Construction Of Recombinant Attenuated Salmonella Typhimurium Harbouring Porcine Rotavirus DNA Vaccine And Study On Immunogenicity

Porcine Rotavirus (PRV) infection is an acute swine disease with eintestinal infectious symptoms, causing severe diarrhea and dehydration, piglets are in high mortality, which causes huge losses to the swine industry. The research of PRV has been focused on the development of a new vaccine that is stable, secure and efficient in preventing and controlling PRV. This study is mainly on the construction of recombinant attenuated Salmonella typhimurium harbouring PRV DNA vaccine and its immunogenicity.1. Cloning and identification of PRV antigen gene VP7 and NSP4According to the gene pool of PRV OSU strain, two pairs of primers were designed, then the entire sequence (1062 bp) of VP7 and the 259-525 site nucleotide sequence (267 bp) of NSP4 were successfully amplified by RT-PCR and TA cloning were also carried out respectively. The sequence analysis showed that the amplified VP7 and NSP4 gene fragments were basically similar with the published standard sequence.2. Prokaryotic expression and immune serum preparation of PRV VP7 and NSP4 geneObtain the correctly identified PRV VP7 and NSP4 gene from T vector by enzyme digestion and insert them to prokaryotic expression vectors respectively to construct the BL21 expression bacteria containing recombinant plasmids pET28a-VP7 and pET32a-NSP4. After IPTG induction, the recombinant bacteria expressed fusion protein of approximately 30 KDa and 28 KDa, and used the purified fusion-protein to immunize rabbits for the preparation of anti-fusion protein serum. High level of specific IgG antibody to fusion-protein of VP7 and NSP4 could be detected in the two prepared serum.3. Construction of recombinant attenuated Salmonella typhimurium delivering PRV VP7 and NSP4 geneReconstruct the eukaryotic expression vector pVAXl and pVAXD in use of the balanced-lethal system, and then the NSP4 and VP7 gene fragments were respectively inserted into the eukaryotic single-expression vector pVAX1-asd and were also both inserted into the eukaryotic dual-expression vector pVAXD-asd to construct the single-expression plasmid and dual-expression plasmid and transform the recombinant plasmids into attenuated Salmonella typhimurium x3730 by electrotransformation, from which the plasmids were extracted and electrotransformed into attenuated Salmonella typhimurium X4550. The recombinant strains were identified by bacteria-colony PCR, the amplified fragments were in the same size with the target gene; the extracted plasmids were identified by enzyme digestion and the digested fragments were proximately in the same size with the target gene and vector fragments respectively, which indicates that the recombinant strain were successfully constructed.4. Biological characteristics of the recombinant attenuated Salmonella typhimurium harbouring PRV VP7 and NSP4 gene and the mice immunization testsExtract plasmids from the recombined attenuated Salmonella typhimurium respectively and transfect them into COS-7 cells, the prepared serum was used as antiserum to carry out the indirect immunofluorescence assay, from which fluorescen was observed and it indicated that the target protein could be expressed in COS-7 cells. The plasmid of recombined Salmonella typhimurium showed good stability in non-resistance factor environment. The transcripted RNA from the target gene could be detected in ileum tissues three days post the mice being orally inoculated. Indirect ELISA detection of serum IgG and intestinal IgA showed that the immunization group of dual-expression plasmid and combined single-expression plasmids induced significantly higher level of serum IgG and intestinal IgA than that in the immunization group of single-expression group.