title: Expression and purification of a secreted functional mouse/human chimaeric antibody against bacterial endotoxin in baculovirus-infected insect cells.
abstract: We have created a mouse/human chimaeric antibody by taking antigen-binding fragment (Fab) genes of a mouse antibody-producing hybridoma with specificity for bacterial endotoxin and joining them to human Ig crystallizing-fragment (Fc) genes using recombinant DNA techniques. This chimaeric antibody has been expressed in Sf21 and High Fivetrade mark (BTI-TN-5B1-4) insect cells using the baculovirus expression system, which may allow the mass production of secretory recombinant antibodies. This was achieved by using infection with a double-recombinant virus containing cDNAs of both the Ig heavy-chain (HC) and light-chain (LC) genes. Prior to recombination, each gene was cloned into the dual-expression baculovirus transfer vector pPLSP2, which permitted the insertion of the LC gene in-frame with the signal peptide of honey bee melittin downstream of the polyhedrin promoter, and of the HC gene in-frame with the signal peptide of Bombyx mori larval serum protein downstream of the p10 promoter. Our results showed that the polypeptide chains were secreted by insect cells and correctly assembled into H(2)L(2) heterodimers containing N-linked carbohydrate at the heavy chain. Furthermore, the recombinant chimaeric antibody exhibited a similar antigen specificity to that of the monoclonal antibody. More importantly, it provides a generic method for the high-level expression of antibodies.
authors.affiliation: National Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Academia Sinica, Beijing 100080, People's Republic of China.
authors.forename: W
authors.initials: W
authors.lastname: Tan
authors.forename: P H
authors.initials: PH
authors.lastname: Lam
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004274:DNA, Recombinant; D004731:Endotoxins / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera
chemical_list: D000906:Antibodies; D004274:DNA, Recombinant; D004731:Endotoxins; D011993:Recombinant Fusion Proteins