1. Preparation and Identification of a Single-chain Variable Fragment Antibody Against Canine Distemper Virus.
- Author
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Yi, Li and Cheng, Shipeng
- Subjects
- *
IMMUNOGLOBULIN synthesis , *CANINE distemper virus , *POLYMERASE chain reaction , *HYBRIDOMAS , *ESCHERICHIA coli - Abstract
The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein (aa 277-471). The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/1N8). After sequence analysis, the scFv/1N8 gene was cloned into the prokaryotic expression vector PET32a with a His-tag. The recombinant scFv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the scFv were determined by indirect ELISA (His-tag) and competitive ELISA. The recombinant scFv/1N8 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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