| Background: Yersinia pestis is the causative agent of plague. Pathogenicity mechanism and host immune reaction are involved in infection and immunity. When accidentally transmitted into human bodies, it is first phagocytosed by neutrophils and macrophages of the local tissue; Bacteria phagocytosed by neutrophils are killed in a short time, while the ones phagocytosed by macrophages may survive and propagate in them; bacteria released from the macrophages develop the capacity of resisting the humoral immunity of the hosts. Y.pestis can be transmitted through lymph fluid and blood to organs of the whole body, causing bubonic plague, pneumonic plague and septicemic plague etc. Among current studies focusing on the mechanism of host immune reaction, humoral immunity is well studied, and some important protective antigens are already found, such as the F1 and the LcrV. More and more evidence indicates that cellular immunity plays an important role in host immunity against y.pestis. For example, mice inoculated by T cells that are stimulated by y.pestis develop a resistence to inhalation infection. There are still proofs of neutrophils and macrophages phagocytosing Y. pestis effectively, but the mechanism of how these two kinds of cells take part in the immune reaction is still unclear. Therefore, study on cellular immunity becomes a hot spot of research recently.Methods: Four plasmids of Y. pestis strain 201 are eliminated to obtain the 201 P-, and then two plasmids are transfered into Y. pestis strain 201 respectively; Plasmid pBC-GFP is a prokaryotic expression vector encoding a green fluorescence protein, while pIRES2 DsRed-Express2 is a eukaryotic expression vector encoding a red fluorescence protein. Neutrophils, macrophages and dendritic cells from spleens are isolated by magnetic beads method. After the mice are inoculated by Y. pestis, three kinds of cells from the spleens are harvested at different time; FCM is used to detect the rates of cells containing Y. pestis. Eukaryotic vectors that express F1 and V are constructed respectively, and then the vectors are transfered into Y. pestis strain 201 P-. After immunization, the antibodies are detected by ELISA to study the possibility of 201P- as a potential DNA vaccine vector against Y. pestis.Results: F1 gene and V gene are cloned into the eukaryotic vector pIRES2 DsRed-Express2 respectively. The eukaryotic vectors containing F1 gene and V gene can express F1 and V antigens normally, which is verified by transfection of 293 cells. Proteins released from cells were tested by Western-Blotting, confirming that they have good immunogenicity. Cultivated macrophages are infected with Y. pestis, and then phagocytosis of bacteria is observed by FCM. After immunization of mice with bacteria, neutrophils, macrophages and dendritic cells from spleens are isolated by magnetic beads method, and then FCM is used to detect the rates of the cells containing Y. pestis. The results indicated that Y. pestis can survive in both macrophages and dendritic cells. Eukaryotic vector can express red fluorescenc in macrophages. The ELISA results showed that the antibody titer of EV76 is as much as that of 201P- after the first inoculation.Conclusion: In this study, two kinds of plasmids that express different fluorescence protein were transferred into Y. pestis strain 201 p- and EV76. The ability for phagocytes to phagocytose constructed Yersinia and the longevity of bacteria in phagocytes were observed in mice. The preliminary results indicated that the virulence of Y. pestis relate with the ability for phagocytes to phagocytose constructed Y. pestis, and that Y. pestis strain 201 p- as a potential vector of DNA vaccine may cause immune response. |
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