A Study on the Detection of Crohn's Disease-Specific Intestinal Protein and Mycobacteria Using Molecular Biological Technique

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(1) Cloning of cDNA and genomic DNA specific for Crohn's disease intestineThe mRNA extracted from the intestinal tissues of Crohn's disease (CD) was reverse transcribed to cDNA.Since the crude protein, obtained by the method of Das et al, turned out to be not specific for the CD intestine from the analysis by Western blotting technique, this protein could not be used for screening to select the CD-specific DNA.Then cDNA described above was annealed to mRNA extracted from normal intestine, and cDNA that did not hybridize to the mRNA was extracted (Subtraction method). We screened cDNA library using this subtracted cDNA and tried to determine the sequence of the selected cDNA,though CD-specific cDNA clone has not been detected. Cloning of CD-specific cDNA using arbitrarily-primed PCR (AP-PCR) is also ongoing, but no CD-specific cDNA clone has been obtained.(2) Detection of the specificity of mycobacteria, yeasts, and viruses for CDUsing the primer specific for Mycobacterium paratuberculo … More sis (M.PTB), nested PCR was performed for the DNA and RNA extracted from the intestinal samples of CD,ulcerative colitis, and controls. No CD-specific PCR product was obtained by agarose gel electrophoresis. For getting higher sensitivity and specificity, both dot and Southern hybridization were methodologically added, but no CD-specific product was detected. These results suggest that M.PTB plays no etiological role in CD.To examine whether yeasts, as food antigen, are involved in the etiology of CD,nested PCR was performed with each primer that recognize Saccharomyces cerevisiae, Candida albicans, and Mucor racemosus. No difference in the detection rate between CD and control was observed. Furthermore, our previous study revealed that there was no difference in the levels of serum antibody to yeasts between CD and control. Taken together, it is suggested that yeasts may play a secondary role in the etiology of CD.Measles, mumpus, and rubella virus were also examined using nested RT-PCR and Southern blotting. No specific PCR product for each virus was detected in the CD intestine, suggesting that these viruses don't play an etiological role in CD. Less