Human interleukin 10-Fc fusion protein, and coding gene and application thereof

A fusion protein, il10-fc technology, applied in human interleukin 10-Fc fusion protein and its coding gene and application field, can solve the problems of side effects of treatment, immunogenicity of Fc fusion protein, etc.

Active Publication Date: 2018-12-07
HANGZHOU BOHU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although this approach is feasible for IL-10 therapy, the human body will produce immunogenicity when the Fc fusion protein is administered repeatedly for a long period of time, which is a potential hidden danger of this type of drug
In addition, if the Fc part retains unwanted biological effector functions, it will lead to additional therapeutic side effects, which is also a potential concern for Fc fusion protein therapy

Method used

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  • Human interleukin 10-Fc fusion protein, and coding gene and application thereof
  • Human interleukin 10-Fc fusion protein, and coding gene and application thereof
  • Human interleukin 10-Fc fusion protein, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: IL10-Fc fusion protein gene construction

[0053] SEQ ID NO: 3 was translated into a DNA sequence, and optimized according to the codon preference of CHO cells to obtain the expression sequence SEQ ID NO: 7 of the IL10-Fc fusion protein. Add NheI restriction site and kozac sequence (SEQ ID No.8) at the 5' end of the optimized sequence, and add stop codon and XhoI restriction site (SEQ ID No.9) at the 3' end to obtain fusion The complete expression cassette of the protein. The complete expression cassette sequence was artificially synthesized and inserted between the NheI and XhoI restriction sites of the pIRES plasmid to obtain the pIRES-IL10-Fc expression plasmid. After the plasmid was linearized, it was electrotransfected into CHO-s cells, and positive clones were screened by adding MSX.

Embodiment 2

[0054] Example 2: Expression and purification of IL10-Fc fusion protein

[0055] For the positive clones obtained in Example 1, after two rounds of limiting dilution, clones with better expression levels were screened out, and after expanded culture, they were inoculated into a 7L fermenter for fed-batch culture to express the target protein. Centrifuge at 4500rpm for 6min after fermentation, collect the supernatant, adjust the pH to 4.0, and store at 4°C.

[0056] The supernatant was first wrapped with a 10KDa ultrafiltration membrane and concentrated by ultrafiltration; then a preliminary affinity chromatography was performed with Mabselect Sure to collect the fusion protein. The mobile phase of affinity chromatography is: A1: 25mM PB+50mM Nacl, pH 7.0, B1: 20mM Gly, pH 3.0, B2: 20mM citric acid buffer, pH 3.0. The chromatographic column was first equilibrated with mobile phase A1. After loading the sample, the impurities were first eluted with mobile phase B1, and then the...

Embodiment 3

[0057] Example 3: In Vivo Half-Life Determination

[0058] Through tail vein injection, rhIL-10 (Rochy Hill Company) and the IL10-Fc fusion protein obtained in Example 2 were respectively injected into SD rats with an average body weight of about 200 g at a dose of 200 ng / Kg body weight). After injection, at different time points (0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96 hours) blood samples were collected by tail-cutting blood method, anticoagulated with heparin sodium, and The collected blood samples were centrifuged at 12000 g for 5 min to collect serum.

[0059] The human IL-10 ELISA kit (purchased from Bender Medsystem Company) was used to detect the content of the fusion protein in the blood samples according to the instructions, and the results were averaged. The results show that the elimination half-life of the IL10-Fc fusion protein prepared by the present invention is 22.6 hours in vivo, while the elimination half-life of human rhIL-10 is 4 hours after tail vei...

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Abstract

The invention relates to the field of genetic engineering medicines, in particular to a human interleukin 10-Fc (IL10-Fc) fusion protein, and a coding gene and application thereof. By replacing aminoacids at multiple positions in an IgG4Fc part, the modified IL10-Fc fusion protein has superior performance over existing Fc fusion proteins, such as increasement of in-vivo stability, elimination ofunnecessary effector functions and reduction of immunogenicity of the fusion proteins in living organisms. The invention also discloses a method for treating diseases by using an IL10-Fc fusion protein medicament, and the method comprises administering a therapeutically effective amount of the medicament to a subject suffering from diseases, wherein the diseases comprise an inflammatory symptom, immune related diseases, fibrosis diseases, cancers and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering medicines, in particular to a human interleukin 10-Fc fusion protein and its encoding gene and application. Background technique [0002] Interleukin-10 (Interleukin-10, IL-10) is a cytokine discovered in 1991, which can regulate the body's inflammation and immune response. It was initially reported that this cytokine can inhibit cytokine secretion, antigen presentation and CD4+ cell activation, and IL-10 can inhibit IL-1α, IL-1β, IL-6, IL -8, the expression of TNF-α, GM-CSF and G-CSF to suppress the immune response, and it also inhibits the production of IFN-γ by NK cells. Although IL-10 is predominantly expressed in macrophages, expression has also been detected in activated T cells, B cells, mast cells and monocytes. In addition to suppressing immune responses, IL-10 also exhibits immunostimulatory properties, including stimulating the proliferation of IL-2 and IL-4-treated thymocytes, enha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/20A61P31/12A61P29/00A61P37/02A61P35/00
CPCA61K38/00A61P29/00A61P31/12A61P35/00A61P37/00A61P43/00C07K14/5428C07K2319/30
Inventor 周亮
Owner HANGZHOU BOHU BIOTECH CO LTD
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