Identification And Properties Of Major Allergen From Chinese Shrimp, Penaeus Chinensis

Shrimp is deeply popular with consumers because of flavor and alimentation. But, it is also one of eight kinds of highly allergenic foods publicized by Food and Agriculture Organization. Penaeus chinensis, a kind of seafood from China, is researched in this paper. In order to obtain relevant data of Chinese shrimp allergen, and lay theoretical base for the seafood allergen control, here the properties of Chinese shrimp are investigated. Its antigenic components are analyzed by SDS-PAGE. Then Western-blotting is used to determine the major allergen of it. A new shrimp allergen was obtained with high performance liquid chromatography (HPLC), and its properties preliminarily are studied by phenol-sulfuric acid reaction, peptide mass fingerprint (PMF) and infra-red (IR) spectrum. The results reveale that the major allergen of Chinese shrimp is molecules-36kD protein. It was a kind of glycoprotein and the total carbohydrate content was 4.4%. 17 segments of identical peptide are retrieved by mascot, andα-Helix is one of secondary structure on Pen c 1. In order to study the molecular biology character of Pen c 1, we design primers according to gene of other shrimp allergens. Employing the method PCR clones pen c 1 gene, and Sequencing. Its open reading frame consists of 825 bp nucleic acids, which encoding 274 amino acids. We download other amino acid sequences of seafood allergen from GenBank, then draw homology and construct phylogenetic tree form by bioinformatics software. The results show that different seafood allergens share a higher amino acid homology, and their relationship more near its homology also higher. Analyzing major IgE-binding regions have been identified in shrimp tropomyosin containing nine-peptide epitopes,find that different seafood allergens share homology epitopes. Besides, we conclude disulphide-bond, isoelectric point (pI), Hydrophobicity, secondary structure, O-glycosylation sites by bioinformatics software online.The component of monosaccharide is analyzed by Gas chromatography. Glycopeptides linkage is analyzed byβ-elimination. Using differential scanning calorimeters(DCS) to analyze thermal stability .The glycosylation sites of Pen c 1 are deterrninded using biological Mass spectrometry. The results reveal that Pen c 1 mainly consists of glucose and seminose. Sugar chain link protein by O-type glycopeptides and boost up heat-stability of Pen c 1. Asn located 101 is N-glycosylation, Thr located 101 is potential N-glycosylation. According to ELISA system established, research infection of sugar chain release and Maillard to antigenicity. The results reveal that N-glycosylation sites and potential O-glycosylation sites are not in the nine-peptide epitopes, sugar chain of Pen c l little effect on the allergic activity.We try to reduce antigenicity of Pen c 1 by Trypsin hydrolysis. The regression model of three factors twice rotation perpendicular regressive design was adopted. The effects of pH, temperature and enzyme to substrate ratio (E:S) on the antigenic activities of Pen c 1 are on study. By F- inspection, T-inspection and applied inspection, the model was verified that it reflected theoretically the intrinsic principle on Pen c 1 hydrolysis by Trypsin. The optimum hydrolysis conditions of Pen c 1 are as follows: pH=8.12, T=43.3℃, E:S = 4.3 U/g for OD.