Ara h2三聚体重组蛋白的制备及其低致敏原性鉴定

Preparation and hypoallergenic characterization of Ara h 2 Trimer

目的制备花生主要过敏原Ara h 2三聚体重组蛋白并检测其过敏原性。方法利用分子生物学的方法将3分子的Ara h 2依次串联起来,并将其整合到原核表达载体pET-32a(+),再转化到感受态Origami中;然后利用IPTG诱导其表达;通过Ni2+亲和层析纯化三聚体重组蛋白;Western-blotting和ELISA检测目的蛋白的过敏原性。结果测序结果表明Trimer成功整合到pET-32a(+)上。三聚体重组蛋白纯化后经SDS-PAGE鉴定,蛋白大小与理论值相符。Western-blotting和ELISA结果表明Trimer与重组的Ara h 2(r-Ara h 2)蛋白相比,结合花生过敏病人混合血清中IgE的能力有所降低。结论成功制备花生主要过敏原Ara h 2三聚体重组蛋白,初步的体外实验表明该重组蛋白具有低致敏原的潜能。

This study is aimed to express and purify the peanut major allergen Ara h 2 Trimer and preliminarily characterize the hypoallergenic of purified recombinant trimer protein.Three Ara h 2 molecules were connected and inserted into the expression vector pET-32a（＋）.Then the vector was transformed into Origami and the protein expression was induced by IPTG.The recombinant trimer protein was purified by Ni2＋ chelating affinity chromatography.And the hypoallergenic of Ara h 2 trimer protein was examined by Western-blotting and ELISA.The ORF containing 1434 bp and encoding 477 amino acids was authenticated to be Ara h 2 Trimer.The recombinant Ara h 2 Trimer protein,induced by IPTG,is consistent with the actual value.Western-blotting and ELISA pointed that the affinity of recombinant Ara h 2 trimer protein to IgE antibodies from pooled peanut-allergic patients serum was significant descended as compared with r-Ara h.All the results indicate that Recombinant Ara h 2 Trimer protein with hypoallergenic is obtained in this study.