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Cloning, expression, and identification of anti-carbofuran single chain Fv gene.
Biotechnol Prog. 2009 Jul-Aug; 25(4):1018-24.BP

Abstract

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.

Authors+Show Affiliations

Key Laboratory of Food Quality and Safety of Guangdong Province, College of Food Science, South China Agricultural University, Guangzhou 510642, PR China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19653307

Citation

Wang, Hong, et al. "Cloning, Expression, and Identification of Anti-carbofuran Single Chain Fv Gene." Biotechnology Progress, vol. 25, no. 4, 2009, pp. 1018-24.
Wang H, Yang J, Liu X, et al. Cloning, expression, and identification of anti-carbofuran single chain Fv gene. Biotechnol Prog. 2009;25(4):1018-24.
Wang, H., Yang, J., Liu, X., Liang, Y., Lei, H., Shen, Y., Xu, X., Sun, Y., Xu, Z., & He, Y. (2009). Cloning, expression, and identification of anti-carbofuran single chain Fv gene. Biotechnology Progress, 25(4), 1018-24. https://doi.org/10.1002/btpr.297
Wang H, et al. Cloning, Expression, and Identification of Anti-carbofuran Single Chain Fv Gene. Biotechnol Prog. 2009 Jul-Aug;25(4):1018-24. PubMed PMID: 19653307.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning, expression, and identification of anti-carbofuran single chain Fv gene. AU - Wang,Hong, AU - Yang,Jinyi, AU - Liu,Xixia, AU - Liang,Yan, AU - Lei,Hongtao, AU - Shen,Yudong, AU - Xu,Xiaoyan, AU - Sun,Yuanming, AU - Xu,Zhenlin, AU - He,Yongsheng, PY - 2009/8/5/entrez PY - 2009/8/5/pubmed PY - 2009/12/16/medline SP - 1018 EP - 24 JF - Biotechnology progress JO - Biotechnol. Prog. VL - 25 IS - 4 N2 - 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. SN - 1520-6033 UR - https://www.unboundmedicine.com/medline/citation/19653307/Cloning_expression_and_identification_of_anti_carbofuran_single_chain_Fv_gene_ L2 - https://doi.org/10.1002/btpr.297 DB - PRIME DP - Unbound Medicine ER -