稀有密码子对人源Id2基因表达的影响  

Effects of rare codon on functional expression of Id2 gene in escherichia coli

作　　者：杨伟[1] 周华[2] 赵沙沙[1] 金丽[1] 郭珍[1] 张绍城[1] 陈检[3] 汪德强[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学重点实验室,重庆400016 [2]重庆医科大学附属第二医院检验科,重庆400016 [3]重庆医科大学附属第一医院心内科,重庆400016

出　　处：《重庆医科大学学报》2013年第4期365-369,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30970563)

摘　　要：目的:通过对人Id2基因的稀有密码子进行突变并构建原核表达重组质粒,在大肠杆菌(escherichia coli,E.coli)中表达Id2蛋白,分析稀有密码子对Id2基因表达的影响。方法:PCR扩增突变Id2基因序列,然后转入E.coli BL21中进行表达,利用镍离子亲和层析、离子交换层析及分子筛纯化。结果:正确构建了Id2基因的基因突变表达质粒,基因突变表达的Id2蛋白在E.coli BL21中为可溶性上清表达,成功纯化了目的蛋白,且分子筛结果显示其在溶液中呈现二聚体状态。结论:稀有密码子对Id2蛋白在E.coli BL21中大量可溶性上清表达有重要影响。Objective：To analyze the effects of rare codon on functional expression of inhibitor of DNA binding and/or differentiation 2（Id2） gene by mutating rare codon of human Id2 protein,constructing expressing recombinant plasmids and expressing Id2 protein with solution in escherichia coli（E.coli） system.Methods：Id2 human gene was amplified by PCR,then Id2 was expressed in E.coli BL21.Plasmids were constructed by mutating rare codes into normal ones.Expressed production was purified by nickel column chromatography,ion-exchange column chromatography and size exclusion chromatography.Results：Expression vector of Id2 was successfully constructed and the expressed aim protein in E.coli BL21 was in a soluble supernatant way.Human recombinant protein was purified and exhibited as homodimers in solutions by the size exclusion chromatography experiments.Conclusion：Rare codons are believed to play a key role in disturbing the soluble supernatant expression of human Id2 protein in E.coli.

关 键 词：ID2 突变 表达 纯化 

分 类 号：Q71[生物学—分子生物学]