Construction and expression of eukaryotic expression vector pLC3-EGFP

【Objective】 To construct eukaryotic expression vector pLC3-EGFP and to observe the expression in human embryonic kidney cell line HEK293T cells.【Methods】 The full length gene of human microtubule-associated protein 1 light chain3(LC3) was amplified from HEK293T by RT-PCR.Then the cDNA fragment was subcloned into pEGFP-N3 vector and the positive clones were selected and sequenced.The recombinant plasmid pLC3-EGFP was transfected into the HEK293T cells using VigoFect transfection agent.Localization of the fusion protein LC3-EGFP was observed under fluorescent microscope,and the expression LC3-EGFP protein was detected by western blotting.【Results】 The PCR amplification yielded a single band of about 378 base pairs,sequence analysis of the PCR products revealed that it was consistent with that of the references published [GenBank(NM_022818)].Furthermore,a recombinant plasmid pLC3-EGFP for eukaryotic expression was also successfully constructed after subcloning.LC3-EGFP fusion protein was expressed in the HEK293T cells and mainly presented in the cytoplasm after transfection with pLC3-EGFP.【Conclusion】 The recombinant eukaryotic expression vector of pLC3-EGFP has been constructed successfully and EGFP tagged LC3 is immediately expressed in the HEK293T cells and localized in the cytoplasm,which lay the foundation for further studying the biological progression and mechanisms of cell autophagy.