Global ETD Search

11	Avaliação da imunogenicidade da proteína BYCr (Boophilus York pro-Cathepsin) expressada por vetores eucariotos. Medeiros, Maria Lúcia Schiaffino January 2008 (has links) O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control. Biotecnologia Imunologia Embriogenese BYCr Vacina de DNA Rhipicephalus (Boophilus) microplus DNA vaccine Eukaryotic expression vector Tick BYC (Boophilus Yolk Pro-Cathepsin) Embryogenesis
12	Avaliação da imunogenicidade da proteína BYCr (Boophilus York pro-Cathepsin) expressada por vetores eucariotos. Medeiros, Maria Lúcia Schiaffino January 2008 (has links) O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control. Biotecnologia Imunologia Embriogenese BYCr Vacina de DNA Rhipicephalus (Boophilus) microplus DNA vaccine Eukaryotic expression vector Tick BYC (Boophilus Yolk Pro-Cathepsin) Embryogenesis
13	Avaliação da imunogenicidade da proteína BYCr (Boophilus York pro-Cathepsin) expressada por vetores eucariotos. Medeiros, Maria Lúcia Schiaffino January 2008 (has links) O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control. Biotecnologia Imunologia Embriogenese BYCr Vacina de DNA Rhipicephalus (Boophilus) microplus DNA vaccine Eukaryotic expression vector Tick BYC (Boophilus Yolk Pro-Cathepsin) Embryogenesis
14	Vlastnosti expresních vektorů pro Corynebacterium glutamicum a jejich využití při studiu faktorů sigma RNA polymerasy / Characteristics of expression vectors for Corynebacterium glutamicum and their use for studies of sigma factors of RNA polymerase Dvořáková, Pavla January 2017 (has links) The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...
15	Chromosomal Integration and In Vivo Transcriptional Optimization of Metabolic Pathways in E. Coli O'Dell, Philip John 26 July 2022 (has links) No description available. Chemical Engineering Microbiology bioproduction E. coli chromosomal integration biosynthetic pathway small molecule production psilocybin norbaeocystin expression vector transposons cas9 homologous recombination transcriptional optimization
16	Construction of an Adenovirus Expression Vector Containing the T4 Den V Gene, Which Can Complement the DNA Repair Deficiency of Xeroderma Pigmentosum Fibroblasts / Construction of an AD 5 Vector Containing the T4 Den V Gene Colicos, Michael, A. 08 1900 (has links) This study demonstrates the use of an adenovirus vector system to study the effect of a DNA repair gene on untransformed human fibroblasts. The bacteriophage T4 pyrimidine dimer DNA glycosylase (den V) gene has been inserted into the E3 region of human adenovirus type 5. The resulting recombinant virus Ad Den V was determined to be producing correctly initiated RNA from the RSV 3' LTR promoter used in the den V expression cartridge inserted into the virus. The effect of the den V gene product on human fibroblasts 'liras examined by assaying for the percent host cell reactivation (%HCR) of Vag production for UV irradiated Ad Den V in comparison to that for a control virus. It was shown that the %HCR was significantly greater for Ad Den V as compared to the control virus in xeroderma pigmentosum (XP) cells. UV survival of adenovirus in XP cells exhibited a two component nature. Introduction of the den V gene into XP group A cells increased the D0 value of the first component of the viral survival curve to a level similar to that of XPC cells, which showed no change in this component irrespective of the presence of the den V gene. It has been suggested that the den V gene is able to partially complement the deficiency in some XP cells because of its small size, allowing it to gain access to the DNA damage site where as the cellular repair enzyme complex can not. Since XPC cells are proficient in their alteration of DNA secondary structure prior to DNA excision repair, these results are consistant with the hypothesis that the first component of UV viral survival curves reflects the pathway involved in accessing the damaged sites. The manuscript of a paper has been included as an appendix. The work theorizes on the origin of mammalian immune system diversity and bacteriophage lambda, and their possible relationship to prokaryotic DNA repair genes. / Thesis / Master of Science (MS)
17	Transcriptional regulation and physiological importance of the kdp-system from the halophilic archaeon Halobacterium salinarum Kixmüller, Dorthe 03 April 2012 (has links) The high affinity, ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum, is highly induced under K+ limitation. In contrast to the well-characterized Kdp system in Escherichia coli, in which the kdpFABC genes are transcriptionally regulated by the sensor kinase/response regulator system KdpD/KdpE, transcriptional regulation of the kdp genes in H. salinarum was unknown due to the absence of halobacterial homologues of KdpD/KdpE. Furthermore, the physiological relevance of the KdpFABC K+ uptake system of H. salinarum was puzzling, since hypersaline habitats usually comprise K+ concentrations which do not induce kdp expression. In order to analyze the regulation of kdp gene expression, it was essential to gain information about the transcriptional unit(s) involved. Northern blotting, primer extension analysis and real-time RT-PCR revealed the presence of a polycistronic leaderless kdpFABCQ transcript with a putative kdp terminator or at least a potential mRNA processing site downstream of kdpQ. Furthermore, promoter truncation studies verified the so far only predicted basal transcription elements together with an upstream-located operator sequence. Since deletions of this putative operator sequence did not lead to a constitutive expression, a further component has to be involved in the regulation of the kdpFABCQ genes. However, truncation and scanning mutagenesis analyses of the kdp promoter as well as translational fusions of a halophilic beta-galactosidase to the kdp promoter excluded an additional regulatory element up- or downstream of the basal transcription elements and in the kdp-coding region. These results lead to speculations of multiple basal transcription factors to be involved. Furthermore, an inducible expression vector (shuttle vector) was constructed based on the promoter of the kdpFABCQ operon due to its, K+-sensitive features. Inducible expression systems are yet not available for H. salinarum. The resulting, replicating vector pKIX is functional and enables a K+-dependent expression from the kdp promoter with rather high induction ratios of 50-fold. Expression levels could further be improved by plasmid- and additional chromosomally encoded kdpQ and mutations generated in the kdp promoter. Since transcript levels from pKIX were found to be independent of differential target genes, the general application of pKIX as an inducible expression system is strongly supported and pKIX could, thus, be made accessible to the scientific community. To decipher the physiological relevance of the halobacterial Kdp system, H. salinarum was encountered to desiccation stress and salt crystal (halite) entombment. Halite crystals grown under non-inducing K+ concentrations with entombed strains of H. salinarum and H. salinarum deleted in the kdpFABCQ genes revealed a significantly reduced survival rate of the deletion strain upon recultivation. Additionally, a kdpFABCQ-inducing desiccation stress could already be determined on agar plates under non-limiting K+ concentrations. Furthermore, the cell morphology of H. salinarum entrapped in halite crystals resembled that of H. salinarum grown under K+-limiting conditions. Therefore, the Kdp system promotes survival of H. salinarum under desiccation stress. Furthermore, the Kdp system could be identified as at least one of the systems important for long-term survival of H. salinarum in halite. Halobacterium Halobacterium salinarum H. salinarum halophile halophil archaea archaeon kdp kdpFABC kdpFABCQ kdp-System ATP Kalium Kaliumaufnahme K+ Halit Salzkristall Austrocknung Trockenstress transkriptionelle Regulation Genexpression pKIX induzierbarer Expressionsvektor Expressionsvektor kdpQ Kdp Komplex halobacterial halophilic kdpFABC genes kdp-system high-affinity ATP-dependent potassium potassium pump K+ uptake desiccation desiccation stress halite salt crystal long-term survival transcriptional regulation gene expression inducible expression vector expression vector Kdp complex ddc:570
18	Investigating Escherichia coli-based Cell Free Protein Expression Systems Gutu, Nicoleta 10 1900 (has links) Synthesizing proteins for use in therapeutics is restrained by, in part, contaminants in in vivo expression systems and limited production capacity of in vitro systems. Cell free expression (CFE) systems have emerged as a potential alternative for protein expression because of the inherently lower contents of contaminants, and their flexible modular design that allows the addition of factors that aid in synthesis of complex products. Here, we investigate and establish an in-house Escherichia coli-based cell free protein synthesis (CFPS) system, explore different CFPS commercial kits, develop assays to test performance of these systems and identify potential rules that dictate expression levels. Using CFE, we were able to test different vectors and conditions of system, as well as scale-up protein synthesis reactions. In conclusion, this work shows that CFPS is a functional and easy-to-use platform and can potentially meet the requirements for the synthesis of therapeutics. cell-free protein expression cell-free protein synthesis cell free protein expression cell free protein synthesis cell-free expression prokaryotic cell-free system E. coli-based cell-free system synthetic biology expression cell-free translation cell-free transcription in vitro synthesis in vitro protein synthesis in vitro expression in vitro protein expression nanobody SARS-CoV-2 anti-SARS-CoV-2 nanobody cell free expression vector cell-free expression vector in-house cell-free synthesis in house cell-free synthesis in-house cell free synthesis in house cell free synthesis cell-free extract

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