一种获得高纯度包涵体蛋白的简便方法  

An Easy Way to Purify the Inclusion Body Protein with High Purity from Prokaryotic Expression Cells

作　　者：刘镕[1] 钟沁萍[1] 蒋明森[1] 董惠芬[1] 

机构地区：[1]武汉大学基础医学院人体寄生虫学教研室,武汉430071

出　　处：《中国寄生虫学与寄生虫病杂志》2011年第5期399-400,F0003,共3页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30872202);武汉大学博士研究生自主科研基金(No.57)~~

摘　　要：以日本血吸虫SjBMP基因部分编码序列构建SjBMP-pET-28a(+)重组原核表达质粒,并转化至大肠埃希菌(E.coli)BL21(DE3)进行原核表达。将经过鉴定的目的蛋白rSjBMP以包涵体形式表达的诱导菌样通过Ni2+-NTA Agarose亲和纯化和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)切胶再纯化。用该纯化蛋白制备免疫血清,用蛋白质印迹(Western blotting)检测其免疫反应性。结果显示,经Ni2+-NTA Agarose亲和纯化和SDS-PAGE切胶再纯化,获得高纯度的目的蛋白,回收率>11.0%。用该纯化蛋白免疫家兔制备免疫血清,获得的血清效价高于1∶1 280;Western blotting检测结果表明,用该免疫血清去识别表达的重组蛋白,出现特异的单一条带,表明该纯化蛋白仍保持其抗原性,可用于免疫学相关实验研究。因此,SDS-PAGE切胶纯化后电渗、透析回收是纯化重组包涵体蛋白有效、简便的方法。To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a（＋） plasmids,and then to transform them into the competent cells E.coli BL21（DE3）,finally a positive clone was used to be induced by IPTG.The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni2＋-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction.The purified protein was used to immune rabbits and make antiserum against the SjBMP,and the antiserum were then used to identify the rSjBMP by Western blotting.The target protein obtained by Ni2＋-NTA Agarose affinity purification was not pure with unspecific proteins,but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure,and the recovery rate was more than 11.0%.Meanwhile,Western blotting was used to identify the recombinant SjBMP protein by antiserum,only a specific single strip appeared,which suggested the protein purified by this method kept its antigenicity,and could be used for common immunological studies.Therefore,the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

关 键 词：重组蛋白 原核表达 包涵体 纯化 

分 类 号：R383.24[医药卫生—医学寄生虫学]