RESUMO
To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.
Assuntos
Baculoviridae/genética , Fragmentos Fc das Imunoglobulinas/biossíntese , Interferon-alfa/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antivirais/metabolismo , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Fusão Gênica , Vetores Genéticos , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Insetos , Interferon alfa-2 , Interferon-alfa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Nanomaterial structure-activity relationships (nano-SARs) for metal oxide nanoparticles (NPs) toxicity were investigated using metrics based on dose-response analysis and consensus self-organizing map clustering. The NP cellular toxicity dataset included toxicity profiles consisting of seven different assays for human bronchial epithelial (BEAS-2B) and murine myeloid (RAW 264.7) cells, over a concentration range of 0.39-100 mg L(-1) and exposure time up to 24 h, for twenty-four different metal oxide NPs. Various nano-SAR building models were evaluated, based on an initial pool of thirty NP descriptors. The conduction band energy and ionic index (often correlated with the hydration enthalpy) were identified as suitable NP descriptors that are consistent with suggested toxicity mechanisms for metal oxide NPs and metal ions. The best performing nano-SAR with the above two descriptors, built with support vector machine (SVM) model and of validated robustness, had a balanced classification accuracy of ~94%. An applicability domain for the present data was established with a reasonable confidence level of 80%. Given the potential role of nano-SARs in decision making, regarding the environmental impact of NPs, the class probabilities provided by the SVM nano-SAR enabled the construction of decision boundaries with respect to toxicity classification under different acceptance levels of false negative relative to false positive predictions.