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Application of human cell factor CCDC134 to autoimmune diseases

An autoimmune and cytokine technology, applied in allergic diseases, disease diagnosis, nervous system diseases, etc., can solve the problems of underdeveloped functions and affect the anti-bacterial immunity of macrophages, so as to reduce infiltration and alleviate EAE The effect of disease

Active Publication Date: 2016-03-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relevant experimental results only show that this cytokine has a certain effect on the proliferation of lymphocytes, and may play a certain role in the development of T lymphocytes and the maturation of B lymphocytes, promote the activation of T lymphocytes, and may affect the anti-inflammatory effect of macrophages. The function of bacterial immunity
[0004] Although some progress has been made in the research of human cytokine CCDC134, its function is still not fully developed. + T cells have a certain role in promoting activation, proliferation and specific killing, and play an anti-tumor effect

Method used

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  • Application of human cell factor CCDC134 to autoimmune diseases
  • Application of human cell factor CCDC134 to autoimmune diseases
  • Application of human cell factor CCDC134 to autoimmune diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Preparation of recombinant human CCDC134 protein

[0049] 1.1 Constructed pMH-CCDC134-his6 fusion protein expression plasmid for expressing CCDC134-his6 fusion protein

[0050] Method: Insert the coding sequence of CCDC134 (SEDIDNO: 1) into the pMH expression vector (Biovector, operated according to the instruction manual) to construct the pMH-CCDC134-his6 expression plasmid (SEQ ID NO: 4), where the sequence of SEQ ID NO: 4 is: start codon atg-IgK signal peptide gene (human immunoglobulin Kappa light chain signal peptide)-CCDC134 coding gene that removes its own signal peptide-6 histag-stop codon tag.

[0051] Result: The sequence of the coding region was correct after DNA sequencing ( figure 1 ).

[0052] 1.2 Perform SDS-PAGE and WB to verify the expression and purification of the target protein

[0053] Method: After the correctness of the plasmid was verified by sequencing, Jiangsu Taicang Minor Hengkang Biotechnology Co., Ltd. was entrusted to prep...

Embodiment 2

[0055] Example 2: Separation and purification of CD4 by recombinant human CCDC134 protein + Effects on T lymphocyte proliferation

[0056] 2.1 Human CD4 + Acquisition of T lymphocytes

[0057] Methods: A 2U concentrated leukocyte blood sample was obtained from Beijing Blood Center. First, normal human peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) were isolated from normal human peripheral blood using lymphocyte separation medium, and then CD4 + T cell positive sorting magnetic beads (refer to the product manual) to obtain CD4 + T lymphocytes; CD4 separated from magnetic beads + Add mouse anti-human CD45RO antibody and rat anti-mouse IgG2a sorting magnetic beads to T lymphocytes to remove CD45RO + cells, the remaining cells are CD45RA + na?ve T lymphocytes. Take a portion out of it (5×10 5 ), and its purity was detected by FACS, the steps are as follows: add 200 μL of pre-cooled blocking solution (5% FBS / PBS, v / v), block at 4 °C for 30 min,...

Embodiment 3

[0063] Example 3: Recombinant human CCDC134 protein to na?veCD4 + Influence of T lymphocyte differentiation process

[0064] 3.1 Isolation of human CD4 +Na?veT cells and Th1, Th2 and Th17 cells induced in vitro, and the role of recombinant human CCDC134 protein in the differentiation process was detected

[0065] Methods: Firstly, na?veT lymphocytes were obtained by magnetic bead sorting; in order to induce the differentiation of na?veT cells in vitro, we cultured them in cells coated with anti-CD3 (1 μg / mL; cloneOKT3) and anti-CD28 (2 μg / mL) ; clone15E8) antibody in cell culture plates, stimulating its activation and proliferation. If Th1 cells are induced, add recombinant human IL-12 (2.5ng / mL), recombinant human IL-2 (10ng / mL) and anti-IL-4 antibody (5μg / mL) to the culture system; if Th2 cells are induced , then add recombinant human IL-4 (12.5ng / mL), recombinant human IL-2 (10ng / mL), anti-human IFN-γ (5μg / mL; cloneB-B) and anti-human IL-10 to the culture system (5μg / mL...

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Abstract

The invention discloses application of a human cell factor CCDC134 to autoimmune diseases. Particularly, genes or proteins of the cell factor are used as an effective component of a detection marker and / or a treatment medicine and are used for preparing a detection reagent and / or a treatment medicine for CD<4+> T cell subset dysfunction mediated autoimmune diseases; and the nucleotide sequence of the proteins is represented as SEQ ID NO: 1 and the amino acid sequence of the proteins is represented as SEQ ID NO: 2. A research shows that the human cell factor CCDC134 can be used for inhibiting the proliferation of naive CD<4+> T cells and also can be used for promoting the naive CD<4+> T cells to be differentiated into Th1 and / or Treg cells, so that the human cell factor CCDC134 can be applied to the CD<4+> T cell subset dysfunction mediated autoimmune diseases.

Description

technical field [0001] The present invention relates to the technical field of molecular biology and immunology, and the new use of cytokines, in particular to human cytokine CCDC134 in the preparation of CD4 + The application as a marker and / or therapeutic drug in related diseases mediated by abnormal T cell function. Background technique [0002] The human cytokine CCDC134 (Coiled-CoilDomainContaining134) is a new molecule with functional activity that was first reported internationally by the inventor of this patent in 2008. In the previous work, it has been confirmed that CCDC134 is a classic secreted protein, which plays an important negative regulatory role in the transcriptional regulation of the MAPK (Mitogen-Activated Protein Kinase) signaling pathway (HuangJ, etal. CellMolLifeSci.2008.65(2):338-349); CCDC134 can also be used as a nuclear protein, together with its interacting protein hADA2a (humanTranscriptionalAdaptor2a) to form a nuclear protein complex, and pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577C12N15/85A61K38/19A61P37/02A61P25/00
CPCA61K38/19A61K48/00G01N33/577G01N33/6893G01N2333/52G01N2800/24G01N2800/285
Inventor 黄晶夏鹏余骉亿巩晓婷肖琳邱晓彦
Owner PEKING UNIV
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