Eosinophil chemotactic factor

A technology of eosinophils and chemotaxis, applied in the field of genetic engineering, which can solve the problem of low-specificity migration of eosinophils and other problems

Inactive Publication Date: 2001-09-05
EFFECTOR CELL INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of them have only a low level of activity that specifically migrates eosinophils

Method used

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  • Eosinophil chemotactic factor
  • Eosinophil chemotactic factor
  • Eosinophil chemotactic factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the preparation of the partial purification of ecalectin and the polyclonal antiserum containing ecalectin specific antibody

[0063] The cell line STO-2 obtained by transforming T cells of eosinophilic patients with human T-cell leukemia virus was used as a source material (Lymphokine Cytokine Research, 10, 481-486, 1991). FACS analysis confirmed that this cell line was derived from T cells, specifically, STO-2 expressed CD2, CD3, CD4, CD5, and CD8, but not CD16, CD19, or Leu7, markers for granulocytes / macrophages and B cells.

[0064] With the addition of 0.1% human serum albumin, 100 U / ml IL-2 (Tokushima Research Institute, Tokushima), 50 μM 2-mercaptoethanol, 100 μg / ml streptomycin, 100 U / ml penicillin and 5 μg / ml amphotericin B STO-2 cells were cultured in SF-02 serum-free medium for 72 hours, the culture supernatant was taken, and the pH was adjusted to 5 with 50 mM sodium acetate buffer. 5 liters of the conditioned medium thus obtained were passed ...

Embodiment 2

[0065] Example 2: Screening of cDNA library and positive clones prepared from T cell line STO-2

[0066] Use ZAP Express TM cDNA Gigapack Cloning Kit (Stratagene, La Jolla, California) was used to prepare the STO-2 cell cDNA expression library. Specifically, Fast Track RNA Isolation Kit (Invitrogen, San Diego, California) was used from 1 × 10 8 Collect about 5 μg poly(A) from each STO-2 cell + mRNA. Using Moloney mouse leukemia virus reverse transcriptase and 50-mer oligodeoxy dT primer (5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3'; containing an XhoI restriction site: SEQ ID NO: 3), synthesize the first DNA strand. This DNA strand synthesis reaction is carried out in the presence of 5-methyl CIP to methylate the XhoI site in the cDNA. An EcoRI linker was ligated to the 5' end of the cDNA. Cut with restriction endonuclease XhoI (Stratagene), and carry out molecular sieve chromatography with 1ml Sephacryl S-500HR (Gibco) gel filtration column (balanced with 0....

Embodiment 3

[0072] Embodiment 3: Determination and analysis of nucleotide sequence

[0073] The DNAs of three independent clones obtained in Example 2 were assayed by the Sanger method (Proceedings of the National Academy of Sciences of the United States of America 74, 5463-5467), and it was found that they encoded the same protein. The DNA consensus sequence and its deduced amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The deduced open reading frame is believed to correspond to amino acids 60 to 1028 in SEQ ID NO: 1, which is predicted to encode a 36 KDa protein consisting of 323 amino acids. The present inventors named the cloned eotaxin as ecalectin. Nucleotides 1576 to 1581 in SEQ ID NO: 1 are a typical polyadenylation signal. In this clone, the 5' untranslated region has 59 nucleotides and the 3' untranslated region has 560 or more nucleotides. A repetitive sequence is often seen in the 3' untranslated region of short half-life transcripts (Cell, 46...

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Abstract

An eosinophil chemotactic factor which is constantly secreted by a T cell clone STO-2 is partly purified and a specific antibody is constructed. By using this antibody, a DNA library originating in the above clone is screened. It is found that ecalectin, which is a galectin consisting of 323 amino acids, has a specific eosinophil chemotactic activity. Use of this galectin makes it possible to provide drugs having an effect of increasing eosinophils in a tissue and a method for screening compounds inhibiting this effect. Moreover, drugs containing as the main ingredient the compounds inhibiting the effect of increasing eosinophils in a tissue, which are obtained by the above-mentioned screening method, are efficacious as preventives or ameliorating agents for various allergic diseases which are typical delayed inflammations caused by eosinophils, for example, bronchial asthma, allergic rhinitis and atopic dermatitis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a DNA encoding a protein with eosinophil chemotactic activity. Background technique [0002] Delayed inflammation by eosinophils has been found to occur in various allergic diseases such as bronchial asthma, allergic rhinitis, atopic rhinitis, and parasitic infections. (Advances in Immunology. 39:177-253, 1986) [0003] Usually, eosinophils are present in small amounts as part of the white blood cells in the blood circulation, but occasionally the number of eosinophils in the blood increases, developing eosinophilia. In tissues, eotaxin induced by several factors mediates the entry of eosinophils in blood from blood vessels into tissues. Therefore, the discovery of factors related to eosinophilia and chemotaxis and their direct or indirect effects can guide the treatment of the above-mentioned allergic diseases. [0004] Peptidergic factors such as eotaxin, granulocyte-macro...

Claims

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Application Information

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IPC IPC(8): A61K38/00C07K14/47
CPCC07K14/4726A61K38/00A61K38/16
Inventor 金ヶ崎士朗松本良二平岛光臣
Owner EFFECTOR CELL INST
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