Objective To amplify defensin gene cDNA of Musca domestica larva, clone it into prokaryotic expression plasmids and express it in Escherichia coli, Methods DNA sequence encoding the defensin mature protein was amplified from Musca domestica larva cDNA library.Then it was cloned into the prokaryotic vector pGEX-4T-1.The transformed clones was screened and identified by sequencing.The recombinant pGEX/MDEF was expressed in E.coli named BL21(DE3). SDS-PAGE was used to identify whether defensin gene was expressed and its molecular weight. Anti-GST monoclonic antibody was used to identify the fusion protein by Western Blot. Results A 140bp length cDNA sequences were obtained from Musca domestica larva cDNA library and identified to be defensin cDNA by sequence analysis.DNA sequence encoding the defensin mature protein encodes a protein of 40 amino acids, the predicted molecular weight of the protein is 4.0 kD.its cDNA was successfully cloned into the pGEX-4T-1 vector,and then.pGEX/ mdef was expressed in BL21(DE3) with molecular weight of about 30kD. Conclusion The defensin gene recombinant prokaryotic expression plasmids was successfully constructed and expressed in E. coli.
China Tropical Medicine