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

(1)克隆克隆该蛋白不能用于筛选CD特异性DNA，然后将上述cDNA与从正常肠中提取的mRNA退火，提取不与mRNA杂交的cDNA（减法）。我们用该cDNA筛选cDNA文库，并试图确定所选cDNA的序列，但尚未检测到CD特异性cDNA克隆。使用任意引物PCR（AP-PCR）克隆CD特异性cDNA也在进行中，但尚未获得CD特异性cDNA克隆。(2)用副结核分枝杆菌特异性引物检测结核分枝杆菌、酵母菌和病毒对CD的特异性 ...更多信息 sis（M.PTB），对从CD、溃疡性结肠炎和对照的肠样品中提取的DNA和RNA进行巢式PCR。琼脂糖凝胶电泳未获得CD特异性PCR产物。为了提高灵敏度和特异性，在方法学上增加了斑点杂交和Southern杂交，但未检测到CD特异性产物。这些结果表明，M.PTB在CD中没有病因学作用。为了检查酵母菌作为食物抗原是否参与CD的病因学，用识别酿酒酵母、白色念珠菌和总状毛霉的每个引物进行巢式PCR。CD和对照组的检出率无差异。此外，我们以前的研究表明，CD和对照组之间的血清酵母抗体水平没有差异。两者合计，这表明，酵母菌可能发挥次要作用的CD的病因。麻疹，腮腺炎，风疹病毒也采用巢式RT-PCR和Southern印迹法进行了检查。在CD肠道中未检测到每种病毒的特异性PCR产物，表明这些病毒在CD中不起病原学作用。少

(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