| Paratuberculosis is a chronic infectious ailment of ruminants caused by Mycobacterium avian subsp.paratuberculosis,leading to granulomatous enteritis,persistent diarrhea,progressive wasting and death.This disease is epidemic worldwide and causes serious economic losses to the cattle and dairy industries.Currently,duo to effective drug treatment is unavailable,vaccine immunization is an important mean of preventing paratuberculosis.The attenuated live bacteria vector vaccine is produced through the chromosome or plasmid of the attenuated live bacteria vector inserted into by the foreign antigen gene,and then stimulates the immune system by presenting the foreign antigen.Thereby,the purpose of preventing one or more diseases is able to achieved.Attenuated Salmonella has become a hotspot of study on vaccine carrier.It has been widely used in the development of vaccines such as viruses and bacteria,and good application prospect has been shown.The technology of bacterial surface display is an effective antigen presentation strategy,which can present foreign antigens on the surface of bacteria.Polypeptide antigens of displayed on the bacteria surface are recognized by the immune system easily.Therefore,surface display technology has broad application prospects in live carrier vaccines.In this study,attenuated Salmonella was used as the carrier,and Ice Nucleation Protein was used as the display platform,which to construct attenuated live vector vaccine.The?Red recombinantion system was firstly used to knock out the outer membrane protease gene ompT of Salmonella,so that the foreign gene could be stably expressed.On the basis of ompT gene deletion,the virulence gene lon is deleted,which further weakens its virulence.Then the target gene map3061c was cloned into a constitutively expressing pSDiGc plasmid and electrotran to attenuated Salmonella.After Western-blot identification,subcellular component extraction,flow cytometry,immunofluorescence and other experimental methods,the target protein was successfully expressed and displayed on the surface of attenuated Salmonella,hence the Salmonella carrier vaccine with surface display of MAP3061c(INP-MAP3061c)was successfully constructed.C57BL/6 mice were immunized with INP-MAP3061c,and inactivated MAP vaccine,Salmonella double deficient strain and PBS groups were used as comparation.The levels of humoral and cellular immunity were analyzed by regularly weighing mice,taking serum and separating spleen lymphocytes to detect antibody level,cytokine transcription and expression level,CD4~+and CD8~+T cell number.Then challenge experiments were performed to evaluate the immune protection effect by detecting changes in cytokines,changes in body weight of mice,and the number of intestinal,liver,and spleen bacteria in mice.The results showed that the antibody levels of INP-MAP3061c vaccine group and inactivated MAP vaccine group were higher than those of control group,which showed that INP-MAP3061c could stimulate the host to produce high level specific antibody;the CD4~+T and CD8~+T cells of INP-MAP3061c vaccine group increased significantly,and the levels of cytokines such as INF-??IL-4?IL-17 and IL-10 increased significantly during the immune period,which was basically consistent with the transcription level of cytokines in spleen,indicating that INP-MAP3061c can mediate the production of cellular immune responses in the host.After the challenge,the body weight of mice in the INP-MAP3061c vaccine group was not significantly different from that of the control group,and the bacterial burdens in the tested organs of the vaccine group were lower than those of the control group.According to this study,the live carrier vaccine with surface display INP-MAP3061c has a better resistance to paratuberculosis,and has laid the foundation for the development of a new paratuberculosis vaccine. |
PDF Full Text Request