EP0534619A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Expression cloning method
Assignee: Rhode Island Hospital
•
Filing: 1992-08-26
•
Granted: —
•
Expires: 2012-08-26
Abstract
Abstract Disclosed is a process for cloning a nucleotide sequence that encodes a protein. The process includes the steps of: preparing a recombinant molecule containing the nucleotide sequence and a vector that includes an expression control element operatively attached at linkage sites to the nucleotide sequence; introducing the recombinant molecule into host cells derived from a multi-cellular organism; identifying, among the host cells into which the recombinant molecule is introduced, cells expressing the protein by a means that specifically detects the protein; attaching the cells expressing the protein to a solid surface; and recovering the recombinant molecule from the attached cells. The same process can also be used to test the specificity of an antibody in its binding to an antigen, when a DNA sequence encoding the antigen is available.
Inventors
- 👤 Junsheng Sang
- 👤 Nancy L. Thompson
- 👤 Douglas C. Hixson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-04 20:09
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/64; C12N15/79; C12N15/85; C12N9/00; C12N9/10; C12N9/12; C12N2800/00; C12N2800/10; C12N2800/108
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
125 words
A process for cloning a nucleotide sequence that encodes a protein, which process comprises the steps of: preparing a recombinant molecule containing said nucleotide sequence and a vector that includes an expression control element operatively attached at linkage sites to said nucleotide sequence; introducing said recombinant molecule into host cells derived from a multi-cellular organism; identifying, among said host cells into which said recombinant molecule is introduced, cells expressing said protein by means that specifically detects said protein, said detecting means being an anitbody or a ligand that specifically binds to said protein and is linked to a fluorescent group, an enzyme, or a radioactive isotope; attaching said cells expressing said protein to a solid surface; and recovering said recombinant molecule from said attached cells.
Claim 2
Dependent Claim
49 words
The process of claim 1, which process further comprises a step of fixing said host cells into which said recombinant molecule is introduced before said identifying step, thereby allowing influx of said detecting means into said fixed host cells while preventing exit of said recombinant molecule and said protein.
Claim 3
Dependent Claim
17 words
The process of claim 2, wherein said fixing step is effected by treatment with a chemical reagent.
Claim 4
Dependent Claim
10 words
The process of claim 3, wherein said reagent is acetone.
Claim 5
Dependent Claim
35 words
The process of claim 1, wherein said host cells into which said recombinant molecule is introduced are cultured in a container the bottom face of which is coated with a matrix before said identifying step.
Claim 6
Dependent Claim
10 words
The process of claim 5, wherein said matrix is collagen.
Claim 7
Dependent Claim
20 words
The process of claim 1, which process further comprises a step of enriching said nucleotide sequence after the recovering step.
Claim 8
Dependent Claim
32 words
The process of claim 7, wherein said nucleotide sequence is enriched by enzymatic amplification with a pair of vector primers that flank said linkage sites between said nucleotide sequence and said vector.
Claim 9
Dependent Claim
12 words
The process of claim 8, wherein said enzymatic amplification is PCR amplification.
Claim 10
Dependent Claim
17 words
The process of claim 1, wherein said recombinant molecule is capable of replication in said host cells.
Claim 11
Dependent Claim
10 words
The process of claim 10, wherein said vector is pCDM8.
Claim 12
Dependent Claim
12 words
The process of claim 1, wherein said host cells are COS cells.
Claim 13
Dependent Claim
15 words
The process of claim 1, wherein said protein is post-translationally modified by said host cells.
Claim 14
Dependent Claim
11 words
The process of claim 13, wherein said post-translational modification is glycosylation.
Claim 15
Dependent Claim
12 words
The process of claim 1, wherein said multi-cellular organism is a mammal.
Claim 16
Dependent Claim
24 words
A process of claim 1, wherein said cells expressing said protein are identified first and, after said identification, are attached to said solid surface.
Claim 17
Dependent Claim
31 words
A process of claim 1, wherein said cells expressing said protein are attached to said solid surface first and, after said attachment, are identified by means that specifically detects said protein.
Claim 18
Dependent Claim
95 words
A process for testing specificity of an antibody in its binding to a protein, which process comprises the steps of: preparing a recombinant molecule containing a nucleotide sequence that encodes said protein and a vector that includes an expression control element operatively attached to said nucleotide sequence; introducing said recombinant molecule into host cells derived from a multi-cellular organism; and detecting, among said host cells into which said recombinant molecule is introduced, cells expressing said protein with said antibody which is detectably labeled to determine specificity of said antibody in its binding to said protein.
Claim 19
Dependent Claim
38 words
The process of claim 18, which process further comprises a step of fixing said host cells into which said recombinant molecule is introduced before said identifying step, thereby allowing influx of said antibody into said fixed host cells.
Claim 20
Dependent Claim
18 words
The process of claim 19, wherein said cellular membranes are rendered semipermeable by treatment with a chemical reagent.
Claim 21
Dependent Claim
10 words
The process of claim 20, wherein said reagent is acetone.
Claim 22
Dependent Claim
11 words
The process of claim 21, wherein said protein is post-translationally modified.
Claim 23
Dependent Claim
11 words
The process of claim 22, wherein said post-translational modification is glycosylation.
Claim 24
Dependent Claim
17 words
The process of claim 18, wherein said recombinant molecule is capable of replication in said host cells.
Claim 25
Dependent Claim
10 words
The process of claim 24, wherein said vector is pCDM8.
Claim 26
Dependent Claim
67 words
A kit for expression cloning a nucleotide sequence or for testing the binding specificity of an antibody to a protein encoded by said nucleotide sequence, said kit comprising: a vector that includes an expression control element; an adaptor compatible to said vector; a pair of vector primers; a container; a cell fixative; and an antibody to which an enzyme, a fluorescent group, or an isotope is linked.
Claim 27
Dependent Claim
18 words
The kit of claim 26, wherein said container is coated with a matrix on its interior bottom surface.
Claim 28
Dependent Claim
25 words
The kit of claim 27, further comprising: digestion buffer; and means for removing or isolating a portion of said matrix coating from the remainder thereof.
Claim 29
Dependent Claim
64 words
An expression cloning kit comprising: a vector that includes an expression control element; an adaptor compatible to said vector; a pair of vector primers; a container; a cell fixative; an absorbent sheet onto which an enzyme substrate is attached; an antibody to which said enzyme is linked; means for removing or isolating a portion of said matrix from the remainder thereof; and digestion buffer.
Claim 30
Dependent Claim
18 words
The kit of claim 29, wherein said container is coated with a matrix on its interior bottom surface.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.