Construction And Evaluation Of The Immune-enhancing DNA Vaccine Of Sereve Acute Respiratory Syndromes Virus

In this research, the polycloned antibody of anti-S1 protein and anti-CTB protein was obtained respectively by S1 protein and CTB protein expressed respectively from prokaryotic expression system firstly. Then S1 gene and CTB gene was respectively inserted into a eukaryotic expression vector, pVAX1, to construct two new plasmids; furthermore, S1-CTB fusion gene linked by a flexible linker was inserted into pVAX1. All three kinds of new recombinant eukaryotic expression plasmids were transfected into BHK-21 cells. Indirect fluorescence assay (IFA) showed that all three kinds of eukaryotic expression plasmids could be expressed transiently in mammalian cells. It provided the theoretical foundation for the following immunization research in vivo.Subseqently, 6 weeks old Kunming mice was used to evaluate the effect of DNA vaccine. The experimental mices were divided into 7 groups, the PBS group inoculated with the PBS buffer, the pVAX group inoculated with the pVAX1 vector, the S1 group inoculated with the pVAX-S1 plasmid, the CTB group inoculated with the pVAX-CTB plasmid, the S1~+CTB group inoculated with the pVAX-S1 and pVAX-CTB plasmid, and the S1-CTB group inoculated with the pVAX-S1-CTB plasmid. The specific proliferation assay of T lymphocytes of spleen and peripheral blood, the population change of CD4~+ and CD8~+ T lymphocytes, CTL activity assay. IL-4 and IFN-γassay, and specific IgG antibody assay were dected. The results showed that, comparing to the controls, both S1~+CTB and S1-CTB could stimulate mices boosting the immune responses of mices obviously. The effect of S1-CTB group is better, but the distance of both was not significant. The transient expression of recombinant plasmids pVAX-S1, pVAX-CTB and pVAX-S1-CTB in BHK-21 cells could be examined.The result of proliferation assay of T lymphocytes showed that , at 42d.p.i, proliferation of two vaccine groups were higher significantly than other controls(P<0.01).The population of CD4~+ T lymphocytes of spleen and peripheral blood reached maximum at 42d.p.i, and CD8~+ T lymphocytes reached maximum at 28 d.p.i.The result of specific CTL respone showed that, at 42d.p.i, fusion vaccine group was the most effective group.The current of anti-S1 protein IgG indicated that fusion vaccine group was higher than any other groups significantly(P<0.01).And the content of IL-4 and IFN-γassay indicated that fusion vaccine group had stronger cytokine stimulated function.Above all, the results showed that when antigen S1 gene was inoculated with CTB gene,it could improve the the host immune respone. The results could provided scientific ground to the further study of biology function of CTB gene,and the theoretical basis to the dicovery of new type immune-enhancing vaccine to SARS.