Recombinant solvent protein derivative, its production and use

A fusion protein and recombinant protein technology, which is applied in the fields of biology, medicine, food and beverage, can solve the problems such as the preparation method and application of recombinant fusion protein derivative immunosuppressants, the side effects of increasing resistance, and the lack of widespread clinical application. Achieve the effects of reduced preparation cost, improved capacity, and long half-life

Inactive Publication Date: 2006-08-16
SHANGHAI CHANGHAI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0035] The first stage (1908-1967): up to 70 years, using radiation or chemical drugs to destroy all differentiated cells indiscriminately. This immunosuppressant has a high incidence of severe infection and death, so it is not widely used clinical
[0036] The second stage (1967-1976): mainly focused on the research on the inhibition of T cells, using polyclonal serum preparations that have an immunosuppressive effect on T cells. Although this immunosuppressant can reduce the activity of various T cells, Improves graft survival, but increases recipient resistance as a side effect
[0052] However, after searching the literature, so far, no relevant reports have been found on the recombinant fusion protein derivatives with high affinity / high biological activity targeting the ICOS co-stimulatory pathway as immunosuppressants and their preparation methods and uses.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0288] Embodiment 1, the preparation method of ICOS120R-IgG1

[0289] ① According to the sequence of the ligand binding region of human ICOS, design primers, clone the required ICOS fragment from the DNA of human peripheral blood mononuclear cells by standard PCR method, and use the site-directed mutagenesis kit to modify the 120th amino acid in the above ICOS fragment Replace it with arginine, and use the same standard PCR method to clone the immunoglobulin constant fragment from the plasmid (pIgplus) containing human IgG Fc; use the SOC-PCR method to connect the required ICOS fragment and the immunoglobulin constant fragment into ICOS120R-IgG1 recombinant gene fragment, and the above recombinant gene fragment and mammalian eukaryotic expression vector pSecTag2 / Hygro A were digested with sfiI and NotI double enzymes, and the required ICOS fragment was inserted into pSecTag2 / Hygro A using the sticky ends of sfiI and NotI double enzymes Among them, pSecTag2 / Hygro A-ICOS120R-IgG...

Embodiment 2

[0293] Embodiment 2, ICOS120R-IgG1 and ICOS ligand binding test

[0294] (1) Principle

[0295] ICOS120R-IgG1 can specifically bind to the ICOS ligand on the surface of its ligand-expressing cells. When the amount of ligand is constant, the amount of ICOS120R-IgG1 binding to its ligand increases with the increase of the content of ICOS120R-IgG1. The fluorescently labeled anti-IgGFc antibody can be combined with the ligand-bound ICOS120R-IgG1, and the fluorescence emitted by the bound anti-IgGFc antibody can be detected by flow cytometry to reflect the combination of ICOS120R-IgG1 and its ligand.

[0296] (2) Operation steps

[0297] After blocking the FcR of Daudi cells with 20% rabbit serum, take 1×10 Daudi cells 6 After washing twice with PBS, add ICOS120R-IgG1 and blank control at two concentrations of 2ug / ml and 10ug / ml respectively, incubate at 37°C for 1 hour, use HRP-labeled anti-human IgG as the detection antibody, and use flow cytometry Assay for ligand binding act...

Embodiment 3

[0300] Embodiment 3, ICOS120R-IgG1 biological activity assay

[0301] (1) Principle

[0302] After lymphocytes are stimulated by antigens, with the participation of co-stimulatory molecules, lymphocytes are activated and exhibit biological activities such as proliferation and secretion of cytokines. Without the participation of ICOS co-stimulatory molecules, the activated lymphocytes will quickly inactivate and even apoptotic. Adding ICOS120R-IgG1 in the process of allogeneic lymphocyte interaction, if lymphocyte proliferation is inhibited or cytokine secretion is inhibited, it reflects that the co-stimulatory pathway of ICOS is blocked, indicating that ICOS120R-IgG1 has the ability to block the ICOS co-stimulatory pathway and can inhibit the proliferative response between allogeneic lymphocytes.

[0303] (2) Operation steps

[0304] Peripheral blood mononuclear cells (2×10 6 / ml) each 50ul, add ICOS120R-IgG1 100ul (final concentration is 100, 50, 25, 12.5, 6.25, 3.125, 0u...

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PUM

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Abstract

A recombinant fused egg albumen derivative, its production and use are disclosed. The procedure is carried out by fusing, expressing, purifying to obtain final product. It can inhibit memory specifically, activate T-type cell and prolong half-life period. It can be used to large-scale prepare immune inhibiting product with high biological activity and safety, treat, diagnose and inspect immune diseases.

Description

technical field [0001] The present invention relates to the technical fields of medicine, biology, food and beverage, in particular to a biotechnology product and its preparation method and use, more specifically to a recombinant fusion protein derivative and its preparation method and use. Background technique [0002] (1) Research progress of autoimmune diseases [0003] 1. Overview [0004] The human immune system has the function of distinguishing "self" from "non-self", and it will not produce an immune response to its own tissues and cells. However, when the human immune system is abnormal, it may also have an immune response to its own tissues and cells, thereby destroying or injuring its own tissues or cells, that is, an autoimmune disease, and one of its important signs is the formation of autoantibodies against its own tissue components , The inspection and control of the autoantibody is an important diagnostic method and the basis of clinical treatment of autoim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/09C12N15/79C12N15/86A61K38/17A61K39/395A61P37/06A61P37/08A61P29/00A61P17/06A61P1/00A61P1/16A61P1/18A61P7/00A61P25/00A61P15/00A61P3/04A61P3/06A61P3/10A61P35/00A61P5/00G01N33/68
Inventor 沈茜王健张鹏杨佳荟张军
Owner SHANGHAI CHANGHAI HOSPITAL
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