| Porcine Rotavirus(PRV) belongs to Reoviridae family, Rotavirus genus. It is one of the major pathogens of porcine viral diarrhea. This disease spreads mainly through intestinal route. The incidence rate can reach up to80%and the mortal rate can reach up to20%among piglets under4weeks old, which poses great threat to domestic hog industry and leads to great economic losses. Recent studies indicate that non-structural protein4of PRV plays an important role in viral replication and pathogenesis. So it is of great significance to study the biological functions of this protein.In this study,112aa-175aa of non-structural protein4of PRV strain JL94was expressed in Rosseta and the135th,136th and138th amino acids were mutated. Both NSP4and mutated NSP4protein were purified and administrated to young mice by the intraperitoneal (IP) route. In order to compare the pathogenicity of the two proteins, diarrhea level was observed and body weights were recorded at different time. The result showed that there was a significant enhancement on the pathogenicity of mutated protein.In addition, Lactobacillus casei393was selected as an antigen delivery vehicle. The main protective antigen VP7and the NSP4were selected as the target genes. The selective gene VP7and NSP4were directional inserted into the surface expression vector pPG-1and secretary expression vector pPG-2to construct recombinant strains pPG-1-VP7/L,.casei393, pPG-1-NSP4-VP7/L.casei393and pPG-2-NSP4-VP7/L.casei393. In order to explore if NSP4can function as mucosal adjuvant in this expression system, three groups of seven female mice were immunized respectively. Every mouse received dose of2×109colony-forming units (c.f.u.) of recombinant strains. The immune protocol was administered on three consecutive days at each10days. Collected serum, stool and vaginal lavage of mice at day1,2,3,5,7,9after each immunization, detected special slgA level in the vaginal lavage, slgA level in the stool and special IgG level in the Serum using indirect ELISA method. Animal experiments were performed to detect the ability of neutralizing enterotoxin of the serum.The results of indirect ELISA showed that the mice immunized with recombinant strains could produce remarkable special slgA level in the vaginal lavage, in the stool of mice and special IgG in the serum of mice. Compared with the control group, discrepancy predominance(P<0.05). There are also differences at the peak of antibody level among immunization groups. Compared with group pPG-1-VP7/L. casei393, group pPG-1-NSP4-VP7/L casei393induced the body to produce higher level of both serum and mucosal antibody, indicating that NSP4could function as mucosal adjuvant. Compared with group pPG-1-VP7/L.casei393, group pPG-2-NSP4-VP7/L.casei393induced the body to produce higher level of both serum and mucosal antibody, indicating that the immune effect of secretary expression is better than that of surface expression. The result of neutralization experiment showed that the number of diarrhea mice in the group with serum was less than that of control group and the increase of average body weight in the group with serum was more than that of control group, indicating that the antibody against NSP4could neutralize part of the toxicity of the enterotoxin. All theses work provided a theoretical foundation for further study on the pathogenesis of porcine rotavirus and established a material foundation for new vaccine of porcine rotavirus. |
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