- 作者: Chen-Hua Chuang Sheng-Chieh Hsu Chia-Ling Hsu Tsui-Chi Hsu Wan-Jr Syu
- 作者服務機構: Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan, ROC
- 中文摘要: --
- 英文摘要: We have previously characterized a monoclonal anti-bod丫(SC1 D7)that is directed to maltose-binding protein(MBP) of Escherichia coli and other closely related enter-is bacteria. SC1 D7 does not cross-react with proteins ineucaryotes and appears to be a highly specific tool inimmunochemical analyses. To better map the epitope,we took advantage of an available plasmid, pMAL-c2,that encodes the E. coli MBP-coding sequence and con-structed plasmids to express MBP fragments. A con-struct containing the N-terminal portion of MBP does notreact with SC1 D7, whereas a second construct express-ing glutathione S-transferase fused with the C-terminalhalf of MBP does react with SC1D7. To precisely definethe epitope, random peptides displayed on M13 wereused to react with SC1 D7. Sequences of reactive pep-tideg were aligned, and a consensus sequence ofXDXRIPX was deduced. This sequence matches MBPwith an amino acid stretch of KDPRIAA. To consolidatethe mapping result, a sequence encoding this epitopewas inserted into an expression vector and the resultingrecombinant protein did react with SC1D7. Thereafter,this epitope was incorporated into a eucaryotic expres-sion plasmid containing a previously defined hepatitisdelta virus epitope for protein tagging. This two-epitope-tagging vector is useful in various molecular analyses.We demonstrate its usage for localization of a bacterialvirulence factor in host cells. This vector should be appli-cable for high-throughput characterization of new openreading frames found in genome sequencing.
- 中文關鍵字: --
- 英文關鍵字: Monoclonal antibody. Epitope. Tag. Immunofluorescence