Construction and expression of prokaryotic expression plasmid of Der f 1 cDNA of Dermatophagoides farinae

Objective To construct and express prokaryotic expression plasmid of Der f 1 cDNA of Dermatophagoides farinae. Me- thods The Der f 1 gene was digested from recombinant plasmid pMD-18T-Der f 1 by restriction endonuclease, and then inserted into expression vector pET-32a (+) by subclone technique. The recombinants were transferred into E.coli DL21 and identified by restriction endonuc- lease digestion and PCR. The expression of the recombinant plasmid pET-32a (+)-Der f 1 in the genetically engineered bacteria was induced by IPTG, and the expression products were analyzed by SDS-PAGE and densitometric scanning. Results The identified results of the positive recombinant plasmids pET32a(+)-Der f 1 by restriction endonuclease digestion and PCR were in accordance with the expected results. Sequence determination analysis showed that the gene homology with the Der f 1 reported in GenBank was 99.5%. The plasmid pET-32a (+)-Der f 1 could express a specific M r 45 000 protein in E.coli BL21, 15% of total protein of recombinant bacterial. Conclusion The prokaryo-tic expression plasmids, which contain Der f 1 cDNA of Dermatophagoides farinae have been constructed successfully. Plasmid pET-32a(+)-Der f 1 can express specific protein in E.coli DL21.