Method for determining bioactivity of recombinant insulin secretagogue and application thereof

A biologically active and insulin-stimulating technology, applied in the field of cytokine biological activity detection, can solve the problems of poor applicability, dangerous experimental operation, large errors, etc., and achieve the effects of simple steps, good safety and high accuracy.

Inactive Publication Date: 2006-10-11
DONGGUAN TAILI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the reported Exendin-4 biological activity detection methods include: using Exendin-4 to stimulate pancreatic beta cell insulin secretion to measure the change of insulin content; using radioimmunoassay (RIA) to measure the change of cAMP level, such as Chinese patent CN1384755A announced a The method of measuring the biological activity of a chemically synth

Method used

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  • Method for determining bioactivity of recombinant insulin secretagogue and application thereof
  • Method for determining bioactivity of recombinant insulin secretagogue and application thereof
  • Method for determining bioactivity of recombinant insulin secretagogue and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Exendin-4 activity assay method

[0022] The method for measuring Exendin-4 activity using RIN-5F cells (purchased from ATCC, catalog number CRL-2058) comprises the steps:

[0023] (1) Get the RIN-5F cells in the logarithmic growth phase, use serum-containing cell culture medium RPMI1640 such as RPMI1640 with 10% fetal bovine serum to make a cell suspension, and adjust the cell density to be (1.6-2.4)×10 4 cells / well, inoculate the cells on the culture plate, and cultivate until the cell confluency reaches more than 70%;

[0024] (2) Remove the above serum-containing cell culture medium, add serum-free cell culture medium, and incubate in the incubator for 1-4 hours. The purpose of this step is to remove the serum and allow the cells to adapt to the stimulus response condition system in advance. cAMP content, which will affect the stimulated production of cAMP, thereby affecting the assay results.

[0025] (3) Using 0.2-1 mmol / L 3-isobutyl-1-methylxanthine (...

Embodiment 2

[0035] The repeatability of embodiment 2 Exendin-4 activity determination method

[0036]Utilize the method for embodiment 1 to measure recombinant Exendin-4 activity, the half-effect response dose (EC) of 10 repeated tests 50 ) and related conversion data are shown in Table 1:

[0037]

[0038] For the statistical analysis of the above test data, the degree of freedom f=n-1=9, check the t value table to get t (9)0.05 =2.262, 10 times Log(X±SD) is 5.6195±0.09083, each experimental data is normally distributed above and below LogX, 10 times X =10 5.6195 = 416389.7 AU / mg; 95% confidence interval = 10 5.52867 ~10 5.71033 =337808.0~513251.2AU / mg, the 95% confidence interval is 81.1%~123.3% of the mean, and the average confidence limit is 1.1%. The above results show that the method used to determine the biological activity of recombinant Exendin-4 is stable and reliable.

Embodiment 3

[0039] Example 3 Comparison of recombinant Exendin-4 and synthetic Exendin-4 activity

[0040] Adopt the method of the present invention, carry out activity analysis to recombinant Exendin-4 and chemically synthesized Exendin-4 (purchased from Bachem, article number: H8730), test data is shown in Table 2, regression operation is shown in Table 3, and activity comparison spectrum is shown in figure 1 .

[0041] Concentration (mol / L)

[0042] Calculation formula: y=A2+(A1-A2) / (1+(x / x0) p )

[0043] The results showed that the activity of synthetic Exendin-4 (EC 50 ) Consistent with literature reports, the dose-response curves of recombinant Exendin-4 and synthetic Exendin-4 are parallel and basically overlap, further illustrating that the method has strong repeatability and high accuracy, and can be used as a biological activity assay method for recombinant Exendin-4.

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Abstract

The invention discloses a method for testing the biological activity of recombination insulinotropic hormone (Exendin-4) and relative application. Wherein, it comprises: using Exendin-4 to activate the cell to make cAMP excrete; the cAMP of cell will be increased quickly; using enzyme immunity method to test the cAMP content in the supernate of cracked cell and the cAmp content has dosage dependency in the supernate of cracked cell, to calculate the biological activity of Exendin-4, while the test cell is RIN-5F cell. The invention can be used to control the quality of recombination insulinotropic hormone (Exendin-4). The invention uses enzyme immunity method to test the biological activity, with simple process, less waste, high accuracy and better safety.

Description

technical field [0001] The invention belongs to the technical field of cytokine biological activity detection, and relates to a method for measuring the biological activity of a recombinant insulin secretagogue (ie Exendin-4) and an application thereof. Background technique [0002] Insulin secretagogue (Exendin-4) is a polypeptide containing 39 amino acids isolated from the oral secretion of Gila monster Heoderma suspectrum (Eng, J., etc., J.Biol.Chem., 265:20259- 62, 1990; Eng, J. et al., J. Biol. Chem., 267:7402-05, 1992). Its amino acid sequence has 53% homology with glucagon-like peptide (GLP-1) (Coke et al., J. Biol. Chem., 268:19650-55, 1993). Chen and Drucker cloned the gene and concluded that it is the expression product of a gene other than the glucagon gene (GLP-1 is processed from proglucagon) (J. Biol. Chem., 272:4108-15, 1997). [0003] A large number of animal model experiments show that Exendin-4 exhibits an anti-diabetic effect similar to GLP-1. Includin...

Claims

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Application Information

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IPC IPC(8): G01N33/53A61K38/22A61P3/10C12Q1/00
Inventor 韩为跃阳勇张仁怀杨立明刘社际何凯黄碧莲刘德林
Owner DONGGUAN TAILI BIOTECH
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