Precementum- and / or cementum - derived chemotactic factor (CCTF) of tooth of mammalia, process for purifying the same, and drug for accelerating adhesion of new connective tissue, comprising the same as active ingredient

Abstract

A precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, characterized in that a molecular weight measured by SDS-PAGE is 67000±1000. A process for purifying a precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, wherein a molecular weight measured by SDS-PAGE is 67000±1000, which comprises collecting precementum and/or cementum from an extracted tooth of Mammalia and immersing them in saline or collagenase-containing saline with stirring to obtain an eluted ingredient, and purifying the eluted ingredient by molecular weight fractionation, ion-exchange adsorption chromatography and hydroxyapatite adsorption chromatography. A drug for accelerating adhesion of new connective tissue, comprising the precementum- and/or cementum-derived chemotactic factor (CCTF) as an active ingredient. [FIG. 1] Gel filtration chromatography P: Positive control (10% FBS-containing DMEM liquid culture medium) A: Negative control (liquid culture medium alone) Fraction A, fraction B [FIG. 2] DEAE-3SW ion-exchange chromatography P: Positive control: 10% FBS-containing DMEM liquid culture medium A: Negative control: DMEM liquid culture medium alone [FIG. 3] Hydroxyapatitie chromatography P: Positive control: 10% FBS-containing DMEM liquid culture medium A: Negative control: DMEM liquid culture medium alone [FIG. 4] [FIG. 5] Western blotting of CCTF using anti-BSP-II antibody [FIG. 6] Western blotting of CCTF using anti-BMP-2 antibody

1 . A precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, characterized in that a molecular weight measured by SDS-PAGE is 67000±1000.  
     
     
         2 . The chemotactic factor (CCTF) according to  claim 1 , which is a glycoprotein containing sugar chains having an amino acid composition of: Asp; 10.6±0.5%, Thr; 3.7±0.3%, Ser; 13.3±0.7%, Glu; 13.8±0.7%, Gly; 23.3±1.2%, Ala; 10.1±0.5%, Cys/2; 3.6±0.3%, Val; 6.7±0.3%, Ile; 3.8±0.3%, Leu; 7.3±0.4% and Lys; 3.8±0.3%, and has an isoelectric point of 6.5±0.5.  
     
     
         3 . The chemotactic factor (CCTF) according to  claim 1  or  2 , wherein Mammalia is bovine.  
     
     
         4 . A process for purifying a precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, which comprises eluting a protein ingredient from precementum and/or cementum of a tooth of Mammalia and purifying the precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia by molecular weight fractionation, ion-exchange adsorption chromatography and hydroxyapatite adsorption chromatography.  
     
     
         5 . A process for purifying the chemotactic factor (CCTF) of  claim 4 , which comprises: 
 (a) collecting precementum and/or cementum from an extracted tooth of Mammalia and immersing them in saline or collagenase-containing saline with stirring, thereby to elute a protein ingredient;    (b) removing an insoluble matter from the eluate in the step (a) by centrifugal separation and filtration, and subjecting the resulting filtrate to a process such as gel filtration or ultrafiltration to obtain a fraction having a molecular weight of 270,000 or more;    (c) adsorbing the fraction obtained in the step (b) on a DEAE ion-exchange resin, collecting a fraction eluted with a 0.2-0.3 mM sodium chloride-containing tris ethanolamine hydrochloride buffer, and concentrating the fraction by desalination; and    (d) adsorbing the concentrated solution obtained in the step (c) on hydroxyapatite equilibrated with a phosphate buffer, and collecting the fraction to be eluted with 80-110 mM phosphoric acid.    
     
     
         6 . The process for purifying the chemotactic factor (CCTF) according to  claim 4  or  5 , wherein Mammalia is bovine.  
     
     
         7 . A drug for accelerating adhesion of new connective tissue, comprising the precementum- and/or cementum-derived chemotactic factor (CCTF) of any one of  claims 1  to  3  as an active ingredient.