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Connective tissue growth factor homologs

a technology of connective tissue and growth factor, which is applied in the direction of immunoglobulins, peptide/protein ingredients, peptides/protein ingredients, etc., can solve the problem of limited current therapy for ligament repair

Inactive Publication Date: 2005-10-27
SHEPPARD PAUL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current therapy for ligament repair is limited to immobilising the damaged tissue, i.e., stapling, or replacing the damaged tissue with synthetic or natural grafts.

Method used

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  • Connective tissue growth factor homologs
  • Connective tissue growth factor homologs
  • Connective tissue growth factor homologs

Examples

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example 1

[0161] Scanning of a translated DNA database using the CTGF family motifs as a query resulted in identification of an EST sequence found to have some homology to the connective tissue growth factor family.

[0162] Plasmid DNA was isolated from a clone corresponding to the EST, which had been designated zCTGF4, and was analyzed for polynucleotide sequence. Upon alignment of the zCTGF4 cDNA and CTGF family, it was revealed that the clone contained a truncated cDNA sequence with an intron at the 5′ end of the sequence.

[0163] Based on the tissue distribution from Northern blot analysis (see Example 2), a cDNA library was constructed from human testis, and used to screen for a full length clone. The library was calculated to contain approximately 106 clones. The master plate containing 80 pools (each pool representing 12 pools of 1.25×104 clones) was screened using PCR to determine whether zCTFG4 cDNA was present. Reactions were set up using: 1 μl of each pool, 20 pmoles each of oligonuc...

example 2

[0167] Northern analyses were performed using Human Multiple Tissue Blots I, II and III from Clontech (Palo Alto, Calif.). A probe was generated from a gel purified PCR product made from ZC14, 883 (SEQ ID NO: 7) and ZC14, 882 (SEQ ID NO: 6) as primers and zCTGF4 as template, that had been radioactively labeled with REDIPRIME™ DNA labeling kit (Amersham, Arlington Heights, Ill.) according to the manufacturer's suggestion. The probe was purified using a NUCTRAP push column (Stratagene). EXPRESSHYB™ (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65° C., and the blots were then washed in 2×SSC and 0.05% SDS at RT, followed by a wash in 0.1×SSC and 0.1% SDS at 50° C. One major transcript was observed at size of 1.4 kb. Signals were present in testis, bone marrow, trachea, kidney, liver, stomach, small intestine, ovary, placenta, prostate and spinal cord. The expression of zCTGF4 was also examin...

example 3

[0168] zCTGF4 was mapped to chromosome 6 using the commercially available “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics, Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel contained DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient). A publicly available WWW server (http: / / www-genome.wi.mit.edu / cgi-bin / contig / rhmapper.pl) allowed mapping relative to the Whitehead Institute / MIT Center for Genome Research's radiation hybrid map of the human genome (the “WICGR” radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.

[0169] For the mapping of zCTGF4 with the “GeneBridge 4 RH Panel”, 20 μl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, Calif.), and used in a “RoboCycler Gradient 96” thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 μl dNT...

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Abstract

This present invention is directed to polypeptide and polynucleotide molecules that encode a new connective tissue growth factor homolog polypeptide. The human polypeptides have been designated zCTGF4 and the mouse orthologs have been designated zCTGF2. The invention also includes antibodies, expression vectors, host cells expressing zCTGF4 and variants. Also included are methods for producing the polypeptides and using the polynucleotides and polypeptides.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 011,859, filed on Nov. 5, 2001, which is a continuation of U.S. patent application Ser. No. 09 / 253,316, filed on Feb. 19, 1999; which claims the benefit of U.S. Provisional Patent Application 60 / 075,300, filed on Feb. 20, 1998, for which claims of benefit are made under 35 U.S.C. ° 119(e)(1), all of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] Connective tissue growth factor (CTGF) is a growth factor expressed by endothelial and fibroblastic cells. Two members of the CTGF family are known, CTGF (U.S. Pat. No. 5,585,270 and U.S. Pat. No. 5,408,040) and CTFG-2 (WO 96 / 01896), incorporated herein by reference. CTGF belongs to a family of growth factors that includes CTGF, CTGF-2, insulin binding proteins (IBP) 1 and 2. These growth factors have a cysteine-rich motif and several other structural protein domains in common, and have been demons...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07K14/475C12N15/09C12Q1/68
CPCC07K14/475
Inventor SHEPPARD, PAULJASPERS, STEPHENGAO, ZEREN
Owner SHEPPARD PAUL
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