经基因工程改造的花生主要过敏原Ara h 2制备及其低致敏原性鉴定  

Preparation and Characterization of the Genetically Engineered Hypoallergenic Derivative of the Major Peanut Allergen Ara h 2

作　　者：易海涛[1,2] 刘芳 贾梦阳[4] 汤慕瑾 曹小勇 夏立新[2,4] 刘志刚[2,4] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060 [2]深圳大学医学院过敏反应与免疫学研究所,广东深圳518060 [3]新疆华世丹药业股份有限公司,新疆乌鲁木齐830000 [4]深圳大学医学院,广东深圳518060 [5]深圳市出入境检验检疫局,广东深圳518045 [6]福田区妇幼保健院,广东深圳518000

出　　处：《江西农业大学学报》2011年第5期993-998,共6页Acta Agriculturae Universitatis Jiangxiensis

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)

摘　　要：构建经基因工程改造的Ara h 2表达载体,表达并纯化该蛋白,鉴定其过敏原性。将花生主要过敏原Arah 2基因序列进行颠换,并将颠换后的序列进行合成,再将合成后的基因克隆到原核表达载体pET-32a(+)上,然后转入Origami宿主表达菌中;利用Isopropylβ-D-1-Thiogalactopyranoside(IPTG)诱导表达;通过Ni2+亲和层析纯化目的蛋白;Western blotting和ELISA鉴定该重组蛋白的过敏原性。测序结果表明合成后的序列成功整合到原核表达载体pET-32a(+)上。重组的目的蛋白纯化后经SDS-PAGE鉴定,蛋白大小与理论值相符。Western blotting和ELISA结果均表明经基因工程改造的Ara h 2(F-Ara h 2)蛋白与重组的Ara h 2(R-Ara h 2)蛋白相比,结合花生过敏患者混合血清中IgE显著降低。成功构建经基因工程改造的Ara h 2表达载体,初步的体外实验表明该基因表达的重组蛋白具有低致敏原性。The genetically engineered major allergen Ara h 2 was expressed and purified and the hypoallergenic of purified recombinant F-Ara h 2 protein was characterized.The Ara h 2 was reassembled and inserted into the expression vector pET-32a（＋）.The vector was transformed into Origami and the protein expression was induced by IPTG.Ni2＋ Chelating affinity chromatography was used to purify the recombinant F-Ara h 2 protein.The hypoallergenic of F-Ara h 2 was examined by Western blotting and ELISA.The ORF which contained 471 bp and encoded 157 amino acids was authenticated to be F-Ara h 2.The recombinant F-Ara h 2 protein induced by IPTG was consistent with the actual value.The affinity between recombinant F-Ara h 2 protein and IgE antibodies from pooled peanut-allergic patients serum was significantly decreased compared with R-Ara h 2 was identified by Western blotting and ELISA.Recombinant F-Ara h 2 protein was hypoallergenic.

关 键 词：基因工程 花生 过敏原 Arah2 低致敏 

分 类 号：Q786[生物学—分子生物学]