金黄色葡萄球菌蛋白AB结构域的基因优化与原核表达及其亲和性鉴定  

Codon optimization,prokaryotic expression and affinity determination of B domain of staphylococcal protein A

作　　者：李迪[1] 曹永生[1] 徐晶晶[1] 王欢[1] 张润祥[1] 王君伟[1] 高明春[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2013年第3期277-282,共6页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2012BAD12B05;2012BAD12B03);现代农业(奶牛)产业技术体系项目(CARS-37);黑龙江省"十二五"科技攻关项目(GA09B302)

摘　　要：为高效表达可特异地与人及牛、山羊、兔、豚鼠、鸡等多种动物免疫球蛋白(Ig)结合的重组蛋白BB-SPA,以金黄色葡萄球菌蛋白A(staphylococcal protein A,SPA)免疫球蛋白结合结构域(B结构域)为基因优化设计对象,人工设计了SPA B结构域基因B-SPA,将其拆分为4条寡聚核苷酸片段形式,并采用重叠延伸PCR法合成该基因,构建重组表达载体pET30a-BB-SPA,转化至E.coli Rosetta(DE3)pLysS中表达重组蛋白BB-SPA,表达的重组蛋白分子质量约为14ku,相对表达量为56%。经Western-blotting与ELISA鉴定,证实了BB-SPA具有良好的生物活性。为多种动物免疫球蛋白的纯化、检测以及诊断试剂的开发提供了物质支持。For efficient expression of recombinant protein BB-SPA which could specifically combined with immunoglobulins（Igs） of human, cattle, goat, rabbit, guinea pig and chicken and so on, B-SPA gene encoding Ig combination domain（B domain） of staphylococcal protein A（SPA） was designed and split to four oligonucleotide fragments. The B-SPA gene was amplified by using overlap extension PCR. Recombi- nant expression vector pET30a-BB-SPA was constructed and transformed into Escherichia coli Rosetta （DE3） pLysS. Recombinant protein BB-SPA with molecular weight about 14 ku was expressed,and its rela- tive expression load was 56 %. Western-blot and ELISA results confirmed that BB-SPA had good biological activity. This study provided material support for purification and detection of a variety of animal immuno- globulins and development of diagnosis reagent.

关 键 词：免疫球蛋白结合分子 密码子优化 亲和层析纯化 活性分析 

分 类 号：S852.611[农业科学—基础兽医学]