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RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog

An insulin-stimulating and fusion protein technology, applied in biological testing, material testing products, cells modified by introducing foreign genetic material, etc., can solve the problems of cumbersome ELISA detection steps, expensive and other problems, and achieve objective and reliable results, small variation, high The effect of applying value

Active Publication Date: 2013-09-11
NAT INST FOR FOOD & DRUG CONTROL
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  • Application Information

AI Technical Summary

Problems solved by technology

Insulin secretory-stimulating peptide fusion protein (Exendin-4-HSA, Byetalog) is a long-acting protein drug developed in China for the treatment of type 2 diabetes. Reagent test kit

Method used

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  • RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog
  • RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog
  • RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog

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Experimental program
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Embodiment Construction

[0021] 1. Materials and methods:

[0022] 1.1 Research Object: Insulin Secretory Stimulating Peptide Fusion Protein

[0023] 1.2 GLP-1R plasmid: purchased from Aurui Dongyuan Gene Co., Ltd.

[0024] pGL4.26 vector: purchased from promega.

[0025] CHO-K1 cells: purchased from ATCC.

[0026] Byetalog standard products and samples: Retained by the Recombinant Technology Product Office of China Institute of Food and Drug Research and Testing.

[0027] 1.3 Reagents:

[0028] Growth medium: F12K+10% fetal bovine serum (GIBCO, #10099)+1% double antibody (GIBCO, #15240)

[0029] Plate medium: Opti-MEM (GIBCO, #31985)

[0030] MegaTran 1.0 transfection reagent: origene, TT200003

[0031] G418: CalBiochem, #345810

[0032] Hygromycin: Cellgro, 30-240-CR

[0033] Selection medium: growth medium + 200ug / ml G418 + 300ug / ml hygromycin

[0034] Glo analysis: promega, E2661

[0035] Luciferase substrate: promega, E2650

[0036] GLP-1R antibody: Abcam, ab39072

[0037] Chemilumine...

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Abstract

The invention provides a novel biological activity determination method of exendin-4-HAS Byetalog. The basic principle of the method is that a GLP-1R (Glucagon-Like Peptide-1R) and a cAMP (cyclic Adenosine Monophosphate) response element (CER) report gene carrier are used for co-transfection of a CHO-K1 cell, and the steps of pressurization, sieving and monoclonal separation and cultivation are carried out to obtain a stable monoclonal cell strain CHOglplr / crec14. After the GLP-1R is combined with a ligand, a series of signal transduction is carried out to stimulate the expression of CRE reporter gene luciferase, and the activity of the Byetalog is quantified by detecting the change of the expression of the luciferase after the Byetalog stimulates. The specific detection steps are as follows: 6000 CHOglplr / crec14 cells are placed on each pore of a 96-pore plate to be cultured for 16-18 hours; the Byetalog with different concentrations is added to stimulate the cells for 6 hours; the activity of the luciferase is detected. The method is simple to operate, sensitive in reaction and small in variability, and has very important meaning to quality control and clinical application of the Byetalog.

Description

Technical field: [0001] The invention relates to the field of biological drug activity detection, and establishes a faster and more accurate reporter gene quantitative method (report gene assay, RGA) for measuring the biological activity of insulin secretion-stimulating peptide fusion protein (Byetalog). Background technique: [0002] The research and application of insulin secretory peptide (Exendin-4) has become one of the research hotspots in the field of diabetes treatment in recent years. Exendin-4 can effectively solve multiple challenges in the current treatment of type 2 diabetes: effectively reduce blood sugar without increasing the risk of hypoglycemia; improve β-cell function; reduce body weight; reduce cardiovascular risk. Insulin secretory-stimulating peptide fusion protein (Exendin-4-HSA, Byetalog) is a long-acting protein drug developed in China for the treatment of type 2 diabetes. Reagent test kit. Exendin-4 is a glucagon-like peptide-1 (GLP-1) analogue. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N5/10
Inventor 王军志饶春明于雷王兰高凯
Owner NAT INST FOR FOOD & DRUG CONTROL
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