| Transmissible gastroenteritis (TGE) of swine is caused by procine transmissible gastroenteritis virus which belongs to the family of coronaviridae. It is characterized as an acute, highly contact intestinal infection. The main clinical symptoms of TGE are vomiting, diarrhea and dehydraton. The research of TGE is focused on the study of its vaccine. In this study, the recombinant attenuated Salmonella typhimurium mediated a bicinstronic DNA vaccine expressing TGEV S gene and pIL-6 genen was constructed, and the immunogencicty of the recombinant attenuated Salmonella typhimurium was also studied1. The construction and Identification of Bicistronic Eukaryotic Vector Expressing TGEV S Gene and pIL-6 GeneFirst, we designed the primer of TGEV S gene and used the genomic DNA pMD19T-S as the templates to reconstructe the T cloning vector of S gene. The S gene was digested and inserted into the dual promoter eukaryotic plasmid of pVAXD in the second MCS. The recombined plasmid pVAXD-S was detected by restriction analysis and PCR. The result showed that the restriction analysis and PCR produced a same size fragment of 2000bp. It proved that the eukaryotic vector pVAXD-S was successfully constructed. Secondly, the primer of pIL-6 was designed and the gene encoding pIL-6 obtained from the plasmid pMD19T-IL6 was used to rebuild the T cloning vector of pIL-6. The pIL-6 gene was digested and inserted into the plasmid pVAXD-S. The plasmid of pVAXD-S-IL6 was identification by restriction analysis and PCR, the result showed that the restriction analysis and PCR produced a same size fragment of 500bp.The plasmid of pVAXD-S-IL6, pVAXD-S and pVAXD were transfected into COS-7 cells. The expression of pVAXD-S-IL6 was demonstrated by indirect RT-PCR and indirect immunofluorescence assay. The 2000bp and 500bp gene were not observed in the parental vector pVAXD. Using the Fluorescence microscope, the recombinant plasmids could be confirmed by indirect immunofluorscence assay. The results showed that the eukaryotic expression plasmids were expressed well in transfected COS-7 cells. The results provided foundation for the further research of recombinant attenuated Salmonella typhimurium.2. Oral immunization of attenuated Salmonella typhimurium harbouring TGEV S gene and pIL-6 gene DNA vaccine induced special antibody productionThe construction and identification of recombinant attenuated Salmonella typhimurium:The constructed eukaryotic expression vector of pVAXD-S-IL6 and pVAXD-S was transformed into attenuated Salmonella typhimurium SL7207 by electroporation. Recombinant attenuated Salmonella typhimurium was identification by restriction analysis and PCR. The 2000bp and 500bp gene fragments were observed in the recombinant attenuated Salmonella typhimurium SL7207 (pVAXD-S-IL6) and 2000bp in the SL7207 (pVAXD-S). The results showed that the recombinant attenuated Salmonella typhimurium was successfully constructed. The stability testing proved that the recombinant attenuated Salmonella typhimurium was more stable in the envirement containing antibiotics. The mice were randomly divided into four groups,8 rats/group, the groups were inoculated orally with SL7207(pVAXD-S-IL6),SL7207(pVAXD-S),SL7207(pVAXD) separately at dosages of 1×109 CFU, while the control group inoculated with PBS of 0.2ml.1-2 days after immunization some of mice showed lassitude, got together and less appetite. Then the mice recovered after a period of time and acted normally. The results showed that the recombinant bacterium was safe to mice at dosage of 1×109CFU.The dividing, immunization, collection samples of mice:the mice were divided into four groups,16rats/group, the groups were inoculated orally with SL7207(pVAXD-S-IL6),SL7207(pVAXD-S),SL7207(pVAXD) separately at dosages of 1×109 CFU and with 3 times at two weeks interval, and the control group with PBS of 0.2ml. Before immunization and 2,4,6 weeks after immunization, the blood and complete small bowels of 3 mice were collected. Meanwhile, splenocytes of 2 mice was collected to test the level ofγ-interferon in each immunization group. Antigen preparation:The bacteria pET32a-Sbc BL21(DE3) were recoveried and identified. The 38KD epitope sites (b,c) of TGEV S protien were expressed. The expressed protein was purified. By SDS-PAGE, the expressed and purified protein appeared a fragment of 38KD, the result showed that the epitope sites (b,c) of TGEV S protien were expressed well. The purified protein was coated as antigen to detect serum and intestinal sample.The result detection:The special serum IgG and intestinal mucosal IgA antibody of TGEV were detected by indirect ELISA. The result indicated that the mice immunized with SL7207(pVAXD-S-IL6,SL7207(pVAXD-S) elicited special antibody of both TGEV and attenuated Salmonella typhimurium (P<0.01). The level of IgA and IgG in group SL7207(pVAXD-S-IL6) was 0.195 and 0.363, the level of IgA and IgG in group SL7207(pVAXD-S) was 0.210 and 0.402 at the week 4. The group of SL7207(pVAXD-S-IL6) were lower than group of SL7207(pVAXD-S) but there was no significant difference (P>0.05), however the level of group SL7207(pVAXD-S-IL6) and SL7207(pVAXD-S) were significantly different from the control group (P<0.01). The level of IgA and IgG in group SL7207(pVAXD-S-IL6) were 0.253 and 0.466, the level of IgA and IgG in group SL7207(pVAXD-S-IL6) were 0.210 and 0.421 at the week 6. The two group were sever significantly different from the control group (P<0.01), the IgA antibody from the group SL7207(pVAXD-S-IL6) was significantly different from the group SL7207(pVAXD-S) (P< 0.01), the IgA antibody from the group SL7207(pVAXD-S-IL6) was sever significantly different from the group SL7207(pVAXD-S) (P<0.01), the IgG antibody from the group SL7207(pVAXD-S-IL6) was significantly different from the group SL7207(pVAXD-S) (P<0.05). The two group were sever significantly different from the control group (P<0.01). At week 6 before collection the sample of mice, the level ofγ-interferon in spleen lymphocytes was tested. The results showed that the group SL7207 (pVAXD-S), SL7207 (pVAXD-S-IL6) y-interferon concentration (about 1100pg/ml) were higher than SL7207 (pVAXD) and immunohistochemistry PBS concentration (about 700 pg/ml). |
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