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Composition and method for prevention and treatment of type I diabetes

一种组合物、糖尿病的技术，应用在化学仪器和方法、生物化学设备和方法、基因治疗等方向，能够解决影响免疫反应、不良反应等问题

Inactive Publication Date: 2008-10-01
王庆华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As with most gene therapies (like CD3 therapy), another limitation of this therapy is that it non-specifically targets self-attacking T cells, which affects many other immune responses and may cause other adverse reactions

Method used

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  • Composition and method for prevention and treatment of type I diabetes
  • Composition and method for prevention and treatment of type I diabetes
  • Composition and method for prevention and treatment of type I diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1: Construction of plasmid

[0102] In order to produce a bioactive agonist GLP-1 with high efficiency, long half-life, and potent peptide energy, we constructed human GLP-1 (7-37) and mouse IgG by overlapping PCR (overlap PCR) 1 -Fc carrier ( figure 1 ). IgG 1 -Fc region contains IgG 1 Constant heavy chain region (CH1, hinge, CH2 and CH3 portions). The IgK secretion leader sequence was fused to the GLP-1 sequence to direct the secretion of the synthetic fusion protein into the cell culture medium. The cDNA sequence encoding the hGHRH / hGLP-1 fusion protein was chemically synthesized and ligated to PCR amplified encoding human IgG 2 -Fc (hinge-CH2-CH3) cDNA sequence and inserted between the Nco I and HindIII sites of the pAVO243 vector to construct the GLP-1 / hIgG-Fc / pAVO243 plasmid. Secretable GLP-1 / hIgG-Fc fusion protein containing IgG 2 - Fc constant heavy chain region (hinge, CH2, CH3). The fusion of the GHRH secretion guiding peptide sequence and t...

Embodiment 2

[0106] Example 2: Mammalian expression of GLP-1 / IgG-Fc fusion protein.

[0107] To assess the ability of the vectors to express and secrete the GLP-1 / IgG-Fc fusion protein, the constructs were transiently transfected into COS-7 cells. 48h after transfection, total RNA was extracted from the transfected cells and the expression effect of the fusion construct was evaluated by RT-PCR. Apply gene-specific primers to detect the expression of the GLP-1 / IgG-Fc fusion construct and the IgG-Fc control construct at the transcriptional level ( figure 2 a). No transcript level expression was detected in untransfected control samples.

[0108] Transfected COS-7 cell lysates and cell culture fluid were examined by Western blotting and anti-mouse antibody to determine the expression of the fusion protein at the translational level. Such as figure 2 As shown in b, Fc fusion proteins were detected in both culture medium and cell lysates. The electrophoretic migration position of the fus...

Embodiment 3

[0112] Example 3: Purification of GLP-1 / IgG-Fc fusion protein from mammalian cell culture fluid.

[0113] The specific method of small-scale extraction and purification is to take 2.5mL culture solution from each well of the 6-well culture plate where mammalian transfected cells are cultured, and transfer it to a buffer solution containing 100mM Tris, pH 8.0 and 150mM NaCl. There was 70 [mu]L of Sephadex 4 fast elution resin (Amersham-Pharmacia, Piscataway, NJ) previously bound to protein G. After overnight incubation at 4°C, rinse with Tris buffer, and then elute the fusion protein from the resin with 30 μL of SDS-containing sample buffer.

[0114] In order to obtain a large amount of fusion protein, protein G Sephadex column can be used for medium-scale protein purification. A column volume of 50ml can purify the culture fluid of COS-7 cells transfected with fusion protein particles grown in a 15cm culture dish. Briefly, 50 mL of DMEM culture medium 48 h after cell transfe...

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Abstract

A composition for the prevention or treatment of type I diabetes in a subject is disclosed, said composition comprises a fusion protein selected from the group consisting of GLP-1 / IgG or derivative or fragment thereof and an Ex4 / IgG or derivative or fragment thereof, and an autoimmune inhibitor that one or more DNA plasmids code one or more proteins, which has the function to improve pancreatic beta-cell reproduction and has the effect to inhibit autoimmune reaction effect of the pancreatic beta-cell.

Description

technical field [0001] The invention provides a composition and method for preventing and treating type I diabetes. In particular, the present invention provides a composition and method for treating type 1 diabetes in a mammal, wherein the composition comprises a GLP-1 or Ex4 fusion protein and one or more enzymes encoding islet β-cell regeneration And reduce islet β-cell autoimmunity protein plasmid. Background technique [0002] Diabetes is one of the major diseases causing human death worldwide. Type I diabetes, the main form of the disease, mainly occurs in young people. The main cause of the disease is that autoimmunity destroys the islet β-cells, which leads to insulin secretion defects and requires exogenous insulin therapy. Insulin therapy is the main intervention in the treatment of type 1 diabetes, however, since insulin therapy does not keep the subject's blood glucose levels within a narrow physiological range, insulin is not a cure and cannot prevent the prog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/28A61K38/26A61K38/51A61K39/00A61K47/48A61P3/10C12N5/06C12N5/08
CPCA61K47/48246A61K38/00A61K39/0008A61K47/48561A61K48/00A61K2039/53A61K2039/6056C07K14/57563C07K14/605C07K2319/30A61K47/64A61K47/6849A61P3/10
Inventor 王庆华杰拉尔德·J·普鲁德霍姆莫汗·库马尔
Owner 王庆华
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