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Nucleic Acids Research, 1993, Vol. 21, No. 12 2921-2929
© 1993

MOLECULAR BIOLOGY

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions

Michael A. Walls, Ku-chuan Hsiao and Linda J. Harris

Bristol-Myers Squibb Pharmaceutical Research Institute—Seattle, Department of Molecular Immunology 3005 First Avenue, Seattle, WA 98121, USA

Received November 6, 1992. Revised April 15, 1993. Accepted April 15, 1993.

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human lg genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.


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