PMID: 2105495Feb 1, 1990Paper

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins

Proceedings of the National Academy of Sciences of the United States of America
V K ChaudharyI Pastan

Abstract

We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.

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Citations

Jul 1, 1995·Journal of Chemical Technology and Biotechnology·B M GrahamW J Harris
Jul 1, 1993·Medicinal Research Reviews·M WilliamsG L Neil
Sep 27, 1993·Journal of Immunological Methods·P J NichollsS M Andrew
Feb 10, 1994·Journal of Immunological Methods·R J Owens, R J Young
Jul 12, 1994·Journal of Immunological Methods·F RubertiA Bradbury
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Jan 1, 1990·Biotechnology Advances·B R Glick
Jan 1, 1989·Current Opinion in Immunology·N S Courtenay-Luck, A A Epenetos
Apr 1, 1991·Current Opinion in Immunology·A C Hiatt
Apr 1, 1991·Current Opinion in Immunology·P Tijssen, A Adam
Apr 1, 1991·Current Opinion in Biotechnology·A Plückthun
Feb 1, 1991·Current Opinion in Biotechnology·R A Gibbs
Aug 1, 1991·Current Opinion in Biotechnology·M J Zoller
Oct 31, 1991·Biochemical and Biophysical Research Communications·V K ChaudharyI Pastan
Dec 5, 1998·Transplantation Proceedings·L LiA G Sheil
May 7, 1999·Immunotechnology : an International Journal of Immunological Engineering·H de HaardB van Gemen
Jan 1, 1991·Bio/technology·S T Jones, M M Bendig
Feb 1, 1997·Nature Biotechnology·M J Coloma, S L Morrison
Oct 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·U BrinkmannI Pastan
Jul 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·J K BatraC R King
Oct 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·J R SawyerF R Blattner
Apr 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·U BrinkmannI Pastan
Aug 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·U BrinkmannI Pastan
Feb 27, 2014·Biotechnology & Genetic Engineering Reviews·Yaghoub SafdariMasoumeh Khalili
Jun 2, 2001·Cancer Biotherapy & Radiopharmaceuticals·A M Wu

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