RESUMO
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the Baculoviridae family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by Spodoptera frugiperda (Sf) insect cells were characterised for the first time. Using S. frugiperda (SfC1B5) cells stably expressing the baculovirus gp64, the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking p6.9 (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101-which may be useful as a protein marker for sEVs.
RESUMO
Humanised antibodies targeting Crimean-Congo Haemorrhagic virus (CCHFV) are needed for the development and standardisation of serological assays. These assays are needed to address a shortfall in available tests that meet regulatory diagnostic standards and to aid surveillance activities to extend knowledge on the distribution of CCHFV. To generate a humanised monoclonal antibody against CCHFV, we have compared two methods: the traditional mouse hybridoma approach with subsequent sequencing and humanisation of antibodies versus a non-animal alternative using a human combinatorial antibody library (HuCAL). Our results demonstrated that the mouse hybridoma followed by humanisation protocol gave higher affinity antibodies. Whilst not yet able to demonstrate the generation of equivalent humanised antibodies without the use of animals, sequencing data enables the subsequent production of recombinant antibodies, thus providing a reduction in future animal usage for this application. Ultimately, our report provides information on development of a humanised standardised control, which can form an important positive control component of serological assays against CCHFV.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Humanos , Animais , Camundongos , Febre Hemorrágica da Crimeia/diagnóstico , Febre Hemorrágica da Crimeia/epidemiologia , Hibridomas , Anticorpos Antivirais , Imunoglobulina GRESUMO
Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.
Assuntos
DNA Viral/metabolismo , Nucleopoliedrovírus/fisiologia , Replicação Viral , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fluorometria , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/virologia , Microscopia de Fluorescência , Células Sf9 , SpodopteraRESUMO
Over 35 years since it was established to make recombinant proteins, the baculovirus expression vector system continues to develop and improve. Early systems for recombinant virus selection were laborious, but better methods were rapidly devised that enabled non-virologists to use baculovirus vectors successfully in a wide range of applications. These applications include multiple gene expression for complex molecules, production of adeno-associated virus-like particles for gene therapy, the use of baculovirus budded virus for the same purpose, numerous potential human and animal vaccines, and for other therapeutic proteins. A number of products for human and veterinary use are now on the market, which attests to the utility of the systems. Despite these successes, baculovirus vectors essentially remain in a relatively primitive state of development. Many proteins, particularly membrane-bound or secreted products, continue to be difficult to produce. Various research groups are working to identify potential areas of improvement, which if combined into an ideal vector might offer considerable advances to the system. This chapter will review some of the most recent reports and highlight those that might have generic application for recombinant protein synthesis in insect cells. We also summarize parallel developments in host cells used for baculovirus expression and how culture conditions can influence protein production.
Assuntos
Baculoviridae/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Animais , Engenharia Genética/métodos , Humanos , Engenharia de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.
Assuntos
Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Nucleopoliedrovírus/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Mariposas , Nucleopoliedrovírus/química , Nucleopoliedrovírus/genética , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet ß cells (EndoC ßH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.
Assuntos
Baculoviridae/genética , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/instrumentação , Células Secretoras de Insulina/virologia , Transdução Genética , Baculoviridae/fisiologia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ilhotas Pancreáticas/virologiaRESUMO
This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles. We describe advances made to the BEVS to allow the rapid and easy production of recombinant viruses and developments to improve protein yield. We finish by describing the application of recombinant BacMam as vectors for the delivery of genes into mammalian and human cells. © 2018 by John Wiley & Sons, Inc.
Assuntos
Baculoviridae/genética , Baculoviridae/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Humanos , Proteínas Recombinantes/genéticaRESUMO
Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.
Assuntos
Baculoviridae/genética , Baculoviridae/metabolismo , Reatores Biológicos , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Humanos , Proteínas Recombinantes/genéticaRESUMO
Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Análise Mutacional de DNA , Insetos , Espectrometria de Massas , Fosforilação , Conformação Proteica , Multimerização Proteica , Proteínas Virais/genéticaRESUMO
The development of baculovirus expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function and regulation in insect cells. Classification of these viruses has also been refined as we learn more about differences in gene content between isolates, how this affects virus structure and their replication in insect larvae. Baculovirus gene expression occurs in an ordered cascade, regulated by early, late and very late gene promoters. There is now a detailed knowledge of these promoter elements and how they interact first with host cell-encoded RNA polymerases and later with virus-encoded enzymes. The composition of this virus RNA polymerase is known. The virus replication process culminates in the very high level expression of both polyhedrin and p10 gene products in the latter stages of infection. It has also been realized that the insect host cell has innate defenses against baculoviruses in the form of an apoptotic response to virus invasion. Baculoviruses counter this by encoding apoptotic-suppressors, which also appear to have a role in determining the host range of the virus. Also of importance to our understanding of baculovirus expression systems is how the virus can accumulate mutations within genes that affect recombinant protein yield in cell culture. The summary in this chapter is not exhaustive, but should provide a good preparation to those wishing to use this highly successful gene expression system.
Assuntos
Baculoviridae/genética , Biologia Molecular/métodos , Animais , Apoptose , Expressão Gênica , Vetores Genéticos/genética , Humanos , Replicação ViralRESUMO
The production of a recombinant baculovirus expression vector normally involves mixing infectious virus DNA with a plasmid-based transfer vector and then co-transfecting insect cells to initiate virus infection. The aim of this chapter is to provide an update on the range of baculovirus transfer vectors currently available. Some of the original transfer vectors developed are now difficult to obtain but generally have been replaced by superior reagents. We focus on those that are available commercially and should be easy to locate. These vectors permit the insertion of single or multiple genes for expression, or the production of proteins with specific peptide tags that aid subsequent protein purification. Others have signal peptide coding regions permitting protein secretion or plasma membrane localization. A table listing the transfer vectors also includes information on the parental virus that should be used with each one. Methods are described for the direct insertion of a recombinant gene into the virus genome without the requirement for a transfer vector. The information provided should enable new users of the system to choose those reagents most suitable for their purposes.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Transfecção/métodos , Clonagem Molecular , Proteínas Recombinantes/genéticaRESUMO
Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.
Assuntos
Baculoviridae/genética , Fracionamento Químico/métodos , DNA Recombinante/genética , DNA Viral/isolamento & purificação , Transfecção/métodos , Animais , Baculoviridae/crescimento & desenvolvimento , DNA Viral/genética , Escherichia coli/genética , Vetores Genéticos/genética , Reação em Cadeia da Polimerase , Células Sf9 , Spodoptera , Transformação Genética , Ensaio de Placa ViralRESUMO
UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.
Assuntos
Actinas/metabolismo , Coinfecção/veterinária , Nucleopoliedrovírus/fisiologia , Spodoptera/virologia , Superinfecção/veterinária , Animais , Núcleo Celular/metabolismo , Coinfecção/metabolismo , Coinfecção/virologia , Citoplasma/metabolismo , Proteínas de Insetos/metabolismo , Nucleopoliedrovírus/genética , Células Sf9 , Spodoptera/metabolismo , Superinfecção/metabolismo , Superinfecção/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombuspascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.
Assuntos
Abelhas/virologia , Sequência Conservada/genética , Vírus de Insetos/genética , Recombinação Genética/genética , Varroidae/virologia , Asas de Animais/virologia , Animais , Sequência de Bases , Quimera , Genoma Viral/genética , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC™) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus. The method relies on homologous recombination in insect cells between a transfer plasmid containing the gene of interest and a replication-deficient bacmid. The gene of interest replaces the bacterial replicon at the polyhedrin locus, simultaneously restoring a virus gene essential for replication, and as only recombinant virus can replicate, no further separation techniques are required. This chapter describes methods for producing and expression testing multiple recombinant baculoviruses on automated platforms using the flashBAC system.
Assuntos
Baculoviridae/metabolismo , Biotecnologia/métodos , Vetores Genéticos/genética , Plasmídeos/genética , Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/genética , Western Blotting , Técnicas de Cultura de Células/métodos , Linhagem Celular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Recombinação Homóloga/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , SpodopteraRESUMO
Baculoviruses have a unique bi-phasic life cycle and powerful promoters, which greatly facilitates their use for recombinant protein expression in insect cells. We have developed an expression system that utilizes homologous recombination in insect cells between a transfer plasmid containing a gene to be expressed and a replication-deficient virus (bacmid). Only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses using robotic liquid handlers. The bacmid has also been genetically optimized for improved protein expression and stability. We describe the application of this system for high level production of recombinant proteins.
Assuntos
Baculoviridae/genética , Expressão Gênica , Vetores Genéticos/genética , Ensaios de Triagem em Larga Escala , Plasmídeos/genética , Spodoptera/metabolismo , Animais , Automação Laboratorial , Linhagem Celular , Vetores Genéticos/química , Recombinação Homóloga , Plasmídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Spodoptera/citologia , Spodoptera/virologia , Replicação Viral/genéticaRESUMO
A laboratory culture of Spodoptera exigua was examined to assess covert (latent or persistent) baculovirus infections and spontaneous disease outbreaks. Two nucleopolyhedrovirus (NPV) species were found to be reactivated from a covert state in a laboratory culture of S. exigua to fully lethal forms. These were identified as S. exigua multinucleopolyhedrovirus (SeMNPV) and Mamestra brassicae NPV (MbNPV) using restriction enzyme analysis of purified viral DNA. Sequence data derived from both overtly and covertly virus-infected insects revealed highly conserved sequences for lef-8, lef-9 and polyhedrin gene sequence (98-100â% nucleotide identity to SeMNPV published sequence). By monitoring spontaneous overt infections and quantifying viral DNA (by quantitative-PCR) in asymptomatic individuals over two generations we identified fluctuating trends in viral DNA levels from covert SeMNPV and MbNPV within an S. exigua host population. Virus levels per insect life stage ranged from 3.51±0.101×10(5) to 0.29±0.036 pg (detection limit at 0.06 pg). Bioassays performed with this culture of larvae showed a differential susceptibility to SeMNPV-like or MbNPV-like viruses, with SeMNPV superinfections being extremely virulent. The data presented has broad implications relating to our understanding of transmission patterns of baculovirus in the environment and the role of covert infections in host-pathogen interaction dynamics.
Assuntos
Baculoviridae/isolamento & purificação , Lepidópteros/virologia , Animais , Baculoviridae/genética , Baculoviridae/patogenicidade , Linhagem Celular , Sequência Conservada , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Análise de Sobrevida , Proteínas Virais/genética , Virulência , Ativação Viral , Latência ViralRESUMO
Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.
Assuntos
Baculoviridae/genética , Proteínas de Membrana/biossíntese , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Animais , Baculoviridae/enzimologia , Catepsinas/genética , Células Cultivadas , Quitinases/genética , Deleção de Genes , Expressão Gênica , Humanos , Insetos/citologia , Proteínas de Membrana/genéticaRESUMO
The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.
Assuntos
Deleção de Genes , Nucleopoliedrovírus/genética , Spodoptera/virologia , Animais , Cromossomos Artificiais Bacterianos/metabolismo , Clonagem Molecular , DNA Viral/genética , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Frequência do Gene , Variação Genética , Vetores Genéticos , Genoma Viral , Genótipo , Larva/genética , Larva/metabolismo , Larva/virologia , Nucleopoliedrovírus/metabolismo , Controle Biológico de Vetores , Filogenia , Análise de Sequência de DNA , Inoculações Seriadas , Spodoptera/genética , Spodoptera/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/genética , Vírion/metabolismo , Replicação Viral/genéticaRESUMO
Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.