RESUMEN
<p><b>OBJECTIVE</b>The purpose of this study was to construct a eukaryotic expression vector for human amelogenin (AMG).</p><p><b>METHODS</b>PCR was performed to amplify the AMG encoding region. Amplified fragments for human AMG were recovered and inserted into eukaryotic expression vectors PsecTaq2A. The recombinant plasmid PsecTaq2A-AMG was constructed and their positive clones were identified.</p><p><b>RESULTS</b>1. Amplified products were checked by electrophoresis and the results were satisfactory. 2. The recombinant plasmid PsecTaq2A-AMG was analyzed by restriction endonuclease mapping and DNA sequencing. The results of sequencing were consistent with those from GenBank.</p><p><b>CONCLUSION</b>The recombinant plasmid PsecTaq2A-AMG was successfully constructed with properly inserted DNA sequence encoding mature amelogenin.</p>
Asunto(s)
Humanos , Amelogenina , Células Clonales , Metabolismo , Clonación Molecular , ADN Recombinante , Genética , Proteínas del Esmalte Dental , Genética , Escherichia coli , Genética , Metabolismo , Células Eucariotas , Metabolismo , Vectores Genéticos , Plásmidos , Genética , Proteínas Recombinantes , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To prepare the polyclonal antibody to amelogenin.</p><p><b>METHODS</b>The fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test.</p><p><b>RESULTS</b>The Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000.</p><p><b>CONCLUSION</b>The antibody prepared in this study can be used for the detection of amelogenin.</p>
Asunto(s)
Animales , Conejos , Amelogenina , Animales Recién Nacidos , Anticuerpos Monoclonales , Alergia e Inmunología , Esmalte Dental , Química , Proteínas del Esmalte Dental , Alergia e Inmunología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular , Alergia e Inmunología , Inmunoglobulina G , Porcinos , Germen Dentario , QuímicaRESUMEN
Objective:To explore whether the recombinant plasmid PcDNA3.1/ sTNFRⅠcould be expressed properly in mice.Methods:The recombinant plasmid PcDNA3.1/sTNFRⅠand the empty vector PcDNA3.1 were directly injected into the right tibialis anterior muscle of each 6 mice respectively. The expression product, sTNFRⅠ, in muscle homogenates was measured by ELISA 7 and 10 days after injection respectively.Results:7 days after injection sTNFR Ⅰ(mg/g) in muscle homogenates in PcDNA3.1/sTNFRⅠ and PcDNA3.1 injected mice was 161.123?4.0 and 69.34?1.489(P