Purification and characterization of a 20 KD recombinant protein of M. Avium SS paratuberculosis to identify a unique protein of M. Avium for serodiagnosis of Crohn's disease
Background: Crohn's Disease (CD), a chronic inflammatory bowel disease (IBD), is thought to be multifactorial, involving an interaction between genetic susceptibility, undefined environmental triggers, and immune-mediated tissue injury. Biochemical and other molecular approaches identified isolates from intestinal tissues of patients with CD as Mycobacterium avium subsp paratuberculosis (MAP), the causative agent of Johne's disease, a granulomatous bowel disease in ruminants similar to CD. MAP has been identified directly in resected tissues of increasing numbers of CD patients at a frequency significantly higher than those of controls. Treatment of CD patients, which depends on the location and severity of disease, complication, and response to previous treatment is most often to control the disease. There is no cure. Diagnosis of this disease requires a series of tests including upper gastrointestinal endoscopy and colonoscopy. These tests are expensive, inconvenient and require hospitalization. Objective: A blood serologic test is sought for diagnosis of CD patients infected with MAP. Methods: The recombinant E. coli clone pBl 1 containing a 1,302 bp MAP DNA insert and expressing a 20 kD protein has been grown, induced by arabinose and then harvested by centrifugation. Protein extracts were prepared, quantitated and then subjected to Isoelectricfocussing (IEF) in ampholyte buffer pH 3-10. Twenty fractions were collected, quantitated and then analyzed on SDS-P AGE by silver staining and Imrnuboblotting. The immunoblots were screened with anti-express IgG monoclonal antibodies. Fractions containing the semipurified 20 KD protein were analyzed by immnoblot against 85 sera specimens with 1:30 dilution (43 CD patients and 42 controls). Both IgG and IgA response in each patient was determined. Results: Of 20 fractions collected, fractions 5 and 6 with a PI ranging from 4.18 to 5.01 reacted with the anti-express IgG antibodies. p20 with a 20 kD molecular weight was confirmed. These fractions contained fewer proteins bands with p20 being dominant. Of 43 CD sera specimens, 74% contained IgG response and only 50% contained IgA response to p20. On the other hand, of 42 controls, only 17% contained IgG and. 50 % contained IgA response.against p20 antigen. Conclusion: p20 reacted with CD IgG sera with frequency much higher than control sera (74% versus 17%) indicating a great potential for using p20 as a reagent in a quantitative ELISA assay for specific diagnosis of CD patients. Additionally, the data add strong support to MAP role in CD pathogenesis.
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Naser, Saleh A.
Bachelor of Science (B.S.)
College of Health and Public Affairs
Molecular Biology and Microbiology
Dissertations, Academic -- Health and Public Affairs;Health and Public Affairs -- Dissertations, Academic
Length of Campus-only Access
Honors in the Major Thesis
Osbourne, Tanisha, "Purification and characterization of a 20 KD recombinant protein of M. Avium SS paratuberculosis to identify a unique protein of M. Avium for serodiagnosis of Crohn's disease" (2001). HIM 1990-2015. 252.