Construction and Expression Identification of myc- Tagged Human ATG12 Eukaryotic Expression Vector

Objective:To construct and express the eukaryotic expression vector of human autophagy related gene 12(ATG12) labeled with myc tag.Methods:Human ATG12 gene was obtained from human mammary gland cDNA library by PCR and cloned into myc vector.Human 293 T cells were transfected with the recombinant plasmid of myc-ATG12 and the expression was detected by Western blot.In addition,immunoprecipitation assay was applied to determine the interaction between myc-ATG12 and flag-ATG3.Results:ATG12 eukaryotic expression vector labeled with myc tag was successfully constructed by double digestion identification.The inserted fragment was confirmed correct by sequencing.The expression of human ATG12 protein in human 293 T cells was identified by Western blot.The immunoprecipitation assay results showed that human ATG12 protein could interact with Flag-ATG3 in vivo.Conclusion:The eukaryotic expression vector of myc-ATG12 was successfully constructed and expressed in human 293 T cells,which had laid foundation for the further study of the role of ATG12 in autophagy.