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Preparation of single chain variable fragment of MG(7) mAb by phage display technology.
World J Gastroenterol. 2001 Aug; 7(4):510-4.WJ

Abstract

AIM

To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator.

METHODS

mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE.

RESULTS

The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32.

CONCLUSION

The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Authors+Show Affiliations

Department of Gastroenterology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

11819819

Citation

Yu, Z C., et al. "Preparation of Single Chain Variable Fragment of MG(7) mAb By Phage Display Technology." World Journal of Gastroenterology, vol. 7, no. 4, 2001, pp. 510-4.
Yu ZC, Ding J, Nie YZ, et al. Preparation of single chain variable fragment of MG(7) mAb by phage display technology. World J Gastroenterol. 2001;7(4):510-4.
Yu, Z. C., Ding, J., Nie, Y. Z., Fan, D. M., & Zhang, X. Y. (2001). Preparation of single chain variable fragment of MG(7) mAb by phage display technology. World Journal of Gastroenterology, 7(4), 510-4.
Yu ZC, et al. Preparation of Single Chain Variable Fragment of MG(7) mAb By Phage Display Technology. World J Gastroenterol. 2001;7(4):510-4. PubMed PMID: 11819819.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Preparation of single chain variable fragment of MG(7) mAb by phage display technology. AU - Yu,Z C, AU - Ding,J, AU - Nie,Y Z, AU - Fan,D M, AU - Zhang,X Y, PY - 2002/1/31/pubmed PY - 2002/6/6/medline PY - 2002/1/31/entrez SP - 510 EP - 4 JF - World journal of gastroenterology JO - World J. Gastroenterol. VL - 7 IS - 4 N2 - AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody. SN - 1007-9327 UR - https://www.unboundmedicine.com/medline/citation/11819819/Preparation_of_single_chain_variable_fragment_of_MG_7__mAb_by_phage_display_technology_ L2 - http://www.wjgnet.com/1007-9327/full/v7/i4/510.htm DB - PRIME DP - Unbound Medicine ER -