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Novel IL23 antagonist

An IL-23, fusion protein technology, applied in allergic diseases, peptide/protein components, drug combinations, etc., can solve problems such as poor stability, difficult endotoxin removal, and short half-life

Inactive Publication Date: 2015-06-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although IL23R-CHR has a certain in vitro activity of inhibiting Th17 differentiation and development, it is expressed in prokaryotes and lacks the protein modification process after eukaryotic transcription, so it has short half-life, poor stability and difficult to remove endotoxins.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Construction of pcDNA3.1(+)-IL23R-CHR / Fc expression vector

[0093] 1) Construction of IL23R-CHR / Fc fusion gene by Overlap PCR

[0094] a) Using the human leukocyte cDNA library or existing related plasmids as templates, amplify the signal peptide (SP, signal peptide) with two pairs of upstream and downstream primers, F(SP) and R(SP), F(CHR) and R(CHR), respectively. ) and IL23R-CHR gene; mRNA was extracted from normal human peripheral blood leukocytes, reverse-transcribed to obtain the first strand of cDNA, and then used as a template to amplify IgG1Fc gene with F (Fc) and R (Fc) as upstream and downstream primers . Using high-fidelity enzyme amplification, Touch-down PCR conditions enhance the efficiency of specific band amplification. Signal peptide, IL23R-CHR and Fc amplification products were separated and identified by agarose gel electrophoresis, and about 80bp, 570bp and 700bp fragments were recovered, respectively.

[0095] Table 1 Primer sequence...

Embodiment 2

[0107] Example 2: Transient transfection expression of rhIL-23R-CHR / Fc protein

[0108] 1) About 24 hours before transfection, subculture the 293T cells again, try to ensure that the cells are in good condition and the density reaches 70-90% at the time of transfection; before transfection, replace the old medium with fresh DMEM culture without serum base.

[0109] 2) According to the different culture devices, prepare a sufficient amount of transfection complex according to the ratio of PEI(ug):plasmid (ug)=2:1 to 5:1; the plasmid and PEI are diluted in an equal volume of opti-MEM respectively, Then add the PEI dilution to the DNA dilution, mix gently, and incubate at room temperature for 10-15 minutes to obtain a PEI / DNA transfection complex of 1 / 10 times the volume of the medium.

[0110] 3) Evenly drop the transfection complex into the culture device, shake gently to mix it with fresh medium, replace with serum-free medium after 4-6 hours of transfection, culture for 3-5 ...

Embodiment 3

[0111] Example 3: Screening of stable expression cell lines of rhIL-23R-CHR / Fc protein

[0112] Take CHO cells in the logarithmic growth phase at an appropriate concentration and add them to the electric shock cup, then add an appropriate amount of linearized expression plasmids, mix and let stand on ice for about 15 minutes, and set the corresponding parameters of the electroporation instrument to complete. Transfer to FBS-containing DMEM-F12 medium for culture (liposomes can also be used to transfect the plasmid), and when the cell growth state is restored, positive cells are screened with 1 mg / ml G418. Observe the state of the cells, change the medium every 3 days, and after 10 to 14 hours of culture, use the limiting dilution method to screen high-expression monoclonal cell lines using a 96-well culture plate. Immunospot method and Western blot can be used to compare the expression levels of different clones for screening ( Figure 4 ).

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Abstract

The invention relates to a recombinant human IL (Interleukin)-23 receptor extracellular region / Fc fusion protein capable of combining with IL-23 and antagonizing a function of IL-23. The fusion protein is characterized in that a fusion protein encoding gene is rich in mammalian cell preferred codons, and is highly expressed by a CHO (Chinese Hamster Ovary) stable transfection cell strain. The protein is formed by fusion of a gene optimized IL-23R-CHR active fragment and a human immune globulin molecule Fc fragment; the defects that the half-life period of the prokaryotic expression IL-23R-CHR fragment is short, the stability is poor and endotoxin is difficult to remove are overcome; the biological activity of IL-23-CHR is reserved; in addition, the fusion protein does not have ADCC (Antibody Dependent Cellular Cytotoxicity) and CDC (Complement Dependent Cytotoxicity) effects, so that the fusion protein is more suitable for treatment of chronic diseases such as an autoimmune disease and a chronic infection; and the contents of molecular design, expression, purification, construction of a stable expression cell strain and disease treatment and the like of the fusion protein are included.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to an IL-23 receptor extracellular region fragment / Fc fusion protein capable of binding IL-23 and antagonizing its action, a cell line stably expressing the fusion protein, and the fusion protein to abnormal Th17 cells Treatment of diseases characterized by activation. Background technique [0002] Th17 cells are a special cell subgroup during the differentiation and development of T cells. They exert immunological functions by secreting the characteristic cytokine IL-17, and are closely related to the pathogenesis of autoimmune diseases and host resistance to extracellular bacterial infections. IL-17 is a pro-inflammatory cytokine that can recruit and activate neutrophils, induce the expression of other inflammatory factors, mediate the infiltration of inflammatory cells in the lesion site and eventually cause tissue damage. Studies have shown that there are inappropriate activatio...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/63C12N5/10C12N1/21A61K38/16A61P37/02C12R1/185
Inventor 高向东郭薇王欣姚文兵王辰郁冬梅陈语聪王宇恒
Owner CHINA PHARM UNIV
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