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In-vitro expression and separation and purification method for chemotactic factor TARC/CCL17

A chemokine and external recombination technology, applied in the field of molecular biology, can solve problems such as complex evaluation methods and SCORAD not widely used, and achieve the effect of reducing production costs and production difficulties

Inactive Publication Date: 2018-07-27
SHENZHEN PKU HKUST MEDICAL CENT
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Problems solved by technology

However, SCORAD is not widely used in clinical practice due to the complexity of the assessment method

Method used

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  • In-vitro expression and separation and purification method for chemotactic factor TARC/CCL17
  • In-vitro expression and separation and purification method for chemotactic factor TARC/CCL17
  • In-vitro expression and separation and purification method for chemotactic factor TARC/CCL17

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Experimental program
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Embodiment Construction

[0015] The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

[0016] 1. Integrate the known cDNA sequence (SEQ ID NO:1) corresponding to TARC / CCL17 into the E. coli vector PETM3C, and transform the constructed vector into E. coli BL21.

[0017] 2. Cultivate the bacteria in a shaker at 37°C and 200rad / min until OD 600 At 0.6-0.8, add IPTG to make the concentration of IPTG in the bacterial solution reach 0.2mmol / L, and place it in a shaker at 37°C and 200rad / min for 16h.

[0018] 3. After centrifuging at 4000g for 20min to collect the bacteria, suspend them in a small amount of solution containing 50mM Tris-HCl and 20mM NaCl.

[0019] 4. After ultrasonically disrupting the cells, centrifuge at 18000g for 20min, and collect the supernatant.

[0020] 5. Use affinity chromatography to purify the protein, the binding solution is 50mM Tris-HCl, 20mM NaCl, 5mM imidazole, the washing soluti...

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Abstract

The invention discloses a method of in-vitro recombinant expression of human thymus activation regulated chemokine TARC / CCL17. In the method, human TARC / CCL17 protein is obtained through fusion expression of escherichia coli; then by means of protease digestion, affinity chromatography, gel chromatography and ion-exchange column chromatography, the expression product is purified to obtain pure human TARC / CCL17 protein. The method achieves massive soluble expression of the human TARC / CCL17 protein, which can be used as an ELISA standard substance. The method greatly reduces production cost anddifficulty of the TARC / CCL17 protein.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for in vitro expression, separation and purification of chemokine TARC / CCL17. Background technique [0002] Atopic dermatitis is a chronic skin disease with a high incidence rate, and the diagnosis of atopic dermatitis is not difficult. To achieve effective treatment of atopic dermatitis, its accurate diagnosis and disease assessment are crucial to reach a broad consensus standard therapy has also been published, as long as the diagnostic method compatible with the standard is selected, it can be made without difficulty. judge. [0003] Regarding the symptoms and severity of atopic dermatitis, although the SCORAD scoring method was introduced to evaluate the symptoms and development of skin manifestations, the severity can be divided into mild, moderate and severe based on this method. However, due to the complexity of the evaluation method, SCORAD has not been widely ...

Claims

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Application Information

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IPC IPC(8): C07K14/52C07K1/22C07K1/18C07K1/16
CPCC07K14/521
Inventor 陈小帆曹恒昌邹彦芬杨旸张李娟于波
Owner SHENZHEN PKU HKUST MEDICAL CENT
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