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

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...

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Main Authors: Chaudhary, V K, Batra, J K, Gallo, M G, Willingham, M C, FitzGerald, D J, Pastan, I
Formato: Artigo
Idioma:en
Publicado em: 1990
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Research Article
Acesso em linha:https://ncbi.nlm.nih.gov/pmc/articles/PMC53411/
https://ncbi.nlm.nih.gov/pubmed/2105495
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spelling pubmed-534112001-08-25 A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins. Chaudhary, V K Batra, J K Gallo, M G Willingham, M C FitzGerald, D J Pastan, I Proc Natl Acad Sci U S A Research Article 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. 1990-02 /pmc/articles/PMC53411/ /pubmed/2105495 Text en
institution US National Library of Medicine
collection PubMed Central
language en
format Article
topic Research Article
spellingShingle Research Article
Chaudhary, V K
Batra, J K
Gallo, M G
Willingham, M C
FitzGerald, D J
Pastan, I
A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
description 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.
author Chaudhary, V K
Batra, J K
Gallo, M G
Willingham, M C
FitzGerald, D J
Pastan, I
author_facet Chaudhary, V K
Batra, J K
Gallo, M G
Willingham, M C
FitzGerald, D J
Pastan, I
author_sort Chaudhary, V K
title A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
title_short A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
title_full A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
title_fullStr A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
title_full_unstemmed A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
title_sort rapid method of cloning functional variable-region antibody genes in escherichia coli as single-chain immunotoxins.
publishDate 1990
url https://ncbi.nlm.nih.gov/pmc/articles/PMC53411/
https://ncbi.nlm.nih.gov/pubmed/2105495
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