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RANKL-Fc fusion protein, preparation method and application thereof

A technology of fusion protein and protein, applied in the field of biomedicine

Active Publication Date: 2009-08-26
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report of RANKL recombinant protein that can reduce the formation of osteoclasts by activating the information transduction system of ITIM in cells to inhibit the information transduction of ITAM.

Method used

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  • RANKL-Fc fusion protein, preparation method and application thereof
  • RANKL-Fc fusion protein, preparation method and application thereof
  • RANKL-Fc fusion protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Expression of fusion protein RANKL-Fc

[0043] The expression process of the RANKL-Fc fusion protein comprises the following steps: synthesizing the gene; inserting the gene into an expression vector, transfecting CHO cells with the expression vector; utilizing Hygromycin to screen and express the CHO engineering cell line of the RANKL-Fc fusion protein, cultivating the cell line, and finally from The RANKL-Fc protein was isolated and purified from the cell culture medium. Specifically include:

[0044] 1) Gene synthesis

[0045] Entrust a professional company of gene synthesis (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) to synthesize the nucleotide sequence shown in SEQ ID NO: 5, in which 5-10 is the restriction site of Nhe I; 11-73 is the encoding Igκ The nucleotide sequence of chain secretion signal peptide; 74-823 is the nucleotide sequence of coding human RANKL extracellular region, and the RANKL part of its coding corresponds to 68...

Embodiment 2

[0052] Example 2 RANKL-Fc fusion protein and RANK (RANKL receptor) binding experiment

[0053] Mononuclear cells were isolated from human peripheral blood using magnetic bead separation technology. Will 1×10 6 After the monocytes were cultured in MCSF and RANKL medium for 72 hours, they were reacted with 5 micrograms of the purified RAKL-Fc fusion protein in Example 1 for 1 hour at 4° C., and 5 microliters of FITC-labeled anti-human RANKL antibody (CALTAG, CA ), fluorescently labeled cells were detected by flow cytometry. The result is as figure 1 As shown, it shows that the fusion protein RIG expressed in Example 1 can combine with RANK, and THP1 cells are FITC positive. figure 1 Among them, SSC-H is the number of particles in the cell, FSC-H is the cell size, FL2-H is PE, and FL1-H is FITC.

Embodiment 3

[0054] Example 3 Binding experiment of RANKL-Fc fusion protein and FcγRII receptor

[0055] HMC-1 is a high-expression cell line screened in Example 1, and expresses FcγRII on the cell surface. HMC-1 cells were cultured in DMEM medium containing 10% fetal bovine serum. Will 1×10 6 HMC-1 cells were reacted with 5 micrograms of the RANKL-Fc fusion protein purified in Example 1 at 4°C for 1 hour, and 5 microliters of FITC-labeled anti-human IgG FITC-labeled antibody (CALTAG, CA) was added, and detected by flow cytometry cell. The result is as figure 2 As shown, it shows that the fusion protein RIG expressed in Example 1 can bind to the FcγRII receptor, and HMC-1 cells are FITC positive. figure 2 Among them, SSC-H is the number of particles in the cell, FSC-H is the cell size, FL2-H is PE, and FL1-H is FITC.

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Abstract

The invention relates to RANKL-Fc fusion protein, a preparation method and the application thereof. The RANKL-Fc fusion protein has P1-P2 or P1-L-P2 structural style; wherein, the P1 is the extracellular region fragment of PANKL protein, P2 is the Fc part of human I g C, and L is linker peptide. The fusion protein provided by the invention can be used for preparing medicines used for treating bone resorption diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a RANKL-Fc fusion protein and its preparation method and application. Background technique [0002] Bone formation and bone resorption are two closely related processes in normal bone metabolism. In patients with osteoporosis, due to the increased activity of osteoclasts, the rate of bone resorption exceeds the rate of bone formation, resulting in osteoporosis, in which there is no bone mineral deficiency. Bone is also a common site of tumor metastasis and invasion. Multiple myeloma, breast cancer, prostate cancer and lung cancer often metastasize to bone. The interaction between tumor cells and osteoblasts stimulates the differentiation of precursor cells in the bone marrow into osteoclasts. Therefore, the number of osteoclasts commonly seen in tumor metastases in the bone increases, and the osteoclastic bone resorption also increases accordingly. Overall, the excessive ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12P21/02A61K38/19A61K47/48A61P19/08A61K47/42
CPCC07K2319/30
Inventor 包骏陈海明祝道成
Owner SHANGHAI KEXIN BIOTECH
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