A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries.
Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.
Sarantopoulos
S
S
Department of Pathology, Boston University School of Medicine, MA 02118.
Kao
C Y
CY
Den
W
W
Sharon
J
J
D000818:Animals; D001483:Base Sequence; D005260:Female; D015723:Gene Library; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region