摘要
碳化二亚胺法合成FLAG-BSA完全抗原,利用杂交瘤技术制备5株分泌抗FLAG标签单克隆抗体的杂交瘤细胞株。制备腹水,并对其进行纯化、分析和鉴定。制备交联抗FLAGmAb亲和层析柱,纯化带FLAG标签的融合蛋白,经SDS-PAGE电泳后薄层扫描分析纯度,用Western-blotting检测mAb对融合蛋白的反应性。结果表明:5株细胞分泌的单抗都为IgG类型、IgG1亚类,腹水效价均高于1∶106,且5株单抗与其它融合蛋白标签无交叉反应性;亲和层析纯化后蛋白纯度达85%以上,5株单抗均可用于融合蛋白的Western-blotting检测,建立了可用于FLAG融合蛋白纯化和检测方法。
Monoclonal Antibodies against FLAG conjugated with BSA by EDC were produced using conventional cell fusion technology. Five high affinity hybridoma lines were screened for specificity to FLAG by ELISA. The ascites were prepared, purified, and analysed. The Immuno-affinity Chromatography crosslinked McAb against FLAG were preparated and applicated in purification of recombinant protein. Western blotting was used to assess the reactivity between anti FLAG mAbs and denatured FLAG fusion protein. The Characteristic indicated that ascites titers were higher than 1： 10^6, and the McAb were all specific to FLAG. The results showed that the purification of recombinant protein were 85% by Immuno-affinity Chromatography and the McAb could be used for Western bloting. A methods to detect and purify FLAG fusion protein ware established.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第1期93-97,共5页
China Biotechnology
关键词
FLAG标签
单克隆抗体
免疫亲和层析
FLAG tag Monoclonal antibodies Immuno-affinity chromatography
