小鼠c-Myc基因的克隆表达及其纯化

发布时间：2018-06-24 00:57

本文选题：c-Myc基因 + 基因克隆　；参考：《中国生物工程杂志》2017年02期

【摘要】：目的:克隆小鼠c-Myc基因,构建原核表达载体并对其进行蛋白表达及纯化。方法:以TetO-FUW-OSKM质粒为模板经PCR扩增c-Myc基因,再通过基因重组技术与pET-3C载体相连转化大肠杆菌DH5α。经抗性筛选,PCR鉴定阳性重组子,测序正确后,重组质粒转化大肠杆菌Rosetta(DE3),IPTG诱导蛋白表达,SDS-PAGE对重组蛋白进行鉴定分析。结果:DNA电泳结果证实PCR扩增产物与预期大小一致,DNA测序结果显示与GenBank公布的c-Myc基因序列一致,IPTG诱导SDS-PAGE电泳后,在分子量64kDa左右出现诱导后蛋白新增条带,与预测的c-Myc分子量大小一致。结论:成功地构建了pET3C-Myc原核表达载体,表达并纯化出了c-Myc蛋白,为今后研究c-Myc蛋白及相关蛋白诱导体细胞成多能干细胞打下基础。
[Abstract]:Aim: to clone mouse c-Myc gene, construct prokaryotic expression vector, express and purify c-Myc gene. Methods: the c-Myc gene was amplified by PCR using TetO-FUW-OSKM plasmid as template, and then transformed into E. coli DH5 伪 by gene recombination with pET-3C vector. The recombinant plasmid was transformed into E. coli Rosetta (DE3) / IPTG induced protein expression by SDS-PAGE. The recombinant protein was identified by SDS-PAGE. Results the results of DNA electrophoresis confirmed that the PCR amplification products were consistent with the expected size. The results of DNA sequencing showed that after SDS-PAGE was induced by IPTG, a new protein band appeared in the molecular weight of about 64kDa, which was consistent with the sequence of c-Myc gene published by GenBank. The molecular weight of c-Myc is consistent with the predicted molecular weight. Conclusion: the prokaryotic expression vector pET3C-Myc was successfully constructed, c-Myc protein was expressed and purified, which laid a foundation for the further study of c-Myc protein and related protein inducing pluripotent stem cells.
【作者单位】：广东医科大学;温州医科大学药学院;
【基金】：广东省自然科学基金(2015A030313527) 广东省省级科技计划项目(2013B031800014) 东莞市社会科技发展项目(2013108101062) 浙江省自然科学基金(LY13H080004)资助项目
【分类号】：Q78

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