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Cytological method for assaying activity of rhGDNF (recombinant human Glial cell line-Derived Neurotrophic Factor)

A neurotrophic factor and glial cell technology, applied in the field of bioengineering, can solve the problems of lack of cytological methods for the detection of recombinant human glial cell-derived neurotrophic factor, and achieve accurate and reliable results

Active Publication Date: 2015-04-08
JIANGSU CELL TECH MEDICAL RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is currently no cytological method that can effectively detect the activity of recombinant human glial cell-derived neurotrophic factor (rhGDNF)

Method used

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  • Cytological method for assaying activity of rhGDNF (recombinant human Glial cell line-Derived Neurotrophic Factor)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A cytological method for measuring the activity of recombinant human glial cell-derived neurotrophic factor, comprising the steps of:

[0040] S1. Using pcDNA3.1- as the expression vector, use restriction enzymes XhoI and Not I to linearize the vector, and then use T4DNA ligase to connect the hGFRα1 cDNA to pcDNA3.1 to construct the expression plasmid pcDNA3.1-hGFRα1, and then use Prepare HEK293 cells expressing hGFRα1 by transfection, the detailed steps are:

[0041] A. Add 2μg pcDNA3.1-hGFRα1 to 110μl Opti-MEM medium, mix gently and place at room temperature for later use;

[0042] B. Add 15 μl Lipofectamine (DNA transfection reagent) to 150 μl Opti-MEM medium, mix gently and place at room temperature for later use;

[0043] C, mixing the DNA-Lipofactamine liquid of step A and step 2B to obtain a DNA-Lipofectamine mixture;

[0044] D. Place the DNA-Lipofectamine obtained in step C at room temperature (25°C) for 30 minutes;

[0045] E. Then add 320 μl of Opti-MEM me...

Embodiment 2

[0061] Other steps are the same as in Example 1, except that in step S3, rhGDNF to be tested is made into a solution to be tested with a concentration of 1.0 ng / μl.

[0062] Results: Comparing F1 and F2, it is found that F1 and F2 are basically equal, which means that the rhGDNF to be tested has no activity.

Embodiment 3

[0064] Other steps are the same as in Examples 1 and 2, except that in step S3, the rhGDNF to be tested is made into a solution to be tested with a concentration of 83.3 ng / μl.

[0065] Results: Comparing F1 and F2, and finding that F1

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Abstract

The invention relates to a cytological method for assaying the activity of an rhGDNF (recombinant human Glial cell line-Derived Neurotrophic Factor). The method comprises the following steps of: constructing expression plasmid pcDNA3.1-hGFR alpha 1, and then preparing an HEK293 cell which stably expresses hGFR alpha 1; constructing expression plasmid pcDNA3.1-hRET, and then preparing an HEK293 cell capable of simultaneously expressing hGFR alpha 1, hRET and a luciferase reporting system; adding the rhGDNF to be assayed into the HEK293 cell capable of simultaneously expressing hGFR alpha 1, hRET and the luciferase reporting system, and assaying fluorescence intensity F1 after culturing; meanwhile, arranging a blank control group in which the rhGDNF is not added, and assaying fluorescence intensity F2 after culturing; and comparing F1 with F2. The method has the advantages that the activity of the rhGDNF can be effectively assayed and the assayed result is accurate and reliable.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a cytological method for measuring the activity of recombinant human glial cell-derived neurotrophic factor. Background technique [0002] Parkinson's disease, first described by Parkinson (1817), is a common neurodegenerative disease in middle-aged and elderly people. Parkinson's disease is the fourth most common neurodegenerative disease in the elderly. In people ≥65 years old, 1% suffer from the disease, compared with 0.4% in > 40 years old crowd, the disease can also occur in childhood or adolescence. Caucasians have the highest incidence rate, followed by yellows. PD is a life-long disease, so far there is no radical cure. Once the disease is diagnosed, life-long treatment is required. Because surgical treatment is expensive and has strict surgical indications, and gene therapy and stem cell therapy are still in the research stage, drug therapy is still It is cur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02
Inventor 曹杰
Owner JIANGSU CELL TECH MEDICAL RES INST CO LTD
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