芝麻主要过敏原油质蛋白的基因克隆和原核表达  

Gene cloning and prokaryotic expression of the major allergen Oleosins from sesame seeds

作　　者：何凡[1] 邬玉兰[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：《中国油料作物学报》2010年第1期41-45,共5页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(30871752);深圳市深港创新圈项目;深圳大学校科研基金(200712;200807);国家质检总局项目

摘　　要：为克隆黑、白芝麻主要过敏原油质蛋白Oleosins的基因并在原核细胞中对其进行表达,本研究分别提取黑、白芝麻的总RNA,逆转录合成cDNA,根据序列设计简并引物,扩增芝麻主要过敏原油质蛋白Oleosins基因的完整开放阅读框,并与pET-28a载体连接,测序鉴定后转化大肠杆菌E.coliBL21(DE3)及用IPTG诱导表达。结果表明克隆获得黑、白芝麻主要过敏原油质蛋白Oleosins的全长基因约为438bp的开放阅读框(包括终止密码子),编码145个氨基酸。所编码的蛋白相对分子质量约为15.5kDa,为小分子量蛋白,等电点为5.18,与理论值相符。序列同源性分析发现其与数据库中已知的黑、白芝麻油质蛋白Oleosins基因同源性很高(GenBank黑、白芝麻油质蛋白基因登录号分别为EU999158和EU999159),并在DE3中高效表达。RT-PCR method was applied to clone the full-length of the major allergen oleosins gene from sesame seeds. The specific primers were designed and the ORF of oleosins from the sesame seeds was amplified and cloned into the expression vector pET-28a. The recombinant oleosins of sesame seeds was expressed in E.coli BL21（DE3）.Results indicated that the cloned cDNA ORF sequence contained 438bp encoding for 145 amino acids sequence.Sequence analysis showed that the encoded protein had a MW 15.5kDa and pI 5.18. The GenBank accession numbers of these clones were EU999158 and EU999159,which were almost identical with oleosins gene from sesame seeds. The deduced protein was therefore regarded as the major allergen of sesame seeds,which will be used to further study sesame seeds related allergy.

关 键 词：芝麻 油质蛋白 过敏原 基因克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]