Improvement of recombinant protein production by an anti-apoptotic protein from hemolymphof Lonomia obliqua

Apoptosis is a major problem in animalcell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotaviruslike-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host celldeath. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein productionof BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able toprotect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinantprotein in BEVS/IC system.

Antiviral activity of the hemolymph of Lonomia obliqua (Lepidoptera: Saturniidae).

Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3+/-1.25 x 10(7) to 2.1+/-1.5 x 10(5) TCID(50)mL(-1)) in measles virus production and a 61-fold reduction (from 2.8+/-1.08 x 10(9) to 4.58+/-1.42 x 10(7)mL(-1)) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response.

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

Gene expression in insect cells is an advantageous system for recombinant protein production, mainly because of its capacity to produce complex proteins with correct post-translational modifications. Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60%. In this work, the kinetic parameters for cell growth and recombinant rabies virus glycoprotein production were determined in cultures of transfected Drosophila melanogaster Schneider 2 (S2) cells expressing recombinant rabies virus glycoprotein (rRVGP), enriched and non-enriched with the hemolymph of Lonomia obliqua (Hb). The highest concentration of rRVGP was achieved at the beginning of the culture enriched with Hb, indicating that the cells produce greater amounts of rRVGP per cell (specific rRVGP concentration) at the early exponential growth phase. After day 8, a decrease in the concentration of rRVGP (ng/mL) was observed, probably due to protein decomposition. The average specific rRVGP production rate (ìrRVGP) was 30 ng rRVGP/107cell.day, higher than that observed in the non-enriched culture.

Gynandromorphism in Periga circumstans Walker (Lepidoptera: Saturniidae)

This note reports the occurrence of gynandromorphism in Periga circumstans Walker in Brazil. The specimen was collected in Igaratá, state of São Paulo. General aspects and photograph of the insect are presented.

Esta comunicação relata a ocorrência de ginandromorfismo em Periga circumstans Walker. O espécime foi coletado em Igaratá, SP. São apresentados aspectos gerais e fotografias do inseto.

Identification and characterization of a baculovirus from Lonomia obliqua (Lepidoptera: Saturniidae).

A baculovirus has been isolated from larvae of Lonomia obliqua, a Saturniidae of medical importance due to a potent toxin found in their spines. Electron Microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra of approximately 1 microm in diameter containing multiple nucleocapsids per envelope. This baculovirus was thus named Lonomia obliqua multicapsid nucleopolyhedrovirus (LoobMNPV). Restriction endonuclease profiles of viral DNA digested with three restriction enzymes were obtained and the genome size was estimated to be 95.52 +/- 2.3 kbp. The polyhedrin gene of LoobMNPV was identified and its DNA sequence was determined. Phylogenetic analysis of the polyhedrin gene showed that the LoobMNPV polyhedrin belongs to group I NPV and that it is closely related to the polyhedrin of the NPV of Amsacta albistriga.