Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph.

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID50/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.

Improved bioprocess with CHO-hTSH cells on higher microcarrier concentration provides higher overall biomass and productivity for rhTSH.

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (°C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph

O baculovírus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), um membro da família Baculoviridae, tem sido amplamente aplicado como biopesticida para o controle da lagarta de veludo, uma praga do campo de cultivo de soja. Os baculovírus são considerados agentes seguros e eficientes para esse fim, pois não infectam vertebrados, sendo seguros para a saúde de humanos e animais, bem como para o meio ambiente. O objetivo deste trabalho foi identificar proteínas obtidas da hemolinfa de Lonomia obliqua com potencial aplicação na otimização da replicação do baculovírus AgMNPV em cultura de células de inseto Sf9. Neste trabalho, a melhoria da cultura celular e replicação viral do baculovírus AgMNPV foi observada quando o meio Grace foi suplementado com 10% (v / v) de soro fetal bovino (FBS), extrato de hemolinfa a 1% (v / v) ou 3 % (v / v) de frações de hemolinfa ou subfrações de hemolinfa obtidas por purificação de hemolinfa por Cromatografia Líquida de Alto Desempenho. A hemolinfa apresentou efeito positivo na síntese de poliedros e replicação aumentada de baculovírus em células de Spodoptera frugiperda (Sf9) (TCID50 / mL), levando à melhora da cultura de células Sf9. O meio Grace suplementado com 10% (v / v) de SFB e 1% (v / v) de hemolinfa proporcionou um aumento da replicação de baculovírus, quando as células foram infectadas com multiplicidade de infecção de 1. Nesse caso, a replicação de baculovírus foi de 6.443,91 vezes maior do que o obtido com o controle: meio Grace suplementado com 10% (v / v) de FBS. Além disso, este trabalho sugere que a hemolinfa de L. obliqua poderia ter uma aplicação interessante em biotecnologia, devido ao aumento da viabilidade das células e à replicação do vírus.

Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production.

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.