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Cytotoxic T lymphocyte-associated antigen-4, (CTLA-4) fusion protein and preparation method and application thereof

A kind of use, protein technology, applied in the direction of biochemical equipment and methods, peptide/protein components, chemical instruments and methods, etc., can solve the problems of no reports, etc., and achieve the effect of inhibiting T cell activation and obvious immunosuppressive effect

Inactive Publication Date: 2012-08-01
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports about CTLA4Ig fusion proteins of humans, pigs, and dogs, however, there are no reports about CTLA4Ig fusion proteins of monkeys

Method used

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  • Cytotoxic T lymphocyte-associated antigen-4, (CTLA-4) fusion protein and preparation method and application thereof
  • Cytotoxic T lymphocyte-associated antigen-4, (CTLA-4) fusion protein and preparation method and application thereof
  • Cytotoxic T lymphocyte-associated antigen-4, (CTLA-4) fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Preparation of fusion protein mmCTLA4Ig

[0042] 1. Construction of recombinant bacteria

[0043] 1. Preparation of peripheral blood mononuclear lymphocytes:

[0044] Peripheral Blood Mononuclear Cells (PBMCs) were centrifuged by Ficoll-Hypaque density gradient method:

[0045] Take 5-15ml of healthy monkey blood anticoagulated with heparin, wash with equal volume of phosphate buffer saline (PBS, 137mM NaCl, 2.68mM KCl, 8.1mM NaCl 2 HPO 4 , 1.47KH 2 PO 4 , pH 7.2) and spread carefully on top of an equal volume of Ficoll (1.077) solution. Centrifuge at 400g for 30min at room temperature, and the solution after centrifugation has four obvious layers, and the second layer of milky white cells from top to bottom is PBMCs. The collected PBMCs were washed twice with PBS, and the cells were resuspended to 1×10 in RPMI 1640 medium containing 100 U / ml penicillin, 100 mg / l tetracycline and 10% fetal bovine serum. 6 Density culture.

[0046] 2. Obtaining rhesu...

Embodiment 2

[0086]Example 2 Optimization of Induced Expression Conditions for the Fusion Protein mmCTLA4Ig of the Present Invention and Identification of Its Biochemical Properties

[0087] 1. Optimization of expression conditions

[0088] Investigate the induction initial cell density, medium pH, the final concentration of added inducer methanol, and induction expression time, and optimize the induction expression conditions of GS115-pPIC 9K-mmCTLA4Ig:

[0089] The GS115-pPIC 9K-mmCTLA4Ig positive clone obtained in Example 1 was inoculated into BMGY medium (buffered glycerol complex medium, 10mg / ml yeast extract, 20mg / ml peptone, 0.4 μg / ml biotin, 0.1mM / ml potassium phosphate buffer, 67mg / ml yeast nitrogen base, 1% glycerol), 28°C, 280rpm and cultured to the logarithmic growth phase (OD600=2-6). Yeast cells were collected and cultured in BMMY medium (buffered methanol complex medium, 10mg / ml yeast extract, 20mg / ml peptone, 0.4μg / ml biotin, 0.1mM / ml potassium phosphate buffer, 67mg / ml ye...

Embodiment 3

[0113] Example 3 Identification of B7-positive cell-binding ability of fusion protein mmCTLA4Ig

[0114] 1. Experimental materials

[0115] Cell lines: Raji (number TcHu44, tumor cell line of human Burkitt's lymphoma) and K562 (number, human chronic myelogenous leukemia cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences, Raji cell line is B7 positive, K562 cell line is B7 Negative cell line.

[0116] 2. Experimental method

[0117] FITC-labeled protein: first use 1M Na 2 CO 3 Adjust the pH of the protein solution to 8.5, then add FITC dissolved in dimethylformamide according to 20 times the molar amount of the protein to be labeled, incubate at room temperature in the dark for 1 hour, and dialyze with PBS buffer at 4°C to remove the FITC that is not bound to the protein. FITC.

[0118] Based on the fact that human CTLA4 can be combined with human B7 molecules, we use FITC to mark the fusion protein mmCTLA4Ig of the present invention (prepared ...

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Abstract

The invention provides a gene, which is a deoxyribonucleic acid (DNA) molecule with the sequence of nucleotide illustrated as the SEQ ID NO:1. A fusion protein of the gene code and a preparation method and application thereof are further provided. The fusion protein mm CTLA4Ig can prevent a CD 28 molecule from being combined with a B7 molecule, further suppresses activation of a T cell, has an obvious immunosuppression function, can be used for preparing an immunosuppressive agent, and is particularly suitable for the immunosuppressive agent for rhesus monkeys.

Description

technical field [0001] The invention relates to the field of immunosuppression, in particular to a fusion protein and its preparation method and application. Background technique [0002] CTLA4, Cytotoxic T Lymhocyte-Associated Antigen 4 (Cytotoxic T Lymhocyte-Associated Antigen 4) and CD28 (Cluster of Differentiation 28), are both immunoglobulins expressed by T lymphocytes, and they are identical in amino acid sequence and protein structure. similar and highly homologous at the gene level. [0003] B7 molecule is an important molecule in the co-stimulatory signaling pathway that mediates T cell activation, and CTLA4 and CD28 are both natural receptors of B7 molecule. CD28 can interact with B7 molecules to transmit, enhance and amplify the immune response, and promote the activation of T cells. The binding force between CTLA4 and B7 is 10 times that of CD28 and B7, and it is easy to combine with B7 molecules to prevent the combination of CD28 molecules and B7 molecules, th...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C12N15/81C12N1/19C07K19/00C12P21/02A61K38/17A61K47/48A61P37/06C12R1/84
Inventor 卢晓风朱升云刘珊万琳杨浩程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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