Purification And Immunogenicity Of VP1 Epitope Embeded S-layer Protein SLP-MultiVP1 Of Multi-type FMDV

Foot-and-mouth disease (FMD) is a highly contagious disease mainly caused by FMDV infection. The capsid of virous was composed with 60 subunites of four structural proteins VP1, VP2, VP3 and VP4. Among these structure proteins, VP1 was the main part that can induce neutralizing antibodies. Once FMD outbreak, it will not only bring huge loss to animal husbandry but also affect stability of economy and polity. Currently, prevention of FMD mainly rely on inactivated virus of all vaccine but many bottleneck problem exist in practice such as tedious purification technology of virus, vaccinated efficacy showed instability. The contents of this study are as follows:1.Constructed pGEX-SLP-MultiVP1 prokaryotic expression genetic vector which contain VP1 epitope was induced by IPTG, successfully obtained the embedded protein which were about 80 kDa.The embedded protein was purified by method of combining Pierce(?) GST Spin Purification Kit with gel slices and obtained the target protein which has relative molecular mass of 54 kDa after of which GST tag protein excised by Prescission Protease. Western blotting test revels that the target protein were able to bind each-type FMDV vaccine positive serum with A, O and Asia I specifically, which proved that purification of the protein was correct. And then, after Kun Ming mice were vaccinated 3 times with the protein(concentration was about 1.12 mg/ml), the serum was isolated from peripheral blood at the 7th day and the result of indirect ELISA showed that embedded protein SLP-MultiVP1 can stimulate mice to produce high titers of specific antibodies(the value of OD450nm was higher than 2.0), which laid foundations for further research progress in immunogenicity of FMD VP1 epitope.2.6 weeks old Kun Ming mice were vaccinated by embedded protein SLP-MultiVP1 which purified and fusion protein SLP and PBS were set as negative control. Kun Ming mice were respectively sacrificed after once、twice and third vaccination, and the spleen were separated and total RNA were immediately extracted to cDNA. Expression levels of IL-4 and IFN-γ were examined by real-time PCR and results were analysed by statistics. The result showed that:expression of cytokine from embedded protein SLP-MultiVP1 vaccinated group were higher than 2 control groups, however, there was no significant difference between two control groups. (P>0.05)3.6 weeks old Kun Ming mice which freshly purchased were randomly separated and peripheral blood was taken from the heart at 7th day after second vaccination. After blood samples were managed, stimulating to produce CD4+T and CD8+T cells by embedded protein SLP-MultiVPl were analysed with flow cytometry and the data was statistically analysed. The result of experiment showed that:single number of CD4+T cells of embedded protein vaccination group was significantly higher than SLP recombinant protein vaccination group and PBS control group (P<0.05).However, SLP fusion protein vaccination group did not have significant difference compared to PBS control group. In addition, the rate of CD4+T/CD8+T from embedded protein SLP-MultiVPl group higher than SLP fusion protein vaccination group and PBS control group in peripheral blood, which showed remarkable difference (P>0.05)This study laid foundations for further research of immune protection test in each-type FMDV VP1 epitope embedded protein SLP-MultiVPl.