ABSTRACT
<p><b>AIM</b>The six copies of RGD sequences present in osteopontin were expressed in the E. coli, and the biological activity of the purified products was studied.</p><p><b>METHODS</b>cDNA fragments containing six copies of RGD sequences were subcloned into prokaryotic expression vector pGEX-3X including GST coding sequence to construct pGEX-3X-RGD plasmids. E. coli DH5alpha transformed by pGEX-3X-RGD(6) plasmid was induced by different IPTG concentrations for different times to identify the optimal induction condition. Induced GST-RGD fusion protein was purified via GST-Sepharose 4B affinity resin.</p><p><b>RESULTS</b>GST-RGD fusion proteins containing six copies of RGD sequences could be successfully induced and were mainly located in inclusion bodies. After being denatured and dialyzed, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. GST-RGD(6) fusion protein could specifically inhibit adhesion and migration of VSMC stimulated by osteopontin, which could be considered as a basis on producing small peptides containing RGD sequences for inhibition of VSMC adhesion and migration.</p><p><b>CONCLUSION</b>The six copies of osteopontin RGD peptide were successfully expressed in E. coli DH5alpha. The purified GST-RGD fusion protein could inhibit the adhesion and migration of VSMC stimulated by osteopontin.</p>
Subject(s)
Animals , Rats , Cell Adhesion , Cell Movement , Escherichia coli , Genetics , Metabolism , Glutathione Transferase , Genetics , Metabolism , Muscle, Smooth, Vascular , Cell Biology , Oligopeptides , Genetics , Metabolism , Osteopontin , Genetics , Metabolism , Rats, Sprague-Dawley , Recombinant Fusion Proteins , Genetics , Metabolism , PharmacologyABSTRACT
The double-antibody-sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection of rLysostaphin in humans had been developed and established through this study. rLysostaphin of high purity ( > 95 % ) produced in Shanghai Hi-Tech United Bio-Technological Research & Development Co., Ltd (SHUBRD) was used to produce a rabbit anti-rLysostaphin polyclonal antibody. The standard curve of rLysostaphin polyclonal antibody that was constructed showed that the lowest range of detection was found at 0. 98 ng of rLysostaphin/mL, and the curve exhibited linearity preferably from 0. 98 to 500 ng of rLysostaphin/mL. When three serum samples of the same batch were assayed for 6 replicates, and more 3 samples from different batches for 6 replicates, the average intra-assay and inter-assay coefficient variances ( CV) were 6. 4% and 6. 5%, respectively. The relative recovery rate was 98.6% when quantitative standard antigens were added to the serum. The present method for detection of rLysostaphin in serum is specific, highly sensitive, highly precise, and exhibited a low CV and will be helpful in the further study of rLysostaphin pharmacokinetics and holds promise in clinical applications.
Subject(s)
Animals , Humans , Male , Rabbits , Antibodies, Monoclonal , Allergy and Immunology , Blotting, Western , Enzyme Stability , Enzyme-Linked Immunosorbent Assay , Methods , Immune Sera , Allergy and Immunology , Lysostaphin , Blood , Allergy and Immunology , Metabolism , Recombinant Proteins , Allergy and Immunology , Metabolism , Reproducibility of Results , TemperatureABSTRACT
Objective:To investigate the role of extracellular signal-regulated kinase 1/2(ERK1/2)signal pathway in the pathogenesis of stress-induced gastric ulcer.Methods:Animal model of stress-induced gastric ulcer was established in rats with water-immersion restraint(WIR)stress.The mucosal activation of ERK1/2 was observed before and 5,15 and 30 min,and 1, 2 and 3.5 h after WIR stress.Some animals were also treated with an intravenous injection of PD98059(1 mg/kg),a specific ERK1/2 inhibitor,1 h prior to WIR stress.Expression of total ERK1/2 and caspase-3 were detected by Western blot analysis; ERK1/2 activity was measured by kinase activity assay using myelin basic protein as a specific substrate.DNA-binding activities of the transcription factors activator protein-1(AP-1)and nuclear factor-?B(NF-?B)were determined by electrophoretic mobility shift assays(EMSA).Mucosal TNF-?and IL-1?mRNA expression was analyzed by Northern blot analysis.The degrees of the gastric mucosal lesions were expressed as ulcer index(UI)and pathological evaluation.Apoptosis in the gastric mucosa was examined by an in situ TdT-mediated dUTP nick-end labeling(TUNEL)method.Results:Activated ERK1/2 was very weakly expressed in the gastric mucosa of normal rats.ERK1/2 was rapidly activated in the gastric mucosa of rats 15 min after WIR stress and the activity reached the maximal after 3.5 h.Pretreatment with PD98059 significantly inhibited ERK1/2 activation,decreased AP-1 and NF-?B activities and TNF-?and IL-1?mRNA expression,and obviously relieved gastric mucosal lesions,accompanied by caspase-3 activation and increased apoptosis.Conclusion:The present results indicate that ERK1/2 activation plays an important role in the development of stress-induced gastric ulcer.