花生过敏原Ara h2.02的克隆、表达及免疫学鉴定  

Cloning,Expression and Characterization of Peanut Allergen Ara h2.02

作　　者：易海涛[1] 刘志刚[1] 闫浩[1] 刘芳[1] 赵郭存[1] 夏立新[1] 

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,广东深圳518060

出　　处：《江西师范大学学报（自然科学版）》2010年第5期531-535,共5页Journal of Jiangxi Normal University(Natural Science Edition)

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);深圳市深港创新圈和深圳大学创新团队基金资助项目

摘　　要：通过提取花生总RNA,设计特异性引物,RT-PCR克隆花生Ara h2.02基因,将反转录的基因连入pMD19-T Simple Vector,提质粒酶切鉴定并测序,将测序正确的片段连入原核表达载体pET-32a(+)上,并转入BL21(DE3)宿主表达菌中,IPTG诱导表达、Western-blotting检测该重组蛋白的免疫原性.测序结果表明:克隆的花生Ara h2.02基因片段全长为519 bp,编码为172个氨基酸,与GenBank中蛋白序列100%相同.诱导表达后的蛋白经SDS-PAGE鉴定,目的蛋白大小与理论值相符.Western-blotting结果表明该蛋白能与花生过敏病人混合血清中的IgE结合,具有免疫原性.成功克隆并表达了花生过敏原Ara h2.02,该基因表达的重组蛋白具有良好的免疫原性.The ORF of Ara h2.02 was cloned and inserted into the expression vector pET-32a（＋）.The vector was transformed into Escherichia coli BL21（DE3） and the protein expression was induced by IPTG.The allergenicity of Ara h2.02 was identified by Western-blotting.The cloned ORF which contained 519 bp and encoded 172 amino acids was authenticated to be Ara h2.02.The recombinant Ara h2.02,induced by IPTG,which is consensus with the actual value.The affinity between recombinant Ara h2.02 and IgE antibodies from pooled peanut-allergic patients serum was identified by Western-blotting.Peanut recombinant Ara h2.02 protein was obtained with allergenicity.

关 键 词：花生 过敏原 Arah2.02 克隆 表达 免疫印迹 

分 类 号：Q785[生物学—分子生物学]