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Fused polypeptide for IL-17 and TNF[alpha], and application thereof

A technology for fusing polypeptides and compositions, which is applied in the directions of hybrid peptides, medical preparations containing active ingredients, peptides, etc., to achieve the effects of relieving clinical symptoms, improving therapeutic effects and significant application value

Active Publication Date: 2015-01-28
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, biological inhibitors targeting single cytokines such as TNFα and IL-17 have been reported at home and abroad, while biological agents that simultaneously resist two cytokines have not been reported at home and abroad

Method used

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  • Fused polypeptide for IL-17 and TNF[alpha], and application thereof
  • Fused polypeptide for IL-17 and TNF[alpha], and application thereof
  • Fused polypeptide for IL-17 and TNF[alpha], and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, Structural Description of IL-17scfv / sTNFRI Fusion Polypeptide

[0035] The fusion polypeptide for hIL-17 and hTNFα provided by the present invention is named as IL-17scfv / sTNFRI fusion polypeptide, which consists of A fragment and B fragment. Fragment A consists of the heavy chain variable region (VH) of the anti-human IL-17 antibody, the connecting peptide (GS linker), the light chain variable region (VL) of the anti-human IL-17 antibody, CH1 and The antibody hinge region (hinge region) between CH2 is composed. Fragment A is shown in sequence 1 of the sequence listing, and its coding gene is shown in sequence 2 of the sequence listing. Fragment B consists of soluble tumor necrosis factor receptor I. Fragment B is shown in sequence 3 of the sequence listing, and its coding gene is shown in sequence 4 of the sequence listing. For the structural composition of the IL-17scfv / sTNFRI fusion polypeptide, see figure 1 .

[0036]

Embodiment 2

[0037] Example 2, Preparation of IL-17scfv / sTNFRI Fusion Polypeptide

[0038] 1. After overlapping PCR ligation of double-stranded DNA molecules shown in sequence 2 and sequence 4 in the sequence listing, insert into the multiple cloning site of pET-27b(+) vector to obtain a recombinant plasmid.

[0039] 2. Introduce the recombinant plasmid obtained in step 1 into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0040] 3. Inoculate the recombinant bacteria obtained in step 2 into LB liquid medium containing 50 μg / mL kanamycin, culture at 37°C and shake at 120r / min until OD 600nm =0.5, add IPTG to make the concentration 0.5mmol / L, shake culture at 37°C and 120r / min for 4h, and collect the bacteria by centrifugation.

[0041] 4. Take the bacterial cells obtained in step 3, ultrasonically disrupt (20KHz, 10 cycles, work for 10 seconds and stop for 1 second in each cycle), centrifuge (12000r / min, 5min) to collect the precipitate (ie inclusion body), wash with solu...

Embodiment 3

[0045] Example 3, Biological Identification and Blocking Activity Detection of IL-17scfv / sTNFRI Fusion Polypeptide

[0046] 1. The ability of IL-17scfv / sTNFRI fusion polypeptide to bind antigen simultaneously

[0047] Spot the mature antigen protein hTNFα directly on the NC membrane (negative control is BSA), block with blocking solution (containing 5% skimmed milk powder) for 2 hours, then incubate with IL-17scfv / sTNFRI fusion polypeptide diluted in blocking solution for 1 hour at 37°C, PBST Wash the membrane 3 times, 10 min each time; then incubate with the mature antigen protein hIL-17 (purchased from OriGene Technologies, LY400795) diluted with blocking solution for 1 h at 37°C, wash the membrane 3 times with PBST, 10 min each time; then add Mouse anti-human IL-17 monoclonal antibody (purchased from eBioscience, 14-7178) and goat anti-mouse secondary antibody (purchased from LifeSpan BioSciences, LS-C60712-2000). Finally, ECL chemiluminescent exposure. Dot-blot results...

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Abstract

The invention discloses a fused polypeptide for IL-17 and TNF[alpha], and an application thereof. The invention provides a fused polypeptide, specifically an IL-17scfv / sTNFR1 fused polypeptide. The fused polypeptide is composed of an A segment and a B segment, wherein the A segment is (a) protein composed of amino acid sequences shown as a sequence 1 in a sequence table or (b) protein that is derived from the sequence 1 by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequences shown as the sequence 1 and has the same functions; and the B segment is (c) protein composed of amino acid sequences shown as a sequence 3 in the sequence table or (d) protein that is derived from the sequence 3 by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequences shown as the sequence 3 and has the same functions. The fused protein provided by the invention can effectively alleviate clinical symptoms of rheumatoid arthritis (RA) and has significant application values for treating the rheumatoid arthritis (RA).

Description

technical field [0001] The present invention relates to a genetically engineered novel bispecific multi-target fusion polypeptide with the activity of simultaneously resisting interleukin-17 and tumor necrosis factor α, in particular to bispecific multi-target fusion polypeptides that block the inflammatory response of autoimmune diseases fusion peptide. Background technique [0002] IL-17 is a powerful pro-inflammatory cytokine and a fine-tuning cytokine of inflammatory response. IL-17 can stimulate keratinocytes, fibroblasts, epithelial cells and endothelial cells to release IL-6, IL-8, prostaglandin E2 (PGE2), metalloproteinase-1 (MMP-1), granulocyte colony-stimulating factor (G -CSF) and monocyte chemoattractant protein (MCP)-1 and other cytokines. At present, it is believed that the mechanism of IL-17 promoting bone destruction in rheumatoid arthritis (RA) may include: inducing the production of TNFα and IL-1 to enhance the destructive effect of joints; increasing the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/395A61K38/17A61P37/02A61P19/02A61P29/00
Inventor 李德山任桂萍阚方明韩晓辉
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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