甲基丙二酰辅酶A变位酶表达载体的构建及初步表达  

The Construction and Expression of Methymalonyl-CoA Mutase(MCM) Expressive Vector

作　　者：常会波[1] 朱彦丽[2] 王立文[2] 李尔珍[2] 刘卓[1] 欧阳胜荣[1] 吴建新[1] 

机构地区：[1]首都儿科研究所生化室,北京100020 [2]首都儿科研究所神经内科,北京100020

出　　处：《现代生物医学进展》2012年第13期2414-2417,共4页Progress in Modern Biomedicine

基　　金：国家重点基础研究发展计划(2007CB511903);北京市优秀人才资助项目(20081D0303200106);首都特色临床医学技术发展研究(D101100050010040)

摘　　要：目的:克隆表达甲基丙二酰辅酶A变位酶(MCM)蛋白,为进一步研究相关基因突变对功能的影响机制奠定基础。方法:自人外周血淋巴细胞中提取总RNA、逆转录,并与pET32a构建融合蛋白原核表达载体;优化蛋白表达诱导条件;经SDS-PAGE、WesternBlot检测目的蛋白的表达。结果:经酶切鉴定并经测序证实获得全长2210bp的甲基丙二酰辅酶A变位酶基因(MUT),并成功构建融合蛋白原核表达载体,SDS-PAGE在102 kDa处获得目的条带,Western Blot检测确定为MCM表达蛋白。结论:成功克隆表达出MCM表达蛋白。Objective： To clone and express MUT gene in order to study the function and mechanism of MUT gene,which lay the foundation for further study of mutations mechanisms.Methods： RNA was extracted from human lymphocytes and transcribed reversely,and MUT gene was inserted into pET32a to construct fusion protein prokaryotic expression vector.The condition for induction and ex-pression was optimized.The fusion protein was detected by SDS-PAGE and Western Blot.Results： MUT gene,of 2210bp,was obtained and identified by sequencing.MCM fusion protein prokaryotic expression vector were constructed successfully.A protein band of about 102 kDa was detected by SDS-PAGE,and the protein was verified to be MCM by Western-Blot.Conclusion： MUT gene was cloned and expressed successfully.

关 键 词：甲基丙二酰辅酶A变位酶(MCM) MUT基因 表达 

分 类 号：Q75[生物学—分子生物学] Q78