›› 2013, Vol. 31 ›› Issue (5): 7-363-366.

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Cloning,Expression and Identification of Der f 7 gene from Dermatophagoides farinae and its Immunological Characteristics

ZHENG Man-yin, WU Yu-lan, YAN Hao, JI Kun-mei, LIU Zhi-gang*   

  1. Allergy and Immunology Institute, Medical School, Shenzhen University, Shenzhen 518060, China
  • Online:2013-10-30 Published:2014-07-24

Abstract: Objective  To clone and express Der f 7 gene of Dermatophagoides farinae, and identify its immunogenicity.  Methods  Total RNA was extracted from D. farinae mites. A reference sequence (Accession No. AY283292) was used to design specific primers. The Der f 7 gene fragment was amplified by RT-PCR, and cloned into pET-32a vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced with IPTG for protein expression. The recombinant protein was purified by Ni2+ chelating affinity chromatography and analyzed by SDS-PAGE and Western blotting.  Results  The Der f 7 gene fragment was about 650 bp, and shared 99% homology with the published one (Accession No. FJ436108). SDS-PAGE result showed its relative molecular weight (Mr) of 23 000. The recombinant protein showed appropriate combination ability with IgE in sera of mite allergic patients.  Conclusion  Der f 7 gene has been expressed in prokaryotic expression system and shows allergenicity.

Key words: Dermatophagoides farinae, Der f 7, Allergen, Expression, Purification