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Novel receptor protein and method for diagnosing inflammatory diseases by using the same

A diagnostic method and protein technology, applied to seven transmembrane receptor proteins and their DNA encoding them, novel seven transmembrane receptor proteins and their DNA encoding them, ligands and blocking ligands and seven transmembrane receptor proteins In the field of membrane receptors, it can solve the problem of low positive rate

Inactive Publication Date: 2001-10-10
ASAHI KASEI KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, rheumatism factor is positive for 71% of early RA within 1 year of onset, but 59% within 6 weeks of onset, and the positive rate is low

Method used

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  • Novel receptor protein and method for diagnosing inflammatory diseases by using the same
  • Novel receptor protein and method for diagnosing inflammatory diseases by using the same
  • Novel receptor protein and method for diagnosing inflammatory diseases by using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Embodiment 1 The acquisition of novel seven transmembrane receptor gene fragments

[0145] The nucleated cells recovered from 1 liter of peripheral blood buffy coat of healthy people were cultured for 14 days at 37° C. under 5% carbon dioxide environment to differentiate into immature dendritic cells. A culture medium containing 10% fetal bovine serum (FBS) (manufactured by Intergen, USA), 100ng / ml human GM-CSF, 50ng / ml human IL-4 and 1× antibiotic-antimycotic agent (100IU / ml penicillin, 100μg / ml ml streptomycin and 0.25 μg / ml amphotericin B) (USA, GIBCO BRL  ) of RPMI1640 medium. This gives 1 x 10 from peripheral leukocytes 7 immature dendritic cells.

[0146] Recover the immature dendritic cells obtained, suspend in 30 ml of PBS (phosphate buffered saline), use the Quick Prep mRNA purification kit (made by Pharmacia Biotech in Sweden), and extract mRNA according to the operating procedures except that the second column purification is not performed . Next, 1 μg ...

Embodiment 2

[0150] Example 2 Acquisition of the full-length gene of the novel seven-transmembrane receptor

[0151] A clone having the full-length cDNA of the seven-transmembrane receptor of the present invention was isolated from a cDNA library derived from human placenta tissue (manufactured by CLONTECH, USA) by plaque hybridization. Specifically, 106 phages were inoculated on a culture plate according to the usual method, and the plaques that appeared were transferred to a nylon filter membrane Hybond N+ (manufactured by Amersham, UK). Place the transferred nylon filter on the filter paper soaked with alkaline buffer (1.5M NaCl / 0.5M NaOH) for 5 minutes for alkaline treatment, and then neutralize the buffer (1.5M NaCl / 0.5M Tris-HCl, pH7 .5) Place on the soaked filter paper for 5 minutes for neutralization. Also, the neutralization treatment was repeated twice. After neutralization, the filter membrane was shaken and washed twice for 5 minutes in 2×SSC solution (1×SSC solution, 0.15M N...

Embodiment 3

[0156] Example 3 Analysis with Northern blot (Northern blotting)

[0157] The expression of mRNA in each organ of the human seven transmembrane receptor C5L2 obtained from the full-length base sequence of the gene in Example 2 was analyzed by Northern blot. As filters for immobilizing RNA from each organ, Multiple Tissue Northern (MTN) blotting filters (Cat. #7757-1, #7759-1, #7760-1, and #7767-1) (manufactured by CLONTECH, USA) were used. As a probe, the NaeI-EcoRI fragment at the 3' end of the C5L2 gene was used in the same manner as in Example 2 32 The form of the P tag is used. Immerse the above filter membrane in a solution containing 5×SSPE (1×SSPE solution is 0.15M NaCl / 10mM NaH 2 PO 4 / 1mM EDTA, pH 7.4), 10×Denhardht solution, 2% SDS, 50% formamide and 100 μg / ml salmon sperm DNA hybridization solution, shaking at 50°C for 2 hours. Next, the probe was added into the hybridization solution, and shaken at 50° C. for 16 hours to perform hybridization.

[0158] The was...

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Abstract

Disclosed are a novel seven-pass transmembrane receptor protein found in immature dendritic cells and a DNA encoding the same. Further, disclosed are a replicable recombinant DNA which comprises a replicable expression vector and, operably inserted therein, the above-mentioned DNA; a cell of a microorganism or cell culture (transformant), which is transformed with the above-mentioned replicable recombinant DNA; a seven-pass transmembrane receptor protein which is produced on the cell surface of the above-mentioned transformant; a method for screening a ligand which binds to the above-mentioned seven-pass transmembrane receptor protein, and a method for screening a substance which inhibits the ligand from binding to the seven-pass transmembrane receptor protein; and an antibody which binds to the above-mentioned seven-pass transmembrane receptor protein. The present invention also discloses a method for the diagnosis of an inflammatory disease, such as rheumatism, which comprises determining the amount of the seven-pass transmembrane receptor protein expressed in human leukocytes.

Description

technical field [0001] The present invention relates to a novel seven-transmembrane receptor protein and DNA encoding it discovered from immature dendritic cells. More specifically, it relates to the human-derived seven-transmembrane receptor protein composed of the amino acid sequence described in Sequence 2 and the DNA encoding it. Using the seven transmembrane receptor protein of the present invention and the DNA encoding it, the screening of substances useful for the treatment or prevention of diseases related to dendritic cell function, the diagnostic methods of these diseases and the manufacture of diagnostic drugs can be completed. In addition, the present invention also relates to a replicable recombinant DNA recombined by inserting the above-mentioned DNA into a replicable expression vector, microorganisms or cultured cells transformed by the above-mentioned replicable recombinant vector, and the seven-transmembrane receptor protein expressed on the cell surface of th...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K16/28C12N1/15C12N1/19C12N1/21C12N15/12
CPCC07K2317/34C07K16/28C07K14/705
Inventor 高桥恒夫大野满春石丸弘菅野仁喜高桥千晶
Owner ASAHI KASEI KK
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