天津科技大学学报2017，Vol.32Issue(2)：7-12,6.DOI:10.13364/j.issn.1672-6510.20160202
绿色荧光蛋白GFP多克隆抗体的制备及在多种细胞中的特异性鉴定
Preparation of Polyclonal Antibody for Green Fluorescent Protein(GFP) and Characterization of its Specificity in Different Cells
摘要
Abstract
GFP is one of the most popular fluorescent proteins used to detect and quantify targeted proteins in living organ-isms.To develop a high sensitive antibody for its detection or signal capture in different kind of organisms,6×His-tagged and GST-tagged prokaryotic expression plasmids,pET28A-gfp and pGEX-2T-gfp,were constructed,by insertinggfpinto the pGEX-2T and pET28A vectors respectively and then were transformed intoEscherichia coli BL21(DE3)for protein expres-sion. 12% SDS-PAGE analysis results showed that the molecular weights of the fusion protein 6×His-GFP and GST-GFP were 2.6×104 and 5.2×104,respectively.The 6×His-GFP fusion protein was purified by affinity adsorption purification to immunize New Zealand white rabbits.The 5th immune serum was collected and the antibody titer was determined to be 1﹕32,000 by ELISA.The antiserum was purified by Protein A affinity adsorption purification and immobilized GST-GFP puri-fication,and the specificity of polyclonal antibodies was evaluated by Western blot in three kind of organisms including chlamydomonas,HEK293 cells and bacteria.Results show that the polyclonal antibody prepared can specifically and pre-cisely bind GFP in all kind of organisms,which can be used for more GFP and GFP related research.关键词
原核表达/蛋白纯化/多克隆抗体/免疫印迹Key words
prokaryotic expression/protein affinity purification/polyclonal antibody/Western blot分类
生物科学引用本文复制引用
董彬,胡贺贺,王晶,孟德梅,樊振川..绿色荧光蛋白GFP多克隆抗体的制备及在多种细胞中的特异性鉴定[J].天津科技大学学报,2017,32(2):7-12,6.基金项目
国际遗传工程与生物技术中心(ICGEB)研究项目(CRP/CHN15-01) （ICGEB）
