Construction of a Baculovirus-Silkworm Multigene Expression System and Its Application on Producing Virus-Like Particles

A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes

Biomaterials Approaches to Combating Oral Biofilms and Dental Disease

Background: Possibilities for biomaterials to impact the dental caries epidemic are reviewed with emphasis placed on novel delivery biomaterials and new therapeutic targets

Testing the optimal defence hypothesis for two indirect defences: extrafloral nectar and volatile organic compounds

Many plants respond to herbivory with an increased production of extrafloral nectar (EFN) and/or volatile organic compounds (VOCs) to attract predatory arthropods as an indirect defensive strategy. In this study, we tested whether these two indirect defences fit the optimal defence hypothesis (ODH), which predicts the within-plant allocation of anti-herbivore defences according to trade-offs between growth and defence. Using jasmonic acid-induced plants of Phaseolus lunatus and Ricinus communis, we tested whether the within-plant distribution pattern of these two indirect defences reflects the fitness value of the respective plant parts. Furthermore, we quantified photosynthetic rates and followed the within-plant transport of assimilates with 13C labelling experiments. EFN secretion and VOC emission were highest in younger leaves. Moreover, the photosynthetic rate increased with leaf age, and pulse-labelling experiments suggested transport of carbon to younger leaves. Our results demonstrate that the ODH can explain the within-plant allocation pattern of both indirect defences studied

Cyanogenesis of Wild Lima Bean (Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences

A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

BACKGROUND: Various animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A) gene. DT-A is known to kill cells by inhibiting protein synthesis. METHODS: An expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6) and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5. RESULTS: H-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis. CONCLUSIONS: Repeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine

Safety and Immunogenicity of H5N1 Influenza Vaccine Based on Baculovirus Surface Display System of Bombyx mori

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Cyr61/CCN1 Displays High-Affinity Binding to the Somatomedin B 1–44 Domain of Vitronectin

OV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed. at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, β-endorphin, and other molecules. domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties