甲基丙二酰辅酶A 变位酶表达载体的构建及初步表达

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。

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

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.