| Bovine paratuberculosis is caused by Mycobacterium paratuberculosis (MP) and it is a kind of chronic wasting disease in ruminants globally, which is called Johne’s disease. This diseas causes chronic and hyperplastic enteritis in ruminants, e.g.cattle, caprine, deer and so on. In addition, it could infect swine, equines etc. Chiadini separated the MP from the patients of Johne’s diseas. The function of Johne’s disease has received extensive attention globlely. Presently, major measures to prevent Johne’s disease are to control the infection and enhance breeding. Management PPD of paratuberculosis is used to quanratine cattle, the positive cattle are isolated and culled. In addition, people use premunitive measures to control this disease. Heat inactivation or forced weakness of the Mycobacterium paratuberculosis to immunize the cattle and caprine has been comployed. This method can depress the incidence rate, However, the serum becomes positive after vaccine inoculation. This interfers with allergies and the detection of serology. Moreover, it produced granuloma at the injection site, as well as sensitive to tuberculin. These effects hamper the allergy detection of bovine tuberculosis, and affect the quarantine of bovines and international trade. Therefore many governments forbid the use of paratuberculosis vaccine. Therefore, a new type of vaccine and diagnostic reagent for bovine paratuberculosis needs to be studied and debeloped. Studies show the Mycobacterium paratuberculosis secretes immunological competent protein in short term culture and opposites in long term culture. Thus nonage secreted protein is the protective antigen. The DNA vaccine can effectively stimulate the organism to produce persistent humoral immunity and cellular immunologic response. Meanwhile, using a DNA vaccine dose not interfere with the allergy detection of bovine paratuberculosis and bovine tuberculosis, So studing a new type of DNA vaccine to prevent bovine paratuberculosis is a new method. In this experiment, we screen the major protective antigen gene-29kD gene of Mycobacterium paratuberculosis in order to study the sensitive, specific diagnostic reagent and prophylaxis preparation, especially the DNA vaccine. The20kD gene was amlolfied from Mycobacterium paratuberculosis by using the PCR technique and cloned into pMD18-T Vector System. We gained a20kD gene of495bp. The recomninant clone was identified by enzyme digestion and PCR indentification. The result indicated that of the Mycobacterium paratuberculosis, the sequential homogeneity reached99%, and the amino acid homogenetity reached99.5%. The preceding analysis indicated that the 20kD gene was very conservative in Mycobacterium paratuberculosis.T develop new preventive preparation of paratuberculosis, the20kD gene linked to recombinant pMD18-T-20kD was subcloned into the eukaryotic expression vector pVAX I in this experiment, and recombinant pVAX Ⅰ-20kD was successfully constructed. The liposome mediated method was used to transfect BHK-21cells with the recombinant plasmid pVAX Ⅰ-20kD, and20kD gene expression was observed by immunofluorescence and RT-PCR technique. BALB/c mice and cavy was vaccinated with pVAX Ⅰ-20kD, and we set up pVAX Ⅰ, normal saline, and ultrasonic antigen of MAP as control. The results of lymphocyte transformation showed that there was no significant difference between pVAX Ⅰ-20kD 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 INF-γ in spleen cell supernatant of immunized mice showed that there was significant difference in contents of INF-y compared with the other groups(P<0.05). The specific antibody against the20kD antigen was detected in immunized mice by indirect ELISA. The allergy of cavy showed that there was extremely significant difference between pVAX Ⅰ-20kD 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 expressing20kD 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. |
PDF Full Text Request