MARCH1真核表达载体的构建及鉴定

Construction,identification,and purification of a eukaryotic expression vector of E3 ubiquitin-protein ligase MARCH1

目的构建MARCH1真核表达载体并进行鉴定和纯化,为研究MARCH1在小鼠树突细胞(DCs)中的生物学作用奠定基础。方法从小鼠尾部提取cDNA作为模板,采用特异的引物,通过聚合酶链反应(PCR)扩增MARCH1cDNA片段,经EcoRⅠ和BamHⅠ双酶切,连接于载体pMB-HA质粒上,构建重组载体,经双酶切和DNA测序分析检测重组载体构建结果,转化大肠埃希菌DH5α感受态菌、筛选阳性克隆、抽提质粒并进行纯化。结果 PCR获得与预期大小一致(约3900bp)的cDNA片段,成功构建重组载体pMB-HA-MARCH1,双酶切及测序鉴定证实MARCH1 cDNA片段正确插入该真核表达载体中。结论成功构建含有MARCH1的真核表达载体,为进一步研究提供了实验基础。

Objective To construct,identify,and purify a eukaryotic expression vector of E3 ubiquitin-protein ligase MARCH1 to provide a basis for further study of the biological effects in dendritic cells（DCs） of mice.Methods cDNA was extracted from the tail of a mouse and used as the template.The MARCH1 cDNA was amplified by polymerase chain reaction（PCR） with the specific primers.After EcoR Ⅰ and BamH Ⅰ double digestion,the MARCH1 cDNA was linked in pMB-HA plasmid to construct recombinant vectors.The construction result of the recombinant vector was identified using double-digestion and DNA sequencing.The recombinant vector was transfected into Escherichia coli DH5α competent cells,positive clones were screened,plasmid was extracted,and then it was purified.Results PCR product with size of 3900 bp cDNA segment was obtained,which was the same as the expected size.The recombinant vector pMB-HA-MARCH1 was identified by double-digestion and DNA sequencing.The findings proved that the MARCH1 cDNA segment was inserted into the eukaryotic expression vector correctly.Conclusion The eukaryotic expression vector of MARCH1 was constructed successfully and provided an experimental basis for further study.