抗HSV-2单链抗体和碱性磷酸酶融合蛋白的制备  

Preparation of anti-HSV-2 single chain variable fragment-alkaline phosphatase fusion protein

作　　者：徐放[1] 遇广辉 王普[1] 

机构地区：[1]浙江工业大学药学院,浙江杭州310032

出　　处：《浙江工业大学学报》2014年第1期45-49,共5页Journal of Zhejiang University of Technology

基　　金：浙江省大学生科技创新活动计划(新苗人才计划)资助项目(2012R403078)

摘　　要：将抗HSV-2糖蛋白B的单链抗体基因亚克隆入原核表达载体pLac-duet-1-AKP中构建了抗HSV-2单链抗体和碱性磷酸酶融合表达的原核表达载体pLac-H3-AKP.转化大肠杆菌BL21(DE3),经IPTG诱导后SDS-PAGE显示在破菌沉淀中有符合预期大小的75kD的表达带.结果表明抗HSV-2单链抗体和碱性磷酸酶融合蛋白在大肠杆菌中主要以没有生物活性的包涵体形式表达.用镍离子螯合层析纯化裂解后的包涵体,并初步探索了影响包涵体复性的因素.复性后的融合蛋白不仅能与HSV-2糖蛋白B抗原特异结合而且具有碱性磷酸酶活性,为快速检出抗原提供了一种有效的方法.The anti-HSV-2 scFv gene was inserted into the expression vector pLac-duet-1-AKP containing AKP gene to construct the recombinant expression vector pLac-H3-AKP.After confirmed by DNA sequencing,the recombinant vector was transformed into Escherichia coli BL21（DE3） and then induced to express the scFv-AKP fusion protein by IPTG.The fusion protein was analyzed by SDS-PAGE,purified by Ni2＋-NTA affinity chromatograph and identified by ELISA under the optimal condition for refolding.The fusion protein existed in a form of inclusion body with an expected molecular mass of about 75 kD.ELISA confirmed that the fusion protein could bind to HSV-2 antigen and show AKP activity.

关 键 词：HSV-2 单链抗体 碱性磷酸酶 融合蛋白 金属螯合层析 蛋白复性 

分 类 号：Q786[生物学—分子生物学]