title: Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.
abstract: The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. 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/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.
authors.affiliation: 1 Department of Veterinary Clinical Medicine, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, PR China.
authors.forename: Qinghe
authors.initials: Q
authors.lastname: Zhu
authors.forename: Donghua
authors.initials: D
authors.lastname: Guo
authors.forename: Li
authors.initials: L
authors.lastname: Feng
authors.forename: Dongbo
authors.initials: D
authors.lastname: Sun
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D006825:Hybridomas / Q000276:immunology*; D007544:Isopropyl Thiogalactoside; D008562:Membrane Glycoproteins / Q000276:immunology*; D009842:Oligopeptides / Q000276:immunology; D053485:Porcine epidemic diarrhea virus / Q000737:chemistry* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D057127:Single-Chain Antibodies; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D007544:Isopropyl Thiogalactoside