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Synovial membrane cell protein

A protein and cell technology, applied in the field of new proteins, can solve the problems of not elucidating the biological characteristics of cells and not knowing the relationship between RA factors

Inactive Publication Date: 2004-04-21
LOCOMOGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Diagnosis of RA based on the detection of RA factors is problematic in terms of specificity in diagnosing the disease, and another problem is that the relationship between RA factors and etiology is still unclear because the system on which antibodies are produced has not been elucidated
However, although studies related to joint synoviocytes are a major aspect of RA research, their cell biology has not yet been elucidated

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0302] [Example 1] Preparation of anti-synovial cell anti-serum

[0303] Anti-syviocyte anti-serum was obtained using synoviocytes prepared by the method described below as an immunogen. Synovial tissue extracted from 10 rheumatoid arthritis (RA) patients via synovectomy was washed with phosphate-buffered saline (PBS) under sterile conditions. The washed tissue was cut into squares with a size of about 5 mm, and digested with 0.25% trypsin / PBS at 37° C. for 20 minutes. Excess tissue pieces were removed from the digested synovial tissue, and the resulting cells were suspended in Dulbecco's modified Eagle medium (Virology, 8, 396, 1959) (10% FCS-DMEM) containing 10% fetal calf serum, at 5% CO 2 , 37°C for 24 hours in a sterile cell culture dish. The culture supernatant was discarded, washed with 10% FCS-DMEM, and unattached cells were removed to obtain cells adhered to the plate, which were synoviocytes obtained from rheumatoid patients (The Journal of Clinical Investigation,...

Embodiment 2

[0306] [Example 2] Gene Cloning of Antigen (Synoviolin) Recognized by Anti-Syvioviocyte Antiserum

[0307] Total RNA from 10 RA patient synoviocytes obtained in Example 1 was extracted using the acid guanidine / phenol chloroform method, and mRNA was purified using poly T beads (Analytical Biochemistry, 162, 159, 1987). A cDNA library of RA patient synoviocytes was prepared by a conventional method using λZAP vector (Stratagene). Using the picoBlue immune screening kit (Stratagene), carry out immune screening with the anti-synovial cell antiserum of Example 1 above ( figure 1 ). The resulting positive clone (phage) was transformed into plasmid pBluescript II SK(+) with helper phage. Using M13PrimerM4 and M13PrimerRV (Takara), based on the dye terminator method (Proc.Natl.Acad.Sci.USA., 74, 5463, 1977), the ABI PRISM377 DNA sequencer (PERKIN ELMER) was used to measure the number of inserted pBluescript II SK (+) The nucleotide sequence of DNA. Determination of the nucleotide...

Embodiment 3

[0308] [Example 3] Partial synovialin recombinant protein expressed in Escherichia coli

[0309] From the cDNA clones obtained by immunoscreening using anti-synoviocytic antiserum, the cDNA encoding a part of synoviolin (1799 bp; SEQ ID NO: 1, pages 1233-3031) was treated with restriction enzymes EcoRI and XhoI bit), and extract the cDNA. The cDNA with the sequence recognized by EcoRI / XhoI at the end was inserted into glutathione S-transferase (GST) fusion protein expression vector pGEX-5X-3 for subcloning. By heat-shocking at 42°C for 45 seconds, pGEX-5X-3 into which a part of synoviolin cDNA was inserted was introduced into BL21 E. coli strain to obtain BL21 / synoviolin-GST gene / pGEX-5X-3. Cultivate the BL21 strain in LB medium containing 0.1 mg / ml ampicillin, add 0.1 mM isopropylthio-β-D-galactoside (IPTG), and culture at 37°C for another 2 hours to induce the fusion protein expression. After washing BL21 recovered by centrifugation with PBS, BL21 was digested with 1 mg / m...

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Abstract

The present invention discloses a novel protein called Synoviolin and a gene that encodes it. This protein is expressed specifically by synovial tissue and also accompanies the presence of an auto-antibody that recognizes this protein in rheumatoid arthritis (RA) patients. The protein according to the present invention and its antibody can be expected to be used as specific diagnostic markers for RA. In addition, the gene or protein according to the present invention may be used to permit the screening of drugs to treat RA. Moreover, the present invention provides synoviolin gene transgenic animals. The transgenic animals according to the present invention can be used as RA model animals in the development of pharmaceuticals to treat RA.

Description

technical field [0001] The present invention relates to a new protein related to rheumatoid arthritis (RA), polynucleotide encoding the protein and application of the protein or polynucleotide. More specifically, the present invention relates to a novel protein that is expected to be used as a specific diagnostic marker for RA. In addition, the present invention also relates to a novel gene, which may provide a new method for the development of drugs for the treatment of RA. Background technique [0002] RA is a systemic chronic inflammation in which abnormal growths are seen in the synovial tissue of the joints. Synoviocytes are fibroblast-like cells that form the epithelioid layers 1 to 6 of the joint synovium and are thought to contribute proteoglycans and hyaluronic acid to the synovial fluid. In the joints of RA patients, hyperplasia of synovial tissue can be observed, as well as symptoms resulting from the multilayered structure and the resulting infiltration of syno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027A61K38/00C07K14/47C07K14/705C12N1/15C12N1/19C12N1/21C12N15/12C12N15/85G01N33/564G01N33/574
CPCA01K2267/0325C07K14/47A01K2227/105A01K2217/05G01N33/574C07K14/705C12N15/8509A01K2217/00A01K2217/075A01K2217/072A01K67/0276G01N33/564G01N2500/00A61K38/00A01K67/0275A01K2207/15C12Y603/02C12Y203/02C12N9/104C12N9/93A61P17/02A61P29/00A61P43/00A61P9/08C12N15/11
Inventor 中岛利博天野徹也
Owner LOCOMOGENE INC
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