title: Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.
abstract: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.
authors.affiliation: 1 Department of Biochemistry, Science and Research Branch, Islamic Azad University , Fars, Iran .
authors.forename: Maryam
authors.initials: M
authors.lastname: Hashemi
authors.forename: Rasool
authors.initials: R
authors.lastname: Madani
authors.forename: Nematollah
authors.initials: N
authors.lastname: Razmi
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D015153:Blotting, Western; D002473:Cell Wall / Q000276:immunology*; D005260:Female; D007106:Immune Sera / Q000737:chemistry; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016927:Mycobacterium avium subsp. paratuberculosis / Q000276:immunology*; D011817:Rabbits
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007106:Immune Sera; D007074:Immunoglobulin G