Cloning & Procaryotic Expressing On The SOD Gene In Mycobacterium Paratuberculosis

Bovine paratuberculosis is caused by Mycobacterium paratuberculosis (MP) and it is a kind ofchronic wasting disease in ruminants globally, which is called Johne's disease. This disease causeschronic and hyperplastic enteritis in ruminants, e.g. cattle, caprine, deer and so on. In addition, it couldinfect swine, equines etc. Chiadini separated the MP from the patients of CD. The function of Johne'sdisease has received extensive attention globlely.Presently, major measures to prevent Johne's diseaseare to control the infection and enhance breeding. management So PPD(puified protein derivative) ofparatuberculosis 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 M.paratuberculosis to immunize the cattle and caprine has been comployed. This method can depress theincidence rate. However, the serum becomes positive after vaccine inoculation. This interferes withallergies and the detection of serology. Moreover, it produced granuloma at the injection site, as well assensitive to tuberculin. These effects hamper the allergy detection of bovine tuberculosis, and affect thequarantine of bovines and international trade. Therefore many governments forbid the use ofparatuberculosis vaccine and BCG to control bovine paratuberculosis or bovine tuberculosis. Therefore,a new type of vaccine and diagnostic reagent for bovine paratuberculosis needs to be studied anddeveloped.Studies show the M. paratuberculosis secretes immunological competent protein in short termculture and opposites in long term culture. Thus nonage secreted protein is the protective antigen. TheDNA vaccine can effectively stimulate the organism to produce persistent humoral immunity andcellular immunologic response. Meanwhile, using a DNA vaccine dose not interfere with the allergydetection of bovine paratuberculosis and bovine tuberculosis. So studying a new type of DNA vaccineto prevent bovine paratuberculosis is a new method.In this experiment, we screen the major protective antigen gene-SOD gene of M. paratuberculosisin order to study the sensitive, specific diagnostic reagent and prophylaxis preparation, especially theDNA vaccine. The SOD gene was amplified from Mycobacterium paratuberculosis C-2 chromosomalDNA by using the PCR technique and cloned into pMD18-T Vector System. We gained a SOD gene of624bp.The recombinant clone was identified byα-complementarity, enzyme digestion and PCRidentification. The result indicated that the recombinant plasmid pMD18-T-SOD was successfullyconstructed. Moreover, through sequential determination and DNASTAR analysis between the clonedSOD gene of M. paratuberculosis C-2 and that of the M.paratuberculosis K-10 strain, the sequentialhomogeneity reached 99%, and the amino acid homogeneity reached 99.5%. The preceding analysisindicated that the SOD gene was very conservative in M. paratuberculosis. The SOD gene that wascloned into a pMD18-T Vector System was subcloned into a pET-28a (+) procaryotic expressionsystem, then the procaryotic expression plasmid pET-28a-SOD was constructed successfully. Theplasmid was transformed into E.coli BL21(DE3) and induced by IPTG.. We gained a 26.5KDa fusionprotein. The expression protein was analyzed by using the Western-blot, which proved that it had theantigenic activity of Mycobacterium paratuberculosis. The research results and expression product could serve as a basis for further studies on the gene-engineering vaccine, subunit vaccine and nucleonicacid vaccine against Bovine Paratuberculosis.