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Ren Jun - One of the best experts on this subject based on the ideXlab platform.
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Gene construction expression and activity identification of Recombinant anti her2 scfv fdt caspase 6
Journal of the Fourth Military Medical University, 2007Co-Authors: Ren JunAbstract:AIM: To construct the Recombinant expression vector of anti-HER2 ScFv/FDT/caspase-6 and investigate its pro-apop- tosisactivity after transfected into gastric cancer SGC7901 cells. METHODS: Anti-HER2 ScFv (e23sFv) was attached to human Recombinant caspase-6 (RC6) Gene by Recombinant PCR, and the Furin recognition site of diphtheria toxin (FDT) served as the linkerto construct e23sFv-FDT-RC6 Recombinant Gene. The Recombinant Gene was cloned into the vector pCMV, and the Recombinant plasmid was transiently transfected into HER2 positive SGC7901 cells and HER2 negative HeLa cells via lipofectamine mediation. MTT assay was used to show how the cell living status was affected by the Gene transfection. The pro-apoptotic activity of Recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that e23sFv-RC6 and e23sFv-FDT-RC6 Genes had been cloned into vector pCMV. After transfection, the expression of fusion protein was found through Western Blot. MTT assays showed that the growth of SGC7901 cells was inhibited. Typical apoptotic changes of the SGC7901 cells transfected with pCMV-e23sFv-FDT-RC6 were seen by indirect immunofluorescent staining. CONCLUSION: The expression of Recombinant anti-HER2 ScFv/FDT/caspase-6 Gene can induce the apoptosis of HER2 positive SGC7901 cells.
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Gene construction, expression and activity identification of Recombinant anti-HER2 ScFv/FDT/caspase-6
Journal of the Fourth Military Medical University, 2007Co-Authors: Ren JunAbstract:AIM: To construct the Recombinant expression vector of anti-HER2 ScFv/FDT/caspase-6 and investigate its pro-apop- tosisactivity after transfected into gastric cancer SGC7901 cells. METHODS: Anti-HER2 ScFv (e23sFv) was attached to human Recombinant caspase-6 (RC6) Gene by Recombinant PCR, and the Furin recognition site of diphtheria toxin (FDT) served as the linkerto construct e23sFv-FDT-RC6 Recombinant Gene. The Recombinant Gene was cloned into the vector pCMV, and the Recombinant plasmid was transiently transfected into HER2 positive SGC7901 cells and HER2 negative HeLa cells via lipofectamine mediation. MTT assay was used to show how the cell living status was affected by the Gene transfection. The pro-apoptotic activity of Recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that e23sFv-RC6 and e23sFv-FDT-RC6 Genes had been cloned into vector pCMV. After transfection, the expression of fusion protein was found through Western Blot. MTT assays showed that the growth of SGC7901 cells was inhibited. Typical apoptotic changes of the SGC7901 cells transfected with pCMV-e23sFv-FDT-RC6 were seen by indirect immunofluorescent staining. CONCLUSION: The expression of Recombinant anti-HER2 ScFv/FDT/caspase-6 Gene can induce the apoptosis of HER2 positive SGC7901 cells.
Karl Bayer - One of the best experts on this subject based on the ideXlab platform.
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Tuning the transcription rate of Recombinant protein in strong Escherichia coli expression systems through repressor titration.
Biotechnology Progress, 2003Co-Authors: Gerald Striedner, Monika Cserjan-puschmann, Florentina Pötschacher, Karl BayerAbstract:The main goal of this work was to develop a strategy that enables tuning of Recombinant Gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize Recombinant Gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of Recombinant Gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of Recombinant Gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed-batch cultivation experiments using E. coli HMS174(DE3)(pET11ahsoD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
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Optimizing Recombinant Microbial Fermentation Processes
BioPharm, 2002Co-Authors: And Franz Clementschitsch, Karl BayerAbstract:A new strategy for controlling Recombinant Gene expression improves efficiency, maximizes host vector exploitation, reduces costs, improves product consistency, and accelerates product development. Continuous feeds of limited amounts of inducer proportional to biomass growth grant optimal control over the ratio between Gene expression and host cell metabolism, providing stable, prolonged Recombinant protein production.
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Optimizing Recombinant microbial fermentation processes: An integrated approach
Biopharm, 2002Co-Authors: Monika Cserjan-puschmann, Reingard Grabheer, Franz Clememtschitsch, Gerald Striedner, Karl BayerAbstract:A new strategy for controlling Recombinant Gene expression improves efficiency, maximizes host vector exploitation, reduces costs, improves product consistency, and accelerates product development. Continuous feeds of limited amounts of inducer proportional to biomass growth grant optimal control over the ratio between Gene expression and host cell metabolism, providing stable, prolonged Recombinant protein production.
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Optimization of Recombinant Gene Expression in Escherichia colia
Annals of the New York Academy of Sciences, 1996Co-Authors: Diethard Mattanovich, Karl Bayer, Robert Weik, Silvia Thim, W. Kramer, Hermann KatingerAbstract:The major targets for improvement of Recombinant expression efficiency in Escherichia coli are Gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing Recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of Recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the Recombinant Gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a Recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.
Bernard Moss - One of the best experts on this subject based on the ideXlab platform.
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elucidating and minimizing the loss by Recombinant vaccinia virus of human immunodeficiency virus Gene expression resulting from spontaneous mutations and positive selection
Journal of Virology, 2009Co-Authors: Linda S Wyatt, Patricia L Earl, Wei Xiao, Jeffrey L Americo, Catherine A Cotter, Jennifer Vogt, Bernard MossAbstract:While characterizing modified vaccinia virus Recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol Genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the Recombinant Gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the Recombinant Gene to a site between essential, highly conserved MVA Genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol Genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary Recombinant vaccines.
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Genetically engineered poxviruses for Recombinant Gene expression vaccination and safety
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Bernard MossAbstract:Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing Genes within the cytoplasm of mammalian cells. As a research tool, Recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of Recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A Genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level Gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.
Hu Yx - One of the best experts on this subject based on the ideXlab platform.
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Anti-cD20scFv/CD80/CD28/zeta specific T lymphocytes eradicate primary chronic lymphocytic leukemia cells in vitro
Chinese journal of applied physiology, 2010Co-Authors: Honglan Qian, Yu K, Shen Zj, Liang B, Luo S, Xing Cy, Hu YxAbstract:Objective:To construct anti-CD20scFv/CD80/CD28/zeta Recombinant Gene modified T cells,test its effectiveness of eradicating CD20 positive primary chronic lymphocytic leukemia (CLL) cells and provide a promising tool for tumor adoptive immunotherapy. Me-thods: The Recombinant vectors were transduced into PA 317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection for one week. Then transduced T lymphocytes and primary CLL cells were cocultured. The status of primary chronic lymphocytic leukemia cells were observed by microscope. The level of IL-2 and IFN-γ in the culture medium were measured. Results: Primary T cells expressing anti-CD20scFv/IgGFc/CD80/CD28/zeta could be constructed successfully. These T cells were able to lyse CD20+ targets and secrete high levels of IL-2(1301.00 pg/ml) and IFN-γ(602.18 pg/ml) in vitro. Conclusion: ①Recombinant Gene modified T cells can be constructed successfully. ②Recombinant Gene modified T cells can specially kill CD20 positive primary CLL cells in vitro.
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Construction of anti-cD20scFv/CD80/CD28/zeta Recombinant Gene modified T cell and research on its targeting cytotoxicity
Chinese Journal of Hematology, 2007Co-Authors: Hu Yx, Yu K, Tan Yx, Shen Zj, Jiang Sf, Qian Hl, Hang B, Shan DmAbstract:To construct anti-CD20scFv/CD80/CD28/zeta Recombinant Gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy. CD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 Gene. The Recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively. The Recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro. Recombinant Gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.
James M. Wilson - One of the best experts on this subject based on the ideXlab platform.
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Cyclophosphamide Diminishes Inflammation and Prolongs TransGene Expression Following Delivery of Adenoviral Vectors to Mouse Liver and Lung
Human Gene Therapy, 1996Co-Authors: Karin Jooss, Yiping Yang, James M. WilsonAbstract:ABSTRACT Immune responses to adenovirus-mediated Gene transfer contribute to the problems of transient Recombinant Gene expression, inflammation, and difficulties with vector readministration. Acti...
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Efficient catheter-mediated Gene transfer into the heart using replication-defective adenovirus.
Gene Therapy, 1994Co-Authors: Eliav Barr, Sandeep K. Tripathy, Kalynych Am, James M. Wilson, Carroll J, Karen Kozarsky, Jeffrey M. LeidenAbstract:The ability to express Recombinant Genes in the coronary vasculature and the myocardium holds promise for the treatment of a number of acquired and inherited cardiovascular diseases. Previous in vivo Gene transfer approaches in the heart have been limited by relatively low efficiencies of Gene transduction. In this report, we demonstrate that catheter-mediated infusion of replication-defective adenovirus into the coronary arterial circulation in vivo represents a novel and efficient method for the induction of Recombinant Gene expression in both the coronary arteries and the myocardium. A single intracoronary infusion of 2 x 10(9) - 1 x 10(10) p.f.u. of adenovirus resulted in high level Recombinant Gene expression in both the coronary arteries and surrounding myocardium of adult rabbits for at least 2 weeks. No inflammatory response or myocardial necrosis was observed following the adenovirus infusions. The polymerase chain reaction (PCR) was used to assess the tissue distribution of infection following intracoronary infusion of adenovirus. Adenovirus DNA was detected by PCR in the livers, kidneys, lungs, brains and testes of animals 5 days after virus infusion. Percutaneous transluminal Gene transfer (PTGT) into the heart by intracoronary infusion of replication-defective adenovirus represents a relatively non-invasive and efficient method of inducing Recombinant Gene expression both in the coronary arterial wall and in the surrounding myocardium.
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cellular immunity to viral antigens limits e1 deleted adenoviruses for Gene therapy
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Yiping Yang, Frederick A Nunes, Klara Berencsi, Emma E Furth, Eva Gonczol, James M. WilsonAbstract:An important limitation that has emerged in the use of adenoviruses for Gene therapy has been loss of Recombinant Gene expression that occurs concurrent with the development of pathology in the organ expressing the transGene. We have used liver-directed approaches to Gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-Generation Recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the Recombinant viral genome express the transGene as desired; however, low-level expression of viral Genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the Genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransGene-containing hepatocytes. These findings suggest approaches for improving Recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral Genes.