抗金黄色葡萄球菌单链抗体(scFv)的原核表达及蛋白纯化  

Prokaryotic Expression and Purification of Single-chain Variable Fragment(scFv)Against Staphylococcus aureus

作　　者：李晶泉[1] 徐永平[1,2] 王熙涛[1] 李媛[1] 王丽丽[1,2] 李晓宇[1,2] 

机构地区：[1]大连理工大学生命科学与技术学院,大连116024 [2]教育部动物性食品安全保障技术工程研究中心,大连116620

出　　处：《生物技术通报》2016年第11期194-201,共8页Biotechnology Bulletin

基　　金：中央高校基本科研业务费(DUT13 JB04);国家海洋公益项目(201405003-3)

摘　　要：旨在将来源于鸡的金黄色葡萄球菌单链抗体(scFv)进行原核诱导表达,获得有抗体活性的目的蛋白。构建含有目的抗体基因的重组质粒,将此质粒进行原核诱导表达并鉴定所获得蛋白的生物活性。结果显示,(1)成功构建了含有金黄色葡萄球菌单链抗体(scFv)的重组质粒p Cold I-scFv,质粒成功转化到大肠杆菌表达菌株中;(2)经过诱导表达后,目的蛋白主要以包涵体的形式存在于沉淀中;(3)使用4 mol/L尿素成功地将包涵体变性溶出;(4)通过柱层析法及透析法获得了纯化及复性效果较好的目的蛋白;(5)间接ELISA鉴定证实所获蛋白具有金黄色葡糖球菌抗体活性。通过质粒构建及原核诱导表达、包涵体溶出和复性等步骤,最终获得了有金黄色葡萄球菌抗体活性的目的蛋白。This work aims to clone and express single-chain variable region fragment（scFv）of Staphylococcus aureus in Escherichia coli,and to obtain the target protein with scFv activity. A plasmid containing target scFv gene was constructed,then transformed into aprokaryotic expression strain to be induced,and the activity of the harvested protein was identified. As results ：（1）Recombinant plasmidp Cold I-scFv was successfully constructed,and transformed into the expression strain of Escherichia coli.（2）After induction,the targetproteins mainly exist in pellet in the form of inclusion bodies.（3）Inclusion body was dissolved successfully using 4 mol/L urea.（4）Purifiedand renatured target protein by column chromatography and dialysis was favorable.（5）The refolded protein showed specific binding activitywith S. aureus in ELISA experiment. Conclusively,the target protein with S. aureus antibody activity was successfully acquired by plasmidconstruction and prokaryotic expression,inclusion body dissolving and refolding.

关 键 词：可溶性表达 包涵体 蛋白纯化 蛋白复性 有活性的蛋白 

分 类 号：Q78[生物学—分子生物学] S852.4[农业科学—基础兽医学]