The Influence On Chicken Macrophage Function Of Yersinia High Pathogenicity Island In Salmonella

By using modern molecular biology, Salmonella carrying HPI genes was successfully screened in this research. After analyzing its genetic structure, carrying forms, expression of associated protein and bacterial growth characteristics, together with discussing the mechanism and significance of Salmonella obtained HPI gene, a full-length HPI gene deletion Salmonella strain was built. Meanwhile, the chicken iNOS indirect ELISA detection method and the chicken IL-12, IL-18, TNF-a Real-time PCR detection method were successfully established. Combining with existing techniques, the relevant indicators of chicken macrophage-like cell line HD11infected by the two Salmonella chosen as the research object were detected and comparative analyzed, and the impact of Salmonella HPI on the immune function of macrophage was comprehensively discussed from view of immunology, cell biology, molecular pathology.The main research contents and results are as follows:First, with the gene-specific primers of Salmonella invA and Yersinia irp2gene, a duplex PCR detection method for Salmonella carrying HPI genes was established and the screening system was established.Using the optimized screening system,3stains of HPI-positive Salmonella were successfully screened from64stains of Salmonella and27samples of mix bacteria.Second, using the microbiology and molecular biology method, the characteristic of HPI-positive Salmonella was analyzed and the source and transfer mechanisms of Salmonella HPI gene were discussed:1、After PCR detection, this research detected that the HPI core region of the3stains of HPI-positive Salmonella existed integrity difference in different degree,which indicated the gene loss during horizontal transfer of HPI,. or caused by the pressure of natural selection after Salmonella obtained complete HPI.2、By the Southern Blot analysis and HPI gene insertion sequence test, combining with the existing literature genomics, this research detected that the HPI of the3stains were obtained from different sources and carried by plasmids.3、With the specific primers which contained homologous flanks of HPI gene under-knockout in Salmonella at5’end and complementary sequences for both sides of the chloramphenicol resistance gene (cat) on pKD3plasmid, the target gene segment was got by PCR amplification. With the help of pKD46plasmid which expressed Exo, Beta and Gam proteins in HPI-positive salmonella, the whole HPI was successfully replaced by part of the target gene segment, which means HPI deletion salmonella was successfully constructed.4、Through the growth observation of the3stains of HPI-positive salmonella in/on the CAS medium (iron deficiency), this research detected that all the3stains can express iron-affinity material similar as Yersinia did, and Western Blot analysis further demonstrated the3stains can express HMWPs induced by low-temperature and low-iron condition, which indicated this3stains of Salmonella inherited the basic functions of HPI.Finally, chicken macrophage-like cell line HD11respectively infected by the HPI-positive salmonella and the HPI knockout strain were chosen as research object, this research comprehensively discussed the influence of Salmonella HPI on macrophage by detecting the relevant indicators and from the view of immunology, cell biology, molecular pathology:1、With the help of recombinant plasmid pET-30a-ChiNOS, Chicken iNOS was successfully expressed in E. coli BL21(DE3). As soon as purification of Chicken iNOS, polyclonal antibody was prepared by immunizing rabbits. Meanwhile, the chicken iNOS indirect ELISA detection method and the chicken IL-12, IL-18, TNF-a Real-time PCR detection method were established.2、By the macrophage phagocytosis test, this research detected that we found the phagocytosis (or adhesion) on Congo-red staining yeast of macrophages infected by HPI-positive salmonella was significantly lower (P<0.05)than the one infected by HPI deletion salmonella during0-2h infection. After2h, although this relation was still kept, but were not significant different (P>0.05). It could be speculated, HPI can enhance the invasion ability of Salmonella against macrophage by competitive binding the corresponding sites on surface of macrophages, or inhibiting macrophage phagocytosis through other mechanisms.3、Salmonella infection could rapidly induced the expression of iNOS, IL-12, IL-18and TNF-a in macrophages, but there was no statistical significance between the two groups of samples (P>0.05), which explained whether Salmonella carried HPI genes was not the main reason for this phenomenon above.