Salmonellae Choleraesuis-mediated DNA And Its Immune Response Against Classical Swine Fever

DNA vaccine can elicit immunization against diseases was authenticated by many researchers previously, but efficiently delivering and expressing of the interest gene into mammal cells is the major obstacle which blocks its application. In this paper, we reported an efficient delivery system mediated by Salmonellae choleraesuis,which protected the immunized rabbits and piglets partially from the challenge of classical swine fever virus and Salmonellae choleraesuis, simultaneously. The major antigenic gene E2 of classical swine fever virus, which consists of the N-terminal signal peptide sequence but without the transmembrane region, was amplified by PCR from CSFV genome cDNA clones. The amplified E2 gene fragment was cloned into T-vector and transformed into E. coli followed by blue-white screening on LB plates, and the pT-E2 plasmids was mini-prepared and sequenced. The sequenced E2 gene was subcloned into eukaryotic expression vector pVAX1 subsesquently and the recombinated pVAXE2 plasmid obtained. Indirect ELISA result demonstrated that the pVAXE2, which was transfected into BHK cell lines, expresses the gene of interest efficiently. Furthermore, the expressed E2 protein shows its antigenic activity. The recombinated pVAXE2 plasmid was transformed into attenuated vaccine strain Salmonellae choleraesuis competent cells to sort the positive recombinated strain on LB plate which contains additionally proper antibiotics. The positive clones were identified by PCR and restricted enzyme digestion. The safety test result reveales that the mice can survive from various doses of immuno-inoculation of S. Choleraesuis from 106 to 109 CFU/ml, respectively. Two groups of mice were immunized with the recombinated Salmonellae choleraesuis strain through oral immunization and muscle injection, respectively. Each group was immunized three times in two-week-interval. The sera antibody was detected by indirect ELISA, the results indicated that the recombinated S. Choleraesuis induced higher level antibodies than pVAXE2 alone, which was encapsulated by spans/glycerol mixture. This suggests that the attenuated vaccine strain of S. Choleraesuis deliveres the recombinated pVAXE2 plasmid efficiently and the E2 gene be expressed in vivo and stimulated the humoral immune response in mice. Furthermore, two groups of rabbits were tested with the same methods described previously.It is showed in the indirect ELISA that the recombinated vaccine strain of S. Choleraesuis efficiently activates the immune system of rabbits. The immunized rabbits were challenged with virulent S. Choleraesuis and strain HCLV of CSFV on 14th day after the 3rd immunization. The challenge results revealed that the immunized rabbits were partially protected, 20% of them can survive from thechallenge of 20 ID50 HCLV and 67% of them from 10 8 CFU /ml virulent S. Choleraesuis. Based on the results that obtained from mice and rabbits immuno-challenge tests, four groups of piglets were immunized through the former routes. ELISA detection showed that the antibody titer is significantly higher in pVAXE2 immunized piglets than that in controls. This suggests that the recombinated pVAXE2 vaccine strain of S. Choleraesuis efficiently mediates the humoral immune response against CSFV and S. Choleraesuis in piglets. All of the immunized piglets were challenged with the virulent strain of CSFV Shimen on 28th day after 3rd immunization. The challenge result showed that none of the pVAXE2-immunized piglets could survive from the challenge of virulent strain of CSFV. It could be drawn from the tests that there are large amount of research works be done at hand and in the coming days. According to the results of our experiments, we present that the attenuated vaccine strain of S. Choleraesuis may be used as an efficient delivery system of CSFV DNA-vaccine and be suitable for delivery of the other eukaryotic expression plasmids.