Prokaryotic expression and antigenic analysis of recombinant thioredoxin reductase GliT of Aspergillus fumigatus

Objective To clone thioredoxin reductase GliT(TR) gene,construct prokaryotic expression vector,prepare recombinant protein and analyze its antigenicity.Methods The cDNA of TR was amplified from total RNA of Aspergillus fumigatus by reverse transcription-PCR.The amplified fragment was cloned into pMD18-T vector and sequenced.The recombinant plasmid pMD18-T/TR was digested by the restriction enzymes,and the target fragment was inserted into pET-28a(+) vector which was transformed into E.coli BL21(DE3).The recombinant TR protein was expressed by IPTG induction.Following the analysis of SDS-PAGE and western blot,the expressed protein was purified by Talon metal affinity resins.Results The recombinant plasmid consisting of full length TR gene was constructed.The recombinant TR protein was expressed richly in E.coli.Western blot showed that the recombinant protein was recognized by the sera from patients with proven invasive aspergillosis.Conclusion The recombinant expression plasmid pET28a(+)/TR was successfully constructed and expressed richly in E.coli BL21(DE3).The recombinant protein showed strong anitgenicity and may be used in the further study as the experimental material.