Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) bearing a...Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-l), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP-1 (200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-l and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9% , release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-l gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-l mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.展开更多
Proteins adsorption at solid surfaces are of paramount important for many natural processes. However, the role of specific water in influencing the adsorption process has not been well understood. We used molecular dy...Proteins adsorption at solid surfaces are of paramount important for many natural processes. However, the role of specific water in influencing the adsorption process has not been well understood. We used molecular dynamics simulation to study the adsorption of BPTI on Au surface in three water environments （dielectric constant model, partial and full solvation models）. The result shows that a fast and strong adsorption can occur in the dielectric environment, which leads to significant structure changes, as confirmed by great deviation from the crystal structure, largely spreading along the Au surface, rapid lose in all secondary structures and the great number of atoms in contact with the surface. Compared to the dielectric model, slower adsorption and fewer changes in the calculated properties above are observed in the partial solvation system since the specific water layer weakens the adsorption effects. However, in the partial solvation system, the adsorption of polar Au surface causes a significant decrease in the specific hydration around the protein, which still results in large structure changes similar to the dielectric system, but with much less adsorption extent. Enough water molecules in the full solvation system could allow the protein to rotate, and to large extent preserve the protein native structure, thus leading to the slowest and weakest adsorption. On the whole, the effects of non-specific and specific solvation on the protein structure and adsorption dynamics are significantly different, highlighting the importance of the specific water molecule in the protein adsorption.展开更多
The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The op...The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The open reading frame(ORF) encoding the CT440 protein from the C.trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase(GST) fusion protein in E.coli XL1-Blue.The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody.After verification by Western blot and immunofluorescence assay(IFA),the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells.Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection.The results showed that the hypothetical protein CT440 was localized in the C.trachomatis inclusion membrane,and was detectable 12 h after chlamydial infection.Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection.In summary,we have identified a new inclusion membrane protein that may be an important candidate for understanding C.trachomatis pathogenesis.展开更多
To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verif...To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.展开更多
基金This program was supported by the National Natural Sciences Foundation of China (No. 39870196).
文摘Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-l), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP-1 (200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-l and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9% , release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-l gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-l mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.
文摘Proteins adsorption at solid surfaces are of paramount important for many natural processes. However, the role of specific water in influencing the adsorption process has not been well understood. We used molecular dynamics simulation to study the adsorption of BPTI on Au surface in three water environments （dielectric constant model, partial and full solvation models）. The result shows that a fast and strong adsorption can occur in the dielectric environment, which leads to significant structure changes, as confirmed by great deviation from the crystal structure, largely spreading along the Au surface, rapid lose in all secondary structures and the great number of atoms in contact with the surface. Compared to the dielectric model, slower adsorption and fewer changes in the calculated properties above are observed in the partial solvation system since the specific water layer weakens the adsorption effects. However, in the partial solvation system, the adsorption of polar Au surface causes a significant decrease in the specific hydration around the protein, which still results in large structure changes similar to the dielectric system, but with much less adsorption extent. Enough water molecules in the full solvation system could allow the protein to rotate, and to large extent preserve the protein native structure, thus leading to the slowest and weakest adsorption. On the whole, the effects of non-specific and specific solvation on the protein structure and adsorption dynamics are significantly different, highlighting the importance of the specific water molecule in the protein adsorption.
基金supported by the National Natural Science Foundation of China (Grant Nos. 30970165 and 81102230)the Hunan Provincial Natu-ral Science Foundation of China (Grant No. 09JJ3059)the Team Project for the Technology Innovation of Higher Education of Hunan Province,China
文摘The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The open reading frame(ORF) encoding the CT440 protein from the C.trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase(GST) fusion protein in E.coli XL1-Blue.The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody.After verification by Western blot and immunofluorescence assay(IFA),the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells.Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection.The results showed that the hypothetical protein CT440 was localized in the C.trachomatis inclusion membrane,and was detectable 12 h after chlamydial infection.Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection.In summary,we have identified a new inclusion membrane protein that may be an important candidate for understanding C.trachomatis pathogenesis.
基金the Scientific Research Project of Shanghai Municipal Health Bureau (2008Y034)the Natural Scientific Research Project of Shanghai(05ZR14086)
文摘To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.
