Human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein and preparation method and application thereof

A technology of human immunoglobulin and heavy chain constant region, applied in the field of biomedicine, can solve the problem of lack of effective drugs for the treatment of allergic diseases, etc.

Active Publication Date: 2015-03-11
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0046] In general, there is still a lack of effective drugs for the treatment of allergic diseases in clinical practice.

Method used

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  • Human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein and preparation method and application thereof
  • Human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein and preparation method and application thereof
  • Human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein and preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0086] The present invention provides a kind of preparation method of human immunoglobulin epsilon heavy chain constant region (hIgE Fc) fragment protein, comprising the following steps:

[0087] (1) operably linking the nucleotide sequence encoding hIgE Fc to the expression vector to obtain a hIgE Fc recombinant expression vector;

[0088] (2) Transcribing the recombinant expression vector in step (1) into CHO cells, and screening to obtain engineered cell lines expressing hIgE Fc;

[0089] (3) cultivating the engineered cell strain obtained in step (2) under suitable conditions;

[0090] (4) Isolating the human immunoglobulin ε heavy chain constant region (hIgE Fc) fragment protein from the culture product in step (3).

[0091] The step of cultivating the engineered cell strain expressing hIgE Fc protein under suitable conditions in step (3) comprises: cultivating the engineered cell strain obtained through screening in a medium that does not contain bovine serum, and the c...

Embodiment 1

[0100] Expression of embodiment 1 hIgE Fc protein

[0101] The expression process of hIgE Fc protein includes the following steps: synthesizing the gene; inserting the gene into the expression vector, transfecting CHO cells with the expression vector; using Hygromycin to screen the CHO engineered cell line expressing the hIgE Fc fusion protein, cultivating the cell line, and finally extracting from the cell culture medium Separation and purification of RANKL-Fc protein. specifically:

[0102] 1) Gene synthesis

[0103] Entrust a professional company for gene synthesis to synthesize the nucleotide sequence shown in SEQ ID NO: 2, and add a BamHI restriction site and a Kozak sequence at the 5' end; add an Xba I restriction site at the 3' end .

[0104] 2) Construction of expression vector

[0105] After obtaining the above-mentioned synthesized gene from Gene Synthesis Company, use BamHI and Xba I to double-enzyme digest the gene and vector pCDNA3.1, recover the digested fra...

Embodiment 2hI

[0110] Example 2 hIgE Fc protein inhibits activation of basophils to release histamine

[0111] Basophils were isolated from the peripheral blood of healthy people, distributed into 96 plates according to 1×106cells / well, and then added Figure 4 The amount of hIgE and hIgE Fc shown in , after 4 hours, carefully aspirate the culture supernatant, and wash twice with calcium and magnesium-free HBSS buffer, anti-stimulate with 100 μL Anti-IgE Fc, after half an hour, remove the supernatant , using histamine ELISA assay kit to detect the concentration of histamine. The percentage of histamine released is the ratio of the amount of histamine released after the cells were stimulated to the amount of histamine released after the same amount of cells were boiled at 100°C for 6 minutes.

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Abstract

The invention relates to a human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein and a preparation method and an application thereof. The human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein is capable of combining an IgE binding site on Fc epsilon RI molecule. The human immune globulin epsilon heavy chain constant region (hIgE Fc) fragment protein can be used for allergy diseases, such as allergy asthma and rhinitis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a human immunoglobulin epsilon heavy chain constant region (hIgE Fc) fragment protein for treating allergic diseases, a preparation method and application. Background technique [0002] Allergic diseases, also known as allergic diseases, are common diseases and can cause various complications during the onset. Common allergic diseases mainly include allergic rhinitis, allergic asthma, and anaphylactic shock. With the deterioration of air and environment around the world, the incidence rate is increasing year by year. The occurrence of allergic diseases is mainly due to the allergic response cells (basophils in the blood and mast cells in the interstitial space) in the body receiving antigenic substances (allergens), such as pollen particles, dust mites, food (milk, egg , shrimp, crab, etc.), drugs (penicillin, sulfonamide, etc.), parasites, etc., release a large amount of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/85A61K39/395A61P37/08
Inventor 孙祥明王红勋祝道成包骏
Owner SHANGHAI KEXIN BIOTECH
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