A kind of humanized anti-interleukin 22 genetic engineering antibody and its application

A humanized and antibody technology, applied in the field of biomedicine, can solve problems such as rapid clearance and ineffective activation of effector systems

Active Publication Date: 2019-01-18
武汉原谷生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are many problems in the application of mouse-derived antibodies in human therapy: (1) they cannot effectively activate the effector systems related to complement and Fc receptors in the human body; (2) they are recognized by the human immune system to produce human anti-mouse antibodies (HAMA ); (3) is quickly cleared in the human circulatory system

Method used

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  • A kind of humanized anti-interleukin 22 genetic engineering antibody and its application
  • A kind of humanized anti-interleukin 22 genetic engineering antibody and its application
  • A kind of humanized anti-interleukin 22 genetic engineering antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of human IL-22

[0038] Using genetic engineering technology to construct a human IL-22 gene expression vector (human IL-22 encoding nucleotide sequence includes SEQ ID NO. 7, and amino acid encoding sequence includes SEQ ID NO. 8). We constructed human IL-22 cDNA into pTXB 1 expression vector, transformed it into competent E. coli BL21 (DE3), prepared a single clone on LB (Amp+) agar plate, and cultured it overnight at 37°C. Transfer to 1L LB training center at the ratio of 1:100 the next day. Shake the bacteria at 37°C to A600=0.5~0.7, add IPTG (1mM) to induce expression. Shake at 37°C for 6-8 hours, centrifuge, and remove the supernatant. Resuspend the bacteria in 50ml Column buffer (Column buffer formula: 20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 7.4 25°C), sonicate, and take the supernatant of the sonicated bacteria and pass through the column . Wash the column with 100ml Column buffer, and then wash it again with 30ml DTT buffer, 30mM (Tris-HC...

Embodiment 2

[0039] Example 2 Screening of humanized anti-IL-22 antibodies

[0040] 1. Screen positive antibodies from phage antibody library

[0041] Phage surface display technology is a new technology established and developed in recent years that uses phage to express foreign genes. It uses the restructured phage as a carrier, and inserts the candidate gene fragments into the gene region of the phage coat protein, so that the foreign polypeptide or protein is expressed and displayed on the phage surface, and then the specific peptide or protein expression is screened by affinity enrichment method. Phage. It realizes the conversion of genotype and phenotype, provides a high-efficiency screening system, and will have a profound impact in many basic and applied research fields. Because this technology has the potential to produce humanized antibodies, it has attracted many scholars to invest in this research, enabling the rapid development of phage antibody library technology, and thus creat...

Embodiment 3

[0047] Example 3 Preparation of recombinant antibodies

[0048] 1. Construction of recombinant antibody plasmid

[0049] The light chain of the obtained Fab antibody was cloned into the pAC-K-Fc vector (cat No. PROGENPR3003), and then the Fd segment of the heavy chain was cloned to construct a recombinant antibody expression vector.

[0050] 2. Transfection and determination of recombinant virus titer:

[0051] Transfect Sf9 cells (Pharmagen Baculo Gold co-transfection kit). For specific transfection methods, see the transfection instructions. After culturing at 27°C for 4-5 days, the supernatant was collected and the virus titer was determined.

[0052] 3. Secretory expression and purification of recombinant antibody IgG:

[0053] Sf9 cells were infected with virus, incubated at 27°C for 2 hours, replaced with SF-900 Ⅱ SFM serum-free medium, cultured at 27°C for 5 days, and the supernatant was collected. The supernatant was purified by affinity chromatography (Amersham, Protein-A affi...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a humanized interleukin-22-resistant genetically engineered antibody and a preparation method and application thereof. By means of the genetic engineering technology and the phage display technology, the antibody capable of being combined with human IL-22 can be directly screened out of a human antibody gene pool, and the antibody gene is obtained and expressed. The antibody can be used for treating autoimmune diseases related to Th17 / IL-17 and detecting IL-22.

Description

Technical field [0001] The invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing a humanized anti-interleukin-22 (IL-22) genetic engineering antibody and its application in Th17 / IL-17 related autoimmune diseases. Background technique [0002] Th17 cells are a newly discovered subset of T cells in recent years, mainly secreting IL-17A, IL-17F, IL-21 and IL-22. The main role of Th17 cells is to participate in host defense against fungi and bacteria, but many studies have found that Th17 / IL-17 is involved in many autoimmune diseases such as psoriasis, lupus erythematosus, multiple sclerosis, inflammatory bowel disease and other diseases and tumors. Play a very important role in development 1 . [0003] The main function of Th17 / IL-17 is to fight against bacterial and fungal infections, but in the process of activating host defenses, it is often accompanied by local inflammatory reactions dominated by inflammatory cell infiltratio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24A61K39/395A61P19/02A61P29/00A61P37/02A61P1/00A61P3/10G01N33/68
CPCC07K16/244C07K2317/565C07K2317/622
Inventor 王晨辉
Owner 武汉原谷生物科技有限责任公司
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