Дисертації з теми "ORF2 protein"
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Guo, Hailong. "Antigenic epitope composition and protectivity of avian hepatitis E virus (avian HEV) ORF2 protein and vertical transmission of avian HEV." [Ames, Iowa : Iowa State University], 2006.
Ankavay, Maliki. "Étude des modifications post-traductionnelles de la protéine de capside ORF2 du virus de l'hépatite E (HEV)." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S013.
Hepatitis E virus (HEV) infection is a major public health problem that affects more than20 million people and kills approximately 70,000 worldwide each year. HEV is the leadingcause of acute viral hepatitis in the world. In France, the seroprevalance of HEV is 22.4% inthe general population. This virus is transmitted by fecal-oral route or by consumption ofundercooked contaminated meat. Very recently, we have described an efficient cell culturesystem to amplify HEV. This system represents a unique tool for characterizing the variousstages of the infectious HEV lifecycle that are very poorly understood to date. As part of mythesis, I focused on the ORF2 capsid protein which is the structural unit of viral particles andis therefore a central player in the HEV lifecycle. ORF2 is a 660 amino acids protein thatcontains a signal peptide and three potential N-glycosylation sites (N1, N2 and N3). My workaimed to identify the role of the ORF2 N-glycosylation in the HEV lifecycle. Our resultsrevealed that the ORF2 protein is N-glycosylated on the N1 and N3 sites, whereas the N2 siteis not N-glycosylated. Also, the N-glycosylation has no significant relevance neither for thestability, oligomerization, antibody recognition of the ORF2 protein nor for viral assemblyand viral infectivity. We also revealed that, the ORF2 protein is translocated into the nucleusof infected cells, independently of N-glycosylation. We identified for the first time afunctional nuclear localization signal (NLS) located at the N-terminus of the ORF2 proteinthat interacts probably with the importin alpha1. Surprisingly, this NLS regulates both nuclearimport and endoplasmic reticulum translocation of the ORF2 protein. Finally, wedemonstrated that, the ORF2 protein possesses three nuclear export signals (NES) located atits C-terminus. The ORF2 protein interacts with the exportin1 and is exported from the cellnuclei. This interaction drives the ORF2 protein from the nucleus to the cytoplasm of theinfected cells. Hence, this study led to new insights into the molecular mechanisms of theORF2 protein and could help to the design of novel antiviral strategies against HEV
Ferrié, Martin. "Étude de la maturation protéolytique et du trafic intracellulaire de la protéine de capside ORF2 du virus de l'hépatite E (HEV)." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS067.
Hepatitis E virus (HEV) infection is a major public health problem, affecting an estimated 100 million people and killing 100,000 every year worldwide. HEV is the leading cause of acute hepatitis worldwide. In France, seroprevalence is 22.4%. This virus is transmitted via the feco-oral route, or by eating contaminated undercooked meat. During my thesis, I focused on the ORF2 capsid protein, which is the structural unit of viral particles and a central player in the HEV lifecycle. ORF2 is a 660 amino acid protein with an N-terminal signal peptide and three potential N-glycosylation sites. During its lifecycle, HEV produces at least three forms of its capsid protein: (i) the ORF2g (for glycosylated) form and (ii) the ORF2c (for cleaved) form, abbreviated ORF2g/c, which are glycosylated forms and massively secreted in culture supernatants or serum from infected patients, and (iii) the ORF2i (for infectious) form, which is not glycosylated and is associated with viral particles.As part of my thesis work, I studied the mechanisms of proteolytic maturation and intracellular trafficking of the ORF2 protein. More specifically, I first showed that ORF2 is imported into the nucleus of infected cells via an Importin-alpha1-dependent mechanism, thanks to an arginine-rich motif located at the N-terminus of ORF2 and named ARM. The ORF2 protein is then exported to the cytoplasm via a CRM1 exportin-dependent mechanism, thanks to three nuclear export sites (NES9, NES10 and NES12) that we have identified in the ORF2 sequence. I have also shown that nucleocytoplasmic trafficking of ORF2 probably regulates the expression of certain antiviral immunity genes in the early stages of infection.Secondly, I participated in the identification and characterization of HEV viral factories. We have shown that the viral proteins ORF1, ORF2 and ORF3, as well as viral RNA, are enriched in vesicular and tubular structures located in the perinuclear regions of infected cells. We have shown that these structures are also enriched in markers of the endosomal recycling compartment (ERC), such as Rab11 and CD71, indicating that HEV viral factories probably derive from the ERC. By performing Rab11 silencing experiments with siRNAs, we confirmed the importance of the ERC in the production of HEV viral particles. Thirdly, I demonstrated that the ORF2i protein, which is associated with the membranes of the secretion pathway, is addressed to viral factories by a mechanism involving the AP-1 adaptor complex.In a fourth step, I characterized the proteolytic maturation mechanisms of the ORF2g/c and ORF2i forms. I showed that furin, a proproprotein convertase of the secretory pathway, is involved in the proteolytic maturation of ORF2g/c glycoproteins. I have also shown that presenilin, which is the catalytic subunit of the macro-complex Gamma-secretase, is involved in the proteolytic maturation of the ORF2i form. Interestingly, I have shown that pharmacological inhibition of presenilin drastically reduces viral infectivity in human hepatocarcinoma lines and in primary human hepatocytes. These data suggest that pharmacological inhibition of presenilin represents a promising antiviral strategy. In conclusion, the results obtained during my thesis provide a better understanding of the mechanisms of subcellular addressing and proteolytic maturation of the ORF2 capsid protein, and pave the way for the development of new therapies to combat HEV
Horridge, Jackie J. "RNA-protein interactions of the adenovirus proteins E1B 55K and E4 Orf6." Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322435.
Baghbadorani, G. Ahmadian. "Analysis of the utilisation of second ORFs in pneumovirus mRNA." Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323416.
Chand, Puran. "Molecular and immunological characterisation of a major envelope protein of capripoxvirus." Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/2774/.
Koyuncu, Emre. "Expression, Purification, And Functional Analysis Of Adenovirus Type 5 E4 Orf3 Protein." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/12605415/index.pdf.
Bowers, Laura Yvonne. "Orf protein modulates phage and bacterial pathways of genetic recombination." Thesis, Durham University, 2008. http://etheses.dur.ac.uk/2345/.
Liedeman, Kerwin. "Transient transgene expression of human Coronavirus nl63 orf3 protein in a baculovirus system." University of the Western Cape, 2020. http://hdl.handle.net/11394/8042.
Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.
Yadav, Kush Kumar. "Genotype 1 hepatitis E virus (HEV) ORF4 protein enhances genotype 3 HEV replication." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574781581580768.
Ali, Ahmed Said. "Investigation of epoxide hydrolase activity in Saccharomyces cerevisiae ORF YNR064c protein." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-203473.
Dickinson, Victoria Jane. "The cloning and subcellular localisation of maize streak virus ORF V1." Thesis, University of Hull, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321050.
Mo, Min, and n/a. "Characterization of an Orf virus RING-H2 protein, B5L : a mimic of cellular anaphase promoting complex subunit 11." University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090220.085825.
Butarbutar, Nunut. "Analysis of yeast codon usage patterns using the movable ORF collection /." Online version of thesis, 2007. http://hdl.handle.net/1850/5700.
Laitinen, Saara. "Family of human oxysterol binding protein homologues : ORP2 is a new regulator of cellular lipid metabolism." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/laa/kansa/vk/laitinen/.
Öhrmalm, Christina. "Functional Characterization of the Cellular Protein p32 : A Protein Regulating Adenovirus Transcription and Splicing Through Targeting of Phosphorylation." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6794.
Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system.
The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation.
Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex.
Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription.
In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.
D'Angelillo, Anna. "Studio dell'attività biologica della proteina ORF-A del virus dell'immunodeficienza felina." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423592.
Il Virus dell'Immunodeficienza Felina (FIV) è un Lentivirus dei non primati che causa nel gatto domestico una sindrome cronica progressiva molto simile alla Sindrome da Immunodeficienza Acquisita (AIDS) generata dal Virus dell'Immunodeficienza Umana di tipo 1 (HIV-1). Le analogie tra FIV ed HIV-1 coinvolgono numerosi aspetti, tra cui l'organizzazione genomica, il ciclo replicativo, i bersagli cellulari ed i meccanismi patogenetici. Per questi motivi FIV ed il suo ospite naturale rappresentano un interessante modello per la ricerca sull'AIDS. Similmente ad HIV-1, il genoma di FIV contiene i geni gag, pol, env che codificano le proteine strutturali ed enzimatiche del virione e i geni accessori e regolatori che giocano un ruolo chiave nella regolazione e nell'infettività virale. A differenza del genoma di HIV-1 caratterizzato dalla presenza di quattro geni accessori, il genoma di FIV ne presenta due: vif ed orf-A. Di questi, il primo codifica il fattore di infettività virale, molto simile a quello di HIV-1, mentre il secondo gene codifica la proteina Orf-A. Il ruolo biologico di Orf-A non è stato ancora chiarito, ma si ritiene rappresenti una proteina accessoria multifunzionale con caratteristiche simili alle proteine accessorie di HIV-1 quali Vpr, Tat e Nef. Il presente lavoro di dottorato si inserisce in un progetto di ricerca più ampio, volto a chiarire la biologia di base di FIV, focalizzandosi sulle funzioni svolte dalla proteina Orf-A. Inizialmente è stata valutata l'espressione della proteina ed il suo coinvolgimento nel ciclo cellulare mediante diversi sistemi di over-espressione. Successivamente sono stati condotti gli stessi esperimenti in un contesto più simile a quello fisiologico. Pertanto, sono stati ottenuti plasmidi basati sul genoma provirale, Orf-A plus e minus, dove l'estremità Carbossi-terminale della proteina Orf-A è stata fusa al tag HA. I vettori sono stati testati sia in cellule umane che feline per valutarne l'espressione ed il coinvolgimento nella progressione del ciclo cellulare. Gli stessi plasmidi sono stati impiegati per valutare i) la localizzazione della proteina Orf-A e della Ciclina B1 target del blocco del ciclo cellulare in saggi di immunofluorescenza; ii) la capacità replicativa del virus; iii) l'incorporazione della proteina Orf-A nelle particelle virali. Nel complesso, i risultati suggeriscono che, laddove la proteina Orf-A è espressa, si verifica un arresto del ciclo cellulare in fase G2/M indicando un ruolo di Orf-A simile a quello descritto per la proteina Vpr di HIV-1. Inoltre, per la prima volta in letteratura, è stata dimostrata l'incorporazione della proteina Orf-A nelle particelle virali. Questo studio contribuisce a dissezionare le funzioni della proteina Orf-A di FIV ed a chiarire i meccanismi alla base della biologia del Virus dell'Immunodeficienza Felina e, più in generale, di quella dei lentivirus
Tan, Joanne Li-Ching, and n/a. "Development of Orf virus as a vaccine vector : manipulation of structural proteins for surface display of immunogenic peptides." University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090427.144304.
Diekmann, Ulrike Verfasser], and Lothar [Akademischer Betreuer] [Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
Diekmann, Ulrike [Verfasser], and Lothar [Akademischer Betreuer] Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
Estmer, Nilsson Camilla. "Viral Control of SR Protein Activity." Doctoral thesis, Uppsala : Acta Universatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5124-1/.
White, Ian Alexander. "Hematopoietic stem cell expansion : under serum free and cytokine-limited conditions using primary endothelial cells transfected with the adenoviral E4-ORF1 gene /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692100331&sid=5&Fmt=2&clientId=8424&RQT=309&VName=PQD.
Mardegan, Catarina. "Caracterização funcional da ORF XAC0239 de Xanthomonas citri subsp. citri /." Jaboticabal, 2018. http://hdl.handle.net/11449/155873.
Coorientador: Helen Alves Penha
Banca: José Belasque Júnior
Banca: Daniel Guariz Pinheiro
A Xanthomonas citri subsp. citri (Xac) é a bactéria causadora da doença cancro cítrico. A doença atinge todas as variedades comerciais de citros e, o entendimento da relação fitopatogênica permitirá conhecer formas mais eficientes de controle da bactéria. Com o sequenciamento e análise do genoma e transcriptoma da Xac, foram identificadas ORFs que, provavelmente, estão envolvidas em processos de ataque e colonização da bactéria. Para iniciar a caracterização de uma destas ORFs foram utilizadas, neste estudo, as estratégias de mutação sítio-dirigida de PCR por sobreposição e extensão e modelagem molecular. A ORF XAC0239, é predita como proteína de membrana plasmática, e por homologia, possivelmente, pode fazer parte de uma proteína transportadora de zinco, além disso, há indícios de que é regulada por um fator sigma. A mutação teve efeito negativo sobre a ação de celulases e motilidade swimming e efeito positivo sobre a formação de biofilme e a multiplicação bacteriana in vivo. Aqui é sugerido que na falta desta proteína, houve pouca absorção de zinco, necessário para a formação de biofilme e consequentemente, para a patogenicidade. Além disso, sugestiona-se que, como subterfúgio, a bactéria superexpressou genes para produção e/ou ação de celulases, tentando atacar o hospedeiro de forma alternativa.
Xanthomonas citri subsp. citri (Xac) causes the citrus canker disease. The disease reaches all commercial varieties of citrus and, the understanding of the phytopathogenic relationship allow knowing the ways of control of bacteria. With sequencing of the Xac genome and the realization of a transcriptome, ORFs have been identified that are probably involved in bacterial attack and colonization processes. To initiate the characterization of one of these ORFs, in this study, the strategies of site-directed mutagenesis by overlap extension PCR and molecular modeling were used. The ORF XAC0239 is predicted as plasma membrane protein, and by homology may possibly be part of a zinc transporter protein, moreover, there are indications that it is regulated by a sigma factor. The mutation had a negative effect at the cellulases action and swimming motility and a positive effect on biofilm formation and in vivo multiplication. Here it is suggested that in the absence of this protein, there was little absorption of zinc, necessary for biofilm formation and consequently for pathogenicity. In addition, it is suggested that, as a subterfuge, the bacteria overexpressed genes for production and / or cellulases action, trying to attack the host on alternative way.
Mestre
Deane, David Leslie. "The characterisation of a GM-CSF and IL-2 inhibitory protein encoded by Orf virus." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23323.
Melling, Michael [Verfasser], and Thomas [Akademischer Betreuer] Dobner. "The influence of SUMOylation on the adenoviral early region 4 protein Orf6/7 / Michael Melling ; Betreuer: Thomas Dobner." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1163013692/34.
Craft, Jennifer Leigh. "Generating an expression construct and soluble protein for characterization studies of a putative RNA m5C methyltransferase, yeast ORF YNLO22c." Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319220.
Department of Biology
Scholz, Kai. "Immunmodulation durch Parapocken-Viren: Identifikation und Analyse funktionaler Viruskomponenten." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2003. http://nbn-resolving.de/urn:nbn:de:swb:14-1061969873968-07930.
Key, Kijona Farthing. "Molecular characterization of the major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) and evaluation of its use for a diagnostic assay, vaccine development, and the examination of quasispecies evolution." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27282.
Ph. D.
Chi, Xiuling. "BIOSYNTHETIC PATHWAY OF THE AMINORIBOSYL COMPONENT OF LIPOPEPTIDYL NUCLEOSIDE ANTIBIOTICS." UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/23.
Huang, Yu-Liang, and 黃有良. "Immuno-protection of porcine circovirus type 2 ORF1 and ORF2 recombinant protein." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/12062489232254298987.
國立屏東科技大學
獸醫學系
92
Porcine circovirus type 2 (PCV2) has been reported to be associated with the development of porcine multisystemic wasting syndrome (PMWS), which often results in the death of pigs due to secondary infection. The genome of PCV2 might contain eleven open reading frames (ORFs). ORF 1 and ORF 2 encode the replication-associated proteins and capsid protein, respectively. In this study, the immunogenicity of E. coli-expressed PCV2 ORF 1 and PCV2 ORF 2 and baculovirus-expressed PCV2 ORF 2 were compared in mice and pigs. In addition, ORF 2 gene with Pseudomonas aeruginosa exotoxin A (expressed as PE407 and PE532) gene added at 5’ terminal, and/or the KDEL amino acids gene (expressed as K) added at 3’ terminal were constructed and expressed in E. coli. A total of seven recombinant proteins including PCV2 ORF1 (21 KDa), PCV2 ORF 2 (54 KDa), PCV2 ORF2K (56 KDa), PE407-ORF2 (81 KDa), PE407-ORF2K (100 KDa), PE532-ORF2 (95 KDa) and PE532-ORF2K (97 KDa) were successfully expressed in E. coli. A recombinant protein of 34 KDa (Bac-PCV2ORF2) was also expressed in baculovirus. Antibodies against PCV2 could be detected when pigs or mice were immunized with PCV2ORF1, PCV2ORF2, PE407-ORF2K and Bac-PCV2ORF2. Among the recombinant proteins tested, Bac-PCV2ORF2 induces the best antibody titer in animals. The aforementioned recombinant proteins can be used as the components for the development of PCV2 subunit vaccine in the future.
Chen, Yu-San, and 陳裕森. "Cloning, Expression, and Antigenesity Analysis of the PCV2 Coat Protein Gene (ORF2)." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/99558729710906776501.
國立宜蘭大學
生物技術研究所碩士班
94
Porcine circovirus type 2 belongs to the Circoviridae family. PCV2 is a small, nonenveloped virus with a single-stranded circular DNA genome of about 1.76 kb. The open reading frame ORF2 of the PCV2 genome encodes the immunogenic structural capsid protein. The expression for this immunogenic protein using full length ORF2 DNA was difficult to obtain in large quantity in prokaryotic expression system. Therefore, an alternative approach was to divide ORF2 DNA into three segments for expression, F1, F2, and F3. It was discovered that expression in E.coli was obtained in segments other than F1, which contained the N-terminal sequences. When F1+F2 (named F6) and F2+F3 (named F5) fragments were cloned and expressed, F6, similar to F1, was not expressed due to the N-terminal sequences. On the other hand, F5 was still expressed in large quantity. When dividing the full length ORF2 into half, F4 with C-terminal sequences was again expressed in large quantity as predicted. Analysis on PCV2 ORF2 full length sequence showed that within F1, there were 21 arginine codons, far more than the 9 arginine codons in F2 and F3 combined. Therefore, five additional constructs with the first 9, 21, 36, 48, 111 nucleotides missing of ORF2 (named F10, F22, F37, F49, and F112 respectively) were cloned and subsequently expressed. It was discovered that PCV2 ORF2 recombinant protein expression increased as arginine codons decreased. When immunizing rat with purified F5 recombinant protein, high antibody titer against PCV2 was induced, as confirmed by the IPMA method in PCV2 infected PK-15 cells. Currently, since PCV2 vaccine is not commercially available, this research project was also aimed for the development of a PCV2 subunit vaccine. Rats were used as an experimental animal model to evaluate the various recombinant proteins, F2, F3, F5, and F49, for their abilities to induce antibodies against PCV2. In addition, F5 and F49 recombinant proteins, in conjunction with ORF1 and/or ORF2 recombinant eukaryotic expression plasmids, were injected in rats to evaluate their abilities to induce antibodies against PCV2. Other than using IPMA for the detection of neutralizing antibodies, I used ELISA to measure the levels of antibodies induced in the collected rat serum of different experimental groups. The result of ELISA indicated that F2 recombinant protein induced the highest level of IgG antibodies in rat. As for neutralizing antibodies, neither the recombinant proteins nor when used in conjunction with recombinant eukaryotic expression plasmids were induced when using rat as an animal model.
"Analysis Of The Line-1 Orf2 Protein Using An Evolutionarily-informed Genetic Approach." Tulane University Digital Library, 2016.
1
Claiborne Magnant Christian
Chowdhury, Soumya Roy. "Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV)." Thesis, 2010. https://etd.iisc.ac.in/handle/2005/1465.
Chowdhury, Soumya Roy. "Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV)." Thesis, 2010. http://hdl.handle.net/2005/1465.
Tseng, Yeu-Yang, and 曾宇揚. "Functional analysis of orf virus OV20.0 protein isoforms." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/19898262310985605669.
國立中興大學
微生物暨公共衛生學研究所
103
Orf virus (ORFV) infection causes pustule and ulceration around muzzle of small ruminants. Although it often occurs with low mobility and mortality, orf may be fatal in juvenile hosts. ORFV, belonging to genus parapoxvirus of the family Poxviridae, is an enveloped virus with double-stranded DNA genome. OV20.0 protein is produced from OV20.0L gene, an E3L ortholog, which is conserved in the genome of most members in Poxviridae. Vaccinia E3 (VV E3) has been studied extensively in these days; however sequences of VV E3 shares low identity with those of OV20.0. According to previous publication, OV20.0L could be translated into to two isoforms, full-length OV20.0 and N-terminal truncated one (sh20). Due to limited information of OV20.0 protein, our research focused on the translation mechanism of the isoform, comparative analysis of their cellular distribution and realizing their functions as well as their contribution to pathogenicity in mice model. First, we proved sh20 was translated from the third start codon of OV20.0L gene. In in vitro and in virus infection condition, both ORFV 20.0 and sh20 can hold the ability to bind double-stranded RNA (dsRNA), sequester the substrate of dsRNA-dependent protein kinase (PKR) that in turns inhibits the activity of downstream factor, eukaryotic initiation factor 2 (eIF2α), and influences cytokine releasing in different levels. Moreover, constructing recombinant virus and animal experiments clarify the role ORFV OV20.0 played in vivo. Although full-length OV20.0 and sh20 shared most functions in vitro, the ORFV recombinant virus which only expressed sh20, was attenuated in vivo. This data implied N-terminus of OV20.0 was required to induce intact pathogenicity in live animals.
Cilloniz, Cristian. "Role of the varicella zoster virus ORF9 protein in viral replication." 2007. http://proquest.umi.com/pqdweb?did=1331418241&sid=10&Fmt=2&clientId=39334&RQT=309&VName=PQD.
Title from PDF title page (viewed on Nov. 27, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Ruyechan, William T. Includes bibliographical references.
Huang, Yu-Yuan, and 黃俞淵. "Characterization of SARS-CoV encoded accessory proteins ORF8a and ORF8." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/74372663873642985655.
國立陽明大學
生物藥學研究所
97
SARS-CoV, a coronavirus, was identified as the etiologic agent for severe acute respiratory syndrome. It is established that human SARS-CoV originated from animals, such as palm civets and raccoon dogs. SARS-CoV genome shares about 99.8% homology with its animal counterpart. The major difference of the two viruses is the 29 nucleotide deletion found in the corresponding open reading frame (ORF) 8 of human SARS-CoV, given rise to ORF8a and ORF8b. Our previous data had shown that ORF8a localizes to mitochondria. Using yeast two-hybrid screening, ORF8a, but not ORF8 and ORF8b, was shown to interact with calcium-modulating cyclophilin ligand (CAML), a protein had been known to regulate the intracellular Ca2+ concentration. Using Hela cell Tet/on system, ORF8a can promote the apoptosis initiated by staurosporine. The main goal of this dissertation is to determine the biological functions of ORF8 and ORF8a that might give better understandings to the pathogenesis of both viruses. With immunofluorescence assay and subcellular fractionation, ORF8a and ORF8 are shown to be located in mitochondria and ER, separately, in the presence of endogenous CAML. However, when we ectopically expressed CAML, both ORF 8a and ORF8 co-localized with exogenous CAML and displayed a similar pattern of distribution, which differed from that in the presence of endogenous CAML. ORF8a was shown to have higher binding capacity for CAML than that of ORF8 in an immunoprecipitation assay, which likely contributed to the redistribution of ORF8a. In light of reports indicating that in SARS patient lung cells showed readily apoptosis than that of enterocytes, although both tissues are permissive for viral infection and growth. We proceeded to evaluate the apoptotic effect of either ORF8a or ORR8 on an alveolar epithelium cell, A549 and an enterocyte, Caco-2. A549 was showed to have DNA fragementation in the presence of ORF8a or ORF8 by propidium iodide staining, while both proteins also facilitated staurosporine-induced apoptosis using TMRM staining assay that targeting at changes in mitochondrial membrane potential. A549 was also showed low grade of apoptosis in the DMSO control group in the presence of ORF8a or ORF8, which coincided with the observed induction of DNA fragmentation by both proteins. Interestingly, Caco-2 with ectopically expressed ORF8a or ORF8 failed to exhibit apoptotic phenotype enhancement regardless of the presence or absence of staurosporine. Currently, the contribution of interaction between CAML and ORF8a or even ORF8 in apoptosis remains unclear. In the future, we will re-examine the above stated observations in both A549 and Caco-2 cells with CAML knockdown for the evaluation of the contribution of the interactions between CAML and ORF8a or CAML and ORF8.
Li, ChiWei, and 李誌偉. "A novel mechanism to regulate IS1550 putative transposase production by ORF1 protein." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/22403527173198471760.
國立陽明大學
醫學生物技術研究所
90
We have previously identified a transposable element, IS1550 from Mycoplasma fermentans. This element encodes two open reading frames, ORF1 and ORF2. Two proteins were detected with the molecular weight of 16 kDa and 50 kDa, respectively, when expression of the IS1550 genes was examined in E. coli T7 expression system. The 16-kDa protein was determined to be encoded by ORF1, whereas the 50-kDa protein was produced by forming a fusion of ORF1 and ORF2, ORF12’, through an efficient —1 translational frameshift mechanism. Our previous studies have confirmed a framseshift signal in IS1550. It contains an essential shifty site AAAAAAG (A6G) which is located near the 3’ end of ORF1, and an enhancing element, the internal inverted repeat (IIR) to form a RNA stem-loop structure, which is located 5 bp downstream of the essential shifty site. In this study, we examined the effect of ORF1 protein on the -1 translational frameshift product by using our previously established dual-reporter gene (lacZ and luc) system. The results showed that ORF1 protein could repress the -1 frameshift product both in vivo and in vitro with an inhibitory effect of about 60 % and 80 %, respectively. We also showed the ORF1 protein could bind to the frameshift signal DNA region. Furthermore, we proved that a truncated mRNA was formed when the ORF1 protein was present. Thus, we proposed that the repression of ORF1 protein on the -1 frameshift product was possibly caused by ORF1 protein binding to the frameshift signal DNA region, blocking the RNA polymerase proceeding, and resulting in the decreasing of the fusion protein (a putative transposase) production.
Wu, Ting-Yun, and 吳亭昀. "Monoclonal Antibody Preparation and Cellular Distribution Analysis of Koi Herpesvirus ORF72 Protein." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/8a7b5r.
Ou, Chi-Ming, and 歐啟明. "Expression and Purification of the Major Envelope Protein of Orf Virus." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/50383988977441905067.
中興大學
獸醫學系暨研究所
95
Abstract Orf is also known as contagious ecthyma, contagiou pustular dermatitis, Scabby mouth and sore mouth. Orf occurs worldwide in sheep and goats, especially in young animal. The disease is characterized by proliferative lesion on the lips, around the nostrils and in the oral mucosa. Orf virus is a member of the Parapoxvirus genus of Poxviridae. The virus particles are ovoid and the viral genome is a linear double-stranded DNA. The viral B2L gene encodes for the highly immunogenic major envelope protein , which is the homologue of vaccinia virus envelope protein antigen p37 . We amplified the B2L gene by polymerase chain reaction (PCR) and cloned the gene into the expression vector pET24a. The plasmid was transformed into the E. coli. strain BL21(DE3) and the expression of recombinant B2L protein was induced by IPTG. The recombinant B2L protein was identified with western blotting and with mass spectrometry analysis. Then, the B2L protein was purified by affinity chromatography. Moreover, we made four truncated constructs to express shorter B2L proteins; although two of these deletion mutants were also expressed, they showed lower level of production. The vaccinia virus envelope protein p37 has phospholipase activity; therefore it has been proposed that the B2L protein has phospholipase function. However, we were not able to detect the phospholipase activity of our purified recombinant protein.
LEE, Tzu-Ying, and 李姿穎. "Molecular Mechanism of the type I interferon antagonist function by SARS Coronavirus ORF6 protein." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/75653793395898183769.
中國醫藥大學
醫學檢驗生物技術學系
96
Abstract Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV).SARS-CoV proteins including the ORF6 protein have been reported to inhibit interferon signaling response. The goal of this study is to identify human SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries and to investigate their interaction on the type I interferon antagonist function. At first, the recombinant ORF6 protein was generated in BL21(DE3) and purified by using IMAC (immobilized mental-affinity chromatography). After five rounds of biopanning, PI-3 kinase-related kinase SMG-1 protein was identified as SARS-CoV ORF6 interacting protein from phage displayed lung cDNA library. Confocal imaging revealed co-localization of SARS-CoV ORF6 protein with SMG-1 protein in HL-CZ cells. In vivo signaling pathway assay and real time RT-PCR showed that single gene expression of SARS-CoV ORF6-expressing cells blocked the INFα/β-induced responses, such as ISRE-responsive firefly luciferase activity and the expression of PKR. However, both gene expression of SARS-CoV ORF6 and SMG-1 had a significantly higher relative activities of INFα/β-induced ISRE-responsive firefly luciferase than single gene expression of SARS-CoV ORF6 in HL-CZ cells. In addition, confocal imaging and Western blotting assays revealed that the translocation of STAT-1 into nucleus and the STAT-1 phophorylation were found in the transfected cells expressing both genes of ORF6 and SMG-1, but not in the ORF 6-expressing cells in response to INFα/β. Therefore, cell expression of PI-3 kinase-related kinase SMG-1 could restore the INFα /β responses in SARS-CoV ORF6 expressing cells. The interaction of SARS CoV ORF6 and SMG-1 may be responsible for the inhibition of type I IFN response by SARS-CoV.
Chao, I.-Chi, and 趙譯棋. "Establishment of Recombinant Orf Virus Expressing Goatpox virus P32 Proteins." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/30630163851365322611.
國立中興大學
微生物暨公共衛生學研究所
103
Orf virus (ORFV), belonging to parapoxvirus, causes skin lesions in infected sheep and goats. Due to the restricted host range, the skin tropism, the absence of systemic virus spread, and the short-lived ORFV vector-specific immunity allowing repeated immunizations, it has been successfully used as a novel viral vector system for expressing foreign antigens to prevent infectious diseases of swine, rabbit, and avian. Hence, study herein aimed to establish ORFV as a bivalent vaccine platform for goat. In 2008, goatpox (GP) outbreaks caused substantial loss in the production and productivity of goats in Taiwan. Considering that control of goatpox virus (GPV) infection relies on foreign vaccine, the goal of current study is to construct a recombinant ORFV that would express goatpox virus P32, the major immunogenic protein. To do so, coding regions of GPV P32 and eGFP that serves as a selection marker were inserted between the flanking sequences, i.e. open reading frame 127 and 128 of the ORFV genome. By homologous recombination, recombinant ORFV with GPV p32 gene (GPV P32-ORFV) was generated. At the present, the presence and expression of GPV P32 gene were confirmed by polymerase chain reaction and western blot analysis, respectively. The immune characteristics of GPV P32-ORFV will be further tested in animal models.
Chorghade, Sandip Gulab. "Studies on Interactions between ARE Binding Proteins and Splicing Factors and their Role in Altered Splicing of PDGF-B ORF." Thesis, 2012. http://etd.iisc.ac.in/handle/2005/3227.
Chorghade, Sandip Gulab. "Studies on Interactions between ARE Binding Proteins and Splicing Factors and their Role in Altered Splicing of PDGF-B ORF." Thesis, 2012. http://hdl.handle.net/2005/3227.
Lin, Wei Li, and 林維莉. "Expression and functional analysis of the porcine circovirus type 2 Rep and ORF3 proteins." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/04865829915580224077.
國立中興大學
微生物暨公共衛生學研究所
100
Porcine circovirus type 2 (PCV2) is a member belongs to the genus Circovirus of the Circoviridae family, and closely associated with a disease syndrome in pigs described as “postweaning multisystemic wasting syndrome” (PMWS) now known as porcine circovirus diseases (PCVD). The genome of PCV2 is a single-stranded circular DNA, and contains three major open reading frames (ORFs). They are ORF1, the rep gene, which encodes the Rep proteins responsible for virus replication; and ORF2, the cap gene, which encodes the immunogenic capsid (Cap) protein; and ORF3 encodes the protein for induce cell apoptosis. Synthesis of Rep protein as the first stage after PCV2 infection, is essential to viral DNA replication. The non-pathogenic type 1 PCV (PCV1) Rep protein has been shown to process the nuclear localization signal (NLS) for protein transport into nucleus and has DNA binding activity for viral DNA replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the PCV2 Rep protein, the defined coding regions of rep gene were cloned, expressed and analyzed the DNA binding activity. The results demonstrated that the full-length Rep (N314) and the deletion mutants coding the N-terminal 110 amino acid residues had the ability to bind to the 22-mer dsDNA fragment of the PCV2 origin sequence minimal binding site (ori MBS) sequence. Furthermore, to determine the region responsible for nuclear localization, several eukaryotic expression plasmids containing various coding regions of Rep protein identical to those expressed in E. coli were constructed. At 48 h post-transfection of plasmids into PK15 cells, cell were fixed, analyzed by indirect immunofluorescence assay (IFA) using mouse immune serum against PCV2 Rep, and observed by florescent microscopy. The full-length Rep protein (N314) and recombinant deletion mutants with 110 amino acid at N-terminal of Rep protein were dominantly localized in the nucleus. Examination of the 110 amino acid (aa) residues of the N-terminal region of PCV2 Rep revealed three clusters of basic aa with homology to other NLS. These results suggest that the utmost 20 residues in the N-terminal region could sufficiently mediate nuclear import of fusion protein, confirming its role as a functional NLS. Recent report showed that PCV2 induced apoptosis in the cultured porcine kidney cell (PK-15) via a viral protein encoded by ORF3. Monocyte/macrophage lineage cells are the major target cells for PCV2 infection. The purpose of this study was to expressed PCV2 ORF3 protein and further analyzing the role of ORF3 in inducing apoptosis in porcine peripheral blood mononuclear cells (PBMC). The full length of PCV2 ORF3 gene DNA fragment was amplification by PCR and suclone onto pET24 plasmid for preparation of the mouse antiserum against recombinant ORF3 protein. To analyze whether ORF3 is able to induce apoptosis, transient expression of the full length and the N- or C-terminal half of ORF3 protein in porcine PBMC was performed. After transfection, the transfected cells were detected by IFA and apoptosis induced by transient expression of viral protein was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling (TUNEL-labeling) which detected the DNA breakage and caspase activity. Quantification of TUNEL-positive cells showed that the percentage of apoptotic cells transfected with pcDNA/ORF3 was significantly higher the C-terminal half of ORF3 retained its ability to induce apoptosis similar to the full length of ORF3. Caspase activity assays demonstrated significant activation of caspase-3, -8, and -9 by the full length and the C-terminal half of ORF3 protein. The results suggest that nuclear localization may be required for apoptosis induction and the C-terminal half region of ORF3 contains either N53-68 or N85-104 regions responsible for nuclear localization. In summary, the functional regions of PCV2 Rep protein responsible for DNA binding activity and nuclear localization appear to be the N-terminal 110 residue portion of the protein. Another nonstructural protein of PCV2, ORF3, is capable of triggering apoptotic response in porcine PBMC maybe associated with the NLS. The apoptotic activity is correlated with the nuclear localization of ORF3, and nuclear localization may play an important role of PCV2 viral protein function and virus life cycle.
Starck, Holger [Verfasser]. "Das RNA-bindende Protein Orb2 und seine regulatorische Funktion in der Spermatogenese von Drosophila melanogaster / vorgelegt von Holger Starck." 2008. http://d-nb.info/988337436/34.
Tanner, Tricia Lynn. "Analysis of the function of two varicella-zoster virus proteins involved in gene regulation IE63 and ORF29 /." 2007. http://proquest.umi.com/pqdweb?did=1240709031&sid=6&Fmt=2&clientId=39334&RQT=309&VName=PQD.
Title from PDF title page (viewed on July 18, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Ruyechan, William T. Includes bibliographical references.
Dan, Tran Quang, and 陳光瑩. "Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/xwceag.
中國文化大學
生物科技研究所
102
Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to be involved in the pathogenesis of PCV2 infection by its apoptotic activity. The ORF3–based antigens thus represent a traditional approach to develop efficacious oral vaccines for immunization against PCV2. In this study, various ORF3-containing expression vectors, namely pGKF/U-ORF3, pGKF/U-L-ORF3, pGKF/U-ORF2-3b, and pGKF/U-L-ORF2-3b, were transformed to tobacco plants in order to produce the respective subunit recombinant vaccines. Following the Agrobacterium tumefaciens-mediated transformation of 200 leaf samples, 20 ORF3, 15 L-ORF3, 14 ORF2-3b, and 14 L-ORF2-3b putative transgenic tobacco plants were regenerated on the selective medium supplied with 200 mg/L kanamycin and showed GUS expression. The expected sequences of respective ORF3-containing trangenes, 359 bp, 369 bp, 1056 bp, and 1439 bp were amplified from genomic DNA of 19 ORF3 (9.5%), 14 L-ORF3 (7.0%), 12 ORF2-3b (6.0%), and 12 L-ORF2-3b (6.0%) GUS-positive transgenic plants, using specific pair of primers in PCR analysis. On the other hand, integration of 1-2 copies of independent ORF3-containing transgenes into tobacco genome was determined by means of Southern hybridization. The mRNA transcripts of particular transgenes were also found in the PCR-positive transgenic plants. Particularly, expression of respective ORF3 fusion proteins was qualitatively examined via immunobloting (Western blot) assay. However, the results have not clearly shown presence of target proteins, due to either low level of expression or interfering background.
Lee, Chia-Chun, and 李佳俊. "Expression of modified ORF 5 protein of PRRSV in the baculovirus system and cell surface display system." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/pefp2t.
Fabrizio, Jacqueline Alba. "Characterising Novel Substrates of the Asparaginyl Hydroxylase FIH." Thesis, 2017. https://hdl.handle.net/2440/132758.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018