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Cloning, expression, and identification of anti‐carbofuran single chain Fv gene
Author(s) -
Wang Hong,
Yang Jinyi,
Liu Xixia,
Liang Yan,
Lei Hongtao,
Shen Yudong,
Xu Xiaoyan,
Sun Yuanming,
Xu Zhenlin,
He Yongsheng
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.297
Subject(s) - microbiology and biotechnology , phagemid , recombinant dna , phage display , panning (audio) , antibody , monoclonal antibody , escherichia coli , immunoglobulin light chain , single chain variable fragment , clone (java method) , chemistry , antigen , biology , peptide library , gene , biochemistry , bacteriophage , peptide sequence , paleontology , zoom , genetics , immunology , lens (geology)
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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009