Establishment of the LC3 Recombinant Vector and Its Expression in Prokaryotic and Eukaryotic Cells

Objective:Microtubule associated protein 1 light chain 3(MAP1LC3,for short LC3)is the first identified autophagosome membrane protein.In this paper,the human LC3 gene was cloned and the Prokaryotic and Eukaryotic recombinant vectors were constructed for further research.Method:The human LC3 gene was cloned from esophagus cancer cell 9706 with RT-PCR and then the pET-32a-LC3 and pEGFP-N3-LC3 recombinant plasmid vectors were constructed.The pET32a-LC3 plasmid was induced by IPTG(1mmol/L)in E.coli cells,while the pEGFP-N3-LC3 plasmid DNA was transiently transfected into the A549 lung cancer cells and one fish cell line(Epithelioma papulosum cyprinid,EPC)cells,respectively.Result:The LC3 protein was expressed in pET32a-LC3 transformed E.coli cells.Fluorescent microscope examination showed that fluorescent signals could be detected in both the cytoplasm and nucleus of tranfected cells after 36 hours.Conclusion:The LC3 recombinant expression vectors were established,which would provide a good cell research model for future vertebrate cell autophagy mechanisms and biological processes.