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

AUTOR(ES)

Walls, M A

RESUMO

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 Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.

Documentos Relacionados

• Analysis of the expression immunoglobulin gene repertoire by screening libraries derived from PCR-amplified cDNA.
• Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions.
• PCR amplification of antibody variable regions using primers that anneal to constant regions.
• The effect of replication errors on the mismatch analysis of PCR-amplified DNA.
• Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection