| Fish roe are rich in yolk protein,which is mainly composed of yolk lipoprotein(Lv),yolk high-phosphorus protein(Pv)and ?'-component(?'-c),all of which belong to glycoprotein.The study found that the main allergen in fish roe is ?-c glycoprotein,which is named "Onc k 5" in the official list of allergens of the World Health Organization and the International Union of Immunology.So far,compared with other food allergens,the structural information of the allergen ?'-c in fish roe is less,and the causes of sensitization of different fish roes have not been systematically studied.This study used sturgeon roe(Acipenser schrenckii)as the research object,focusing on the structural characterization of ?'-c glycoprotein in sturgeon roe,exploring the cross-reactivity of allergenic proteins between different species,and verifying their antigenicity from bioinformatics.Below are key research findings:1.The polysaccharide and protein content of the ?'-c protein from sturgron roe were determined after separation and purification.The polysaccharide content was 4.87%and the protein content was 33.2%.The N-glycans in the glycoprotein were released by PNGase F enzyme.After complete methylation,seven N-glycans were identified by ion mass spectrometry,including three high mannose types(Hex3HexNAc2,Hex4HexNAc2,Hex5HexNAc2);four complex types(Hex3HexNAc3,Hex3HexNAc4,Hex3HexNAcs,Hex3HexNAc6),the cleavage mode is mainly glycosidic bond cleavage,and important cross-ring rupture also occurs.The trypsin digested product was isolated and identified by HILIC-ion trap-MSn.The structure of N-glycopeptide was identified by automatic acquisition of MS2 and manual collection of MS3.N78 glycosylation site was identified,and the peptide sequence was SHGQATQVKVNGEAIS,The glycosylation site is modified by two N-glycans:Hex3HexNAc5 and Hex3HexNAc62.A method for preparing polyclonal antibody against ?'-c protein in sturgeon roe was established,and an indirect ELISA method was optimized.The serum antibody titer was between 40,000-48,000,which met the experimental requirements.The preferred antigen coating concentration is 100 ng/mL,the primary antibody dilution factor is 1:8000,and the secondary antibody dilution is 1:40000.The competitive inhibition ELISA experiment confirmed that there was an immunological cross reaction between the ?'-c protein of sturgeon roe and crucian roe,perch roe,snakehead roe,catfish roe,cyprinus carpio roe.The cross-reaction rates were 43.47%,87.12%,92.46%,60.54%,and 59.11%,respectively3.The physical and chemical properties and structural characteristics of the ?'-c protein from sturgeon roe were analyzed by bioinformatics software.The results showed that the ?'-c protein of sturgeon roe was C959H1521N259O317S11,containing 201 amino acid residues,the relative molecular mass was 22103.83 Da,the isoelectric point was 6.76,and the Aliphatic index was 70.25.The instability index(II)is 34.93 and has a hydrophilicity(GRAVY)of-0.487,which is a hydrophilic stable protein.Using a variety of software to predict the epitope of ?'-c protein,and referring to the physical and chemical properties of the protein,the linear epitope of ?--c protein in sturgeon roe was finally determined to be 46-52,69-78,84-90,137-151,the glycosylation site N78 in the glycoprotein,located in the predicted linear epitope region of the ?'-c protein of the sturgeon roe.The bioinformatics online software was used to predict the ?'-c protein domain and amino acid sequence alignment in different fish roe.The results showed that the ?'-c proteins from different sources belonged to the VWD region and were highly conserved in biological evolution.The sequence of ?'-c protein in different species was strong,and there were multiple regions with the same amino acid composition.It shows high homology and is prone to immune cross-reactivity. |
PDF Full Text Request