Cloning, expression, and identification of immunological activity of an allergenic protein in tartary buckwheat.
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 degrees 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.
Wang
Zhuanhua
Z
Institute of Biotechnology, Shanxi University, Taiyuan, China. zhwang@sxu.edu.cn
Wang
Lan
L
Chang
Wenjun
W
Li
Yuying
Y
Zhang
Zheng
Z
Wieslander
Gunilla
G
Norback
Dan
D
D052179:Antigens, Plant / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019612:Fagopyrum / Q000235:genetics* / Q000378:metabolism; D007073:Immunoglobulin E / Q000276:immunology; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA
D052179:Antigens, Plant; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E