Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) is a rather new ’soft ionization’ techniques. Because of its high sensitivity and accuracy, it has been widely used in dete...Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) is a rather new ’soft ionization’ techniques. Because of its high sensitivity and accuracy, it has been widely used in detection and characterization of macromolecules such as peptides, proteins and olignucleotides. It also has been applied successfully in the analysis of posttranslational modification of proteins, such as phosphorylation and glycosylation. Compared with the conventional chemical methods, mass spectrometry is simple, rapid and does not require radiolabeling. In this research, a systematic method to analyse glycoprotein by MALDI-TOF-MS was developed. A practical sample-recombinant human erythropoietin was analysed by the method. The molecular weight, content of carbohydrate and glycosylation sites of the glycoprotein were determined by MALDI-TOF-MS, combined with the endo-glycosidase digestion and peptide mapping. The experimental result shows that MALDI-TOF-MS is a very powerful technique in the characterization of glycosylation proteins.展开更多
Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera...Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co- transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.展开更多
文摘Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) is a rather new ’soft ionization’ techniques. Because of its high sensitivity and accuracy, it has been widely used in detection and characterization of macromolecules such as peptides, proteins and olignucleotides. It also has been applied successfully in the analysis of posttranslational modification of proteins, such as phosphorylation and glycosylation. Compared with the conventional chemical methods, mass spectrometry is simple, rapid and does not require radiolabeling. In this research, a systematic method to analyse glycoprotein by MALDI-TOF-MS was developed. A practical sample-recombinant human erythropoietin was analysed by the method. The molecular weight, content of carbohydrate and glycosylation sites of the glycoprotein were determined by MALDI-TOF-MS, combined with the endo-glycosidase digestion and peptide mapping. The experimental result shows that MALDI-TOF-MS is a very powerful technique in the characterization of glycosylation proteins.
文摘Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co- transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.
