摘要
目的优化表达条件后纯化出切除GST标签的Met-PGEX-4T-1蛋白,制备出假单胞菌来源的多克隆抗体.方法将蛋氨酸酶(L-methionine gamma-lyase,Me)t的基因克隆至pBSK载体中,形成重组质粒pBSK-Met,采用基因工程技术将pBSK-Met与PGEX-4T-1载体连接,形成Met-PGEX.利用大肠杆菌表达系统诱导表达出Met-PGEX,采用亲和层析法过柱纯化Met-PGEX,凝血酶切除分子量为26KD的GST标签,免疫新西兰兔制备出蛋氨酸酶多克隆抗体Met PcAb,双向免疫扩散实验测多克隆抗体效价为1:128,间接Elisa法测效价1:1×105.结果成功表达纯化出纯度大于90%的Met-PGEX并制备出多克隆抗体.结论所制备的多克隆抗体可用于检测不同来源重组蛋氨酸酶抗原差异性变化.
Objective To optimize the expression conditions,purify Metase-PGEX-4T-1 protein cleaved GST tag and product polyclonal antibody by Pseudomonas putida.Methods Recombinant plasmid of pBSK-Metase was obtained by combining L-methionine gamma-lyase and PBSK.Met-PGEX was prepared connecting to PBSK-Metase and PGEX-4T-1 by using gene engineering technique.Met-PGEX was expressed inducible by Escherichia coli expression system.The expressed protein was purified by GST SefinoseTM Resin affinity chromatography.The GST tag with the relative molecular mass of 26KD was cleaved by thrombin.The titer of new Zealand white rabbit antisrum was 1：128 on double immunodiffusion,and up to 1：1×105by indirect ELISA.Results The Met-PGEX with a purity of over 90% was successfully expressed and purified.Conclusions The polyclonal antibody prepared could be used to detect the changes from different origin recombinant L-methionine gamma-lyase.
出处
《昆明医科大学学报》
CAS
2013年第3期8-11,共4页
Journal of Kunming Medical University
基金
国家自然科学基金资助项目(30760287)
云南省科技厅自然科学基金资助项目(2009CC022)
关键词
蛋氨酸酶GST标签
多抗制备
L-methionine gamma-lyase
GST tag
Polyclonal antibody production
