克氏原螯虾主要变应原原肌球蛋白的一个片段区基因的克隆表达、纯化及免疫原性鉴定  

The Cloning Expression,Purification and Characterization of a Fragment of Red Swamp Crayfish Major Allergen Tropomyosin

作　　者：易海涛[1] 夏立新[1] 刘芳[1] 闫浩[1] 黄钟[1] 汤慕瑾[1] 陈大玮[1] 刘志刚[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）》2011年第3期280-285,共6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(SZ2008105);深圳市深港创新圈项目;深圳大学创新团队基金(200904)资助项目

摘　　要：克隆克氏原螯虾主要过敏原原肌球蛋白的一个片段区基因,表达并纯化该蛋白,检测其免疫原性.提取克氏原螯虾总RNA,设计特异性引物,RT-PCR克隆得到目的基因;将目的基因连入pMD19-T载体,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;通过Ni2+亲和层析(FPLC)纯化目的蛋白;利用Western-blotting和ELISA检测该重组蛋白的免疫原性.克隆获得目的基因,其片段长为291 bp,编码97个氨基酸.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结果表明该蛋白与克氏原螯虾过敏病人混合血清中IgE结合,具有免疫原性.To clone,express and purify a fragment of red swamp crayfish major allergen tropomyosin and preliminarily characterize the allergenicity of purified recombinant protein.The interest gene 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.Ni2＋ chelating affinity chromatography was used to purify the recombinant protein.The allergenicity of interest protein was examined by Western-blotting and ELISA.The cloned interest gene which contained 291 bp and encoded 97 amino acids was authenticated to be the very fragment of red swamp crayfish major allergen tropomyosin.The recombinant interest protein,induced by IPTG,which is consistent with the actual value.The affinity between recombinant interest protein and IgE antibodies from pooled red swamp crayfish allergic patients serum was identified by Western-blotting and ELISA.

关 键 词：克氏原螯虾 原肌球蛋白 变应原 克隆表达 纯化 免疫印迹 酶联免疫吸附测定 

分 类 号：Q785[生物学—分子生物学]