Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Targovnik, Alexandra Marisa; Villaverde, Marcela Solange; Arregui, Mariana Bernadett; Fogar, M. N.; Taboga, Oscar; Glikin, Gerardo C.; Finocchiaro, Liliana M.E.; Cascone, Osvaldo; Miranda, Magdalena

doi:10.1016/j.procbio.2014.03.013

Cited by 5 publications

(6 citation statements)

References 39 publications

(58 reference statements)

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“…DomIIIHFBI expression level in R. nu larvae was 4.5 mg per g of larva according to the gel densitometry analysis, higher than that reached for other recombinant proteins previously produced in our laboratory with the same expression system: wheat germ agglutinin (346.6 ± 88.5 µg per g of larva) [43], feline interferon alpha (116 ± 6.3 µg per g of larva) [44] and Influenza A H1N1 neuraminidase (1.2 mg per g of larva) [45].…”

Section: Accepted Manuscriptmentioning

confidence: 54%

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Smith

Targovnik

Cerezo

et al. 2017

Protein Expression and Purification

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Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus. The antigen was expressed as a fusion to hydrophobin I (DomIIIHFBI) to easily purify it by an aqueous two-phase system (ATPS) and to efficiently immobilize it in immunoassay plates. A high level of recombinant DomIIIHFBI was obtained in R. nu, where yields reached 4.5 mg per g of larva. Also, we were able to purify DomIIIHFBI by an ATPS with 2% of Triton X-114, reaching a yield of 73% and purity higher than 80% in a single purification step. The recombinant DomIIIHFBI was efficiently immobilized in hydrophobic surface plates. The immunoassay we developed with the immobilized antigen was able to detect IgG specific for dengue virus type 2 in serum samples and not for other serotypes.

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Section: Accepted Manuscriptmentioning

confidence: 54%

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Smith

Targovnik

Cerezo

et al. 2017

Protein Expression and Purification

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“…In comparison with the process in S. frugiperda larvae previously reported by Targovnik et al (2014) some differences could be remarked: using the same inoculum of recombinant baculovirus for the larval infection, the optimal day of harvest for rFeIFNβ was 4 DPI, while for rFeIFNα it was 5 DPI. Each cytokine presented different levels of expression in crude S. frugiperda extracts (7.5 × 10 5 and 1.1 × 10 6 UI mL −1 ), demonstrating that the yield of the platform varies even among two cytokines belonging to type I IFN family and both with feline origin.…”

Section: Purificationmentioning

confidence: 83%

“…First, to determine rFeIFNβ expression and localization, we infected Sf9 cells with AcMNPV-FeIFNβ at MOIs of 0.05, 2 and 5 and analyzed the culture supernatants on different DPI. Although the Sf9 cell line used was adapted to serum-free medium which would facilitate further purification of product, we added 1% FBS, because it not only led to a higher cell growth rate, but also generated a tenfold increase in IFN expression (Targovnik et al 2014). The expression kinetics curve showed that the rFeIFNβ antiviral activity increased gradually, achieving a maximum at 3 DPI (7.5 ± 1.8 × 10 4 IU mL −1 ) at MOIs 0.05 and 2, higher than at MOI 5 (5 × 10 4 IU mL −1 ).…”

Section: Expression Of Rfeifnβ In Insect Cell Lines and Larvaementioning

confidence: 99%

“…Second, we achieved the expression of rFeIFNβ directly in S. frugiperda larvae. The inoculum of AcMNPV-FeIFNβ used for infection was 5 × 10 5 PFU per larva, because we have previously shown that 1 × 10 6 PFU result lethal for the larvae, while 1 × 10 5 PFU result in poor protein expression levels (Targovnik et al 2014). Larvae started showing signs of infection, such as motility and appetite loss, at 1 DPI.…”

Section: Expression Of Rfeifnβ In Insect Cell Lines and Larvaementioning

confidence: 99%

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Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus

et al. 2018

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Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ was recovered after purification with a specific activity of 1 × 10 IU mg. By this method, we obtained 8.9 × 10 IU of purified rFeIFNβ per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 10 IU mL, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNβ was confirmed by peptide--glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNβ production in laboratory research or veterinary medicine applications.

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“…El IFN-γ expresado en E.coli se acumula en los cuerpos de inclusión y la extracción desde cuerpos de inclusión implica pasos experimentales adicionales para un re-plegamiento de proteínas a fin de obtener una conformación correcta. También se ha logrado expresar IFNs de diferentes especies en sistemas que realizan glicosilación diferente a la de células de mamíferos, como ser las células de insecto [103] y las levaduras [104]. En todos los casos de obtuvieron proteínas con actividad biológica semejante a la de sus homólogos naturales.…”

Section: Expresión De Interferones Recombinantesunclassified

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity

Quintana

Barone

Forlenza

et al. 2018

Journal of Virological Methods

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Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.

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Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Cited by 5 publications

References 39 publications

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity

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