人自噬相关基因5真核表达载体的构建及表达鉴定

Construction and Expression Identification of Flag-Tagged Human ATG5 Eukaryotic Expression Vector

目的:构建带Flag标签的自噬相关基因5(ATG5)的真核表达载体,获得Flag-ATG5融合蛋白。方法:以人乳腺文库为模板,采用PCR技术扩增人ATG5编码序列,将其插入pc DNA3-Flag载体,转染人胚肾293T细胞后,Western印迹检测其表达情况;将该重组质粒与ATG12表达质粒共转染293T细胞,通过免疫共沉淀法检测2个蛋白的相互作用。结果:Flag-ATG5真核表达质粒构建成功,转染293T细胞后融合蛋白获得表达,其相对分子质量约33×103;Flag-ATG5可与Myc-ATG12结合。结论:构建了带Flag标签的人ATG5真核表达载体,为进一步研究ATG5在自噬中的功能奠定了基础。

Objective： To construct and express the eukaryotic expression vector of human autophagy related gene 5（ATGS）. Methods： Human ATG5 gene was obtained from human mammary gland cDNA library by PCR and cloned into vector pcDNA3-Flag. Human 293T cells were transfected with the recombinant plasmid Flag- ATG5 and the expression was detected by Western blotting. In addition, assay was applied to determine the inter- action between Flag-ATG5 and Myc-ATG12. Results： ATG5 eukaryotic expression vector was confirmed by dou- ble restriction enzyme digestion and inserted fragment sequencing. The expression of human ATG5 protein with rel- ative molecular of 33×10^3 in human 293T cells was identified by Western blotting. In addition, immunoprecipita- tion results showed that human ATG5 protein could interact with Myc-ATG12 in vivo. Conclusion： The eukaryotic expression vector of Flag-ATG5 was constructed and expressed in human 293T cells, which had laid foundation for the further study of the role of ATG5 in autophagy.