鹰嘴桃过敏原的鉴定及其生物学特性分析

Identification and Analysis of Biological Characteristics of Allergens in Olecranon Peach

【目的】鹰嘴桃是岭南地区的特色水果,因其良好的风味和品质被评为“岭南十大佳果”。通过分析鹰嘴桃过敏原蛋白的抗原表位,为过敏原重组抗原的制备提供研究基础,也为阐述食品加工过程中鹰嘴桃过敏原的特性变化和致敏特性提供研究依据。【方法】利用磷酸缓冲液提取鹰嘴桃冻干粉中的粗蛋白,采用SDS-PAGE蛋白电泳方法鉴定并联合蛋白质谱方法分析粗蛋白中存在的过敏原,通过蛋白数据库UniProt进行筛选比对,并采用生物信息学方法分析致敏蛋白的理化性质、空间结构和抗原表位等生物学特性。【结果】从鹰嘴桃中鉴定出7种致敏蛋白(A0A251RBV3、P86888、M5X697、M5WV03、M5WTQ8、Q2I6V8、Q9LED1),主要归属于病程相关蛋白(Pru p 1)、类甜蛋白(Pru p 2)、非特异性脂质转移蛋白家族(Pru p 3)和赤霉素调节蛋白(Pru p 7)4类过敏原蛋白。7种鹰嘴桃过敏原蛋白具有较高稳定性,分子量为6.91~26.04 kD,脂肪族氨基酸指数为29.37~81.54。M5WTQ8和Q2I6V8过敏原蛋白为酸性蛋白,其余过敏原蛋白为碱性蛋白。除Q9LED1蛋白外,其余过敏原蛋白均为亲水蛋白。筛选抗原表位、亲水性和柔韧性大于0且表面可及性大于1的区域,并结合二、三级蛋白结构分析蛋白键能较低的区域,获得鹰嘴桃过敏原的抗原表位分别为Pru p 1(EIP、GSQ、KEN、NL、KG、EIK、HPD)、Pru p 2(TGDQKPQ、SP、NQ、PPNDKPETCPPT、DDKSS、RP)、Pru p 3(RT、VN)和Pru p 7(AGY、GTYGN、LKNSKGN)。【结论】通过对鹰嘴桃过敏原蛋白的结构、亲水性、表面可及性、柔韧性和抗原指数进行分析,获得过敏原蛋白的多个抗原表位,可为鹰嘴桃过敏原在食品加工中的致敏特性研究提供研究基础。

【Objective】The olecranon peach is known as one of the characteristic fruits in Lingnan region because of its good flavor and quality,and it is listed as“The top ten best fruits in Lingnan”.However,less studies on its allergens have been reported.By analyzing the epitope of allergen protein,it would provide a research basis for the preparation of the recombinant antigen of the allergen,and also provide a research basis for explaining the characteristic changes and sensitization characteristics of the allergen in the process of food processing.【Method】Phosphate buffer saline was used to extract the crude protein from the lyophilized powder of olecranon peach,and the allergens in the crude protein were identified by SDSPAGE protein electrophoresis and analyzed by mass spectrometry.The allergens were screened and compared by the protein database of UniProt,and the physicochemical properties,spatial structure and antigenic epitopes of the allergens were analyzed by bioinformatics methods.【Result】Seven kinds of allergenic proteins including A0A251RBV3,P86888,M5X697,M5WV03,M5WTQ8,Q2I6V8,and Q9LED1 were identified from olecranon peach,which belonged to the four classes of allergenic proteins,namely,pathogenesis-related proteins(Pru p 1),thaumatin-like proteins(Pru p 2),non-specific lipid transfer proteins(Pru p 3)and gibberellin-regulated proteins(Pru p 7).The seven kinds of allergenic proteins in olecranon peach had high stability,their molecular weight is 6.91-26.04 kD,with the aliphatic index between 29.37 and 81.54.M5WTQ8 and Q2I6V8 allergen proteins were acidic proteins,while the remaining allergen proteins were alkaline proteins.Except for Q9LED1 protein,all other allergen proteins were hydrophilic proteins.The regions with antigenic epitopes,hydrophilicity and flexibility greater than 0 and surface accessibility greater than 1 were screened,and regions with low protein bonding energy were analyzed by binding the secondary and tertiary structures of proteins to obtain the antigenic epitopes of allergens,including Pru p1(EIP,GSQ,KEN,NL,KG,EIK,HPD),Pru p 2(TGDQKPQ,SP,NQ,PPNDKPETCPPT,DDKSS,RP),Pru p 3(RT,VN)and Pru p 7(AGY,GTYGN,LKNSKGN).【Conclusion】By analyzing the structure,hydrophilicity,surface accessibility,flexibility and antigen index of olecranon peach,multiple epitopes of 7 allergens are obtained,which can provide a research basis for the sensitization characteristics of olecranon peach allergens in food processing.