Preparation and application of human fibroblast growth factor 21 fusion protein and mutant of human fibroblast growth factor 21 fusion protein

A human fibroblast and fusion protein technology, which is applied in the preparation method of peptides, medical preparations without active ingredients, and medical preparations containing active ingredients, etc., to achieve the effects of improving affinity, reducing injection frequency and reducing pain.

Inactive Publication Date: 2012-07-11
北京康明百奥科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Xinqing Huang et al. used diethylnitrosamine to act on transgenic mice expressing FGF21 in large quantities in hepatocytes. Surprisingly, compared with nude mice, transgenic mice could significantly delay the occurrence of adenomas, although both were in There is not much difference in the occurrence time of liver cancer, but the results also prove that FGF21 does not have the notoriety of cancer factors like other members of the FGF family
[0009] Alexei Kharitonenkov found that FGF21 can phosphorylate FGFR-1 (fibroblast growth factor receptor 1) and FGFR-2 (fibroblast growth factor receptor 2) when studying the biological function of FGF21, but this process does not The involvement of heparin is not required, which is inconsistent with previous understanding of FGF family members

Method used

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  • Preparation and application of human fibroblast growth factor 21 fusion protein and mutant of human fibroblast growth factor 21 fusion protein
  • Preparation and application of human fibroblast growth factor 21 fusion protein and mutant of human fibroblast growth factor 21 fusion protein
  • Preparation and application of human fibroblast growth factor 21 fusion protein and mutant of human fibroblast growth factor 21 fusion protein

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Experimental program
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Embodiment 1

[0068] Embodiment 1: the construction of carrier

[0069] A vector encoding fusion protein containing FGF21 and its mutants and IgG2-Fc and its mutants was constructed by overlapping PCR. The IgG2-Fc region contains an IgG2 constant heavy chain (hinge-CH2-CH3 portion). The murine IgK secretion leader sequence was fused to the FGF21 sequence in order to direct the secretion of the synthetic fusion protein into the culture medium. The cDNA encoding the FGF21 / hIgG-Fc fusion protein was chemically synthesized and ligated to the PCR-amplified product encoding human IgG2-Fc (hinge, CH2, and CH3), then inserted into the EcoRV and BamHI of the pcDNA3.1 vector The FGF21 / hIgG-Fc-pcDNA3.1 vector was constructed between the sites. Secretable FGF21 / hIgG-Fc and its mutant fusion proteins contain an IgG2 constant heavy chain (hinge-CH2-CH3). The ERBITUX heavy chain secretion guide peptide sequence (IGH) is fused to the FGF21 sequence to guide the secretion of the synthesized protein into ...

Embodiment 2

[0076] Example 2: CHO expression of FGF21 / IgG-Fc and its mutant fusion proteins

[0077] FGF21 / IgG-Fc or IgG-Fc cDNA was transfected into CHO-S cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The process is to first make the density 2.5×10 per well 5 Individual CHO-S cells were grown in 6-well cell culture plates. Serum-free and antibiotic-free DMEM (Invitrogen) containing 4 µg of FGF21 / IgG-Fc cDNA and 10 µl of transfected liposomes was added for culture. After 6 hours of transfection, switch to full culture medium. Culture medium and cells were collected 48h after transfection. In order to express FGF21 / IgG-Fc fusion protein on a large scale, CHO-S cells grown in a 150mm culture dish were related with cationic transfection reagent, poly-ethyleneimine (Poly-ethyleneimine; PEI, 25kDa) and 80μg. cDNA transfection. Dilute DNA and PEI with 150mM Nacl respectively, mix and incubate for 20min. Then the DNA / PEI complex was added to the cells and incubated for 6 h in ser...

Embodiment 3

[0079] Example 3: Purification of fusion proteins from mammalian cell cultures

[0080] Medium-scale protein purification can be performed using a Sephadex A column. A column volume of 50 ml can purify the conditioned medium of CHO-S cells transfected with FGF21 / IgG-Fc and its mutant fusion vector grown in 15 cm dishes. 50 ml of DMEM culture medium at 48 h after cell transfection, or cells stably expressing fusion protein were collected and added to a 1 mL full volume of Protein A Sephadex (Amersham-Pharmacia). Add 1% TritonX-100 and incubate overnight at 4°C. The protein was then eluted from the resin by washing with 1% TritonX-100 in PBS buffer, then with 150 mM NaCl, and finally with 1 mM NaCl glycine (pH 2.7). The eluate was immediately neutralized with Tris buffer (pH 9.0) and the purified protein was desalted with a PD-10 gel column (Amersham-Pharmacia) and eluted with PBS. As seen in the figure ( figure 2 ), the two-step elution method can elute most of the fusion pr...

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Abstract

The invention provides a fusion protein used for detecting and treating human I-type diabetes mellitus and human II-type diabetes mellitus and obesity as well as a preparation and an application of the fusion protein. The fusion protein contains a human FGF21 or a mutant thereof and a human IgG2-Fc (hinge region -CH2-CH3) or a human IgG2-Fc mutant ( T250Q/M428L and T307Q/N434A), can improve the living condition of the obesity patients, reduce the blood sugar level of diabetics, effectively reduce aggregation and degradation of the FGF21 protein, and meanwhile prolong in vivo half-life period of the FGF21, and especially through the fusion of the FGF21 and the mutant and the IgG-Fc (T250Q/M428L and T307Q/N434A) mutant, the affinity with FcRn is enhanced, and in vivo half-life period of the FGF21 is further prolonged.

Description

Field of Invention [0001] The present invention relates to preventive and therapeutic drugs for human diabetes and obesity. Specifically, the present invention provides a fusion protein for detecting and treating human type I or II diabetes mellitus and obesity, and a preparation method and application thereof. The fusion protein contains human FGF21 or its mutant and human IgG2-Fc (hinge region-CH2-CH3) or IgG2-Fc mutant (T250Q / M428L; T307Q / N434A), which can effectively improve the survival of obese patients, Reduce the blood sugar level of diabetic patients, enhance insulin sensitivity, and increase the half-life of FGF21 in vivo, especially the fusion protein of FGF21 and its mutant and IgG-Fc (T250Q / M428L; T307Q / N434A) mutant, enhance the affinity with FcRn, further Increases the in vivo half-life of FGF21. Background technique [0002] The human FGF21 gene was first reported in 2000 by Testuya Nishimura et al. The gene is located in the 5' flanking region of the huma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/16C12N15/81C12N15/70C12N15/85A61K38/18A61K47/48A61K48/00A61P3/04A61P3/10
Inventor 张海涛
Owner 北京康明百奥科技有限公司
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