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Abstract
The rotavirus nonstructural glycoprotein, NSP4, has been identified as the first viral enterotoxin capable of inducing diarrhea. To investigate the biological function of NSP4 in the inflammatory process, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by RT-PCR, cloned into TA vector, and subsequently subcloned into pET23b expression plasmid. The expression of NSP4 protein was determined by SDS-PAGE and Western blotting, then, the protein was purified by affinity chromatography on Ni-NTA-agarose column. The inflammatory effects of NSP4, namely, production of nitric oxide (NO), proinflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α), and prostaglandin E2 (PGE2), was evaluated using NSP4-stimulated RAW 264.7 murine macrophages and compared with those observed after stimulation with lipopolysaccharide (LPS). The levels of IL-1β, IL-6, and TNF-α were significantly increased, and those of NO and PGE2 also increased in NSP4-stimulated RAW 264.7 cells. These findings indicate that NSP4 plays an important role in the inflammatory response observed during rotavirus infection.
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Figure 1.
Expression of recombinant NSP4. Recombinant NSP4 was expressed by induction with IPTG, as shown by western blotting: lane 1~3: 0, 0.1, and 1 mM IPTG at 37°C, respectively; lane 4~5: 0 and 1 mM IPTG at 37°C, 2% EtOH; and lane 6~8: 0, 0.1, and 1 mM IPTG at 22°C, respectively.
Figure 2.
Purification of recombinant NSP4 after the suspension and dissolution of inclusion bodies. (A) 12% SDS-PAGE of the fraction obtained by Ni-ion affinity chromatography; lane 1: total denatured lysates; lane 2, denatured lysis supernatant; lane 3, precipi-tate; lane 4, flow-through; lane 5~6, sequential column washings; lane 7, elution of the recombinant NSP4 protein from the column; lane 8, concentration of the recombinant NSP4 protein; and lane 9, protein molecular weight marker (Invitrogen, USA). (B) Western blotting using the monoclonal anti-His antibody of each fraction obtained during purification. Lane 1~8, as in SDS PAGE (panel A).
Figure 3.
Viability of RAW 264.7 cells after 24-h induction with NSP4. Viability was determined using the colorimetric MTT assay.
Figure 4.
Effects of NSP4 on the levels of NO and PGE2 in RAW 264.7 cells. Cells were treated for 24 h with 1, 5, 10, 50, 100, and 500 pM of various NSP4s. The concentrations of nitrite and PGE2 were measured as described in the Materials and Methods. The results are expressed as the mean ± standard error from three independent experiments.
Figure 5.
Effect of NSP4 on the levels of IL-1β, IL-6, IL-10, and TNF-α in RAW 264.7 cells. The concentrations of IL-1β, IL-6, IL-10, and TNF-α released into the medium were determined by performing ELISA with the culture supernatant. They were measured as described in the Materials and Methods. Error bars shows the mean ± standard deviation of three measurements.
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