Construction of prokaryotic expression vector with human autophagy-related LC3 B gene and its activity detection / 军事医学

ABSTRACT

Objective To construct the prokaryotic expression vector of human autophagy-related LC3B gene,obtain the GST-LC3B recombinant plasmid , purify the GST-LC3B fusion protein and identify its activity in vitro.Methods Human LC3B coding region was amplified from the human mammary gland cDNA by PCR and inserted into the prokaryotic expres -sion vector pGEX-KG.The recombinant plasmid pGEX-KG-LC3B was transformed into E.coli Rossate.The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis .The function of the purified protein GST-LC3B was detected by GST pull-down assay.Results About 400 bp of the LC3B coding region was successfully amplified from the mammary gland library by PCR and inserted into pGEX -KG.The result of double diges-tion and sequencing showed that the GST-LC3B recombinant plasmid was successfully obtained .The GST-LC3B fusion pro-tein of about 40 000 (Mr) was successfully purified and identified by SDS-PAGE and Western blotting analysis.GST pull-down assay showed that GST-LC3B could interact with Atg4B, which identified its known function .Conclusion The pro-karyotic expression vector of GST-LC3B is constructed successfully , which will facilitate further research on the function of LC3B in autophagy.