刺苋花粉泛过敏原Profilin的抗原性评估与三级结构分析

Allergenicity evaluation and three dimensional structure analysis of pollen panallergen Profilin from Amaranthus spinosus L.

目的克隆刺苋花粉中Profilin蛋白基因，分析同源序列中不同性质氨基酸对抗原性及三级结构的贡献。方法基于生物信息学分析已知泛过敏原Profilin的氨基酸序列并获得核心代表序列，以之为基础设计合成引物，采用Touchdown PCR技术从刺苋花粉的eDNA池中进行基因克隆，并经菌落PCR和双酶切验证；采用生物信息学软件MULTIPRED及SWISS-MODEL在线软件对所得基因编码蛋白进行抗原性评估及三级结构模拟。结果从刺苋中获得两个序列不同的泛过敏原基因，分别命名为PRF7和PRF23。蛋白质三级结构显示：PRF7与已知的泛过敏原Profilin存在少数氨基酸的差异，其空间结构与抗原性没有明显变化；而PRF23与北方豚草花粉泛过敏原Q64LH0之间相似程度较低，而空间结构也呈现明显的差异；尽管Q64LH0与PRF23的全序列抗原性均值差异无统计学意义，受一些区段上不同性质的氨基酸改变的影响，PRF23在这些区段上的抗原性显著低于Q64LH0的抗原性。结论基于Q64LH0和PRF23同源氨基酸序列的抗原性评估及三级结构分析，获得了不同性质氨基酸对抗原性及三级结构的贡献等信息，映射了南方花粉过敏症发生率较低的原因所在，也为过敏原遗传改良过程中进行氨基酸置换指明了方向。

Objective To clone and characterize Profilin encoding genes in Amaranthus spinosus and to analyze the contribution of different amino acids in isoallergens to allergen antigenicity and tertiary structure. Methods The primers were designed according to the core sequences which were obtained by bioinformatic analysis of the known Profilin amino acid sequences, followed by gene cloning from the Amaranthus spinosus cDNA pool and subsequent confirmation by double-digestion, colony PCR and DNA sequencing. Antigenicity evaluation and tertiary structural modeling of the encoded protein were accomplished by online software MULTIPRED and SWISS-MODEL, respectively. Results Two Panallergenic genes, named as PRF7 and PRF23, were acquired from Amaranthus spinosus. Sequence and structure analysis demonstrated that there was some discrepancy in tertiary structures of the encoded proteins, besides distinct difference in their amino acid sequences. PRF7 exhibited high homology with panallergen Profilins Q64LHO, with the identities 98%, whereas the homology of PRF23 and Q9XF42 （apple allergen） was 81%. Q64LHO and PRF23 were modeled as 3nulA （Q42449） and lg.SuB （Q9LEI8）, respectively. PRF23 exhibited distinct three dimensional structural difference in certain fragments compared with Q64LHO and other Profilins. Though the average values of antigenicity displayed no difference between Q64LHO and PRF23 on whole sequences, the antigenicity of PRF23 on certain fragments was obviously lower than that of Q64LHO because of the alteration of some amino acids with different characters, implying the cause of lower incidence of hay fever in South China than in North China. Conclusion Based on sequence analysis, antigenicity evaluation and tertiary structural modeling for Q64LHO and PRF23, we obtained lots of useful information about the contribution of different amino acids to antigenicity and protein structures, thus would facilitate allergen genetic improvement by amino acid replacement.