| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Complete amino acid sequence of four novel allergens from third stage larva of Anisakis simplex were determined by immunoblotting using expressed proteins and Anisakis allergic patient's sera, and cDNA sequencing in positive clones. From the results of homology search, three of four allergens were identified as SXP/RAL-2 protein family, serine protease inhibitor, and AS14 (an antigen from swine roundworm Ascaris suum)-like protein, and unknown protein. Positive IgE reaction against each recombinant allergen was recognized 35, 14, 31, and 44% in Anisakis allergic patients, respectively.
Overlapping peptides consisting of 15 or 16 residues were synthesized based on the primary structure of a major allergen, Ani s 1. Three IgE-reactive peptides were found out by an enzyme-linked immunosorbent assay (ELISA) in 32 kinds of peptides. Important amino acids as IgE-binding epitopes were identified by comparing with shortened peptides and analogous peptides of the three peptides.
A simplified isolation method of Ani s 1 by an affinity chromatography utilizing polyclonal antibody obtained from Ani s 1-immunized rabbit was successfully developed. Natural Ani s 1 was purified by a reverse phase HPLC followed after this immunochromatography. Twenty five micro grams of natural Ani s 1 (nAni s 1) were obtained from 1 gram of worms only two chromatographic steps. Moreover, yield was also raised about 15 times than the previous report by us. Allergenicity of recombinant Ani s 1 (rAni s 1) was compared with that of nAni s 1 by ELISA. rAni s 1 was nearly maintained allergenicity of nAni s 1, i.e., experimental value of {(rAni s 1)/(nAni s 1) X 100} was about 80%. From consideration of this allergenic titer, rAnis1is estimated to be a substitutive diagnostic-tool for confirmed diagnosis of Anisakis allergy.
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