Cloning,expression,and analysis of the Mal f 6 allergen from Dermatophagoides farinae in China

Objectives To acquire the cDNA of the Der f Mal f 6 gene(Mal f 6) from Dermatophagoides farinae in China and construct a prokaryotic expression system.Methods Total RNA was extracted from D.farinae.The Mal f 6 gene was amplified with RT-PCR and cloned into the pCold TF DNA vector.The pCold TF-Mal f 6 was then transformed into E.coli BL21 and induced by IPTG to express the gene.It was then identified with SDS-PAGE.Bioinformatics tools were used to analyze the physiochemical properties,secondary structure,and function of Mal f 6.Results The Mal f 6 gene was obtained by RT-PCR and was 495 bp in length.The constructed plasmid pCold TF-Mal f 6 was then transformed into E.coli BL21 and induced to express the gene.SDS-PAGE detected a specific band in whole cells,supernatant,and precipitate containing pCold TF-Mal f 6.Bioinformatics indicated that the allergen consisted of 164 amino acids and had a molecular weight of 17.7 KDa.Its secondary structure consisted of an alpha helix(4.88%),extended strand(37.8%),and random coil(57.32%).The Mal f 6 allergen is predicted to be a hydrophilic cytoplasmic protein and have peptidyl-prolylcis-trans isomerase activity.Conclusion The cDNA coding for Mal f 6 allergen of D.farinae was cloned and successfully expressed.This provides a foundation for further research and large-scale production for clinical treatment of allergic disorders.