RESUMEN
Matrix attachment region (MAR) is DNA fragment that can bind to the nuclear matrix. In order to isolate the MAR fragment from the halotolerant green alga Dunaliella salina, we created a library of randomly obtained MAR from D. salina. Firstly the intact nuclei were released using 0.5% Triton X-100, then purification was carried out by discontinuous centrifugation using 30% and 70% Percoll gradients. Histones of nuclear matrices were removed using 25mmol/L lithium dioodosalicylate, the DNAs not closely associated with the matrices were removed using restriction enzymes. The remained matrices DNAs were digested by proteinase K, extracted with phenol/chloroform and precipitated with ethanol, and then cut with four kinds of restriction enzymes, the resulting DNAs were subsequently ligated to pUC18-vector and transferred to E. coli JM109 strains, DNA sequencing showed that the DNA fragments had the features of MAR DNA fragments.
Asunto(s)
Chlorophyta/genética , ADN de Algas/genética , ADN de Algas/aislamiento & purificación , Regiones de Fijación a la Matriz/genéticaRESUMEN
Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.
Asunto(s)
Membrana Corioalantoides/irrigación sanguínea , Colágeno Tipo IV/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
The mouse canstatin and its N-domain cDNA were amplified from total RNA of mouse liver by RT- PCR and cloned into vector pMD18-T for sequencing. Prokaryotic expression vectors pET/Can and pET/Can-N were constructed and expressed in E.coli BL21(DE3) with induction of IPTG.. Mouse canstatin cDNA is 684bp in length encoding 227 amino acids. The sequences of both cDNA and amino acids share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. N-domain of mouse canstatin is the same amino acid sequence as that of human canstatin. In the present study, prokaryotic expression vector pET/Can and pET/Can-N were expressed in E.coli BL21 with amount of 35% and 18% of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. This work has laid down the basis for further study of its angiogenic activity and potential application for tumor dormancy therapy.
Asunto(s)
Inhibidores de la Angiogénesis/biosíntesis , Colágeno Tipo IV/biosíntesis , Fragmentos de Péptidos/biosíntesis , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/genética , Animales , Secuencia de Bases , Clonación Molecular , Colágeno Tipo IV/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos , Humanos , Ratones , Fragmentos de Péptidos/genética , Plásmidos , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The present study is to obtain heat shock protein 70a cDNA fragment from Dunaliella salina. Two pairs of degenerate primers were designed according to conserved motifs of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG of the homologous amino acid sequences and used to amplify hsp70a cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique. The resulting PCR products were inserted into T-vector then transformed into JM109. Ten colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Three of ten nucleotide sequences were obtained,of which there was 372 bp coding 126 amino acids. The sequences shared high homology with hsp70a,with identity 96% to Chlamydomonas reinhardtii, 94% to Petunia, 93% to Pisumsativum, 92% to tomato, 92% to human,90% to Drosophila and 89% to yeast respectively. It can be concluded that the cloned sequence is putatively hsp70a cDNA fragment from Dunaliella salina.
