Immune Research Of Mycobacterium Avium Ssp. Paratuberculosis SOD Gene In Experimental Animal

The paratuberculosis is also known as Johne's disease,which is caused by Mycobacterium avium ssp. paratuberculosis(MAP),and it is a kind of chronic diarrhea and emaciation disease mainly in ruminants.This disease is widespread in the world,and it causes serious damage in those countries which have developed dairy and beef cattle industry.Now it is becoming one of the most serious infectious diseases in animal husbandry industry.In addition,MAP has been separated from Crohn's patients,so Johne's disease has attracted wide attention all over the world.At present,there is no effective therapeutic drug,and detection,isolation or elimination has been the major prevention measures,that is to quarantine PPD(purified protein derivative) of MAP,and to isolate and eliminate the positive individuals.In addition,vaccination has been used to prevent the paratuberculosis,that is to use vaccine made by MAP of heat inactivation or being weakened to inoculate sheep and cattle as to decrease morbidity.But after being inoculated,the serum turned to be positive,and allergic reaction and serological detection were interfered to cause tuberculin sensitive.So new prevention vaccine and diagnostic reagent are urgently needed as to promote health development of animal husbandry. Some studies indicated that the secretory proteins in the culture filtrate of MAP can effectively induce the resistance and immuno protection to the MAP.SOD is one of the main secretory proteins of MAP.DNA vaccine can effectively stimulate permanent humoral immunity and cell-mediated immunity,and the activation of CTL and killing the target cells are the main mechanisms of DNA vaccine,which do not disturb the allergy detection and it is necessary for the prevention of MAP infection.To develop new preventive preparation of paratuberculosis,especially the DNA vaccine,the SOD gene linked to recombinant pGEM-T-SOD was subcloned into the eukaryotic expression vector pVAX1 in this experiment,and recombinant pVAX1-SOD was successfully constructed.The liposome mediated method was used to transfect BHK-21 cells with the recombinant plasmid pVAXl-SOD,and SOD gene expression was observed by immunofluorescence and RT-PCR technique.BALB/c mice and cavy was vaccinated with pVAXl-SOD,and we set up pVAXl,normal saline,and ultrasonic antigen of MAP as control.The results of lymphocyte transformation showed that there was no significant difference between pVAXl-SOD and control groups(P>0.05).The detection results of CD₃⁺T, CD₄⁺T,and CD₈⁺T cells in mice spleen demonstrated that there was no significant difference in CD₄⁺T,and CD₈⁺T cell numbers among the three groups(P>0.05),and there was difference in CD₃⁺T cell numbers,but not significant(P>0.05).The detection results of IFN-γin spleen cell supernatant of immunized mice showed that there was significant difference in contents of IFN-γcompared with the other two groups(P < 0.05).The specific antibody against the SOD antigen was detected in immunized mice by indirect ELISA.The allergy of cavy showed that there was extremely significant difference between pVAX1-SOD group and ultrasonic antigen of MAP group(P < 0.01),and it was suggested that recombinant plasmid immune had no interference to the allergy detection.The results of this experiment showed that the eukaryotic vector expressing SOD gene was successfully constructed and this vector induced humoral immunity and cell-mediated immunity in the experimental animal.These results laid a foundation for further research to the immune protection as to develop DNA vaccine to prevent paratuberculosis.