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Method for expressing human IgE high affinity receptor protein in vitro

A receptor protein, affinity technology, applied in the field of molecular biology, can solve the problem of difficulty in obtaining patient cooperation, and achieve the effect of reducing production cost and production difficulty

Inactive Publication Date: 2014-12-17
SHENZHEN PKU HKUST MEDICAL CENT
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Problems solved by technology

However, this method is difficult to implement clinically, and it is difficult to obtain the cooperation of patients.

Method used

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  • Method for expressing human IgE high affinity receptor protein in vitro
  • Method for expressing human IgE high affinity receptor protein in vitro
  • Method for expressing human IgE high affinity receptor protein in vitro

Examples

Experimental program
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Embodiment Construction

[0013] The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

[0014] 1. After PCR amplification of the cDNA sequence (SEQ ID NO: 1) corresponding to the known IgE high affinity receptor protein ( figure 1 ), integrated into the E. coli vector PET32M ( figure 2 ), and transform the constructed vector into Escherichia coli BL21.

[0015] 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.

[0016] 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.

[0017] 4. After ultrasonically disrupting the cells, centrifuge at 18000g for 20min, and collect the supernatant.

[0018] 5. Use affinity chromatography to purify ...

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Abstract

The invention discloses a method for expressing human IgE high-affinity receptor protein in vitro. The method is used for expressing the human IgE high-affinity receptor protein by virtue of escherichia coli fusion; next, the expressed human IgE high-affinity receptor protein is purified by use of a method comprising protease digestion, affinity chromatography, gel chromatography and ion-exchange column chromatography so that the pure IgE high-affinity receptor protein can be obtained. Large-scale soluble expression of the human IgE high-affinity receptor protein in vitro can be realized by use of the method, and therefore, the production cost and the production difficulty of the IgE high-affinity receptor protein are greatly reduced.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for expressing human IgE high-affinity receptor protein in vitro. Background technique [0002] Chronic urticaria is one of the common diseases in dermatology. It has the characteristics of complex etiology, repeated symptoms, and long course of disease. Most patients cannot find a clear cause and are considered "idiopathic". About 40% of the disease course can exceed 10 years Although not life-threatening, the pain and trouble it brings and its impact on work and social life are no less than heart disease. The annual number of cases in Shenzhen exceeds 10,000. Conventional anti-allergic treatment (such as antihistamine drugs) is often ineffective for a considerable number of patients. These patients belong to autoimmune chronic urticaria, and there are anti-FcεR Iα (IgE receptor) autoantibodies or (and) anti-IgE in serum Autoantibodies, the pathogenesis is based on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/705C07K1/22C07K1/18C07K1/16
Inventor 魏准张伟杨旸于波万峻张杰
Owner SHENZHEN PKU HKUST MEDICAL CENT
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