Cloning, Expression, and Identification of Immunological Activity of an Allergenic Protein in Tartary Buckwheat

DOI Web Site Web Site 23 References
  • WANG Zhuanhua
    Institute of Biotechnology, Shanxi University
  • WANG Lan
    Institute of Biotechnology, Shanxi University
  • CHANG Wenjun
    Institute of Biotechnology, Shanxi University
  • LI Yuying
    Institute of Biotechnology, Shanxi University
  • ZHANG Zheng
    Institute of Biotechnology, Shanxi University
  • WIESLANDER Gunilla
    Department of Medical Sciences/Occupational and Environment Medicine, Uppsala University, University Hospital
  • NORBACK Dan
    Department of Medical Sciences/Occupational and Environment Medicine, Uppsala University, University Hospital

Search this article

Abstract

A cDNA fragment encoding a 24 kDa allergenic protein in tartary buckwheat was obtained using reverse transcription PCR, 3′-rapid amplification of cDNA ends (RACE), and nest PCR. The cDNA clone contained 768 nucleotides, including 588 nucleotides in the open reading frame (ORF) and 180 nucleotides in the 3′-terminal sequence. The ORF encoded a functional protein of 195 amino acids. It shared 95% and 93% nucleotide homology with the allergenic storage protein and a legumin-like protein from common buckwheat respectively. The encoding region was expressed in host strain Escherichia coli BL21 (DE3) induced by IPTG at 28 °C. The inclusion bodies of recombinant protein obtained were analyzed by western blot and purified by affinity chromatography. The purity of target protein reached above 95%. After they were refolded by step-wise dialysis, 68% of the inclusion bodies reached soluble state. An analysis of immunological activity showed that the recombinant protein had a specific IgE binding activity. This is the first report of the molecular cloning and expression of the major allergen from tartary buckwheat.

Journal

References(23)*help

See more

Keywords

Details 詳細情報について

Report a problem

Back to top