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Recombinant solvent protein, its production and use

A fusion protein and recombinant protein technology, which is applied in the fields of biology, food, medicine, and beverage, and can solve the problems of undiscovered recombinant fusion protein immunosuppressant preparation method and use, high incidence rate, and not widely used clinically.

Inactive Publication Date: 2008-10-29
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
[0051] According to literature search, so far, no relevant reports have been found on the recombinant fusion protein targeting the ICOS costimulatory pathway as an immunosuppressant and its preparation method and use.

Method used

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  • Recombinant solvent protein, its production and use
  • Recombinant solvent protein, its production and use
  • Recombinant solvent protein, its production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] Embodiment 1, the preparation method of hICOS-IgG1

[0269] Sources of main reagents:

[0270] Lipofectamine2000 and TRIZOL reagents were purchased from Invitrogen; various tool enzymes were purchased from Takara; hygromycin was purchased from Shanghai Pufei Biotechnology Company; DNA purification and recovery kits and plasmid DNA extraction kits were purchased from Vertejie; IL-2 , IFN-γELISA detection kit was purchased from Jingmei Biotechnology Company. The pMD-18T cloning vector was purchased from TAKARA Company, the pSecTag2 / Hygro A expression vector was purchased from Invitrogen Company, the pGL-3-Basic cloning vector, and the plasmid pMD-18T-Ig carrying the Fc gene of immunoglobulin were preserved in our laboratory; serum-free Medium EX302 was purchased from JRH Company of the United States; protein A affinity purification kit was purchased from Sigma Company of the United States; 293 cells were purchased from Institute of Cells, Chinese Academy of Sciences.

...

Embodiment 2

[0295] Embodiment 2, hICOS-IgG1 and ICOS ligand binding test

[0296] (1) Principle

[0297] hICOS-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 hICOS-IgG1 binding to its ligand increases with the increase of hICOS-IgG1 content. The fluorescently labeled anti-IgGFc antibody can be combined with the ligand-bound hICOS-IgG1, and the fluorescence emitted by the bound anti-IgGFc antibody can be detected by flow cytometry to reflect the combination of hICOS-IgG1 and its ligand.

[0298] (2) Operation steps

[0299] After blocking the FcR of Daudi cells with 20% rabbit serum, take 1×10 Daudi cells 6 After washing twice with PBS, add 2ug / ml, 10ug / ml two concentrations of hICOS-IgG1 and blank control 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 activity.

[0300] (3) Results

[0...

Embodiment 3

[0302] Embodiment 3, hICOS-IgG1 biological activity assay

[0303] (1) Principle

[0304] 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 hICOS-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 hICOS-IgG1 has the ability to block the ICOS co-stimulatory pathway and can inhibit the proliferative response between allogeneic lymphocytes.

[0305] (2) Operation steps

[0306] Peripheral blood mononuclear cells (2×10 6 / ml) each 50ul, add hICOS-IgG1 100ul (final concentration is 209,105,53,27,14,7,0ug / ml) that contains the 1...

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Abstract

A recombinant fused egg albumen, its production are 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 safety and specificity, 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 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 autoimmune diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/79A61K38/16A61P37/02A61P37/06A61P29/00A61P3/10A61P3/04A61P3/06A61P1/00A61P1/16A61P1/18A61P15/00A61P7/00A61P17/06A61P25/00A61P5/00A61P35/00A61P31/12A61P27/02A61P19/02
Inventor 沈茜王健张鹏杨佳荟张军
Owner SHANGHAI CHANGHAI HOSPITAL
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