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Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire

a gene repertoire and variable domain technology, applied in the field of new cloning methods of immunological gene repertoire, can solve the problem of limited multiplexing ability of pcr-based amplification, and achieve the effect of increasing the size and diversity of the protein library

Inactive Publication Date: 2009-02-12
SORRENTO THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a method for amplifying and cloning immunological genes, specifically the variable domain sequences of immunoglobulins. The method uses non-PCR amplification methods, such as DNA-dependent RNA polymerase driven RNA transcription, strand displacement amplification, and ligase chain reaction. These methods provide faster amplification and cloning of rare targets compared to PCR-based amplification methods. The resulting amplified sequences can be used for further peptide / protein synthesis and in vitro or in vivo translation. The invention also provides a novel method for generating a library of the immunoglobulin gene repertoire. Overall, the invention improves the amplification and cloning of the immunological genes and receptors.

Problems solved by technology

Secondly, the RNA transcriptional amplification is linear and does not result in preferential amplification, which is a major problem associated with PCR-based amplification reactions.
Thirdly, the RNA transcriptional amplification process can be applied to amplify multiple sequences simultaneously with a capacity of amplifying at least more than ten to twenty sequences at a time in a single reaction, while the PCR-based amplification has limited ability for multiplexing with a typical limit of amplifying less than ten, most often less than five sequences in a single group reaction (Gao et al., supra).

Method used

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  • Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire
  • Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire
  • Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire

Examples

Experimental program
Comparison scheme
Effect test

example 1

Gene-Specific Oliggnucleotide and RNA Promoter-Linked Primer Selection

[0153]The nucleotide sequences coding for the human immunoglobulin complimentary determining region (CDR) are highly variable (Marks, J. D. et al., J. Mol. Biol., 222:581-597 (1991); Haidaris, C. G. et al., 257:185-202 (2001); Welschof, M. et al., J. Immunol. Methods, 179:203-214 (1995); Marks, J. D. et al., Eur. J. Immunol., 21:985-991 (1991); and Haard, H. J. D. et al., J. Biol. Chem., 274:18218-18230 (1999)). However, there are several regions of conserved sequences that flank the human VH domains, containing substantially conserved nucleotide sequences, i.e., sequences that will hybridize to the same primer sequence in a number of different genes. Therefore, gene-specific oligonucleotide primers can be selected for both gene-specific primers and the promoter-linked primers and synthesized to hybridize to the conserved sequences for reverse transcription, double stranding and RNA transcription reactions as desc...

example 2

Preparation of Source mRNAs Containing the VH and VL Gene Repertoire

[0160]Total cellular RNA was prepared from the blood cells collected from a pool of patients using the RNA preparation methods well known in the art as described by Chomczynski et al., Anal Biochem., 162:156-159 (1987) and the RNA isolation kit produced by QIAGEN GmbH (Hilden, Germany).

[0161]Messenger RNA (mRNA) enriched for sequences containing long poly A tracts was prepared from the total cellular RNA using methods described in Maniatis et al., eds., “Molecular Cloning: A Laboratory Press Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982). Briefly, the total RNA isolated from the blood cells prepared as described above was resuspended in 1 ml of DEPC-H2O and maintained at 65° C. for 5 minutes. One ml of 2× high salt loading buffer consisting of 100 mM Tris-HCl, 1 M sodium chloride, 2.0 mM disodium ethylenediamine tetraacetic acid (EDTA) at pH 7.5, and 0.2% sodium dodecyl sulfate (SDS) w...

example 3

Transcriptional Amplification of the VH Gene Repertoire

[0163]Transcriptional amplification is performed using the scheme as depicted in FIG. 4. In detail, about 5-10 μg of poly(A)+ mRNAs in DEPC-treated water were first hybridized (annealed) with 1 μM antisense primer mixture comprising equal amounts of antisense EcoRI & stop codon-linked HJH primers, for example, aHJH-1, 5′-dTGG AAT GAA TTCGAT TGC TAGTCAGAC GGT GAC CAG GGT GCC-3′ (SEQ ID NO:_); aHJH-2, 5′-dTGG AAT GAA TTC GAT TGC TAG TCAGAC GGT GAC CAT TGT CCC-3′ (SEQ ID NO:_); aHJH-3, 5′-dTGG AAT GAA TTC GAT TGC TAG TCA GAC GGT GAC CAG GGT TCC-3′ (SEQ ID NO:_); and aHJH-4, 5′-dTGG AAT GAA TTC GAT TGC TAG TCAGAC GGT GAC CGT GGT CCC-3′ (SEQ ID NO:_) as listed in Table 1 (the EcoRI site and stop codons in three different frames are underlined), at 65° C. for 5 minutes and then cooled down to room temperature. The mixture was subsequently added to a reverse transcription (RT) reaction admixture (20 μl) on ice, comprising 2 μl of 10× b...

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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 contemplates 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.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. patent application Ser. No. 10 / 477,907, filed Nov. 13, 2003, which is incorporated herein by reference in its entirety for all purposes.TECHNICAL FIELD[0002]The present invention relates to methods for amplifying and cloning variable region or domain sequences of the immunological genes, generating libraries of immunological gene repertoire and isolating a gene coding for an antigen-combining molecule such as antibody or immunoglobulin.BACKGROUND OF THE INVENTION[0003]A dozen or so monoclonal antibodies have been approved by the Food and Drug Administration (FDA) as human therapeutics including Orthoclone OKT3 for allograft rejection, ReoPro (abciximab) for adjunct treatment of percutaneous coronary intervention (PCI) including balloon angioplasty, atherectomy and stent placement, Rituxan for Non-Hodgkin's lymphoma, Simulet and Zenapax for organ rejection prophylaxis, Remicade for Rheumatoid ar...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/02C12Q1/68C40B40/08C40B40/10C40B50/06C07K16/00C12P19/34
CPCC07K16/00C07K16/005C12P19/34C07K2317/622C07K2317/21
Inventor JI, HENRY
Owner SORRENTO THERAPEUTICS INC
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