凡纳滨对虾过敏原蛋白Lit v 1.2的原核表达与纯化  

Expression and purification of Litopenaeus vannamei allergen protein Lit v1. 2

作　　者：陈惠芳[1] 赖荷[1] 黄于艺[1] 邹泽红[1] 何颖[1] 陶爱林[1] 李文[1] 

机构地区：[1]广州医科大学附属第二医院广东省过敏反应与免疫重点实验室/呼吸疾病国家重点实验室,广州510260

出　　处：《中国免疫学杂志》2015年第12期1659-1662,共4页Chinese Journal of Immunology

基　　金：国家重大科技专项(2014ZX08011-005B);广东高校科技创新项目(2013KJCX148);广州市科技攻关(201300000159)项目的资助

摘　　要：目的:诱导表达凡纳滨对虾(Litopenaeus vannamei)过敏原蛋白Lit v 1.2,并利用6-His标签获得纯化蛋白。方法:用NdeⅠ和PstⅠ双酶切实验室前期构建保存的p GEM-T-Lit v 1.2质粒,将目的基因与表达载体p ET44a连接,转入表达菌株Rosetta,通过氨苄青霉素(Amp)抗性基因筛选,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得Lit v 1.2蛋白。通过亲和层析纯化蛋白,SDS-PAGE凝胶电泳检测纯化效果。结果:成功构建了重组质粒p ET44a-Lit v 1.2,SDS-PAGE结果显示Lit v1.2基因在大肠杆菌Rosetta中获得高效的可溶性表达,蛋白质分子量与理论值相符,且获得的目标蛋白纯度高。结论:本研究通过原核表达及亲和层析获得高产量、高纯度的凡纳滨对虾过敏原蛋白Lit v 1.2,为进一步研究Lit v 1.2的免疫学特性奠定基础。Objective： To obtain purified recombinant Litopenaeus vannamei allergen protein Lit v 1. 2. Methods： The target gene of Lit v 1. 2 was inserted into clone vector p GEM-T and then ligated to the expression vector p ET44 a. The p ET44a-Liv 1. 2 was transformed into Rosetta and screened by ampicillin resistance. The recombinant protein was expressed by IPTG induction. The protein was purified by 6-His tag affinity chromatography and the purification was analyzed by SDS-PAGE gel electrophoresis. Results： The expression plasmid p ET44a-Lit v 1. 2 was constructed. SDS-PAGE showed that expressed Lit v 1. 2 was efficient and soluble in E. coli Rosetta. The protein molecular weight was consistent with the theoretical value. The highly purified target protein was obtained. Conclusion： In this study,we successfully gained highly purified recombinant allergen protein Lit v 1. 2 which was expressed in prokaryotic system and purified by affinity chromatography column. The purified Lit v 1. 2 protein will facilitate us to further study its role in immunological responses.

关 键 词：凡纳滨对虾 过敏原 原肌球蛋白 重组表达 蛋白纯化 

分 类 号：R392.11[医药卫生—免疫学]