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Donald L Jarvis - One of the best experts on this subject based on the ideXlab platform.
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Baculovirus insect cell expression systems
Methods in Enzymology, 2009Co-Authors: Donald L JarvisAbstract:In the early 1980s, the first-published reports of Baculovirus-mediated foreign gene expression stimulated great interest in the use of Baculovirus-insect cell systems for recombinant protein production. Initially, this system appeared to be the first that would be able to provide the high production levels associated with bacterial systems and the eukaryotic protein processing capabilities associated with mammalian systems. Experience and an increased understanding of basic insect cell biology have shown that these early expectations were not completely realistic. Nevertheless, Baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. Furthermore, important technological advances over the past 20 years have improved upon the original methods developed for the isolation of Baculovirus expression vectors, which were inefficient, required at least some specialized expertise and, therefore, induced some frustration among those who used the original Baculovirus-insect cell expression system. Today, virtually any investigator with basic molecular biology training can relatively quickly and efficiently isolate a recombinant Baculovirus vector and use it to produce their favorite protein in an insect cell culture. This chapter will begin with background information on the basic Baculovirus-insect cell expression system and will then focus on recent developments that have greatly facilitated the ability of an average investigator to take advantage of its attributes.
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protein n glycosylation in the Baculovirus insect cell expression system and engineering of insect cells to produce mammalianized recombinant glycoproteins
Advances in Virus Research, 2006Co-Authors: Robert L. Harrison, Donald L JarvisAbstract:Abstract Baculovirus expression vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value. The insect cells that serve as hosts for Baculovirus vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N ‐glycosylation in mammalian cells. However, while mammalian cells produce compositionally more complex N ‐glycans containing terminal sialic acids, insect cells mostly produce simpler N ‐glycans with terminal mannose residues. This structural difference between insect and mammalian N ‐glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans. These features obviously compromise the biomedical value of recombinant glycoproteins produced in the Baculovirus expression vector system. Thus, much effort has been expended to characterize the potential and limits of N ‐glycosylation in insect cell systems. Discoveries from this research have led to the engineering of insect N ‐glycosylation pathways for assembly of mammalian‐style glycans on Baculovirus‐expressed glycoproteins. This chapter summarizes our knowledge of insect N ‐glycosylation pathways and describes efforts to engineer Baculovirus vectors and insect cell lines to overcome the limits of insect cell glycosylation. In addition, we consider other possible strategies for improving glycosylation in insect cells.
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Baculovirus as versatile vectors for protein expression in insect and mammalian cells
Nature Biotechnology, 2005Co-Authors: Thomas A Kost, Patrick J Condreay, Donald L JarvisAbstract:Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in Baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, Baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of Baculovirus technology.
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mammalian glycosyltransferase expression allows sialoglycoprotein production by Baculovirus infected insect cells
Protein Expression and Purification, 2001Co-Authors: Neungseon Seo, Jason R Hollister, Donald L JarvisAbstract:Abstract The Baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N -glycoprotein sialylation by the Baculovirus-insect cell system. This was accomplished by using a novel Baculovirus vector designed to express a mammalian α2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian β1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the Baculovirus-insect cell expression system can be metabolically engineered for N -glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.
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novel Baculovirus expression vectors that provide sialylation of recombinant glycoproteins in lepidopteran insect cells
Journal of Virology, 2001Co-Authors: Donald L Jarvis, Dale Howe, Jared J AumillerAbstract:: This report describes novel Baculovirus vectors designed to express mammalian beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes at early times after infection. Sf9 cells infected with these viral vectors, unlike cells infected with a wild-type Baculovirus, produced a sialylated viral glycoprotein during the late phase of infection. Thus, the two mammalian glycosyltransferases encoded by these viral vectors are necessary and sufficient for sialylation of a foreign glycoprotein in insect cells under the conditions used in this study. While some of the new Baculovirus vectors described in this study produced less, one produced wild-type levels of infectious budded virus progeny.
Oliveira, Virgínia Carla - One of the best experts on this subject based on the ideXlab platform.
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A ação de proteínas virais supressoras de silenciamento gênico na patogenicidade de um Baculovírus
2011Co-Authors: Oliveira, Virgínia CarlaAbstract:O silenciamento gênico, ou RNA de interferência (RNAi), atua como um mecanismo de defesa contra infecções virais em organismos eucarióticos. Ao longo da evolução, os vírus adquiriram proteínas específicas com a capacidade de suprimir o silenciamento de RNA em diferentes pontos do processo. Como por exemplo, as proteínas AC2 de begomovírus, NS1 do vírus da gripe (Influenza A) e NSs de um tospovírus (Tomato spotted wilt virus - TSWV). Neste estudo, pretendeu-se induzir mecanismos de supressão de silenciamento gênico através da expressão heteróloga de AC2, NS1 e NSs por baculovírus AcMNPV – Autographa californica multiple nucleopolyhedrovirus, a fim de analisar o efeito na replicação viral em diferentes células de inseto. Assim, no primeiro capítulo, foi feita uma revisão sobre o silenciamento gênico, a ação de proteínas virais supressoras de RNAi e o uso potencial de baculovírus recombinantes como agentes bioinseticidas melhorados. O segundo capítulo descreve a clonagem dos genes AC2, NS1 e NSs, a construção dos baculovírus recombinantes (vAcAC2, vAcNS1 e vAcNSs) e os ensaios preliminares de infecção em célula e em larvas de inseto. Nesses ensaios, o vAcNSs (contendo o gene NSs) foi o recombinante com maior influência na replicação do baculovírus selvagem AcMNPV. Por sua vez, o terceiro capítulo relata a ação da proteína supressora de silenciamento NSs de TSWV, expressa pelo baculovírus vAcNSs, em três linhagens hospedeiras: uma permissiva, derivada de Trichoplusia ni (BTI-Tn-5B1-4); outra semipermissiva, derivada de Anticarsia gemmatalis (UFL-AG-286); e, uma linhagem não-permissiva, derivada de Bombyx mori (BM-5). Os resultados mostraram que vAcNSs, em células semipermissivas, obteve maior eficiência na replicação quando comparado ao selvagem, pois produziu mais vírus extracelulares; em uma linhagem celular não-permissiva, causou efeito citopático, enquanto a infecção com o tipo selvagem AcMNPV não provocou nenhuma alteração morfológica; aumentou a produção de poliedros do baculovírus tipo selvagem em todas as linhagens de células testadas; aumentou fortemente a expressão da proteína fluorescente verde (EGFP) nas células semipermissivas e em hemócitos de A. gemmatalis quando co-infectado com um AcMNPV recombinante contendo o gene egfp. A análise de microscopia confocal revelou que a NSs acumulou em abundância no citoplasma de células permissivas e semipermissivas. Em contraste, a NSs foi detectada no núcleo da célula não-permissiva. A ausência de moléculas curtas de RNA (siRNA) de transcritos de egfp em linhagens semipermissivas e permissivas indica atividade de supressão do silenciamento gênico. Por outro lado, vAcNSs não suprimiu o RNAi na linhagem celular nãopermissiva. Por fim, o quarto capítulo investiga a ação bioinseticida do baculovírus vAcNSs em larvas de Spodoptera frugiperda e Anticarsia gemmatalis - um hospedeiro permissivo e outro semi-permissivo, respectivamente. Não houve diferença estatisticamente significativa entre a DL50 do vAcNSs para S. frugiperda e A. gemmatalis, quando comparada à DL50 observada para o tipo selvagem AcMNPV. Entretanto, o TL50 foi significativamente diferente, com valores menores para vAcNSs em S. frugiperda [5,62 dias com 1 p.f.u. e 4,82 dias com 105 p.f.u.] e em A. gemmatalis [7,46 dias com 1 p.f.u. e 3,2 dias com 105 p.f.u.] quando em comparação com o TL50 do AcMNPV em S. frugiperda [8,5 dias com 1 p.f.u. e 7,52 dias com 105 p.f.u.] e em A. gemmatalis [20,11 dias com 1 p.f.u. e 7,34 dias com 105 p.f.u.]. Estes resultados corroboram os dados observados in vitro, indicando que a proteína NSs de TSWV aumenta a replicação do baculovírus e pode contribuir para gerar bioinseticidas mais eficientes. ______________________________________________________________________________ ABSTRACTGene silencing, or RNA interference (RNAi), works as a eukaryotic defense mechanism against viral infections. During evolutionary time viruses acquired specific proteins, which are able to halt the silencing process in different steps of its biochemical pathway e.g. the AC2 protein from Begomovirus, the NS1 protein from influenza (Influenza A) and the NSs protein from Tomato spotted wilt virus (TSWV). In this study we sought generation and evaluation of the gene-silencing suppression effect of three different genes, AC2 from begomovirus, NS1 from Influenza A and NSs from TSWV, via heterologous expression by recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV, genus AlphaBaculovirus, family Baculoviridae) in different insect cell lines. In the first chapter, a review concerning gene silencing, suppression of gene silencing by viral proteins and the potential use of recombinant Baculovirus as bio-insecticidal agents suitable for biological control is presented. The second chapter describes the cloning strategies designed for the construction of recombinant Baculoviruses carrying the AC2, NS1 and NSs genes (vAcAC2, vAcNS1 and vAcNSs, respectively) as well as the results of preliminary insect cell infection assays. In these preliminary assays, vAcNSs had more influence on wild type AcMNPV replication than vAcAC2 and vAcNS1. The third chapter shows the effect of the protein NSs from TSWV on virus replication in different host insect cell lines: one permissive, Trichoplusia ni (BTI-Tn-5B1-4); other semi-permissive, Anticarsia gemmatalis (UFL-AG-286); and a non-permissive cell line, Bombyx mori (BM-5). Results showed that infection of the semi-permissive cell line by vAcNSs was more efficient than infection by wild type AcMNPV, since production of budded virus was higher. In the non-permissive cell line, vAcNSs was able to produce cytopathic effects whereas no morphological alteration was found when wild type AcMNPV was inoculated. When vAcNSs and wild type AcMNPV were co-inoculated, production of polyhedra was enhanced despite the insect cell line used. Co-infection of vAcNSs and a recombinant AcMNPV carrying the egfp gene was also evaluated. In the semi-permissive cell line and in A. gemmatalis hemocytes (permissive cell line) co-infection greatly increased enhanced green fluorescent protein (EGFP) expression. Northern blotting assays showed absence of small interfering RNA (siRNA) molecules associated to egfp transcripts in the permissive and semi-permissive cell lines, which indicates suppression of gene silencing activity by the NSs protein. On the other hand, vAcNSs was not able to suppress the siRNA production in the non-permissive cell line. Confocal microscopy analysis showed that the NSs protein accumulated abundantly in the cytoplasm of permissive and semipermissive infected cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Finally, chapter four presents the study of the bio-insecticidal activity of vAcNSs on Spodoptera frugiperda and Anticarsia gemmatalis larvae, a permissive and semipermissive host, respectively. The vAcNSs LD50 for S. frugiperda and A. gemmatalis was not statistically different from wild-type AcMNPV. However, the LT50 values were significantly smaller for vAcNSs in S. frugiperda [5.62 days with 1 p.f.u. and 4.82 days with 105 p.f.u.] and A. gemmatalis [7.46 days with 1 p.f.u. and 3.20 days with 105 p.f.u.) when compared to the LT50 for AcMNPV in S. frugiperda [8.5 days with 1 p.f.u. and 7.52 days with 105 p.f.u.] and A. gemmatalis [20.11 days with 1 p.f.u. and 7.34 days with 105 p.f.u.]. These in vivo results are in accordance with the data observed in vitro indicating that the protein NSs from TSWV could efficiently improve Baculovirus replication and be used to generate more effective bioinsecticides
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A ação de proteínas virais supressoras de silenciamento gênico na patogenicidade de um Baculovírus
2010Co-Authors: Oliveira, Virgínia CarlaAbstract:Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, 2010.O silenciamento gênico, ou RNA de interferência (RNAi), atua como um mecanismo de defesa contra infecções virais em organismos eucarióticos. Ao longo da evolução, os vírus adquiriram proteínas específicas com a capacidade de suprimir o silenciamento de RNA em diferentes pontos do processo. Como por exemplo, as proteínas AC2 de begomovírus, NS1 do vírus da gripe (Influenza A) e NSs de um tospovírus (Tomato spotted wilt virus - TSWV). Neste estudo, pretendeu-se induzir mecanismos de supressão de silenciamento gênico através da expressão heteróloga de AC2, NS1 e NSs por baculovírus AcMNPV – Autographa californica multiple nucleopolyhedrovirus, a fim de analisar o efeito na replicação viral em diferentes células de inseto. Assim, no primeiro capítulo, foi feita uma revisão sobre o silenciamento gênico, a ação de proteínas virais supressoras de RNAi e o uso potencial de baculovírus recombinantes como agentes bioinseticidas melhorados. O segundo capítulo descreve a clonagem dos genes AC2, NS1 e NSs, a construção dos baculovírus recombinantes (vAcAC2, vAcNS1 e vAcNSs) e os ensaios preliminares de infecção em célula e em larvas de inseto. Nesses ensaios, o vAcNSs (contendo o gene NSs) foi o recombinante com maior influência na replicação do baculovírus selvagem AcMNPV. Por sua vez, o terceiro capítulo relata a ação da proteína supressora de silenciamento NSs de TSWV, expressa pelo baculovírus vAcNSs, em três linhagens hospedeiras: uma permissiva, derivada de Trichoplusia ni (BTI-Tn-5B1-4); outra semipermissiva, derivada de Anticarsia gemmatalis (UFL-AG-286); e, uma linhagem não-permissiva, derivada de Bombyx mori (BM-5). Os resultados mostraram que vAcNSs, em células semipermissivas, obteve maior eficiência na replicação quando comparado ao selvagem, pois produziu mais vírus extracelulares; em uma linhagem celular não-permissiva, causou efeito citopático, enquanto a infecção com o tipo selvagem AcMNPV não provocou nenhuma alteração morfológica; aumentou a produção de poliedros do baculovírus tipo selvagem em todas as linhagens de células testadas; aumentou fortemente a expressão da proteína fluorescente verde (EGFP) nas células semipermissivas e em hemócitos de A. gemmatalis quando co-infectado com um AcMNPV recombinante contendo o gene egfp. A análise de microscopia confocal revelou que a NSs acumulou em abundância no citoplasma de células permissivas e semipermissivas. Em contraste, a NSs foi detectada no núcleo da célula não-permissiva. A ausência de moléculas curtas de RNA (siRNA) de transcritos de egfp em linhagens semipermissivas e permissivas indica atividade de supressão do silenciamento gênico. Por outro lado, vAcNSs não suprimiu o RNAi na linhagem celular nãopermissiva. Por fim, o quarto capítulo investiga a ação bioinseticida do baculovírus vAcNSs em larvas de Spodoptera frugiperda e Anticarsia gemmatalis - um hospedeiro permissivo e outro semi-permissivo, respectivamente. Não houve diferença estatisticamente significativa entre a DL50 do vAcNSs para S. frugiperda e A. gemmatalis, quando comparada à DL50 observada para o tipo selvagem AcMNPV. Entretanto, o TL50 foi significativamente diferente, com valores menores para vAcNSs em S. frugiperda [5,62 dias com 1 p.f.u. e 4,82 dias com 105 p.f.u.] e em A. gemmatalis [7,46 dias com 1 p.f.u. e 3,2 dias com 105 p.f.u.] quando em comparação com o TL50 do AcMNPV em S. frugiperda [8,5 dias com 1 p.f.u. e 7,52 dias com 105 p.f.u.] e em A. gemmatalis [20,11 dias com 1 p.f.u. e 7,34 dias com 105 p.f.u.]. Estes resultados corroboram os dados observados in vitro, indicando que a proteína NSs de TSWV aumenta a replicação do baculovírus e pode contribuir para gerar bioinseticidas mais eficientes. ______________________________________________________________________________ ABSTRACTGene silencing, or RNA interference (RNAi), works as a eukaryotic defense mechanism against viral infections. During evolutionary time viruses acquired specific proteins, which are able to halt the silencing process in different steps of its biochemical pathway e.g. the AC2 protein from Begomovirus, the NS1 protein from influenza (Influenza A) and the NSs protein from Tomato spotted wilt virus (TSWV). In this study we sought generation and evaluation of the gene-silencing suppression effect of three different genes, AC2 from begomovirus, NS1 from Influenza A and NSs from TSWV, via heterologous expression by recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV, genus AlphaBaculovirus, family Baculoviridae) in different insect cell lines. In the first chapter, a review concerning gene silencing, suppression of gene silencing by viral proteins and the potential use of recombinant Baculovirus as bio-insecticidal agents suitable for biological control is presented. The second chapter describes the cloning strategies designed for the construction of recombinant Baculoviruses carrying the AC2, NS1 and NSs genes (vAcAC2, vAcNS1 and vAcNSs, respectively) as well as the results of preliminary insect cell infection assays. In these preliminary assays, vAcNSs had more influence on wild type AcMNPV replication than vAcAC2 and vAcNS1. The third chapter shows the effect of the protein NSs from TSWV on virus replication in different host insect cell lines: one permissive, Trichoplusia ni (BTI-Tn-5B1-4); other semi-permissive, Anticarsia gemmatalis (UFL-AG-286); and a non-permissive cell line, Bombyx mori (BM-5). Results showed that infection of the semi-permissive cell line by vAcNSs was more efficient than infection by wild type AcMNPV, since production of budded virus was higher. In the non-permissive cell line, vAcNSs was able to produce cytopathic effects whereas no morphological alteration was found when wild type AcMNPV was inoculated. When vAcNSs and wild type AcMNPV were co-inoculated, production of polyhedra was enhanced despite the insect cell line used. Co-infection of vAcNSs and a recombinant AcMNPV carrying the egfp gene was also evaluated. In the semi-permissive cell line and in A. gemmatalis hemocytes (permissive cell line) co-infection greatly increased enhanced green fluorescent protein (EGFP) expression. Northern blotting assays showed absence of small interfering RNA (siRNA) molecules associated to egfp transcripts in the permissive and semi-permissive cell lines, which indicates suppression of gene silencing activity by the NSs protein. On the other hand, vAcNSs was not able to suppress the siRNA production in the non-permissive cell line. Confocal microscopy analysis showed that the NSs protein accumulated abundantly in the cytoplasm of permissive and semipermissive infected cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Finally, chapter four presents the study of the bio-insecticidal activity of vAcNSs on Spodoptera frugiperda and Anticarsia gemmatalis larvae, a permissive and semipermissive host, respectively. The vAcNSs LD50 for S. frugiperda and A. gemmatalis was not statistically different from wild-type AcMNPV. However, the LT50 values were significantly smaller for vAcNSs in S. frugiperda [5.62 days with 1 p.f.u. and 4.82 days with 105 p.f.u.] and A. gemmatalis [7.46 days with 1 p.f.u. and 3.20 days with 105 p.f.u.) when compared to the LT50 for AcMNPV in S. frugiperda [8.5 days with 1 p.f.u. and 7.52 days with 105 p.f.u.] and A. gemmatalis [20.11 days with 1 p.f.u. and 7.34 days with 105 p.f.u.]. These in vivo results are in accordance with the data observed in vitro indicating that the protein NSs from TSWV could efficiently improve Baculovirus replication and be used to generate more effective bioinsecticides
Chaves, Lorena Carvalho De Souza - One of the best experts on this subject based on the ideXlab platform.
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Uso de baculovírus como ferrramenta para produção de antígenos vacinais e “virus like particles” (VLPs)
2016Co-Authors: Chaves, Lorena Carvalho De SouzaAbstract:Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.Os baculovírus são vírus de insetos amplamente utilizados no controle biológico de pragas agrícolas e também como ferramenta para expressão de proteínas heterólogas. Os baixos custos, a segurança na manipulação e as diferentes formas de expressão de proteínas tornam os baculovírus e células de inseto uma escolha eficaz para a correta expressão de antígenos vacinais e produção de VLPs (“Virus like particles”). No capítulo 1 deste trabalho, foi avaliado o potencial imunogênico de BVs (“Budded virus”) recombinantes contendo o EDIII (Domínio III da proteína de envelope do vírus da Febre amarela – FA) fusionado à proteína GP64 dos baculovírus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) e Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV); e também de uma massa protéica gerada pela fusão de EDIII com a proteína poliedrina de AcMNPV. O EDIII interage com os receptores celulares e contém os epítopos reconhecidos pelos anticorpos neutralizantes, sendo assim alvo para a produção de uma vacina de subunidade. Foi mostrado neste trabalho, a confirmação da correta expressão das proteínas recombinantes (GP64 + EDIII e Poliedrina + EDIII) por Western blot, microscopia de luz e confocal. No caso do EDIII fusionado à GP64 de AcMNPV e AgMNPV, as partículas virais recombinantes foram purificadas e inoculadas em camundongos. O teste de proliferação de linfócitos indicou uma maior proliferação em camundongos inoculados com o vírus recombinante contendo o EDIII fusionado à proteína GP64 do baculovírus AgMNPV quando comparado com o controle LPS. Testes complementares serão feitos, para avaliar o perfil da resposta imunológica e confirmar a obtenção de um antígeno vacinal contra FA. No capítulo 2, o sistema baculovírus de expressão foi utilizado para expressar o precursor da proteína GAG (Pr55) de HIV-1. A expressão de Pr55 HIV-1 é suficiente para a montagem de VLPs na membrana plasmática da célula do hospedeiro. Porém, a proteína GP64 de baculovírus é altamente imunogênica e também é expressa na superfície de células infectadas e normalmente está presente na superfície da VLP. Neste trabalho, mostramos que não é possível separar BVs e VLPs produzidos em um mesmo ciclo de infecção por baculovírus mesmo usando diferentes metodologias de separação. Então, utilizando um sistema que consegue produzir baculovírus recombinantes livres de GP64, foi construído o vírus recombinante vGAGHIV-1 GP64 null e utilizado na infecção de células Sf9. Por Western blot foi observado a perda do sinal da proteína GP64 de baculovírus no extrato de células infectadas com esse vírus recombinante. Essas mesmas células foram visualizadas por microscopia eletrônica de transmissão (MET), mostrando que mesmo quando GP64 não está presente (o que resulta na ausência de produção de BVs), o brotamento de VLPs continua a acontecer. Essa técnica mostrou-se eficiente para a produção de VLPs livres de partículas ou proteínas baculovirais. Este trabalho confirma a eficiência do uso do sistema de expressão baseado em baculovírus para a expressão de proteínas e VLPs de interesse famacológico.Baculoviruses are insect viruses widely used in the biological control of agricultural pests and as a tool for heterologous protein expression. Their low production costs, security in manipulation and the many ways of protein expression, make Baculoviruses and insect cells an effective choice for the efficient expression of vaccine antigens and VLPs (Virus like particles) production. In chapter 1 of this work, it was evaluated the immunogenic potential of recombinant BVs (Budded virus) containing EDIII (Yellow fever virus -YF- envelope protein domain III) fused to the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) GP64 protein; and also of a protein mass generated by the fusion of the EDIII and AcMNPV polyhedrin protein. The EDIII interacts with cell receptors and contains epitopes recognized by neutralizing antibodies, being the target for the production of a subunit vaccine. We showed in this work the confirmation of the correct recombinant protein expression (GP64 + EDIII and Polyhedrin + EDIII) by Western blot, optical and confocal microscopy. In the case of the EDIII fused with AcMNPV and AgMNPV GP64, the recombinant viral particles were purified and inoculated in mice. The lymphocyte proliferation assay showed a higher proliferation in mice immunized with recombinant virus containing the EDIII fused with the GP64 from the AgMNPV Baculovirus comparing to the LPS control. Complementary assays will be carried out in order to evaluate the immunological response pattern and to confirm the production of a vaccine antigen against YF. In chapter 2, the Baculovirus expression system was used to express the GAG protein precursor (Pr55) of HIV-1. The Pr55 expression is sufficient to VLP assembly on the cell host plasma membrane. However, the GP64 Baculovirus protein is highly immunogenic and is also expressed on the surface of infected cells and is also present on the surface of the VLP. In this work, we showed that is not possible to separate BVs and VLPs produced in the same Baculovirus infection cycle using different separation methodologies. Then, using a system able to produce GP64 free recombinant Baculovirus, the recombinant virus vGAGHIV-1 GP64 null was produced and used to infect Sf9 cells. By Western blot analyses, it was shown that the Baculovirus protein GP64 signal was missing in the recombinant virus infected cell extract. These same cells were observed by transmission electron microscopy (MET), showing that even when the GP64 is absent (which results in the absence of BVs production), VLPs budding continues to happen. This technique was shown to be efficient for VLPs production, free of baculvirus particles or proteins. This work confirms the efficiency of the Baculovirus expression system for protein expression and VLPs of pharmacological interest
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Uso de baculovírus como ferrramenta para produção de antígenos vacinais e “virus like particles” (VLPs)
2016Co-Authors: Chaves, Lorena Carvalho De SouzaAbstract:Os baculovírus são vírus de insetos amplamente utilizados no controle biológico de pragas agrícolas e também como ferramenta para expressão de proteínas heterólogas. Os baixos custos, a segurança na manipulação e as diferentes formas de expressão de proteínas tornam os baculovírus e células de inseto uma escolha eficaz para a correta expressão de antígenos vacinais e produção de VLPs (“Virus like particles”). No capítulo 1 deste trabalho, foi avaliado o potencial imunogênico de BVs (“Budded virus”) recombinantes contendo o EDIII (Domínio III da proteína de envelope do vírus da Febre amarela – FA) fusionado à proteína GP64 dos baculovírus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) e Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV); e também de uma massa protéica gerada pela fusão de EDIII com a proteína poliedrina de AcMNPV. O EDIII interage com os receptores celulares e contém os epítopos reconhecidos pelos anticorpos neutralizantes, sendo assim alvo para a produção de uma vacina de subunidade. Foi mostrado neste trabalho, a confirmação da correta expressão das proteínas recombinantes (GP64 + EDIII e Poliedrina + EDIII) por Western blot, microscopia de luz e confocal. No caso do EDIII fusionado à GP64 de AcMNPV e AgMNPV, as partículas virais recombinantes foram purificadas e inoculadas em camundongos. O teste de proliferação de linfócitos indicou uma maior proliferação em camundongos inoculados com o vírus recombinante contendo o EDIII fusionado à proteína GP64 do baculovírus AgMNPV quando comparado com o controle LPS. Testes complementares serão feitos, para avaliar o perfil da resposta imunológica e confirmar a obtenção de um antígeno vacinal contra FA. No capítulo 2, o sistema baculovírus de expressão foi utilizado para expressar o precursor da proteína GAG (Pr55) de HIV-1. A expressão de Pr55 HIV-1 é suficiente para a montagem de VLPs na membrana plasmática da célula do hospedeiro. Porém, a proteína GP64 de baculovírus é altamente imunogênica e também é expressa na superfície de células infectadas e normalmente está presente na superfície da VLP. Neste trabalho, mostramos que não é possível separar BVs e VLPs produzidos em um mesmo ciclo de infecção por baculovírus mesmo usando diferentes metodologias de separação. Então, utilizando um sistema que consegue produzir baculovírus recombinantes livres de GP64, foi construído o vírus recombinante vGAGHIV-1 GP64 null e utilizado na infecção de células Sf9. Por Western blot foi observado a perda do sinal da proteína GP64 de baculovírus no extrato de células infectadas com esse vírus recombinante. Essas mesmas células foram visualizadas por microscopia eletrônica de transmissão (MET), mostrando que mesmo quando GP64 não está presente (o que resulta na ausência de produção de BVs), o brotamento de VLPs continua a acontecer. Essa técnica mostrou-se eficiente para a produção de VLPs livres de partículas ou proteínas baculovirais. Este trabalho confirma a eficiência do uso do sistema de expressão baseado em baculovírus para a expressão de proteínas e VLPs de interesse famacológico. _________________________________________________________________________________________________ ABSTRACTBaculoviruses are insect viruses widely used in the biological control of agricultural pests and as a tool for heterologous protein expression. Their low production costs, security in manipulation and the many ways of protein expression, make Baculoviruses and insect cells an effective choice for the efficient expression of vaccine antigens and VLPs (Virus like particles) production. In chapter 1 of this work, it was evaluated the immunogenic potential of recombinant BVs (Budded virus) containing EDIII (Yellow fever virus -YF- envelope protein domain III) fused to the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) GP64 protein; and also of a protein mass generated by the fusion of the EDIII and AcMNPV polyhedrin protein. The EDIII interacts with cell receptors and contains epitopes recognized by neutralizing antibodies, being the target for the production of a subunit vaccine. We showed in this work the confirmation of the correct recombinant protein expression (GP64 + EDIII and Polyhedrin + EDIII) by Western blot, optical and confocal microscopy. In the case of the EDIII fused with AcMNPV and AgMNPV GP64, the recombinant viral particles were purified and inoculated in mice. The lymphocyte proliferation assay showed a higher proliferation in mice immunized with recombinant virus containing the EDIII fused with the GP64 from the AgMNPV Baculovirus comparing to the LPS control. Complementary assays will be carried out in order to evaluate the immunological response pattern and to confirm the production of a vaccine antigen against YF. In chapter 2, the Baculovirus expression system was used to express the GAG protein precursor (Pr55) of HIV-1. The Pr55 expression is sufficient to VLP assembly on the cell host plasma membrane. However, the GP64 Baculovirus protein is highly immunogenic and is also expressed on the surface of infected cells and is also present on the surface of the VLP. In this work, we showed that is not possible to separate BVs and VLPs produced in the same Baculovirus infection cycle using different separation methodologies. Then, using a system able to produce GP64 free recombinant Baculovirus, the recombinant virus vGAGHIV-1 GP64 null was produced and used to infect Sf9 cells. By Western blot analyses, it was shown that the Baculovirus protein GP64 signal was missing in the recombinant virus infected cell extract. These same cells were observed by transmission electron microscopy (MET), showing that even when the GP64 is absent (which results in the absence of BVs production), VLPs budding continues to happen. This technique was shown to be efficient for VLPs production, free of baculvirus particles or proteins. This work confirms the efficiency of the Baculovirus expression system for protein expression and VLPs of pharmacological interest
Daniel Mendes Pereira Ardisson De ,araújo - One of the best experts on this subject based on the ideXlab platform.
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Genômica, evolução e caracterização funcional de genes de baculovírus
2016Co-Authors: Daniel Mendes Pereira Ardisson De ,araújoAbstract:Baculovirus são vírus de DNA dupla-fita circular capazes de infectar oralmente o estágio larval de insetos. Atualmente, são usados para o controle biológico de insetos praga e como vetores de expressão de proteínas heterólogas. Pouco é sabido das bases moleculares da interação do vírus com o hospedeiro e de sua evolução. Os fatores limitantes estão associados ao número de genomas sequenciados bem como a restrição do cultivo in vitro de várias espécies virais. De fato, a base para o início de quaisquer estudos moleculares mais detalhados de novas espécies de baculovírus ou de isolados certamente se inicia com o sequenciamento do genoma completo e com o estudo de genes encontrados. Dessa forma, neste trabalho, vários genomas de baculovírus isolados no Brasil foram sequenciados e descritos. Sequenciamos e descrevemos baculovírus isolados do mandarová-da-mandicoca, da broca da cana-de-açúcar, do bicho da seda, da lagarta polífaga Helicoverpa armigera, do mandarová-do-mate entre outros. Concomitante à descrição do genoma, caracterizamos estruturalmente algumas espécies, avaliamos a taxa de mortalidade em situações controladas de infecção, bem como caracterizamos alguns genes que permitiram um entendimento evolutivo mais amplo das espécies descritas e de sua interação com o hospedeiro. Descrevemos o primeiro inibidor de serino protease de baculovírus capaz de bloquear a imunidade inata do inseto hospedeiro e causar proteção ao patógeno. Encontramos o primeiro betabaculovírus com uma proteína de fusão de envelope de alphabaculovírus, a gp64 e caracterizamos sua funcionalidade. Além disso, mostramos pela primeira vez o papel de genes envolvidos no metabolismo de nucleotídeo e sua capacidade de alterar o desempenho viral. Em conclusão, baculovírus apresentam plasticidade genômica com aquisições proeminentes de genes de vários organismos como outros vírus de insetos, bactérias e plantas. Além disso, perdas de genes ancestrais e duplicação são eventos recorrentes. Tanto a genômica quanto o estudo molecular básico de baculovírus tem contribuído para a compreensão de doenças associadas a humanos como câncer e doenças virais cujo agente etiológico apresenta genoma com DNA dupla-fita ou que infectam primariamente o intestino médio de insetos, como herpesvírus e arboviroses, respectivamente. ______________________________________________________________________________________________ ABSTRACTBaculoviruses are circular double-stranded DNA viruses that are orally infectious to larval stages of insects. Nowadays, they are used as biological control agents of agricultural and forest pests and as vector for heterologous protein expression. The understanding of both the molecular basis and the evolution of the virus/host interaction is scarce due to the few numbers of sequenced genomes and the restriction in cultivating several virus species in vitro. In fact, the beginning of any molecular study of new Baculovirus species or isolates certainly pervades the whole genome sequencing. Therefore, in this work, several genomes of Baculoviruses isolated in Brazil were sequenced and described. We sequenced and described Baculoviruses isolated from subject cadavers of the cassava hornworm (Erinnyis ello), the sugar cane borer (Diatraea saccharalis), the silkworm (Bombyx mori), the bollworm (Helicoverpa armigera), and the mate hornworm (Perigonia lusca). Together with the genome description, we characterized structurally some species, evaluated the mortality in controlled infections, and characterized as well some genes to better understand the novel species and their interaction with the host. We described the first baculoviral serine protease inhibitor capable of blocking the insect immunity response and causing pathogen protection. We found the first betaBaculovirus harboring an alphaBaculovirus envelope fusion protein, a gp64 and we characterized its functionality. Furthermore, we have shown for the first time a role of genes related to nucleotide metabolism and it ability of altering the virus fitness. In conclusion, Baculoviruses present genomic plasticity with great and recurrent acquisition of genes from several organisms including other insect viruses, bacteria, and plant. Moreover, ancestral gene losses and duplication are common events in Baculovirus evolution. Both genomics and molecular biology of Baculovirus have contributed to the comprehension of human-associated diseases such as cancer and viral whereas the etiologic agent presents dsDNA genome or infects primarily the insect midgut like herpexviruses and arboviruses, respectively
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Genômica, evolução e caracterização funcional de genes de baculovírus
'Biblioteca Central da UNB', 2015Co-Authors: Daniel Mendes Pereira Ardisson De ,araújoAbstract:Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015.Orientador estrangeiro: Dr. Rollie J. ClemBaculovirus são vírus de DNA dupla-fita circular capazes de infectar oralmente o estágio larval de insetos. Atualmente, são usados para o controle biológico de insetos praga e como vetores de expressão de proteínas heterólogas. Pouco é sabido das bases moleculares da interação do vírus com o hospedeiro e de sua evolução. Os fatores limitantes estão associados ao número de genomas sequenciados bem como a restrição do cultivo in vitro de várias espécies virais. De fato, a base para o início de quaisquer estudos moleculares mais detalhados de novas espécies de baculovírus ou de isolados certamente se inicia com o sequenciamento do genoma completo e com o estudo de genes encontrados. Dessa forma, neste trabalho, vários genomas de baculovírus isolados no Brasil foram sequenciados e descritos. Sequenciamos e descrevemos baculovírus isolados do mandarová-da-mandicoca, da broca da cana-de-açúcar, do bicho da seda, da lagarta polífaga Helicoverpa armigera, do mandarová-do-mate entre outros. Concomitante à descrição do genoma, caracterizamos estruturalmente algumas espécies, avaliamos a taxa de mortalidade em situações controladas de infecção, bem como caracterizamos alguns genes que permitiram um entendimento evolutivo mais amplo das espécies descritas e de sua interação com o hospedeiro. Descrevemos o primeiro inibidor de serino protease de baculovírus capaz de bloquear a imunidade inata do inseto hospedeiro e causar proteção ao patógeno. Encontramos o primeiro betabaculovírus com uma proteína de fusão de envelope de alphabaculovírus, a gp64 e caracterizamos sua funcionalidade. Além disso, mostramos pela primeira vez o papel de genes envolvidos no metabolismo de nucleotídeo e sua capacidade de alterar o desempenho viral. Em conclusão, baculovírus apresentam plasticidade genômica com aquisições proeminentes de genes de vários organismos como outros vírus de insetos, bactérias e plantas. Além disso, perdas de genes ancestrais e duplicação são eventos recorrentes. Tanto a genômica quanto o estudo molecular básico de baculovírus tem contribuído para a compreensão de doenças associadas a humanos como câncer e doenças virais cujo agente etiológico apresenta genoma com DNA dupla-fita ou que infectam primariamente o intestino médio de insetos, como herpesvírus e arboviroses, respectivamente.Baculoviruses are circular double-stranded DNA viruses that are orally infectious to larval stages of insects. Nowadays, they are used as biological control agents of agricultural and forest pests and as vector for heterologous protein expression. The understanding of both the molecular basis and the evolution of the virus/host interaction is scarce due to the few numbers of sequenced genomes and the restriction in cultivating several virus species in vitro. In fact, the beginning of any molecular study of new Baculovirus species or isolates certainly pervades the whole genome sequencing. Therefore, in this work, several genomes of Baculoviruses isolated in Brazil were sequenced and described. We sequenced and described Baculoviruses isolated from subject cadavers of the cassava hornworm (Erinnyis ello), the sugar cane borer (Diatraea saccharalis), the silkworm (Bombyx mori), the bollworm (Helicoverpa armigera), and the mate hornworm (Perigonia lusca). Together with the genome description, we characterized structurally some species, evaluated the mortality in controlled infections, and characterized as well some genes to better understand the novel species and their interaction with the host. We described the first baculoviral serine protease inhibitor capable of blocking the insect immunity response and causing pathogen protection. We found the first betaBaculovirus harboring an alphaBaculovirus envelope fusion protein, a gp64 and we characterized its functionality. Furthermore, we have shown for the first time a role of genes related to nucleotide metabolism and it ability of altering the virus fitness. In conclusion, Baculoviruses present genomic plasticity with great and recurrent acquisition of genes from several organisms including other insect viruses, bacteria, and plant. Moreover, ancestral gene losses and duplication are common events in Baculovirus evolution. Both genomics and molecular biology of Baculovirus have contributed to the comprehension of human-associated diseases such as cancer and viral whereas the etiologic agent presents dsDNA genome or infects primarily the insect midgut like herpexviruses and arboviruses, respectively
Salvador Herrero - One of the best experts on this subject based on the ideXlab platform.
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improvement of Baculovirus as protein expression vector and as biopesticide by crispr cas9 editing
Biotechnology and Bioengineering, 2019Co-Authors: Veronica Pazminoibarra, Adria Mengualmarti, Alexandra Marisa Targovnik, Salvador HerreroAbstract:The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of Baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the Baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve Baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the Baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in Baculovirus, contributing to the engineering of Baculovirus as a protein expression vector as well as a biological control agent.
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improvement of Baculovirus as protein expression vector and as biopesticide by crispr cas9 editing
bioRxiv, 2019Co-Authors: Veronica Pazminoibarra, Adria Mengualmarti, Alexandra Marisa Targovnik, Salvador HerreroAbstract:Abstract The CRISPR (Clustered Regularly Interspaced Short Palindromic repeats) system associated Cas9 endonuclease is a molecular tool that enables specific sequence edition with high efficiency. The edition using CRISPR/Cas9 system has been successfully reported in small and large viral genomes. In this study, we have explored the use of CRISPR/Cas9 system for the edition of the Baculovirus genome. We have shown that the delivering of Cas9-sgRNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knocks-out as well as knocks-in into the Baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve Baculovirus as protein expression vector and as biopesticide, we attempted to knock-out several genes from a recombinant AcMNPV form used in the Baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knocks-in specific regions of the viral genome. Analysis of the generated mutants revealed that the edition efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the rate of edition ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for the edition of Baculovirus contributing to the engineering of Baculovirus as protein expression vector as well as a biological control agent.
