Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression of Sf-caspase-1 and Tn-caspase-1.

The baculovirus expression vector system (BEVS) is an attractive manufacturing platform for recombinant protein production in insect cells. However, baculovirus infection commonly induces host apoptosis in 3-4 days which would subsequently terminate the protein expression. Previous studies have proved that protein production by BEVS can be elevated in apoptosis-suppressed insect cells. We also developed a baculovirus vector in our previous report to inhibit the apoptosis and improve protein production in Sf9 cells. In this study, we designed five short hairpin RNA (shRNA) expression cassettes targeting a conserved region in Spodoptera frugiperda caspase-1 (Sf-caspase-1) and Trichoplusia ni caspase-1 (Tn-caspase-1), and found that introduction of C to T mutations within the stem region of the expression cassette was beneficial for the heterologous protein expression. One of the improved shRNA expression cassettes was knocked into a bacmid with the deletion of several nonessential genes. The novel baculovirus vector demonstrated the ability to suppress cell apoptosis in both Sf9 and High Five cells, and exhibited superior recombinant protein productivity of intracellularly expressed GFP and firefly luciferase and secreted glycoprotein OD-Fc. The antiapoptotic baculovirus vector developed in this study could serve as a useful tool for the protein production in scientific research and pharmaceutical industries.

Immersion immunization with recombinant baculoviruses displaying cyprinid herpesvirus 2 membrane proteins induced protective immunity in gibel carp.

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp.

Novel naïve Bayes classification models for predicting the chemical Ames mutagenicity.

Prediction of drug candidates for mutagenicity is a regulatory requirement since mutagenic compounds could pose a toxic risk to humans. The aim of this investigation was to develop a novel prediction model of mutagenicity by using a naïve Bayes classifier. The established model was validated by the internal 5-fold cross validation and external test sets. For comparison, the recursive partitioning classifier prediction model was also established and other various reported prediction models of mutagenicity were collected. Among these methods, the prediction performance of naïve Bayes classifier established here displayed very well and stable, which yielded average overall prediction accuracies for the internal 5-fold cross validation of the training set and external test set I set were 89.1±0.4% and 77.3±1.5%, respectively. The concordance of the external test set II with 446 marketed drugs was 90.9±0.3%. In addition, four simple molecular descriptors (e.g., Apol, No. of H donors, Num-Rings and Wiener) related to mutagenicity and five representative substructures of mutagens (e.g., aromatic nitro, hydroxyl amine, nitroso, aromatic amine and N-methyl-N-methylenemethanaminum) produced by ECFP_14 fingerprints were identified. We hope the established naïve Bayes prediction model can be applied to risk assessment processes; and the obtained important information of mutagenic chemicals can guide the design of chemical libraries for hit and lead optimization.

TRAF5-mediated Lys-63-linked Polyubiquitination Plays an Essential Role in Positive Regulation of RORγt in Promoting IL-17A Expression.

Retinoid-related orphan nuclear receptor γt (RORγt) is a key transcription factor for the development and function of Th17 cells. In this study, we show that tumor necrosis factor receptor-associated factor 5 (TRAF5), known as an E3 ubiquitin protein ligase and signal transducer, interacts with and ubiquitinates RORγt via Lys-63-linked polyubiquitination. TRAF5 stabilizes the RORγt protein level depending on its RING finger domain. Depletion of TRAF5 in Th17 cells destabilizes RORγt protein and down-regulates Th17-related genes, including IL-17A, an inflammatory cytokine involved in pathogenic mechanisms of several autoimmune diseases such as systemic lupus erythematosus. Moreover, up-regulation of the TRAF5 mRNA level was found in systemic lupus erythematosus patient CD4(+) T cells. Our findings reveal a direct link between TRAF5-mediated ubiquitination and RORγt protein regulation, which may aggravate inflammatory progress and provide new therapeutic drug targets for autoimmune diseases.