Construction, expression, purification, refold and activity assay of a specific scFv fragment against foot and mouth disease virus.
An active form of a single-chain antibody (scFv) from the murine monoclonal antibody (mAb) 1C7, which is specific for type O foot and mouth disease virus (FMDV), was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. VH-(Gly4Ser)3-VL genes were screened by phage display technology. The sequencing results showed that the VH gene of scFv was composed of germline VH76-1BG-DFL16.1-JH4 and the VL gene of scFv consisted of germline bw20-JK2. The resultant scFv gene was cloned to the pPRoEX HTc vector and expressed in E. coli as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8 mol/L urea. The expressed scFv fusion proteins were purified by nickel-nitrilotriacetic acid and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay. The result revealed that the 1C7 scFv conserved the same characteristics of specific recognition and binding to type O FMDV as the parental 1C7 mAb.
ShengFeng
Cao
C
Biotechnology Institute, School of Agriculture and Biology, Shanghai JiaoTong University, Shanghai 201101, People's Republic of China. Cshengfeng@Sohu.com
Ping
Lu
L
Tao
Suen
S
Xin
Wang
W
GuoFeng
Wang
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029544:Foot-and-Mouth Disease Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012367:RNA, Viral; D011994:Recombinant Proteins