小鼠MUC1基因的克隆及其在大肠杆菌DH5α中的表达

目的构建表达小鼠MUC1基因的工程菌,并纯化重组小鼠MUC1融合蛋白。方法通过RT-PCR扩增小鼠MUC1基因片段,连入pMAL-p2原核表达载体,并转化大肠杆菌DH5,αIPTG诱导表达,经Western blot鉴定,Amylose Resin亲和层析纯化。结果获得了小鼠MUC1 708bp DNA片段,并构建了pMAL-mMUC1表达载体。表达产物相对分子质量约为67000,经Western blot鉴定,与兔抗MBP多克隆抗体产生特异性反应。纯化的融合蛋白在SDS-PAGE图谱上呈单一条带。结论成功构建了表达小鼠MUC1蛋白的工程菌。

Gene Cloning and Expression of Mouse MUC1 in E. coli DH5α

Objective To establish a recombinant E. coli strain for expression of mouse MUC1 gene and purify the expreased fusion protein. Methods Amplify mouse MUC1 gene fragment by RT-PCR,then insert into prokaryotic expression vector pMAL-p2 and transform to E. coli DHSct for expression under induction of IPTG. Identify the expreased product by Western blot and purify by Amylose Resin affinity chromatography. Results The MUC1 DNA fragment at a length of 708 bp were amplified, and recombinant plasmid pMAL-mMUC1 was constructed correctly as proved by restriction analysis. SDS-PAGE proved that the fusion protein MUC1-MBP,with a relative molecular mass of about 67 000, was expressed. Western blot showed specific reaction of expressed product with rabbit anti-MBP polyclonal antibody. The purified fusion protein showed a single band on SDS-PAGE profile. Conclusion The recombinant E. coil strain for expression of mouse MUC1 protein was successfully established.