Epitopes Mapping Of The Main Egg Allergens Identified By The Sera From Chinese Population Of Egg Allergy

Egg is one of the main allergic food that can cause allergies in infants and children.The incidence of egg allergy in children under 5 years old is about 2%.The material basis of egg allergy is the allergenic epitopes in egg allergens.Epitopes,also known as the antigenic determinants,are the special chemical groups in allergens that binding to antibodies or are recognized by the cell surface receptors and induced the immune re-sponse.According to the spatial structure,epitopes can be divided into linear epitopes and conformational epitopes.According to the different recognition receptor cells,epitopes can be divided into T-cell epitopes and B-cell epitopes.The conformational epitopes are more easily destroyed than linear epitopes due to the changes in the spatial structure of the allergens caused by processing.B-cell epitopes are the epitopes that can be recognized and bound to each other by the B-cell surface receptors or the spe-cific antibodies secreted by B cells.Two types of linear B-cell epitopes are important in allergic reactions,which are named the linear B-cell IgG-binding epitopes and linear B-cell IgE-binding epitopes.In addition,epitopes are specific,and the epitope identi-fication results will be different according to the different study populations.Therefore,mapping the linear B-cell epitopes of egg allergen for the Chinese population is of great significance for a deeper understanding of egg allergy in China.At present,the epitopes mapping of ovotransferrin using the Chinese egg-allergic patients has been carried out,while the epitopes of the other three main allergens in egg white have yet to be identified.In this study,the linear B-cell epitopes of ovomucoid,ovalbumin and lysozyme were first predicted by bioinformatics technology and then identified using the peptide scanning mapping technology based on the Chinese egg-allergic population,and the dominant epitopes of lysozyme were further identified.The main research methods,results and conclusions were as follows:（1）Five bioinformatics tools,which were DNAStar,Immunomedicine Group,Bepi Pred,Bcepred and ABCpred,were chosen to analyze the amino acid sequence of three egg allergens to forecast the potential linear B-cell epitopes.Six linear B-cell epitopes of ovomucoid were predicted by bioinformatics tools,which were AA8-32（FPNATDKEGKDVLVCNKDLRPICGT）,AA52-58（TNISKEH）,AA66-90（VPMNC SSYANTTSEDGKVMVLCNRA）,AA102-104（YDN）,AA113-144（VEQGASVDK RHDGGCRKELAAVSVDCSEYPKP）and AA148-161（AEDRPLCGSDNKTY）.Fourteen linear B-cell epitopes of ovalbumin were predicted,which were AA42-45（V YLG）,AA47-51（KDSTR）,AA58-60（VRF）,AA70-78（IEAQCGTSV）,AA92-99（TK PNDVYS）,AA110-124（ERYPILPEYLQCVKE）,AA140-142（DQA）,AA151-154（E SQT）,AA158-168（IRNVLQPSSVD）,AA188-194（AFKDEDT）,AA199-208（FRVT EQESKP）,AA264-269（KLTEWT）,AA272-278（NVMEERK）and AA287-296（KME EKYNLTS）.Four linear B-cell epitopes of lysozyme were predicted,which were AA18-21（DNYR）,AA39-51（NTQATNRNTDGST）,AA62-78（WWCNDGRTPGSR NLCNI）and AA109-117（VAWRNRCKG）.（2）The specific antibodies in 78 sera of the Chinese egg-allergic patients were screened by indirect ELISA.The positive rates of s IgE were ovomucoid（30/78,38.4%）,ovalbumin（37/78,47.4%）,ovotransferrin（24/78,30.7%）and lysozyme（36/78,46.1%）,and the positive rates of s IgG were ovomucoid（69/78,88.4%）,ovalbumin（70/78,89.7%）,ovotransferrin（50/78,64.1%）and lysozyme（61/78,78.2%）,respectively.（3）According to the principle of peptide scanning,the peptide fragments that can specifically bind to IgE and IgG in the sera pool of ovomucoid-allergic patients were identified using the dot-blot method.Nine linear B-cell IgE-binding epitopes and four IgG-binding epitopes of ovomucoid were identified,which were AA6-16（SRFPNAT DEKG）,AA15-22（KGKDVLVC）,AA24-34（KDLRPICGTDG）,AA33-43（DGVTY TNDCLL）,AA48-58（IEFGTNISKEH）,AA75-85（NTTSEDGKVMV）,AA84-94（M VLCNRAFNPV）,AA123-133（HDGGCRKELAA）and AA171-181（VVESNGTLTL S）for the IgE-binding epitopes,and AA75-85（NTTSEDGKVMV）,AA84-94（MVLC NRAFNPV）,AA144-154（PDCTAEDRPLC）and AA174-184（SNGTLTLSHFG）for the IgG-binding epitopes,respectively.Among these two kinds of epitopes,AA75-85（NTTSEDGKVMV）,AA84-94（MVLCNRAFNPV）and AA174-181（SNGTLTLS）were their common epitopes.In addition,epitope AA75-85 was the new IgG-binding epitope that had not been reported.（4）According to the principle of peptide scanning,the peptide fragments that can specifically bind to IgE and IgG in the sera pool of ovalbumin-allergic patients were identified using the dot-blot method.Four linear B-cell IgE-binding epitopes and four IgG-binding epitopes of ovalbumin were identified,which were AA84-91（LRDILNQ I）,AA129-139（GLEPINFQTAA）,AA213-220（YQIGLFRV）and AA360-370（RADH PFLFCIK）for the IgE-binding epitopes,and AA27-37（NIFYCPIAIMS）,AA72-82（A QCGTSVNVHS）,AA291-301（KYNLTSVLMAM）and AA360-370（RADHPFLFCI K）for the IgG-binding epitopes,respectively.Among these two kinds of epitopes,AA360-370（RADHPFLFCIK）was their common epitope.In addition,epitopes AA84-91,AA213-220 and AA360-370 were the new IgE-binding epitopes that had not been reported,and epitopes AA27-37,AA72-82,AA291-301 and AA360-370 were the new IgG-binding epitopes that had not been reported.（5）According to the principle of peptide scanning,the peptide fragments that can specifically bind to IgE and IgG in the sera pool of lysozyme-allergic patients were identified using the dot-blot method.Seven linear B-cell IgE-binding epitopes and six IgG-binding epitopes of lysozyme were identified,which were AA3-13（FGRCELAA AMK）,AA18-22（DNYRG）,AA30-34（CAAKF）,AA63-73（WCNDGRTPGSR）,AA75-79（LCNIP）,AA84-91（LSSDITAS）and AA115-117（CKG）for the IgE-binding epitopes,and AA3-13（FGRCELAAAMK）,AA31-36（AAKFES）,AA57-64（INSRW WCN）,AA75-79（LCNIP）,AA88-93（ITASVN）and AA115-117（CKG）for the IgG-binding epitopes,respectively.Among these two kinds of epitopes,AA3～13（FGRCE LAAAMK）,AA31-34（AAKF）,AA75-79（LCNIP）,AA88-91（ITAS）and AA 115-117（CKG）were their common epitopes.In addition,epitopes AA30-34 and AA84-91 were the new IgE-binding epitopes that had not been reported,and epitopes AA3-13,AA31-36,AA57-64,AA75-79,AA88-93 and AA115-117 were the new IgG-binding epitopes that had not been reported.（6）According to the principle of peptide scanning,the peptide fragments that can specifically bind to IgE and IgG in the single serum of lysozyme-allergic patients were identified using the dot-blot method.Three dominant linear B-cell IgE-binding epitopes and two dominant IgG-binding epitopes of lysozyme were identified,which were AA24-34（SLGNWVCAAKF）,AA81-91（SALLSSDITAS）and AA102-112（GNGM NAWVAWR）for the dominant IgE-binding epitopes,and AA30-37（CAAKFESN）and AA84-94（LSSDITASVNC）for the dominant IgG-binding epitopes,respectively.Among these two kinds of epitopes,AA30-34（CAAKF）and AA84-94（LSSDITAS）were their common epitopes.（7）The key amino acids in the dominant linear B-cell epitopes of lysozyme were predicted using Bioedit software.Twenty-one key amino acids in the linear B-cell IgE-binding epitope were predicted,which were ²⁴S,²⁵L,²⁸W,²⁹V,^31-32A,³⁴F,⁸¹S,⁸²A,^83-84L,^85-86S,⁹⁰A,⁹¹S,¹⁰⁵M,¹⁰⁷A,¹⁰⁸W,¹⁰⁹V,¹¹⁰A and ¹¹¹W,respectively.Six key amino acids in the linear B-cell IgG-binding epitopes were predicted,which were ³⁴F,³⁵E,³⁶S,^85-86S and ⁹¹S,respectively.In conclusion,the linear B-cell IgE-binding epitopes and IgG-binding epitopes of ovomucoid,ovalbumin,lysozyme,and the dominant epitopes of lysozyme were mapped using the sera from the Chinese egg-allergic population in this thesis,which filled the domestic gap,and provided important information for the further understand-ing of the structure and allergenicity of egg allergens.