重组蛋白A在大肠杆菌中的分泌表达及活性测定  

High Level Secretory Expression and Biological Assay of Recombinant Protein A in E.coli

作　　者：张秀华[1] 刘素丽 刘彬[1] 邢芸[1] 范豪 曹荣月[1] 刘景晶[1] 

机构地区：[1]中国药科大学天然药物活性物质与功能国家重点实验室,江苏南京210009 [2]浙江一就生物医药有限公司,浙江平湖314200

出　　处：《药物生物技术》2012年第1期11-15,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(30772570;30872393);中央高校基本科研业务费专项资金资助(JKY2009021;JKQ2009022)

摘　　要：利用大肠杆菌表达系统表达金黄色葡萄球菌蛋白A,根据大肠杆菌密码子偏好性对金黄色葡萄球菌蛋白A的基因序列进行密码子优化,在N端加入L-门冬酰胺酶(L-AsPsII)信号肽简并基因序列使其分泌表达,将全合成的蛋白A基因片段插入到pET-28a载体中,转化入BL21(DE3)。乳糖诱导使其表达,其中80%以上目的蛋白位于胞外,表达量达到375 mg/L,剩余目的蛋白位于周质。将胞外目的蛋白超滤浓缩,通过DEAE阴离子交换剂,一步纯化后得到的蛋白,在SDS-PAGE上显示为约32 k的单一条带,纯度达95%以上。利用酶联免疫吸附试验(ELISA)检测重组蛋白A活性较高,能与小鼠、兔及羊IgG抗体高效结合,且沸水煮10 min后仍能保持同IgG结合的活性。To express staphylococcal protein A（SPA） in E.coli expression system at a high level,L-AsPsII signal peptide was added ahead of protein A gene containing E,D,A,B,C domains.Synthetic recombinant protein A gene,of which the rare codon was substituted,was inserted into pET28a and then transformed into E.coli BL-21（DE3）.The recombinant pET28a-Pro A plasmid was expressed at a high level in BL21（DE3） by lactose induction,more than 80% of the recombinant protein A was secreted into the culture medium and the expression level was up to 375 mg/L.The culture medium containing protein A was ultrafiltrated and purified by DEAE.Using the one step purification,the purity of target protein was up to 95% and there was only one band at 32 ku by SDS-PAGE.The recombinant protein A had a high activity detected by ELISA,even boiled for 10 min,and it also had high affinity with different IgGs from mouse,rabbit and goat.

关 键 词：重组蛋白A 大肠杆菌 分泌表达 酶联免疫反应 

分 类 号：Q51[生物学—生物化学]