RESUMO
GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl beta-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.
Assuntos
Humanos , Sequência de Bases , Escherichia coli , Genética , Metabolismo , Espaço Extracelular , Metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes , Genética , Fatores de Necrose Tumoral , GenéticaRESUMO
Objective To investigate the effects of the traditional herb-epimedium on the expression of interleukin-6 (IL-6) mRNA in bone of ovariectomized rat. Methods Forty female rats were randomly allocated into 4 groups, 10 in each: ovariectomized (OVX) group, sham operation group, OVX followed by epimedium (group 3)or nilestriol (group 4) for 3 months respectively. All rats were then sacrificed,and total RNA were directly isolated from their right tibia. Interleukin-6 mRNA expression was detected by relative semiquantitative reverse transcription-polymerase chain reaction technique. Lumbar bone mineral density (BMD) was measured by dual-energy X ray absorptiometry before sacrifice. Results The BMD of epimedium group was significantly higher than that in the OVX group ( P