During the experimental procedures the animals were monitored daily and were kept with free access to food and water. Mice found in a moribund condition (fatigue and “hunchback-like posture”) were selleck inhibitor instantly euthanized. This project was approved by The Animal Research Ethics Committee of Ume? University, Sweden. Evaluation of immune response To evaluate and compare the immune responses after vaccination and infection, eight animals were vaccinated with cDNA encoding N, four with cDNA containing the open reading frame of the GN/GC poly-protein and two groups, each containing four animals, were immunised with either the GN or the GC construct. To analyse the immune responses after infection, one group consisting of nine mice were infected with 2.4 �� 104 PFU of RVFV. At day 14 p.i.
the animals were euthanized and samples collected. As negative controls, four mice were immunised with the pcDNA3.1 vector without insert and another four mice were injected with sterile PBS and kept under the same conditions. Challenge study A total of 30 mice were used in the challenge study, eight of which were vaccinated with cDNA encoding the RVFV N protein and eight with the GN/GC construct. As controls, eight animals were vaccinated with an irrelevant gene (encoding the N protein of the Puumala virus, PUU-N) and six animals with pcDNA 3.1 vectors without insert. After four rounds of immunisations, half of the mice of each vaccination group were challenged with 2.4 �� 103 and half with 2.4 �� 104 PFU of RVFV. Blood samples were collected every alternate day until the end of the experiment at day 17 p.
i. Antigen production and purification For antigen production and prokaryotic expression, cDNA encoding the full-length N protein (aa 1�C245) of RVFV was ligated into pET-14b (Novagen, Darmstadt, Germany) and cDNA encoding truncated N derivatives, N1 (aa 1�C100), N2 (aa 71�C170), N3 (aa 141�C245), N1/2 (aa 1�C170) and N2/3 (aa 71�C245), were inserted into pET101/D-TOPO? or pET151/D TOPO? (Invitrogen). The primer sequences are shown in Table Table11. DNA constructs expressing the N protein and truncated N derivatives were expressed in Escherichia coli (E. coli) BL21 DE3 (Invitrogen). Briefly, transformed bacteria were grown in Luria-Bertani media supplemented with 100 ��g/ml carbencillin to OD A600 of 0.7. Expression of the antigens was induced by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM. The purification of the full length Dacomitinib N protein expressed from a poly-histidine-fusion vector was performed with metal chelating chromatography using Ni-NTA Agarose (Qiagen GmbH, Hilden, Germany), essentially as described previously .