Human IgE (immunoglobulin E) Fab (fragment ab) against human and coding gene and application thereof

A technology of antibodies and fragments, applied in the fields of antibodies, genetic engineering, plant genetic improvement, etc., can solve problems such as adverse reactions, achieve the effects of inhibiting allergic reactions, mast cell degranulation and histamine release

Active Publication Date: 2014-01-08
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In June 2003, the humanized anti-IgE antibody drug Sorel (omalizumab) was approved by the US Food and Drug Administration (FDA) for the treatment of allergic asthma; source, so sometimes there will be serious adverse reactions, the US FDA only recommends the use of patients over 12 years old

Method used

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  • Human IgE (immunoglobulin E) Fab (fragment ab) against human and coding gene and application thereof
  • Human IgE (immunoglobulin E) Fab (fragment ab) against human and coding gene and application thereof
  • Human IgE (immunoglobulin E) Fab (fragment ab) against human and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of Natural Human Immunoglobulin Gene Library

[0038] Get the peripheral anticoagulant blood of 300 healthy people (every person 5ml), separate lymphocyte with Ficoll-Paque (Pharmacia, Uppsala, Sweden), extract total RNA with kit (QIAGEN GmbH, Hilden, Germany); Gene-Amp RNA PCR Kit (Perkin-Elmer Cetus, Norwalk, Conn) was reverse-transcribed into cDNA with Oligo (dT) 16, and the upstream and downstream primers (Invitrogen) of the conserved sequence of the human IgG light and heavy chain variable region (Table 1) 1) for PCR amplification of immunoglobulin γ, κ, λ chain genes; the PCR products were purified by a kit (QIAGEN GmbH, Hilden, Germany), and then digested with AscI and NheI (NEW ENGLAND BioLabs) respectively. chain and lambda chain products. The κ chain and λ chain products after digestion were combined with the human immunoglobulin Fab expression vector pFab-His2 (such as figure 1shown), followed by electrotransformation into Escherichia...

Embodiment 3

[0047] Example 3. Screening of human immunoglobulin G against human IgE

[0048] Take 9×10 7 Transform 100 μl JM109 Escherichia coli with DNA 10ng of antibody library independent of clonal titer, spread the bacterial solution on Luria broth (10g sodii chloridum, 10g tryptone, 5g yeast extract / L, PH 7) plate (containing 50 μg ampicillin / ml), cultured at 37°C for 7 hours, to be cloned (about 5×10 3 Clones / 90mm diameter plate) when the diameter is about 0.3mm, cover the plate with a nitrocellulose membrane (Armacia / Pharmacia) with a diameter of 82mm. After the clones are completely transferred to the membrane, place the membrane in LB containing 1.0mM IPTG On the plate, induce expression at 30°C for 6 hours, and then use lysozyme, DNase and bovine serum albumin (100mM Tris-HCl [pH 7], 150mM NaCl, 5mM MgCl 2 , 1.5% BSA, 1mg of DNase, 40mg lysozyme / ml) to lyse the membrane; wash to remove residual bacterial fragments etc. on the membrane, block with bovine serum albumin (BSA), a...

Embodiment 4

[0055] Example 4. Characteristic Analysis of Anti-Human IgE Antibody Fab Fragment Heavy and Light Chain Genes

[0056] Take the plasmids of positive clones and digest them with AscI-NdeI and SfiI-NotI restriction endonucleases respectively to obtain the light chain and heavy chain genes, and then connect them with the sequencing vectors CV-2 and CV-1 modified by enzyme digestion, and transform Escherichia coli JM109, respectively extract the plasmid DNA containing the light chain or heavy chain gene, and use the M13Reverse primer (5'-GGATAACAATTTCACACAGG-3') for sequencing. The sequencing work is done by Invitrogen, and the amino acid sequence is calculated by Vector NTI 10 software , performed homology analysis with IgBlast (Table 4), and divided the CDR and FR of the light chain variable region and heavy chain variable region of positive clones according to the Kabat system (such as Figure 4 , 5 shown).

[0057] Table 4 Gene homology analysis of positive clones

[0058] ...

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Abstract

The invention belongs to the technical field of biotechnology and relates to a human IgE (immunoglobulin E) Fab (fragment ab) against human and a coding gene and application thereof. In the invention, a recombined IgE C3-C4 protein is used for screening a natural human immunoglobulin gene library, and a human antibody Fab of IgE against human is screened from the library through ELISA (enzyme-linked immunosorbent assay) sequencing analysis and the like; extensive expression purification and further identification prove that the antibody Fab of human IgE against human is fully human and without Fc (fragment c), has an effect of inhibiting IgE mediated anaphylaxis without activating complements or causing human immune response or other pathological lesions and can be used for preparing antibody drugs for treating allergic diseases caused by IgE.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a humanized anti-human IgE antibody Fab fragment, a coding gene and its use, in particular to the application of a natural human immunoglobulin gene library to screen the anti-human IgE humanized antibody Fab fragment, coding gene and its application. use. Background technique [0002] In recent years, the incidence of allergic diseases mediated by immunoglobulin E (IgE) has increased year by year, and it has greatly endangered people's health. In August 2005, the World Allergy Organization (WAO) announced the survey results of more than 30 countries around the world on the first allergic disease day. Allergic diseases mediated by globulin E (IgE), such as food allergy, drug allergy, allergic rhinitis, asthma, etc. Marone et al first isolated IgE from the serum of patients with allergic diseases in 1966, and the World Health Organization (WHO) confirmed it as a new immunoglobulin in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/42C12N15/13C12N15/63C12N1/21C12N1/19A61K39/395A61P37/08
Inventor 程训佳杨彬蔡俊龙曹伯良付永锋
Owner FUDAN UNIV
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