pTWIN1-EgAgB8/3自剪切融合蛋白原核表达载体的构建及表达  

Construction and expression of protein self-splicing prokaryotic expression vector pTWIN1-EgAgB8/3

作　　者：张瑞妮[1] 吾拉木·马木提[2] 于春洋[2] 法蒂玛·木特力甫[1] 

机构地区：[1]新疆医科大学基础医学院寄生虫教研室,乌鲁木齐830011 [2]新疆医科大学第一附属医院包虫病重点实验室

出　　处：《中国人兽共患病学报》2013年第2期154-158,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(No.81160202)资助~~

摘　　要：目的表达获得较纯的细粒棘球绦虫AgB8/3重组抗原(rEgAgB8/3)。方法根据GeneBank登陆号(AF362442)下载目的基因核酸序列,利用DNAman软件设计引物,对EgAgB8/3编码分泌型多肽片段的核酸序列进行PCR扩增,测序鉴定其正确性后定向连入原核表达质粒pTWIN1上,并转化至大肠杆菌ER2566,IPTG诱导表达CBD-intein1-EgAgB8/3融合蛋白后进行纯化,用SDS-PAGE电泳和western blot分析鉴定融合蛋白与目的蛋白rEgAgB8/3的表达量和纯度。结果成功克隆获得EgAgB8/3基因目的片段和具有蛋白自剪切功能的重组表达载体pTWIN1-EgAgB8/3,并鉴定其表达的融合蛋白主要以可溶形式存在;构建的重组载体中融合标签的几丁质结合域(CBD)和内含肽(intein1)分别使亲和层析纯化和融合标签自剪切一步完成。结论成功的构建重组表达载体pTWIN1-EgAgB8/3,获得高表达融合蛋白CBD-intein1-EgAgB8/3,并经简单后续处理即可获得含极少任何额外氨基酸的可溶性目的蛋白rEgAgB8/3,尽可能保证了其原有的结构和活性,为抗rEgAgB8/3特异性单克隆抗体的快速制备奠定了基础。The objective was to establish expression vector pTWIN1-AgB8/3 of a self-splicing prokaryotic expression system and obtain pure recombinant antigen of Echinococcu. granulosus AgB8/3 （rEgAgBS/3）. The specific primers were de- signed according to the published nucleotide sequence of EgAgB8/3 deposited in the GenBank database（AF362442）. The nucle- otide sequence corresponding to the secreted form of EgAgBS/3 was amplified by PCR, and the product was directionally liga- ted into Prokaryotic Expression Vector pTWIN1. The ligation was transferred into the competent cell ER2566, the fusion pro- tein CBIYinteinl-EgAgB8/3 was expressed by inducing with IPTG, and the target protein rEgAgBS/3 was purified by chitin binding affinity purification column. The expression product and the target protein were analyzed by SDS-PAGE and western blot. Results showed that the EgAgBS/3 gene was successfully cloned and ligated into pTWIN1 to form protein self-splicing prokaryotic recombimant expression vector pTWlN1-EgAgBS/3. The SD^PAGE and western blot analysis showed that the fu- sion protein CBD-inteinl-EgAgB8/3 was expressed as soluble form. The chitin binding domain （CBD） and inteinl were re- moved from target protein rEgAgBS/3 at one step during the chitin column affinity purification. It suggested that the high level of soluble target protein rEgAgBS/3 with few additional amino acid residue was successfully purified by simple treatment after expression, which may allow us to keep the original amino acid sequence and possible activity of the target protein and facilitate the process of production of anti-rEgAgB8/3 monoclonal antibodies.

关 键 词：棘球绦虫 EgAgB8/3 pTWIN1表达载体 诱导表达纯化 WESTERN BLOT 

分 类 号：R383.13[医药卫生—医学寄生虫学]