Expression of CVB3 VP1 protein in prokaryotic system and study on its immune effects

Objective:To express coxsackievirus group B3 VP1 protein in prokaryotic cells and to observe its immunogenecity.Methods:The encoding sequence for coxsackievirus group B3 VP1 antigen was cloned into pET-his and expressed in Escherichia coli BL21(DE3)pLysS,and the recombinant VP1 protein was purified from the germ.BALB/c mice were divided into experimental group and the control group randomly.The mice in experimental group were intraperitoneally given the recombinant protein,while the control group of mice was injected with PBS.The mice were immunized for 3 times at 3 week interval.Two weeks after each immunization,the serum was collected and the titres of CVB3-specific neutralizing antibodies were detected by neutralization assay;Three weeks after the last immunization,the lymphocytic proliferation activity and specific CTL cytotoxic activity of three mice of each immunized group were tested by CCK-8;Furthermore,three mice of each group were challenged with 3LD50 CVB3 and sacrificed on the 7th day in order to evaluate the virus titres in blood;The rest twelve mice of each group were challenged with lethal CVB3 to observe the protecting effect of the vaccination.Results:Titres of CVB3-specific neutralizing antibodies in sera of mice in experimental group were growing along with the immune times (P0.05).Mice immunized with the protein produced higher titers of neutralizing antibody (70.79±1.31) after the third immunization,when compared with those of PBS group (P0.05).The non-specific proliferative activity of lymphocate in the protein-immunized group was more evident than that of PBS group (P0.05),but there were no significant(P0.05) differences in specific proliferative activity or CTL cytotoxic activity between the two groups.After challenged with CVB3,the virus titers of blood in protein-immunized group were significantly lower than that of the PBS control group.Survival rates of the mice in protein-immunized group were 33.33%,while there was no surviving mice in the PBS group.The difference between the two groups was significant (P0.05).Conclusion:The VP1 protein vaccination increases highly humoral immune responses and provises more effective protections for the immunized mice from death after challenged with lethal CVB3.