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Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3.
World J Gastroenterol. 2002 Apr; 8(2):258-62.WJ

Abstract

AIM

To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.

METHODS

mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced.

RESULTS

The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type.

CONCLUSION

The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Authors+Show Affiliations

Department of Biochemistry & Molecular Biology, Third Military Medical University, Chongqing 400038, China. hefengtian@163.netNo 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

Language

eng

PubMed ID

11925603

Citation

He, Feng-Tian, et al. "Expression and Identification of Recombinant Soluble Single-chain Variable Fragment of Monoclonal Antibody MC3." World Journal of Gastroenterology, vol. 8, no. 2, 2002, pp. 258-62.
He FT, Nie YZ, Chen BJ, et al. Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3. World J Gastroenterol. 2002;8(2):258-62.
He, F. T., Nie, Y. Z., Chen, B. J., Qiao, T. D., Fan, D. M., Li, R. F., Kang, Y. S., & Zhang, Y. (2002). Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3. World Journal of Gastroenterology, 8(2), 258-62.
He FT, et al. Expression and Identification of Recombinant Soluble Single-chain Variable Fragment of Monoclonal Antibody MC3. World J Gastroenterol. 2002;8(2):258-62. PubMed PMID: 11925603.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3. AU - He,Feng-Tian, AU - Nie,Yong-Zhan, AU - Chen,Bao-Jun, AU - Qiao,Tai-Dong, AU - Fan,Dai-Ming, AU - Li,Rong-Fen, AU - Kang,Yun-Sheng, AU - Zhang,Yan, PY - 2002/4/2/pubmed PY - 2002/12/4/medline PY - 2002/4/2/entrez SP - 258 EP - 62 JF - World journal of gastroenterology JO - World J. Gastroenterol. VL - 8 IS - 2 N2 - AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source. SN - 1007-9327 UR - https://www.unboundmedicine.com/medline/citation/11925603/Expression_and_identification_of_recombinant_soluble_single_chain_variable_fragment_of_monoclonal_antibody_MC3_ L2 - http://www.wjgnet.com/1007-9327/full/v8/i2/258.htm DB - PRIME DP - Unbound Medicine ER -