重叠PCR克隆Exendin-4的基因及序列分析  

Clone of Exendin-4 cDNA gene by overlap extension PCR and its sequence analysis

作　　者：王俊红[1,2] 郭永红[3] 张静[1] 徐静[1] 谢璇[1] 杨广笑 王全颖[4] 王枫[2] 

机构地区：[1]西安交通大学医学院第二附属医院内分泌科,陕西西安710004 [2]第四军医大学营养与食品卫生教研室,陕西西安710032 [3]西安交通大学医学院第二附属医院感染科,陕西西安710004 [4]西安华广生物工程有限公司,陕西西安710025

出　　处：《中国医药导报》2013年第15期13-15,共3页China Medical Herald

基　　金：陕西省科学技术研究发展计划项目(编号2011K14-06-04)

摘　　要：目的克隆Exendin-4 39肽氨基酸序列基因编码区cDNA。方法根据Exendin-4氨基酸序列编码cDNA,设计正、负向引物/模板链,利用重叠延伸PCR法扩增出Exendin-4编码区基因DNA片段;将获得的基因片段插入pGEM-T-Easy质粒中;转化到大肠杆菌DH5α后挑选阳性克隆,利用限制性内切酶及核苷酸序列分析技术鉴定重组质粒。结果经质粒DNA酶切分析及序列测定,获得了Exendin-4 DNA片段序列。结论本研究成功克隆了Exendin-4 cDNA,为下一步构建Exendin-4真核表达载体打下基础,也为Exendin-4基因治疗糖尿病提供了前提条件。Objective To clone and identity the Exendin-4 cDNA of 39 peptides amino acid sequences of the genes encoding area.Methods The forward and reverse primer were designed according to the Exendin-4 cDNA by comple mentary primer/template PCR.The Exendin-4 open reading frame（ORF） was cloned by overlap extension PCR.The Exendin-4 cDNA fragment was inserted in pGEM-T-Easy vector after digested with restriction enzyme and DNA sequencing.The positive cloning was chosen after the recombinant vectors transforming to Escherichia coli DH5 α.Recombinant plasmid was identified by restriction enzymes and nucleotide sequence analysis technology.Results The Exendin-4 cDNA clone was confirmed by restriction enzyme digestion and DNA sequencing.Conclusion The Exendin-4 cDNA clone is constructed successfully in this study.It provide biases for constructing Exendin-4 eukaryotic expression vector and diabetes gene therapy.

关 键 词：EXENDIN-4 重叠延伸PCR 基因克隆 

分 类 号：Q78[生物学—分子生物学]