原的氨基酸序列为模板,参考大肠杆菌表达系统的密码子偏好性以及所用表达载体(pGEX-3x)的特点,经计算机分析、设计人胰岛素原基因为8个寡核苷酸片段,以重叠延伸PCR(SOE-PCR)技术体外延伸获得完整的人胰岛素原基因,分子克隆后测定所得基因的核苷酸序列。结果经限制性内切酶分析确定为重组克隆的质粒进行核苷酸序列分析,结果表明,我们所克隆的基因序列完全符合我们先前的设计。结论SOE-PCR技术可用于体外寡核苷酸片段的延伸并获得完整长度的基因。TaqDNA聚合酶延伸反应前以Klenow片段处理可提高较短覆盖末端或较低专一性末端正确延伸的成功率。Objective To design and molecular cloning of the human proinsulin gene which is suitable for Escherichia coli expression system.Methods According to the amino acid sequence of the human proinsulin from the USA gene bank （GenBank）,the codon bias of E.coli K12 expression system and the characteristics of the expression vector （pGEX-3x）,8 overlapping oligonucleotide fragments were designed through computer analysis and then synthesized for SOE-PCR.Results The recombinant plasmids identified by restriction enzyme analysis were further sequenced.The results showed that a proinsulin gene completely in line with our previous design was cloned.Conclusion SOE-PCR technique can be used in vitro oligonucleotide extension and obtain full-length genes.Prior to the Taq DNA polymerase extension reaction,the Klenow fragment treatment can improve the extension accuracy of the shorter overlapping ends and of the lower specific ends.

关 键 词：套叠PCR(SOE-PCR) 人胰岛素原 Klenow片段 基因克隆 

分 类 号：TQ467.32[化学工程—制药化工]