The Construction And Immune Efficiency Study Of Attenuated Salmonella Typhimurium Harbouring VP4-VP7 Genes Of GPRV

Giant Panda Rotavirus (GPRV) caused by the young pandas (5-11 months) infectious and refractory diarrhea, finally lead to multiple organ failure and death, for the Giant Panda brought enormous threat to the population expansion. At present the mainly mean to strengthen management is to prevent rotavirus infection. But due to environmental factors the effect is not ideal, therefore immune becames the main measures and the research spot. The study is mainly on the construction of recombinant attenuated Salmonella typhimurium harbouring VP4 and VP7 double fusion genes of GPRV vaccine and its immunogenicity.1. Cloning and identification of GPRV antigen gene VP4 and VP7According to GPRV, CH-1 strain, VP4 and VP7 gene sequences in the GenBank, the entire sequence of GPRV-VP4 (2331bp) and GPRV-VP7 (981bp) nucleotide sequences were successfully amplified by RT-PCR amplification. And T-A cloning were also carried out respectively. The sequence analysis showed that GPRV-VP4 and GPRV-VP7 gene were right.2. Construction of the attenuated Salmonella typhimurium harbouring VP4 and VP7 double fusion genes of GPRVChoose the eukaryotic expression plasmid pVAXl, and then the GPRV-VP4 and GPRV-VP7 gene were inserted into the eukaryotic single-expression vector respectively and both into the dual-expression vector to construct the single-expression plasmid and dual-expression plasmid, and transform the recombinant plasmids into attenuated salmonella typhimurium S.L7207. The recombinant strains were identified by PCR, restriction enzyme cutting and sequences identification, which indicates that the recombinant strain were successfully constructed; Extract plasmids from recombined strains respectively and transfect them into IPEC-J2 cells, and it showed that the recombined plasmids could be expressed in IPEC-J2 cells by RT-PCR, the indirect immunofluorescence test and the sequence analysis.3. Immunological characteristics of the DNA vaccine harbouring GPRV-VP4-VP7The stability of recombinant strain S.L 7207/pVAXl-VP4-VP7 cultured in vitro was detected. BALB/c mice were inoculated orally with the recombinant strain at different dosages to analyse the transcription, safety and security in vivo. Antibody and cytokine in serum were detected. Lymphocyte proliferation activity was detected by MTT method. The results showed that the recombinant strains were stable cultured with Kanamycin Resistance medium in vitro. The VP4 and VP7 genes were detected in the intestine of the BALB/c mice after 3 days inoculated orally with the recombinant strain at dosage of 1×108CFU, and the strain was stable in vivo in 15 days. Compared with the control group, the change of antibodies and the spleen lymphocyte proliferation has obviously difference (P<0.05). Cytokine detection in serum shows that the recombinant strain has good immunogenicity.All the results show that the attenuated Salmonella typhimurium S.L 7207 /pVAX1-VP4-VF7 in the test is safety, stability and immunogenicity. It could probably serve as a vaccine against Giant panda Rotavirus.