| The experiment are divided two stages: one is the prepration of Porcine Rotavirus Group A Vp6 GeneEngineering Lactobacillus, the other is detection of Protective Immunity. Vp6 gene of Porcine Rotavirus Awas amplified by the reverse transcription polymerase chain reaction (RT-PCR), the product of which wasan approximately 1172bp cDNA segment. The purified Vp6 gene was sub cloned into the vector pW425et.Thus, the recombinant pW425et-Vp6 was constructed, which then was transformed into the competenceE.coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on which approximately 48kD fusion protein was observed. The protein was further analyzed by Western blot, which indicated that theprotein was reactive with the antibody of Rotavirus A. The recombinant pW425et-Vp6 was constructed, which then was transformed into the competence Lactobacillus mutant. Selection the positive mutant byPCR and digesting plasmid. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on whichapproximately 48 kD fusion protein was observed. The protein was further analyzed by Western blot, which indicated that the protein was reactive with the antibody of Rotavirus A. The Lactobacillus mutantwhich was transformed into pW425et-Vp6 is the vaccine which is we need.The BABL/c mice used for immunization were divided into A, B, C, D four groups, 20individuals/group. Group A was control group, Group B was feed with acidophilic lactobacilli, Group Cwas feed with E. coliX13, Group D was feed with genetically engineering vaccine of Lactobacillus and themice in each group were feed three times one day. After buying, all mice were feed at regular time and thedosage was the same. Blood samples collected from mice at 0 14 28 42d after first feeding and thosecollected from mice of each group at 0 14 28 42d, were used for detecting antibody levels by indirectELISA and the differences in number of CD3~+, CD4~+, CD8~+, CD4~+ /CD8~+ T cells in peripheral bloodlymphocyte and CD19~+ B cellsThe results indicated: after immunization, the specifically antibody of porcine rotavirus A significantlyincreased compared with the control group. The differences in number of CD3~+, CD4~+, CD8~+, CD4~+/CD8~+, CD 19~+B cells in peripheral blood lymphocyte was significantly. In membrana mucosa, the vaccinemake the epithelium secrete a lot of SIgA, and the organism received valid protection to against porcinerotavirus A. |
