Cloning, expression, and identification of anti-carbofuran single chain Fv gene.
Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly(4)Ser)(3). The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC(50) value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF.
Wang
Hong
H
Key Laboratory of Food Quality and Safety of Guangdong Province, College of Food Science, South China Agricultural University, Guangzhou 510642, PR China.
Yang
Jinyi
J
Liu
Xixia
X
Liang
Yan
Y
Lei
Hongtao
H
Shen
Yudong
Y
Xu
Xiaoyan
X
Sun
Yuanming
Y
Xu
Zhenlin
Z
He
Yongsheng
Y
D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D000918:Antibody Specificity; D002235:Carbofuran / Q000032:analysis* / Q000276:immunology; D003001:Cloning, Molecular; D015870:Gene Expression; D007118:Immunoassay; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D002235:Carbofuran