Method for cloning variable domain sequences of immunological gene repertoire
AssigneeSan Diego Antibody
InventorHenry Ji
Life of this patent
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Abstract
The present invention relates to a non-PCR (polymerase chain reaction) process, particularly a transcription-based amplification method, for amplifying and cloning sequences containing a variable domain sequence such as an immunoglobulin variable domain sequence from the immunological gene repertoire. The present invention comtemplates the expression of antibody library in either in an in vivo expression vector or in an in vitro transcription/translation system. Isolation of a gene coding for a receptor having the ability to bind a preselected ligand and receptors produced by the gene isolated by the method is also contemplated.
Claims18
A process for isolating a nucleic acid encoding a protein that binds to a target antigen, comprising:
- (a) exposing a plurality of nucleic acids encoding a plurality of different proteins that bind a plurality of different antigens to a pluratity of promoter-linked primers in a single reaction mixture under conditions suitable to produce a DNA comprising a nucleic acid that functions as an RNA polymerase promoter, wherein said promoter-linked primers consist of an RNA polymerase promoter sequence and a gene-specific sequence;
- (b) transcribing said DNA of step (a) with a suitable RNA polymerase to produce plural RNAs;
- (c) reverse transcribing said plural RNAs to produce plural reverse transcribed DNA;
- (d) exposing said plural reverse transcribed DNA to said plurality of promoter-linked primers under conditions suitable to produce additional DNA comprising nucleic acid that functions as an RNA polymerase promoter;
- (e) transcribing said additional DNA of step (d) with a suitable RNA polymerase to produce additional plural RNAs;
- (f) cloning said DNA, RNA or functional parts thereof under conditions that enable expression of said protein;
- (g) expressing proteins encoded by said cloned DNA or RNA of step (f);
- and
- (h) screening said expressed proteins with said target antigen to identify a protein that specifically binds thereto, thereby identifying a nucleic acid encoding a protein that binds to said antigen.
A process for making a library of proteins that bind to antigens, comprising:
- (a) exposing a plurality of nucleic acids encoding different proteins that bind different antigens to a plurality of promoter-linked primers in a single reaction mixture under conditions suitable to produce a DNA comprising a nucleic acid that functions as an RNA polymerase promoter, wherein said promoter-linked primers consist of an RNA polymerase promoter sequence and a gene-specific sequence;
- (b) transcribing the DNA sequence of step (a) with a suitable RNA polymerase to produce plural RNAs;
- (c) reverse transcribing said plural RNAs to produce plural reverse transcribed DNA;
- (d) exposing said plural reverse transcribed DNA to said plurality of promoter-linked primers under conditions suitable to produce additional DNA comprising nucleic acid that functions as an RNA polymerase promoter;
- (e) transcribing said additional DNA of step (d) with a suitable RNA polymerase to produce additional plural RNAs;
- (f) cloning said DNA sequence or said RNA sequence or functional parts thereof in an expression system;
- and
- (g) expressing proteins encoded by the cloned DNA sequence or RNA sequence or fragment thereof to form a library of expressed proteins capable of binding different antigens.
Dependencies are inferred from the claim text.