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Recombinant anti human IgE monoclonal antibody and preparation method and usage thereof

A monoclonal antibody and heavy chain technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc.

Inactive Publication Date: 2007-10-31
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xolair was used to treat asthma after it was launched on the market, and achieved certain therapeutic effects, but there are still many asthma patients who are unresponsive to the treatment of Xolair. Therefore, an alternative with stronger specificity and higher affinity is urgently needed clinically. product

Method used

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  • Recombinant anti human IgE monoclonal antibody and preparation method and usage thereof
  • Recombinant anti human IgE monoclonal antibody and preparation method and usage thereof
  • Recombinant anti human IgE monoclonal antibody and preparation method and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Screening the antibody gene variable region of IgE from the antibody library

[0041] 1) Construction of mouse antibody library

[0042] Human IgE (obtained by affinity chromatography of 8033 cell culture supernatant) and Freund's adjuvant were used to immunize Balb / C mice. After four times of immunization, the mouse serum was diluted 1:500 and showed a strong positive reaction with human IgE. Mouse spleen according to Marks et al. J. Mol. Biol., 222, 581-597. Hoogenboom and Winter, J. Mol. Biol., 227, 381-388. Haidaris CG et al. J Immunol Methods. 2001 Nov 1;257 (1-2): 185-202, method described in Griffiths, A.D. et al. EMBO J., 13, 3245-3260 (1994). Nissim, A. et al. EMBO J., 13, 692-698 (1994) , to construct a murine antibody library.

[0043] 2) screening

[0044] Add 1 ml of the revived antibody library strain to 14 ml of fresh LB medium, and culture in a 50 ml Erlenmeyer flask at 37°C for 16 hours.

[0045] Centrifuge at a high speed of 12000rpm for ...

Embodiment 2

[0063] Example 2 Expression Intensity of Antibody Genes in CHO Cells

[0064] The 12 high-expression candidate clones obtained from the above screening were cultured in a 10 cm tissue culture dish, and the expression level of the antibody was determined by ELISA as described below. Coat goat anti-mouse IgG (Fc) on an ELISA plate, overnight at 4°C, block with 2% BSA at 37°C for 2 hours, add the culture supernatant to be tested and a standard (mouse IgG1), incubate at 37°C for 2 hours, add HRP-goat anti-mouse IgG (κ) for binding reaction, incubated at 37°C for 1 hour, added TMB at 37°C for 10 minutes, and finally washed with H 2 SO 4 Termination reaction, test A 450 value. Table 1 below shows the expression levels of the 12 candidate clones obtained from the above screening.

[0065] Cell line number

[0066]As can be seen from Table 1 above, 8G1, 9B3 and 5H3 have very high expression levels.

Embodiment 3

[0067] Example 3 DNA sequencing of anti-IgE monoclonal antibody gene

[0068] According to the pedigree, DNA sequencing was performed on the anti-tumor necrosis factor receptor antibody gene of the 8G1 cell line obtained above. The results are as follows: SEQ ID NO: 3 shows its light chain variable region gene sequence (5' to 3', 387bp), and its deduced amino acid sequence is shown in SEQ ID NO: 1; SEQ ID NO: 3 shows the 8G1 heavy Chain variable region gene sequence ((5', to 3', 426 bp), its deduced amino acid sequence is shown in SEQ ID NO:2.

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Abstract

The invention discloses a restructuring antihuman IgE monoclonal antibody, which is characterized by the following: possessing amino acid sequence of SEQ ID NO: 1 in light chain variable zone; possessing amino acid sequence of weight chain variable zone of SEQ ID NO: 2 in weight chain variable zone. This invention also discloses a DNA molecule to encode monoclonal antibody, a carrier to express the monoclonal antibody and eukaryotic host cell transformed by the expression carrier. This invention also discloses an usage to prepare I type hypersensitivity disease medicine of restructuring antihuman IgE monoclonal antibody.

Description

technical field [0001] The present invention relates to the technical field of antibody medicines, in particular to a new recombinant anti-human IgE monoclonal antibody and the preparation method and application of the monoclonal antibody. Background technique [0002] Hypersensitivity reaction refers to an abnormal or pathological immune response produced by the body after being stimulated by a certain antigenic substance. It often manifests as increased immune reactivity, and it often occurs when the body is exposed to the same antigen again. Hypersensitivity and immune response are essentially the body's specific immune response to certain antigenic substances, but the former mainly manifests as tissue damage and (or) physiological dysfunction, while the latter mainly manifests as physiological defense effects. At present, hypersensitivity, allergy (allergy) and anaphylaxis (anaphylaxis) are used interchangeably, but most tend to use hypersensitivity. [0003] The occurr...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63C12N15/79C12P21/08A61K39/395A61P37/06
Inventor 郭亚军候盛王海华钱卫珠
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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