| Porcine reproductive and respiratory syndrome(PRRS)，caused by porcine Reproductive and respiratory syndrome virus(PRRSV),which is characterized by severe reproductive failure in sows，and respiratory distress in piglets and growing pigs，is really a new viral disease of swine．PRRS has been one of the most economically important diseases of the global swine industry．In addition, as an intracellular invasive bacterium, Salmonella can effectively present antigen, stimulate specific humoral and cellular immune response against both Salmonella and heterologous antigen, stimulate mucosal and systemic immune at the same time. To study attenuated Salmonella delivering PRRSV ORF5gene vaccine, provide new methods to solve problems. Thus, the study will provide new breakthrough ideas for PRRSV genetic engineering vaccine and expression of exogenous gene at appropriate level.（1）The PRRSV ORF5gene was expressed in prokaryotic The PRRSV ORF5gene with N-terminal signal peptide sequence deleted was clonedand inserted into the pET-28a vector to construct recombinant expression plasmid pET-28a-dORF5,then the recombinant plasmid was transformed into E.coli BL21(DE3) and induced to express by IPTG. Expression of PRRSV GP5protein was purified by nickel-affinity chromatography and refolded, prokaryotic expression of PRRSV GP5set foundation for further study on the biological function of GP5.（2）Biological characteristics of the recombinant PRRSV ORF5gene attenuated Salmonella choleraesuis C78-1strain The recombinant strain of Δcrp asdC78-1(pYA-dORF5) was identified by GP5protein expression, phenotypic traits, stability, growth curve and safety in vitro. The growth ability of recombinant strain of crp asdC78-1(pYA-dORF5) in circumstance without DAP was persisted. The ability of fermentation for maltose, xylose, arabinose was lost, but the recombinant can ferment glucose. Compared with C78-1strain, the growth of recombinant strain of crp asdC78-1(pYA-dORF5) was reduced significantly, but the growth was almost the same like that of crp asdC78-1. And the gene of dORF5can be transmitted stably in the liquid nutrient medium of Luria Bertani after60generation; Furthermore, the successful expression of dORF5gene was confirmed by SDS-PAGE. The expression product was verified with better reactionogenicity by Western-blot. The KM mouse lethal experiment showed that the virulence of C78-1was492times higher than the recombinant strain of crp asdC78-1(pYA-dORF5), and the virulence of C500was1.31times higher than the recombinant strain of crp asdC78-1(pYA-dORF5) through oral infection.（3）preliminary study of immunological characteristics of the recombinant PRRSV ORF5gene attenuated Salmonella choleraesuis C78-1strain Mice were orally immunized with The recombinant strain of crp asdC78-1(pYA-dORF5).The recombinant strain of crp asd C78-1(pYA-dORF5) could be detected in mice liver30days post immuneization, indicating it can survive vivo and stimulate host immune response for a long time．The recombinant strain of crp asdC78-1(pYA-dORF5) could induce a high titre of antibodies，In mouse immunization trial，All immunized groups induced IgG Ab against crp asd C78-1mutant and GP5. The levels of IgG rose from the first week and approached the peak at the third week. The IgG level against crp asd C78-1mutant was higher than that of GP5in the same serum. the OD value of immuned mice could reach to0.788,but OD value of the bacterium as negative control and PBS control group only reach to0.022and0.0218. At two weeks after secondary immunization. and the secreted IgA could be detected in mouse liquor entericus at four weeks after secondary immunization. The OD value of the secreted IgA in the recombinant strain had a significantly higher than that in the bacterium as negative control and PBS control group, are respectively10.58,0.55and2.73.The lymphocyte transformation experiment show that the restructuring can stimulate mice to produce strong cellular immune response. These hinted that the recombinant oral vaccine could induce better cellular immune response at the same time. This suggested that the recombinant vaccine could induce a immunological reponse against PRRSV- Salmonella choleraesuis．... |
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