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OBJECTIVE To construct a eukaryotic expression vector of human autophagy maker protein P62 labeled with myc tag, and detect its biological function. METHODS Human P62 gene was amplified from human breast DNA library by PCR and cloned into pXJ-40-myc vector. HEK293T cells were transfected with the recombinant plasmid myc-p62. Western blotting was conducted to detect the fusion protein expression and the effect on the phosphorylation of extracellular signal-regulated kinase (ERK). RESULTS Double enzyme identification and sequencing result showed that P62 eukaryotic expression vector labeled with myc tag was successfully constructed and the inserted fragment was correct. Western blotting indicated that the fusion protein was successfully expressed and was able to inhibit ERK phosphorylation. CONCLUSIONS The eukaryotic expression vector of myc-p62 was successfully constructed and proved to have an inhibitory effect on ERK phosphorylation.
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