Rapid immunoselection cloning method
AssigneeThe General Hospital Corporation
InventorsBrian Seed, Alejandro Aruffo
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Abstract
A simple and highly efficient method for cloning cDNAs from mammalian expression libraries based on transient expression in mammalian host cells has been discovered. Novel expression vectors allowing highly efficient construction of mammalian cDNA libraries are disclosed. The cloning method of the invention which has been used to clone genes for cell surface antigens of human lymphocytes, has general application in gene cloning. Cell surface antigens cloned according to the present invention have been purified, and the nucleotide and amino acid sequences determined. These antigens have diagnostic and therapeutic utility in immune-mediated infections in mammals, including humans.
Claims16
A method for cloning a cDNA encoding a cell surface antigen, said method comprising the steps of:
- (a) preparing a cDNA expression library from cells expressing a gene encoding a cell surface antigen in an expression vector, wherein said expression vector comprises at least one origin of replication functional in a bacterium, a promoter sequence functional in mammalian cells, splice and polyadenylation signals functional in mammalian cells and each operably linked to a cDNA insert in each member of said library, a replication origin functional in mammalian cells and a cloning site for the insertion of a cDNA insert;
- (b) introducing said eDNA expression library into mammalian tissue culture cells;
- (c) culturing said cells into which said eDNA expression library has been introduced under conditions allowing expression of said cell surface antigen by said cells;
- (d) exposing the cultured cells of step (c) to a first antibody directed against said cell surface antigen;
- (e) thereafter exposing the cells of step (d) to a substrate coated with a second antibody directed against said first antibody, thereby causing said cells expressing said cell surface antigen to which said first antibody is bound in step (d) to adhere to said substrate;
- and (f) separating adherent and nonadherent cells to produce s sub-library of adherent cells;
- and (g) isolating a cDNA clone encoding said cell surface antigen from the sub-library of step (f).
Dependencies are inferred from the claim text.