مقالههای Azam Bolhassani
توجه: محتویات این صفحه به صورت خودکار پردازش شده و مقالههای نویسندگانی با تشابه اسمی، همگی در بخش یکسان نمایش داده میشوند.
۱Evaluation of Cell Penetrating Peptide Delivery System on HPV16E7 Expression in Three Types of Cell Line
اطلاعات انتشار: Iranian Journal of Biotechnology، سيزدهم،شماره۱(پياپي ۴۹)، ۲۰۱۵، سال ۰
تعداد صفحات: ۸
Background: The poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non–viral delivery systems. Objectives: The aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non–covalent complexes with nucleic acids for delivery of pEGFP–E7 as a model antigen in vitro.Materials and Methods: DNA construct encoding HPV16 E7 (pEGFP–E7) was prepared in large scale with high purity. MPG peptide\ DNA complexes were prepared at different N\P (nitrogen\phosphate) ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide–mediated E7–GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP–positive cells by flow cytometry. Furthermore, E7–GFP expression was confirmed by western blot analysis.Results: The cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS–7 cells transfected by MPG–based nanoparticles at an N\P ratio of 15:1 showed the highest transfection efficiency and gene expression. Conclusions: The results indicated that the efficient gene expression depends on both cell type and N\P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG–based nanoparticles as a potent gene delivery system.
اطلاعات انتشار: Iranian Journal of Biotechnology، سيزدهم،شماره۳(پياپي ۵۱)، ۲۰۱۵، سال ۰
تعداد صفحات: ۶
Background: Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses.Objectives: In current study, the efficient generation of recombinant core and core–E1E2 proteins was developed in bacterial expression systems. Materials and Methods: The expression of HCV core and core–E1E2 proteins was performed using prokaryotic pET–28a and pQE–30 expression systems in BL21\ Rosetta, and M15 strains, respectively. The protein expression and identification were detected by SDS–PAGE and western blotting using anti–His antibody. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. Results: The data showed a clear band of ~ 573 bp for HCV core and ~ 2238 bp for core–E1E2 genes in agarose gel. Moreover, a ~ 21 kDa band of core protein and a ~ 83 kDa band of core–E1E2 protein were revealed in SDS–PAGE and Western blotting. The affinity chromatography could not purify the core and core–E1E2 proteins completely, because of low affinity to Ni–NTA bead in comparison with reverse staining method. Conclusions: This study is the first report for purification of HCV core and core–E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE–30 bacterial system.
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