Scaled procaryotic cell and eucaryotic cell internal antibody and antigen library construction and screening

A eukaryotic cell and library construction technology, applied in biochemical equipment and methods, material inspection products, instruments, etc., can solve the problems of non-immunity and achieve high affinity and high specificity effects

Inactive Publication Date: 2003-07-16
韩泽广
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Embodiment 1

[0042] Embodiment 1: Embodiment 1 is to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention (the same applies to the following embodiments). A prokaryotic or eukaryotic in vivo antibody library construction method suitable for large-scale antibody screening, comprising the following steps: (1) collecting immune-related tissues and cells such as peripheral blood, lymph nodes, spleen, bone marrow, and liver; RNA is isolated from the tissues and cells taken, and cDNA is obtained by reverse transcription; (3) DNA primers for the heavy chain variable region, light chain variable region and the third constant region of the immunoglobulin are respectively designed, and in the obtained cDNA Amplify the immunoglobulin heavy chain variable region, light chain variable region and the third constant region; (4) molecular biology and genetics ...

Embodiment 2

[0043] Embodiment 2: construct the high-abundance, high-diversity prokaryotic cell and eukaryotic cell in vivo antigen library suitable for large-scale antibody screening, comprising the following steps: (1) from the organ, tissue, cell (including cell Strains), blood, cell components, etc. to extract RNA. (2) RNA can be processed by a variety of methods, including: a. After reverse transcription of RNA, ultrasonic waves will randomly break into cDNA with a length of 500 bp; b. Use RNA as a template and use mixed pairs of 5' ends as random primers , Polymerase chain reaction (PCR) amplifies RNA, and takes the amplified product with a length of 500bp; c. First use the RNA cap structure to design PCR primers or antibodies to enrich the full-length cDNA, and then use the above method to process; d. According to the research Need, use conserved sequences to design non-random primers and PCR to amplify specific gene families or genes containing specific functional domains (domain) ...

Embodiment 3

[0044] Example 3: A method for large-scale screening of specific antibodies in prokaryotic cells and eukaryotic cells, including the following steps: (1) Mixing the constructed antigen library with the constructed antibody library in a certain ratio, such as the number of antigen plasmids is equal to the number of antibody plasmids Count 109 times; (2) add the competent escherichia coli or yeast that contains reporter gene and carry out co-transformation; (3) will spread the escherichia coli gained from step (2) on containing antibiotic and can play chemical with β-galactosidase The bacterial culture plate of reagent X-gal that reaction presents color, hatches overnight; Or the yeast that obtains is in the X-gal culture plate of auxotrophy, hatches overnight; (4) what can grow on culture plate and have color reaction is positive ( Bacteria or yeast) clones containing antigen genes and corresponding antibodies or antibody fragments such as scFv, Fv and Fab, etc. The intensity o...

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Abstract

A process for configuring multi-type, high-titer, high-abundance and high-diversity antibody libraries and antigen libraries in procaryotic cell and eukaryotic cell is disclosed, which features use of procaryotic and eukaryotic cell reproduction expression. Its screening features that according to the DNA sequence of antigen epitope gene, the all or part of the open reading frames for antigen gene and the special protein coding gene are combined to form fusion gene for screening the high-specificity and-affinity antibody.

Description

1. Technical field [0001] The present invention relates to the fields of genomics, proteomics, molecular biology, immunology, cell biology, antibody engineering, genetic engineering and clinical medicine such as disease treatment and diagnosis. Specifically, the present invention relates to prokaryotic and eukaryotic cells For example, large-scale antibody and antigen libraries have been constructed in Escherichia coli and yeast, and large-scale screening of high-specificity and high-affinity antibody screening methods is carried out based on the basic principle of antigen-antibody interaction. 2. Background technology [0002] There are many methods used in the past to generate antibodies. Such as injecting antigens into animals such as rabbits to produce polyclonal antibodies, or immunizing animals such as mice combined with hybridoma technology to prepare monoclonal antibodies (for example, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur.J Immunol. 6:511, 1976; K...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/569G01N33/68
Inventor 韩泽广刘锋张新段煜
Owner 韩泽广
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