Expression and immunogenicity of secreted forms of bovine ephemeral fever virus glycoproteins applied to subunit vaccine development.

AIMS: Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive or suffer from genetic instability. Therefore, we designed constructs encoding C-terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high-level antigen expression, correct glycosylation pattern and enable further simple purification with the V5 epitope tag. METHODS AND RESULTS: In this study, synthetic biology was employed to create membrane-bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media using a highly effective, single-step method. The V5 epitope tag was genetically fused to the C-termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C-terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover the immunogenicity was confirmed in mice test. CONCLUSIONS: The immuno-dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live-cell images. Our strategy presented the expression of secreted, epitope-tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK-21 cells. This indicates that high-level expression of secreted G glycoprotein is a feasible strategy for large-scale production of vaccines and improving vaccine efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The antigen expression strategy designed in this study can produce high-quality recombinant protein and reduce the amount of antigen used in the vaccine.

[The 2A region of the foot-and-mouth disease virus]. / Le peptide 2A du virus de la fièvre aphteuse.

Translational control of viral gene expression is a fundamental process essential for the vitality of all viruses. The 2A region of the foot-and-mouth disease virus encodes a sequence that mediates self-processing by a novel translational effect « ribosomal skipping ¼, rather than proteolysis. The 2A acts as an esterase within the eukaryotic ribosome to hydrolyze the link between the nascent polypeptide and the t-RNA in the P-site rather than form the Gly-Pro peptide bond at the 2A/2B junction. Translation can continue after hydrolysis, so sequences upstream and downstream of 2A emerge as distinct polypeptides in a fixed stochiometry. Use of 2A and 2A-like peptides to link the sequences encoding several proteins in the same open reading frame has led to their increasing use as important tools in biotechnology and biomedicine.

Human salivary glucose analysis by high-performance ion-exchange chromatography and pulsed amperometric detection.

The aim of this project was to develop an analytical procedure with the required level of sensitivity for the determination of glucose concentrations in small volumes of unstimulated fasting whole saliva. The technique involves high-performance ion-exchange chromatography at high pH and pulsed amperometric detection. It has a high level of reproducibility, a sensitivity as low as 0.1 mumol/l and requires only 50 microliters samples (sensitivity = 0.002 pmol). Inhibition of glucose metabolism, by procedures such as collection into 0.1% (w/v) sodium fluoride, was shown to be essential if accurate results are to be obtained. Collection on to ice followed by storage at -20 degrees C was shown to be unsuitable and resulted in glucose loss by degradation. There were inter- and intraindividual variations in the glucose concentration in unstimulated mixed saliva (range; 0.02-0.4 mmol/l). The procedure can be used for the analysis of other salivary carbohydrates and for monitoring the clearance of dietary carbohydrates from the mouth.

Translating the message.

The "one gene = one protein" model for understanding the genome is well-known. But, as with most biological "rules", it has its exceptions. The viral family that brings polio, the common cold and foot-and-mouth disease translates its entire genome into one long "polyprotein". The study of this translation strategy is bringing to light new targets for fighting disease and new techniques for the biotech industry.

Genomic instability in haematopoietic cells of F1 generation mice of irradiated male parents.

Preconceptional paternal irradiation has been implicated as a causal factor in childhood cancer and it has been suggested that this exposure to radiation may play a role in the occurrence of childhood leukaemia clusters in the vicinity of nuclear installations. Using a transgenic mouse system employing a lambda shuttle vector allowing mutations (in the lacI gene) to be analysed in vitro, we have investigated the possibility that preconceptional paternal irradiation can lead to such transgenerational transmission of genomic instability. We have examined the mutation frequencies in vector recovered from the bone-marrow cells of the F1 offspring of male parents exposed to doses of gamma-rays of 0.1-4 Gy. Our results show that as parental dose increases there is a trend towards higher mutation frequency in vector recovered from the DNA of bone-marrow of F1 progeny. At 4 Gy the frequency of mutations was increased by a factor of approximately two (control mutation frequency, 2.39 x 10(-5); mutation frequency in offspring of 4 Gy male group, 4.26 x 10(-5); P < 0.001). We were unable to confirm reports of spermatogenesis stage sensitivity. The 2-fold increase in mutation frequency was evident in offspring derived from stored spermatozoa (irradiated transgenic males mated with unirradiated non-transgenic females 1-7 days after irradiation). Our data indicates that there exists a route for transgenerational transmission of factor(s) leading to genomic instability in F1 progeny, resulting from preconceptional paternal irradiation.

Human salivary sugar clearance after sugar rinses and intake of foodstuffs.

The aetiology of dental caries is in part related to the retention time of dietary carbohydrates in the oral cavity and their subsequent metabolism by the oral bacteria. Salivary clearance of fermentable carbohydrates from three different foodstuffs was examined in 5 subjects and analyses performed by high-performance anion-exchange chromatography with pulsed amperometric detection. The clearance of glucose, fructose, sucrose, maltose and sorbitol rinses was studied as well as that of chocolate bars, white bread and bananas. Of the sugar rinses studied, sucrose was removed from saliva most rapidly whilst appreciable levels of sorbitol remained even after 1 h. Clearance of residual carbohydrates from bananas and chocolate bars seemed marginally faster than in the case of bread, but sucrose levels still tended to fall more quickly than other carbohydrates studied. Surprisingly, carbohydrate residues from the three foods studied were still present in the mouth even 1 h after ingestion, which is longer than has hitherto been reported.