Prokaryotic Expression of Rat LC3 Protein and Preparation of Its Polyclonal Antibody

Microtubule-associated protein 1 light chain 3-β(LC3),which located on autophagosome membranes in mammalian cells,is a distinctive mark to detect autophage.The LC3 gene was amplified by RT-PCR from the template of total RNA extracted from SD rat brain and then cloned into the pMD-18T vector.The correct identified recombinant was used as a template for the amplification of LC3 gene by PCR.The product was inserted into the prokaryotic expression vector pUC18,named pUC18-LC3 and transformed into E.coli BL21(DE3).The recombinant protein LC3 was expressed.Following SDS-PAGE,the objective band was reclaimed.Anti-LC3 serum was also prepared in New Zealand rabbit with purified reclaimed protein.The results of western blot showed that the polyclonal antibodies could recognize LC3 specifically.