title: A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
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.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: V K
authors.initials: VK
authors.lastname: Chaudhary
authors.forename: J K
authors.initials: JK
authors.lastname: Batra
authors.forename: M G
authors.initials: MG
authors.lastname: Gallo
authors.forename: M C
authors.initials: MC
authors.lastname: Willingham
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D000922:Immunotoxins; D007256:Information Systems; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D015183:Restriction Mapping
chemical_list: D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011994:Recombinant Proteins