US5506126A
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Rapid immunoselection cloning method
Assignee: General Hospital Corp
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Filing: 1993-10-18
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Granted: 1996-04-09
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Expires: 2013-10-18
Abstract
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.
Inventors
- 👤 Brian Seed
- 👤 Alejandro Aruffo
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 09:38
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/70578; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/63; C12N15/79; C12N15/85; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56966; G01N33/56972; A; A61; A61K; A61K38/00; C12N2800/00; C12N2800/10; C12N2800/108; C12N2830/00; C12N2830/15; C12N2830/38; C12N2830/60; C12N2840/00; C12N2840/44
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
229 words
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).
Claim 2
Independent Claim
229 words
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).
Claim 3
Dependent Claim
17 words
The method of claim 1, wherein said cell surface antigen is expressed by mammalian T lymphocyte cells.
Claim 4
Dependent Claim
38 words
The method of claim 1, wherein said cDNA expression library is prepared from RNA extracted from a cell selected from the group consisting of a cell line and tumor cells and inserted into an SV40 origin-based expression vector.
Claim 5
Dependent Claim
20 words
The method of claim 3, wherein said expression vector is selected from the group consisting of piH3, piH3M and CDM8.
Claim 6
Dependent Claim
14 words
The method of claim 3, wherein said cell line is a human cell line.
Claim 7
Dependent Claim
19 words
The method of claim 5, wherein said cell line is selected from the group consisting of HPB--ALL and JY.
Claim 8
Dependent Claim
13 words
The method of claim 3, wherein said tumor cells are human tumor cells.
Claim 9
Dependent Claim
15 words
The method of claim 7, wherein said human tumor cells are human adrenal tumor cells.
Claim 10
Dependent Claim
22 words
The method of claim 1, wherein said mammalian tissue culture cells are selected from the group consisting of COS or WOP cells.
Claim 11
Dependent Claim
38 words
The method of claim 1, wherein said cDNA expression library is prepared from RNA extracted from a cell selected from the group consisting of a cell line and tumor cells and inserted into a cytomegalovirus origin-based expression vector.
Claim 12
Dependent Claim
14 words
The method of claim 10, wherein said cell line is a human cell line.
Claim 13
Dependent Claim
20 words
The method of claim 11, wherein said human cell line is selected from the group consisting of HBP--ALL and JY.
Claim 14
Dependent Claim
14 words
The method of claim 11, wherein said tumor cell is a human tumor cell.
Claim 15
Dependent Claim
16 words
The method of claim 13, wherein said human tumor cell is a human adrenal tumor cell.
Claim 16
Dependent Claim
86 words
The method of claim 1 wherein the replication origin signal functional in mammalian cells is from a virus, and said expression vector comprising a polylinker having two BstXI sites, said sites being identical in nucleotide sequence, inverted in orientation with respect to each other, and separated from one another by an intervening nucleotide sequence such that the intervening nucleotide sequence can be excised by BstXI digestion wherein the sequence of the two BstXI sites does not allow self-ligation after the expression vector is cleaved with BstXI.
Claim 17
Dependent Claim
11 words
The method of claim 15 wherein said expression vector is piH3M.
References Cited (Backward Citations)
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