Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 174
Filter
1.
Article in English | WPRIM | ID: wpr-816639

ABSTRACT

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that produces an acute, usually non-fatal, febrile illness including Mayaro fever. Like other alphaviruses, the MAYV E1 and E2 envelope glycoproteins are major viral surface antigens that play a key role in host recognition and infection. Here, we report expression and purification methods for recombinant MAYV E1 (rE1) and rE2 using a baculovirus system. Enzyme-linked immunosorbent assays (ELISA) revealed that rE1 and rE2 were antigenic and reacted with human anti-MAYV IgG and IgM. Cross-reactivity was also confirmed with human anti-Chikungunya virus (CHIKV) IgG and IgM. Furthermore, we developed an immunochromatographic strip test (IST) with rE2 to diagnose MAYV infection. Thus, purified rE2 may be valuable tool for rapidly diagnosing MAYV infection.


Subject(s)
Alphavirus , Antigens, Surface , Baculoviridae , Enzyme-Linked Immunosorbent Assay , Fever , Glycoproteins , Humans , Immunoglobulin G , Immunoglobulin M
2.
Rev. argent. microbiol ; 51(4): 381-385, dic. 2019. graf
Article in English | LILACS | ID: biblio-1057404

ABSTRACT

Abstract The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is an important maize pest. Due to the environmental impact and emergence of resistance caused by chemical pesticides and transgenic events, the use of baculoviruses becomes a safe and useful alternative for its control in integrated pest management strategies. Here we report the identification of a novel isolate of a granulovirus of S. frugiperda native to the central region of Argentina, named SfGV ARG. We observed that larvae infected with SfGV ARG showed a yellowish coloration, swollen body and, in some cases, severe lesions in the last abdominal segments. We confirmed the identity of the isolate by sequencing fragments of the lef-8, lef-9 and granulin genes and by calculating evolutionary distances using the Kimura-2-Parameter model. SfGV ARG DNA restriction pattern allowed to estimate a genome of at least 135 kb.


Resumen La oruga militar tardía, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), es una plaga importante del maíz. Debido al impacto ambiental y a la aparición de resistencia causados por los pesticidas químicos y los eventos transgénicos, el uso de baculovirus resulta una alternativa útil y saludable para su control en estrategias de manejo integrado de plagas. En este trabajo reportamos la identificación de un nuevo aislamiento del granulovirus de la S. frugiperda nativo de la región central de Argentina, SfGV ARG. Se observó que larvas infectadas con SfGV ARG mostraron coloración amarillenta, hinchazón y, en algunos casos, lesiones graves en los últimos segmentos abdominales. Se confirmó la identidad del aislamiento por secuenciación de fragmentos de los genes lef-8, lef-9y granulina, y por cálculo de distancias evolutivas usando el parámetro de Kimura-2. El patrón de restricción generado con el ADN genómico de SfGV ARG permitió estimar un tamaño de genoma de al menos 135 kb.


Subject(s)
Pest Control, Biological/methods , Spodoptera/parasitology , Granulovirus/isolation & purification , Pesticides , Argentina , Baculoviridae/isolation & purification , Agricultural Pests
3.
Chinese Journal of Biotechnology ; (12): 1135-1142, 2019.
Article in Chinese | WPRIM | ID: wpr-771814

ABSTRACT

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.


Subject(s)
Animals , Baculoviridae , Genetic Vectors , Humans , Recombinant Proteins , Sf9 Cells , Spodoptera
4.
Chinese Journal of Biotechnology ; (12): 577-588, 2019.
Article in Chinese | WPRIM | ID: wpr-771351

ABSTRACT

Baculovirus expression vector system (BEVS) has been successfully applied to the over-expression of various proteins, thus providing sufficient materials for vaccine research. Compared to other systems, BEVS has many advantages: baculovirus solely being parasitic in invertebrates, the resultant products conferring high safety to mammalian, high expression level of recombinant proteins, preferable folding for eukaryotic protein, proper post-translational modification required for biological function, suitable for multiple genes co-expression and large-scale production with serum-free culture media. To better understand the advantages and prospective of BEVS for the vaccine research, this article will review the development of BEVS and its application on vaccine research.


Subject(s)
Animals , Baculoviridae , Genetic Vectors , Prospective Studies , Recombinant Proteins , Vaccines
5.
Article in English | WPRIM | ID: wpr-758963

ABSTRACT

The Schmallenberg virus (SBV) is an orthobunyavirus that causes abortions, stillbirths, and congenital defects in pregnant sheep and cattle. Inactivated or live attenuated vaccines have been developed in endemic countries, but there is still interest in the development of SBV vaccines that would allow Differentiating Infected from Vaccinated Animals (DIVA). Therefore, an attempt was made to develop novel DIVA-compatible SBV vaccines using SBV glycoproteins expressed in baculovirus. All vaccines and phosphate buffered saline (PBS) controls were prepared with adjuvant and administered subcutaneously to cattle at 6 month of age. The first trial included 2 groups of animals vaccinated with either carboxyl-terminus glycoprotein (Gc) or PBS and boosted after 2 weeks. In the second trial, 3 groups of cattle were administered either Gc, Gc and amino-terminus glycoprotein (Gn), or PBS with a booster vaccination after 3 weeks. The animals were challenged with SBV 9 days after the booster vaccination in the first study, and 3 weeks after the booster vaccination in the second study. Using a SBV Gc-specific enzyme-linked immunosorbent assay, antibodies were first detected in serum samples 14 days after the first vaccination in both trials, and peaked on days 7 and 9 after the booster in the first and second trials, respectively. Low titers of neutralizing antibodies were detected in serum from only 3/6 and 2/4 animals in the first and second trial, respectively, at 14 days after the first vaccination. The titers increased 2 to 3-fold after the booster vaccination. SBV-specific RNA was detected in the serum and selective tissues in all animals after SBV challenge independent of vaccination status. The SBV candidate vaccines neither prevented viremia nor conferred protection against SBV infection.


Subject(s)
Animals , Antibodies , Antibodies, Neutralizing , Baculoviridae , Cattle , Congenital Abnormalities , Enzyme-Linked Immunosorbent Assay , Glycoproteins , Orthobunyavirus , RNA , Sheep , Stillbirth , Vaccination , Vaccines , Vaccines, Attenuated , Vaccines, Subunit , Viremia
6.
Article in English | WPRIM | ID: wpr-719490

ABSTRACT

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.


Subject(s)
Animals , Baculoviridae , Blotting, Western , Discrimination, Psychological , Enzyme-Linked Immunosorbent Assay , Insecta , Molecular Weight , Newcastle disease virus , Newcastle Disease , Nucleoproteins , Sensitivity and Specificity
7.
Article in English | WPRIM | ID: wpr-719488

ABSTRACT

PURPOSE: Respiratory syncytial virus (RSV) can cause serious respiratory illnesses such as pneumonia, asthma, and bronchiolitis in infants and elderly or immunocompromised individuals. An RSV vaccine has yet to be developed; only prophylactic anti-RSV antibody is commercially available. So, we investigated whether our vaccine candidate is able to induce type 1 CD4+ T helper (Th1), CD8+ T-cell responses, and protective immunity without vaccine-enhanced disease (VED) against RSV. MATERIALS AND METHODS: We used RSV G protein fragment (Gcf A) with recombinant baculovirus capable of expressing the RSV M2 protein (Bac M2) as a vaccine candidate, and injected this vaccine (Gcf A/Bac M2) intramuscularly, and challenged with RSV intranasally into mice. Enzyme-linked immunosorbent assay, flow cytometry, plaque assay, and weight measurement were performed to confirm humoral immunity, cellular immunity, and protective immunity. RESULTS: The Gcf A/Bac M2 formulation induced a stronger IgG response to Gcf A than Gcf A inoculation alone, and the ratio of IgG1/IgG2a indicated that the responses shifted predominantly to Th1. In addition, both RSV G-specific Th1 responses and RSV M2-specific CD8+ T-cell responses were induced, and G protein-associated eosinophilic infiltration was suppressed compared to the control group. Moreover, the Gcf A/Bac M2 group showed effective protection after an RSV challenge. CONCLUSION: Bac M2 could serve as a vaccine with intrinsic adjuvant activity, and the Gcf A/Bac M2 shows promise as a vaccine candidate for inducing protective immunity without inciting VED.


Subject(s)
Aged , Animals , Asthma , Baculoviridae , Bronchiolitis , Enzyme-Linked Immunosorbent Assay , Eosinophils , Flow Cytometry , GTP-Binding Proteins , Humans , Immunity, Cellular , Immunity, Humoral , Immunoglobulin G , Infant , Mice , Pneumonia , Respiratory Syncytial Viruses , T-Lymphocytes
8.
Article in English | WPRIM | ID: wpr-719485

ABSTRACT

PURPOSE: Enzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein. MATERIALS AND METHODS: rHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice. RESULTS: Our ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model. CONCLUSION: An ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.


Subject(s)
Animals , Antibodies , Antibodies, Neutralizing , Baculoviridae , Diagnosis , Enzyme-Linked Immunosorbent Assay , Hemagglutination , Humans , Influenza A virus , Influenza Vaccines , Influenza, Human , Methods , Mice , Models, Animal , Orthomyxoviridae , Pandemics , Vaccination
9.
Article in English | WPRIM | ID: wpr-742221

ABSTRACT

We developed a Rapid Diagnostic Test (RDT) kit for detecting IgG/IgM antibodies against Zika virus (ZIKV) using monoclonal antibodies to the envelope (E) and non-structural protein 1 (NS1) of ZIKV. These proteins were produced using baculovirus expression vector with Sf9 cells. Monoclonal antibodies J2G7 to NS1 and J5E1 to E protein were selected and conjugated with colloidal gold to produce the Zika IgG/IgM RDT kit (Zika RDT). Comparisons with ELISA, plaque reduction neutralization test (PRNT), and PCR were done to investigate the analytical sensitivity of Zika RDT, which resulted in 100% identical results. Sensitivity and specificity of Zika RDT in a field test was determined using positive and negative samples from Brazil and Korea. The diagnostic accuracy of Zika RDT was fairly high; sensitivity and specificity for IgG was 99.0 and 99.3%, respectively, while for IgM it was 96.7 and 98.7%, respectively. Cross reaction with dengue virus was evaluated using anti-Dengue Mixed Titer Performance Panel (PVD201), in which the Zika RDT showed cross-reactions with DENV in 16.7% and 5.6% in IgG and IgM, respectively. Cross reactions were not observed with West Nile, yellow fever, and hepatitis C virus infected sera. Zika RDT kit is very simple to use, rapid to assay, and very sensitive, and highly specific. Therefore, it would serve as a choice of method for point-of-care diagnosis and large scale surveys of ZIKV infection under clinical or field conditions worldwide in endemic areas.


Subject(s)
Antibodies , Antibodies, Monoclonal , Baculoviridae , Brazil , Cross Reactions , Dengue Virus , Diagnosis , Diagnostic Tests, Routine , Enzyme-Linked Immunosorbent Assay , Flavivirus , Gold Colloid , Hepacivirus , Immunoglobulin G , Immunoglobulin M , Korea , Methods , Neutralization Tests , Point-of-Care Systems , Polymerase Chain Reaction , Reagent Kits, Diagnostic , Sensitivity and Specificity , Sf9 Cells , Yellow Fever , Zika Virus
10.
Biosci. j. (Online) ; 33(3): 703-712, may/jun. 2017. ilus
Article in English | LILACS | ID: biblio-966229

ABSTRACT

Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.


Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.


Subject(s)
Bombyx , Baculoviridae , Nucleocapsid , Lepidoptera , Malpighian Tubules
11.
Article in English | WPRIM | ID: wpr-115771

ABSTRACT

Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.


Subject(s)
Animals , Antibodies , Baculoviridae , Blotting, Western , Certification , Enterovirus B, Human , Enzyme-Linked Immunosorbent Assay , Foot-and-Mouth Disease , Mass Spectrometry , Methods , Microscopy, Electron , Neutralization Tests , Serologic Tests , Swine Vesicular Disease , Swine , Virus Diseases
12.
Article in English | WPRIM | ID: wpr-139534

ABSTRACT

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.


Subject(s)
Animals , Antibodies , Asian Continental Ancestry Group , Baculoviridae , Cell Culture Techniques , Chromatography , Dogs , Enzyme-Linked Immunosorbent Assay , Humans , Nucleoproteins , Rabies virus , Rabies , Sensitivity and Specificity , Vaccination
13.
Article in English | WPRIM | ID: wpr-139531

ABSTRACT

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.


Subject(s)
Animals , Antibodies , Asian Continental Ancestry Group , Baculoviridae , Cell Culture Techniques , Chromatography , Dogs , Enzyme-Linked Immunosorbent Assay , Humans , Nucleoproteins , Rabies virus , Rabies , Sensitivity and Specificity , Vaccination
14.
Article in English | WPRIM | ID: wpr-50089

ABSTRACT

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.


Subject(s)
Animals , Baculoviridae , Blotting, Western , Clone Cells , Cloning, Organism , Enzyme-Linked Immunosorbent Assay , Influenza, Human , Insecta , Mice , Microscopy, Electron, Transmission , Toxoplasma , Toxoplasmosis , Virion
15.
Article in English | WPRIM | ID: wpr-43943

ABSTRACT

PURPOSE: Nitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment. RESULTS: Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses. CONCLUSION: Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.


Subject(s)
Animal Experimentation , Antibody Formation , Baculoviridae , Collodion , Filtration , Hemagglutinins , Influenza, Human , Membranes , Neuraminidase , Orthomyxoviridae , Sucrose , Ultracentrifugation , Vaccines
16.
Mem. Inst. Oswaldo Cruz ; 111(8): 535-538, Aug. 2016. graf
Article in English | LILACS | ID: lil-788999

ABSTRACT

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.


Subject(s)
Baculoviridae/chemistry , Baculoviridae/metabolism , Hepatitis A virus/chemistry , Viral Proteins/biosynthesis , Baculoviridae/genetics , Gene Expression Regulation, Viral , Genetic Vectors , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solubility , Viral Proteins/chemistry , Viral Proteins/genetics
17.
National Journal of Andrology ; (12): 591-595, 2016.
Article in Chinese | WPRIM | ID: wpr-262349

ABSTRACT

<p><b>Objective</b>To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells.</p><p><b>METHODS</b>Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy.</p><p><b>RESULTS</b>The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells.</p><p><b>CONCLUSIONS</b>Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.</p>


Subject(s)
Animals , Baculoviridae , Blotting, Western , DNA, Complementary , Genetic Vectors , Green Fluorescent Proteins , Mice , Plasmids , Polymerase Chain Reaction , Proteins , Genetics , Metabolism , Recombinant Fusion Proteins , Sf9 Cells , Transfection
18.
Chinese Journal of Virology ; (6): 93-100, 2016.
Article in Chinese | WPRIM | ID: wpr-296211

ABSTRACT

Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses.


Subject(s)
Animals , Baculoviridae , Genetics , Physiology , Insecta , Virology , Viral Proteins , Genetics , Metabolism , Virion , Genetics , Physiology
19.
Rio de Janeiro; s.n; 2016. xxiii, 165 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-971516

ABSTRACT

O vírus da hepatite A (HAV) é o principal agente etiológico das hepatites virais agudas e estima-se que ocasione 1,5 milhão de novas infecções no mundo anualmente. Apesar da eficácia apresentada pelas vacinas comerciais, a replicação do HAV em cultura de células é lenta e apresenta baixo rendimento, o que torna a sua produção difícil e dispendiosa. Nesse contexto, o uso de proteínas recombinantes do HAV pode representar uma alternativa ao modelo de vacina existente. O presente trabalho teve como objetivo expressar e avaliar a imunogenicidade das partículas virais destituídas de ácido nucleico (VLPs) e da proteína VP1 do HAV. As VLPs foram geradas a partir do sistema baculovírus/células de inseto através da infecção de células de inseto com baculovírus recombinantes contendo as regiões P1-2A e P3 do genoma do HAV. A poliproteína P1-2A foi expressa, clivada e se organizou em estruturas que continham epitopos conformacionais de neutralização. Partículas com características similares ao HAV foram identificadas por microscopia eletrônica, sugerindo que as proteínas expressas se montaram em VLPs. Em paralelo, a VP1 foi expressa nos sistemas baculovírus/células de inseto e Escherichia coli. Níveis mais elevados de expressão foram obtidos em E. coli, o que consequentemente aumentou a taxa de recuperação da proteína purificada. A VP1 derivada de E. coli foi utilizada nos ensaios de antigenicidade e mostrou reatividade frente aos soros de pacientes infectados pelo HAV, o que demonstra seu potencial como um marcador para diagnóstico. Para os estudos de imunogenicidade, a VP1 derivada de E. coli foi combinada a dois adjuvantes, o hidróxido de alumínio (Al(OH)3) e o adjuvante a base de saponina...


The hepatitis A virus (HAV) is the primary etiological agent of acute viral hepatitis and it is estimated to cause 1.5 million of new infections each year worldwide. Despite the effectiveness of commercial vaccines, the replication of HAV in cell culture is slow and haslow yield, which makes it difficult and expensive to manufacture. In this context, the use of recombinant HAV proteins could represent an alternative model to the existing vaccines. This study aimed to express and evaluate the immunogenicity of virus-like-particles (VLPs) and VP1 protein of HAV. The VLPs were generated using the baculovirus/insect cell expression system by the infection of insect cells with recombinant baculovirus containing HAV P1-2A and P3 regions. The P1-2A polyprotein was successfully expressed, cleaved and organized into structures that contained neutralizing conformational epitopes. HAV-like particles were identified by electron microscopy, suggesting that the expressed proteins were assembled into VLPs. In parallel, VP1 was expressed in both baculovirus/insect cell and Escherichia coliexpression systems. Higher protein levels were obtained in E. coli, which consequently increased the recovery rate after protein purification. The E. coli-derived VP1 was then used in antigenicity assays and showed reactivity against sera from patients infected with HAV, demonstrating its potential as a diagnostic marker. For immunogenicity studies, the E. coli-derived VP1 was combined with two adjuvants, aluminum hydroxide (Al(OH)3) and the saponin-based adjuvant...


Subject(s)
Humans , Hepatitis A virus , Baculoviridae , Escherichia coli , Immunogenetics , Adjuvants, Immunologic
20.
Protein & Cell ; (12): 373-382, 2016.
Article in English | WPRIM | ID: wpr-757133

ABSTRACT

The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.


Subject(s)
Amino Acid Sequence , Animals , Baculoviridae , Genetics , Codon , Gene Expression Profiling , Gene Expression Regulation , Glycosylation , Molecular Sequence Data , Protein Sorting Signals , Genetics , Recombinant Proteins , Genetics , Sf9 Cells , Spodoptera , Genetics
SELECTION OF CITATIONS
SEARCH DETAIL