Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.
We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.
Norderhaug
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Department of Vaccinology, National Institute of Public Health, Oslo, Norway.
Olafsen
T
T
Michaelsen
T E
TE
Sandlie
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I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019556:COS Cells; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins