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1.
Front Cell Infect Microbiol ; 11: 735191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34660343

RESUMO

Pathogens require physical contact with the mucosal surface of the host organism to initiate infection and as such, vaccines eliciting both mucosal and systemic immune responses would be promising. Studies involving the use of recombinant baculoviruses (rBVs) as mucosal vaccines are severely lacking despite their inherently safe nature, especially against pathogens of global importance such as Toxoplasma gondii. Here, we generated rBVs displaying T. gondii rhoptry protein 4 (ROP4) and evaluated their protective efficacy in BALB/c mice following immunization via intranasal (IN) and oral routes. IN immunization with the ROP4-expressing rBVs elicited higher levels of parasite-specific IgA antibody responses compared to oral immunization. Upon challenge infection with a lethal dose of T. gondii ME49, IN immunization elicited significantly higher parasite-specific antibody responses in the mucosal tissues such as intestines, feces, vaginal samples, and brain than oral immunization. Marked increases in IgG and IgA antibody-secreting cell (ASC) responses were observed from intranasally immunized mice. IN immunization elicited significantly enhanced induction of CD4+, CD8+ T cells, and germinal center B (GC B) cell responses from secondary lymphoid organs while limiting the production of the inflammatory cytokines IFN-γ and IL-6 in the brain, all of which contributed to protecting mice against T. gondii lethal challenge infection. Our findings suggest that IN delivery of ROP4 rBVs induced better mucosal and systemic immunity against the lethal T. gondii challenge infection compared to oral immunization.


Assuntos
Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias , Toxoplasma , Administração através da Mucosa , Animais , Anticorpos Antiprotozoários , Baculoviridae/genética , Linfócitos T CD8-Positivos , Citocinas , Feminino , Imunoglobulina G , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Vacinas Protozoárias/imunologia , Toxoplasma/genética
2.
Appl Microbiol Biotechnol ; 105(21-22): 8195-8226, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34618205

RESUMO

Baculoviruses are insect pathogens widely used as biotechnological tools in different fields of life sciences and technologies. The particular biology of these entities (biosafety viruses 1; large circular double-stranded DNA genomes, infective per se; generally of narrow host range on insect larvae; many of the latter being pests in agriculture) and the availability of molecular-biology procedures (e.g., genetic engineering to edit their genomes) and cellular resources (availability of cell lines that grow under in vitro culture conditions) have enabled the application of baculoviruses as active ingredients in pest control, as systems for the expression of recombinant proteins (Baculovirus Expression Vector Systems-BEVS) and as viral vectors for gene delivery in mammals or to display antigenic proteins (Baculoviruses applied on mammals-BacMam). Accordingly, BEVS and BacMam technologies have been introduced in academia because of their availability as commercial systems and ease of use and have also reached the human pharmaceutical industry, as incomparable tools in the development of biological products such as diagnostic kits, vaccines, protein therapies, and-though still in the conceptual stage involving animal models-gene therapies. Among all the baculovirus species, the Autographa californica multiple nucleopolyhedrovirus has been the most highly exploited in the above utilities for the human-biotechnology field. This review highlights the main achievements (in their different stages of development) of the use of BEVS and BacMam technologies for the generation of products for infectious and noninfectious human diseases. KEY POINTS: • Baculoviruses can assist as biotechnological tools in human health problems. • Vaccines and diagnosis reagents produced in the baculovirus platform are described. • The use of recombinant baculovirus for gene therapy-based treatment is reviewed.


Assuntos
Baculoviridae , Vetores Genéticos , Animais , Baculoviridae/genética , Linhagem Celular , Humanos , Insetos , Proteínas Recombinantes/genética
3.
Microbiol Spectr ; 9(2): e0053721, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34668746

RESUMO

UV light, more specifically UV-C light at a wavelength of 254 nm, is often used to disinfect surfaces, air, and liquids. In early 2020, at the cusp of the COVID-19 pandemic, UV light was identified as an efficient means of eliminating coronaviruses; however, the variability in published sensitivity data is evidence of the need for experimental rigor to accurately quantify the effectiveness of this technique. In the current study, reliable and reproducible UV techniques have been adopted, including accurate measurement of light intensity, consideration of fluid UV absorbance, and confirmation of uniform dose delivery, including dose verification using an established biological target (T1UV bacteriophage) and a resistant recombinant virus (baculovirus). The experimental results establish the UV sensitivity of SARS-CoV-2, HCoV-229E, HCoV-OC43, and mouse hepatitis virus (MHV) and highlight the potential for surrogate viruses for disinfection studies. All four coronaviruses were found to be easily inactivated by 254 nm irradiation, with UV sensitivities of 1.7, 1.8, 1.7, and 1.2 mJ/cm2/log10 reduction for SARS-CoV-2, HCoV-229E, HCoV-OC43, and MHV, respectively. Similar UV sensitivities for these species demonstrate the capacity for HCoV-OC43, HCoV-229E, and MHV to be considered surrogates for SARS-CoV-2 in UV-inactivation studies, greatly reducing hazards and simplifying procedures for future experimental studies. IMPORTANCE Disinfection of SARS-CoV-2 is of particular importance due to the global COVID-19 pandemic. UV-C irradiation is a compelling disinfection technique because it can be applied to surfaces, air, and water and is commonly used in drinking water and wastewater treatment facilities. UV inactivation depends on the dose received by an organism, regardless of the intensity of the light source or the optical properties of the medium in which it is suspended. The 254 nm irradiation sensitivity was accurately determined using benchmark methodology and a collimated beam apparatus for four coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-OC43, and MHV), a surrogate indicator organism (T1UV), and a resistant recombinant virus (baculovirus vector). Considering the light distribution across the sample surface, the attenuation of light intensity with fluid depth, the optical absorbance of the fluid, and the sample uniformity due to mixing enable accurate measurement of the fundamental inactivation kinetics and UV sensitivity.


Assuntos
Coronavirus Humano 229E/efeitos da radiação , Coronavirus Humano OC43/efeitos da radiação , Vírus da Hepatite Murina/efeitos da radiação , SARS-CoV-2/efeitos da radiação , Raios Ultravioleta , Animais , Baculoviridae/efeitos da radiação , COVID-19/prevenção & controle , Linhagem Celular , Chlorocebus aethiops , Desinfecção/métodos , Humanos , Células Vero
4.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2786-2793, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472296

RESUMO

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.


Assuntos
Baculoviridae , Galinhas , Animais , Baculoviridae/genética , Ligante de CD40/genética , Clonagem Molecular , Vetores Genéticos/genética , Proteínas Recombinantes/genética
5.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360656

RESUMO

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.


Assuntos
Antivirais/farmacologia , Baculoviridae/genética , Fusão Celular , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Febre de Chikungunya/virologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Ensaios de Triagem em Larga Escala , Mosquitos Vetores , Células Sf9 , Proteínas do Envelope Viral/genética
6.
Viruses ; 13(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34372591

RESUMO

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1-660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens' immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394-608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394-608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461-544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.


Assuntos
Antígenos Virais/imunologia , Vírus da Hepatite E/imunologia , Fases de Leitura Aberta/genética , Proteínas Virais/genética , Animais , Baculoviridae/genética , Baculoviridae/imunologia , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/química , Vírus da Hepatite E/genética , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta/imunologia , Suínos , Proteínas Virais/imunologia
7.
Mar Biotechnol (NY) ; 23(4): 517-528, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34241714

RESUMO

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.


Assuntos
Linhagem Celular , Penaeidae/fisiologia , Transdução Genética/métodos , Animais , Baculoviridae , Carcinógenos , Linfócitos/fisiologia , Penaeidae/genética
8.
Mol Biotechnol ; 63(12): 1223-1234, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34304364

RESUMO

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Baculoviridae/genética , Baculoviridae/imunologia , Biotecnologia , Bombyx/genética , Bombyx/imunologia , Células Cultivadas , Expressão Gênica , Hemolinfa/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/biossíntese , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , SARS-CoV-2/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Elife ; 102021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34263726

RESUMO

Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests have been well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests have not previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a densovirus (HaDV2) is associated with its enhanced growth and tolerance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested tolerance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These findings suggest that exposure to Bt-crops has selected for beneficial interactions between the target pest and a mutualistic microorganism that enhances its performance on Bt-crops under field conditions.


Assuntos
Bacillus thuringiensis , Densovirus , Gossypium , Inseticidas , Animais , Toxinas de Bacillus thuringiensis , Baculoviridae , China , Endotoxinas , Proteínas Hemolisinas , Insetos , Resistência a Inseticidas , Mariposas , Plantas Geneticamente Modificadas , Simbiose
10.
Virus Genes ; 57(5): 459-463, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34185196

RESUMO

Baculovirus infection modulates the chromatin states and gene expression of host insect cells. Here we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of H3 trimethylated at Lys4 (H3K4me3) histone modification in Bombyx mori nucleopolyhedrovirus-infected Bombyx mori cells. The ChIP-seq data revealed the changes of the genome-wide distribution and accumulation of euchromatic histone marks in host insect cells during the progression of baculovirus infection.


Assuntos
Bombyx/genética , Cromatina/genética , Histonas/genética , Nucleopoliedrovírus/genética , Animais , Baculoviridae/genética , Baculoviridae/patogenicidade , Bombyx/virologia , Cromatina/virologia , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas/genética , Nucleopoliedrovírus/patogenicidade , Processamento de Proteína Pós-Traducional/genética
11.
Methods Enzymol ; 653: 3-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34099177

RESUMO

Despite major advances in methodologies for membrane protein production over the last two decades, there remain challenging protein complexes that are technically difficult to yield by conventional recombinant expression methods. A large number of these proteins are multimeric membrane proteins from eukaryotic species, which are required to pass through stringent quality control mechanisms of host cells for proper folding and complex assembly. Here, we describe the development procedure to improve the production efficiency of multi-oligomeric membrane protein complexes in insect cells and recombinant baculovirus, which involves screening of promoters, enhancers, and untranslated regions for expression levels, using calcium homeostasis modulator (CALHM) and N-methyl-d-aspartate receptor (NMDAR) proteins as examples. We demonstrate that our insect cell expression strategy is effective in expression of both multi-homomeric CALHM proteins and multi-heteromeric NMDARs.


Assuntos
Baculoviridae , Proteínas de Membrana , Animais , Baculoviridae/genética , Insetos , Proteínas de Membrana/genética , Receptores de N-Metil-D-Aspartato
12.
Methods Mol Biol ; 2268: 119-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085265

RESUMO

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.


Assuntos
Baculoviridae/genética , Carbocianinas/química , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Receptor Tipo 4 de Melanocortina/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Ligação Competitiva , Bioensaio/métodos , Humanos , Cinética , Ligantes , Ligação Proteica , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Células Sf9
13.
Methods Mol Biol ; 2268: 179-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085269

RESUMO

Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.


Assuntos
Baculoviridae/metabolismo , Gonadotropina Coriônica/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores do LH/metabolismo , Animais , Baculoviridae/genética , Técnicas Biossensoriais/métodos , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Substâncias para o Controle da Reprodução/farmacologia , Transdução de Sinais
14.
Viruses ; 13(5)2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066413

RESUMO

Viruses rely on host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Infection of the silkworm, Bombyx mori, by Bombyx mori nucleopolyhedrovirus (BmNPV), has been studied extensively in the past to unravel interactions between baculoviruses and their lepidopteran hosts. To understand the interaction between the host metabolic responses and BmNPV infection, we analyzed global metabolic changes associated with BmNPV infection in silkworm hemolymph. Our metabolic profiling data suggests that amino acid metabolism is strikingly altered during a time course of BmNPV infection. Amino acid consumption is increased during BmNPV infection at 24 h post infection (hpi), but their abundance recovered at 72 hpi. Central carbon metabolism, on the other hand, particularly glycolysis and glutaminolysis, did not show obvious changes during BmNPV infection. Pharmacologically inhibiting the glycolytic pathway and glutaminolysis also failed to reduce BmNPV replication, revealing that glycolysis and glutaminolysis are not essential during BmNPV infection. This study reveals a unique amino acid utilization process that is implemented during BmNPV infection. Our metabolomic analysis of BmNPV-infected silkworm provides insights as to how baculoviruses induce alterations in host metabolism during systemic infection.


Assuntos
Aminoácidos/metabolismo , Baculoviridae/fisiologia , Bombyx/metabolismo , Bombyx/virologia , Hemolinfa/metabolismo , Hemolinfa/virologia , Metabolômica , Animais , Bombyx/genética , Cromatografia Líquida , Biologia Computacional/métodos , Metabolismo Energético , Perfilação da Expressão Gênica , Glicólise , Interações Hospedeiro-Patógeno , Metaboloma , Metabolômica/métodos , Espectrometria de Massas em Tandem
15.
Biotechnol J ; 16(8): e2100008, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34176228

RESUMO

Western equine encephalitis virus (WEEV) causes lethal encephalitis in humans and equines, and it poses a serious public health threat in many countries. Therefore, the development of an efficient vaccine remains an important challenge for the prevention of WEEV infection. This study presents the first description of WEEV virus-like particles (VLPs) generated from insect cells using recombinant baculoviruses. WEEV VLPs with 206 adjuvant could trigger a strong cellular immune response; increase the levels of IL-2, IL-4 and IFN-γ; and induce a high level of neutralizing antibodies against WEEV in mice. These data showed that the insect cell-baculovirus system is suitable for the production of WEEV VLPs and that these VLPs could elicit the strong immunogenicity in mice. These results suggest a new, nonreplicating, and effective vaccine candidate against WEEV infection.


Assuntos
Baculoviridae , Vírus da Encefalite Equina do Oeste , Animais , Anticorpos Antivirais , Baculoviridae/genética , Vírus da Encefalite Equina do Oeste/genética , Cavalos , Imunidade , Imunização , Insetos , Camundongos
16.
Methods Mol Biol ; 2305: 141-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950388

RESUMO

Baculovirus expression vector systems (BEVS) are widely used to produce heterologous proteins for a wide range of applications. Developed more than 30 years ago, BEVS have been constantly modified to improve product quality and ease-of-use. Plasmid reagents were tailored and engineered to facilitate introduction of heterologous genes into baculoviral genomes. At the same time, detrimental modalities such as genes encoding proteases or apoptotic factors were removed to improve protein yield. Advances in DNA synthesis and manipulation now enable the engineering of part or whole synthetic baculovirus genomes, opening up new avenues to redesign and tailor the system to specific applications. Here, we describe a simple protocol for designing and constructing baculovirus genomes comprising segments of synthetic DNA through the use of iterative Red/ET homologous recombination reactions.


Assuntos
Baculoviridae/genética , Biotecnologia/métodos , Vetores Genéticos , Cromossomos Artificiais Bacterianos/genética , Engenharia Genética , Genoma Viral , Recombinação Homóloga , Plasmídeos , Biologia Sintética/métodos
17.
Int J Biol Macromol ; 183: 1607-1620, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34029585

RESUMO

Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.


Assuntos
Antineoplásicos/farmacologia , Fibrinolíticos/farmacologia , Serpinas/genética , Serpinas/metabolismo , Aranhas/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Células Sf9 , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/genética , Tripsina
18.
Viruses ; 13(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916308

RESUMO

Mink refractory diarrhea is a seasonal disease that occurs in many mink farms in China. Mink circovirus (MiCV) has been recognized as the causative agent of the disease. The aim of the study was to develop a subunit vaccine against mink refractory diarrhea. A recombinant baculovirus strain expressing the capsid protein was constructed using the baculovirus expression vector system (BEVS). A subunit vaccine was developed based on the capsid protein with appropriate adjuvant. Then, a field trial was carried out in two districts in order to evaluate the efficiency of the subunit vaccine. The field trial indicated that in total, only 1.8% of the minks developed typical diarrhea in the vaccinated group compared with 74.5% in the control group. The vaccination could significantly reduce the infection rate of MiCV among the mink herds and could restrain the virus' shedding from feces. Furthermore, the vaccinated group had a higher average litter size in the following year compared to the control group. Collectively, the results indicated that the subunit vaccine based on the capsid protein can provide reliable protection against MiCV infection.


Assuntos
Anticorpos Antivirais/sangue , Baculoviridae/genética , Proteínas do Capsídeo/genética , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Diarreia/prevenção & controle , Vison/virologia , Vacinas Virais/imunologia , Animais , Capsídeo/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , China , Infecções por Circoviridae/imunologia , Circovirus/genética , Diarreia/virologia , Feminino , Masculino , Vacinas de Subunidades/imunologia , Vacinas Virais/administração & dosagem
19.
Enzyme Microb Technol ; 146: 109759, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33812558

RESUMO

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.


Assuntos
Hialuronoglucosaminidase , Fosfolipase D , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Domínio Catalítico , Humanos , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Insetos/metabolismo , Diester Fosfórico Hidrolases
20.
BMC Genomics ; 22(1): 226, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789582

RESUMO

BACKGROUND: Short interspersed nuclear elements (SINEs) belong to non-long terminal repeat (non-LTR) retrotransposons, which can mobilize dependent on the help of counterpart long interspersed nuclear elements (LINEs). Although 234 SINEs have been identified so far, only 23 are from insect species (SINEbase: http://sines.eimb.ru/ ). RESULTS: Here, five SINEs were identified from the genome of Plutella xylostella, among which PxSE1, PxSE2 and PxSE3 were tRNA-derived SINEs, PxSE4 and PxSE5 were 5S RNA-derived SINEs. A total of 18 related SINEs were further identified in 13 lepidopteran insects and a baculovirus. The 3'-tail of PxSE5 shares highly identity with that of LINE retrotransposon, PxLINE1. The analysis of relative age distribution profiles revealed that PxSE1 is a relatively young retrotransposon in the genome of P. xylostella and was generated by recent explosive amplification. Integration pattern analysis showed that SINEs in P. xylostella prefer to insert into or accumulate in introns and regions 5 kb downstream of genes. In particular, the PxSE1-like element, SlNPVSE1, in Spodoptera litura nucleopolyhedrovirus II genome is highly identical to SfSE1 in Spodoptera frugiperda, SlittSE1 in Spodoptera littoralis, and SlituSE1 in Spodoptera litura, suggesting the occurrence of horizontal transfer. CONCLUSIONS: Lepidopteran insect genomes harbor a diversity of SINEs. The retrotransposition activity and copy number of these SINEs varies considerably between host lineages and SINE lineages. Host-parasite interactions facilitate the horizontal transfer of SINE between baculovirus and its lepidopteran hosts.


Assuntos
Baculoviridae , Elementos Nucleotídeos Curtos e Dispersos , Animais , Baculoviridae/genética , Evolução Molecular , Insetos , Elementos Nucleotídeos Longos e Dispersos/genética , Filogenia , Elementos Nucleotídeos Curtos e Dispersos/genética
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