摘要
构建序列重新组合的Ara h 2表达载体,表达并纯化该蛋白,鉴定其低致敏原性。将Ara h 2基因进行合理的缺失和重排,并将重排后的基因T-Ara h 2克隆到原核表达载体pET-32a(+)上,然后转入Origami宿主表达菌中;再用IPTG诱导其表达;通过Ni2+亲和层析(FPLC)纯化目的蛋白;Western-blotting和ELISA鉴定该重组蛋白的低致敏原性。测序结果表明重排后的序列成功克隆到原核表达载体pET-32a(+)上。重组蛋白纯化后经SDS-PAGE鉴定,目的蛋白大小与理论值相符。Western-blotting和ELISA结果均表明:T-Ara h 2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显著降低。成功构建了基因重排的Ara h 2表达载体,该重组蛋白具有良好的低致敏原性。
The present study aims to express and purify a novel peanut major allergen Ara h 2 and analyze the hypoallergenic of purified recombinant T-Ara h 2 protein. The Ara h 2 was initiadly reassembled and then inserted in to the expression vector pET-32a（＋）. The vector was transformed into Escherichia coll. Origami and the protein expression was induced by IPTG. Niz＋ chelating affinity chromatography was used to purify the recombinant T-Ara h 2 protein. The hypoallergenic of T-Ara h 2 was analyzed by Westernblotting and ELISA. The ORF （containing 453 bp and encoded 151 amino acids） was confirmed to be T-Ara h 2. The molecular weight recombinant T-Ara h 2 protein is consistent with the predicted value. The affinity （between recombinant T-Ara h 2 protein and IgE antibodies from pooled peanutallergic patients serum） was significanly decrease compared with R-Ara h 2 in this process. Recombinant T-Ara h 2 protein was obtained with hypoallergenic.
出处
《南昌大学学报（理科版）》
CAS
北大核心
2011年第3期279-283,共5页
Journal of Nanchang University(Natural Science)
基金
国家自然科学基金(30871752)
深圳出入境检验检疫局科技计划项目(SZ2008105)
深圳大学创新团队基金资助项目(200904)
关键词
花生
设计和鉴定
过敏原
ARA
H
2
低致敏原衍生物
arachzs hypogaea L.
designing and characterization~ allergen~ Ara h 2 ~ hypoallergenic derivative
