RESUMO
CTGF (connective-tissue growth factor) has been characterized as an extracellular-matrix-associated protein that modulates basic-fibroblast-growth-factor signalling and angiogenesis. In the present paper, the cloning of the ctgf gene from human umbilical-vein endothelial cells and expression of the protein in Escherichia coli as an N-terminal hexahistidine fusion protein is described. Recombinant human CTGF (rhCTGF) was expressed and purified so that we could investigate its effect on the proliferation of human embryo fibroblast KMB-17 and NIH3T3 cells. The results indicated not only that the protein was properly folded, but also that it had the same specific activity and stability as the native protein. Furthermore, we administered this recombinant protein in a non-human primate [rhesus monkey (Macaca mulatta)] burn-wound model and report the clinical findings and structural effects. Epitheliotrophic effects were conspicuous in wounded tissues at 10-100 ng of CTGF/cm(2), suggesting that administered rhCTGF can play a normal physiological role in wound repairing in a non-human primate model.
Assuntos
Queimaduras/tratamento farmacológico , Queimaduras/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Proteínas Imediatamente Precoces/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Cicatrização/efeitos dos fármacos , Animais , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo , Modelos Animais de Doenças , Fibroblastos/citologia , Humanos , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Células NIH 3T3 , Proteínas Recombinantes/administração & dosagem , Resultado do TratamentoRESUMO
The interaction between virus and receptor is a process that mimics physiological ligand binding receptors and induces signal transduction. In the investigation of the interaction between HSV1 (Herpes Simplex virus 1) and human fibroblasts via virus binding to its receptor complex on cellular membranes, the HTRP (human transcription regulator protein), a protein encoded by an immediate-early gene of cellular response against the specific stimulation of HSV1 binding, was cloned from a cDNA library established from early gene response mRNA. The localization of HTRP expressed as a fusion polypeptide with a fluorescent protein in HeLa cells was confirmed to be the nucleus. The results of a yeast two-hybrid experiment indicated that HTRP is indeed involved in the interaction with the SAP (mSin3-associate polypeptide) complex via SAP30. A pull-down test and Western blotting in vitro, and immunoprecipitation in vivo also provided evidence in support of this result. The interaction of HTRP with SAP30 in its conserved domain implies that this protein family, as the products of immediate-early genes, comprise functional molecules involved in the transcriptional regulation of cells, which might be related to the inhibition of some cell survival genes.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/virologia , Herpesvirus Humano 1/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Sialiltransferases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Sobrevivência Celular , Células Cultivadas , Clonagem Molecular , DNA Complementar/metabolismo , Biblioteca Gênica , Genes Precoces , Glutationa Transferase/metabolismo , Células HeLa , Histona Desacetilases/fisiologia , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Sialiltransferases/química , Fatores de Tempo , Fatores de Transcrição , Transcrição Gênica , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
The specific binding of virus to receptor is essentially the interaction of ligand and receptor. This binding is able to induce cellular primary gene response that has various regulatory function in cells. Different immediate-early gene responses could be induced with different binding virus. The different gene responses were observed as poliovirus and HSV-I bind to human diploid cells. These differences showed that early expression of protooncogenes were enhanced and/or suppressed in the primary gene responses.
RESUMO
The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.