花生是较常见的重要食物过敏原之一.本研究采用石油醚对花生进行脱脂处理,利用硫酸铵盐析、透析、葡聚糖凝胶(Sephadex G-150)分离纯化花生蛋白,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)法确定花生主要蛋白分子量为15、20、35和60 ku左右,Western-Blotting免疫印迹试验确定花生过敏原性蛋白分子量为63.5 ku;通过质谱测序得到31条有效蛋白肽段,其中5条肽段的谱图数较高,占总谱图数的49.6％,分别是KLEYDPRCVYDTGAT、AFNAEFNEIRRVLLEE、DNVIDQIEKQAK、PYSPSQDPDRRDPY和QDPYSPSQDPDR,并以此推断这些肽段为花生致敏蛋白Ara h 1和Ara h 2的主要肽段过敏序列,成功运用蛋白肽段推断与之对应的蛋白质种类,为进一步明确花生过敏原的致敏机理奠定基础.但花生过敏蛋白构象结构复杂,需要通过其他手段进一步开展研究工作.
Isolation,extraction and predictive analysis of peanut allergenic protein
Peanut is one of the most common and important food allergens.In this paper,peanut oil was degreased by petroleum ether and purified by ammonium sulfate salting out,dialysis and Sephadex G-150 dextran gel column.The molecular weight of peanut protein was determined by SDS-PAGE gel electrophoresis:15,20,35 and 60 ku.Western-Blotting immunoblot test showed that peanut allergy was around 63.5 ku.31 effective protein peptides were obtained by mass spectrometry sequencing.Among them,5 peptides had higher number of spectra,namely,KLEYDPRCVYDTGAT,AFNAEFNEIRRVLLEE,DNVIDQIEKQAK,PYSPSQDPDRRDPY and QDPYSPSQDPDR,which accounted for 49.6％of the total number of spectra.Based on this,it is inferred that those peptides are the main peptide allergic sequences of peanut sensitizing proteins Ara h 1 and Ara h 2.The protein peptides are successfully used to infer the corresponding protein types,which lays a foundation for further clarifying the sensitization mechanism of peanut allergens.Since the conformational structure of peanut allergic protein is complex,further research work needs to be carried out by other means
peanutallergic proteinseparation and purificationprotein peptide
NETL
NSTL
万方数据
