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

利用分子生物学技术检测克罗恩病特异性肠道蛋白和分枝杆菌的研究

• 批准号: 05404029
• 负责人: YOSHIDA Yutaka
• 金额: $ 7.87万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05404029/
• 关键词: Crohn's disease; Crohn's disease-specific intestinal protein; Crohn's desease-specific cDNA; PCR; mycobacteria; measles virus; yeast; Mycobacterium paratuberculosis; 麻疹ウィルス; モノクローナル抗体; 麻疹; DNAクローニング

(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

(1)克罗恩病肠道特异性cDNA和基因组DNA的克隆从克罗恩病(CD)肠组织中提取mRNA，将其反转录为cDNA。采用Das等方法获得的粗蛋白为通过Western blotting技术分析，该蛋白对CD肠道不具有特异性，因此不能用于筛选CD特异性DNA。然后将上述cDNA与从正常组织中提取的mRNA退火提取未与 mRNA 杂交的 cDNA（扣除法），并尝试确定所选 cDNA 的序列，但尚未检测到 CD 特异性克隆。使用任意引物PCR（AP-PCR）的CD特异性cDNA也在进行中，但尚未获得CD特异性cDNA克隆。（2）分枝杆菌、酵母菌和酵母菌的特异性检测使用副结核分枝杆菌sis（M.PTB）特异性引物，对CD、溃疡性结肠炎肠道样本中提取的DNA和RNA进行巢式PCR，未获得CD特异性PCR产物。为了获得更高的灵敏度和特异性，在方法上添加了点杂交和Southern杂交，但没有检测到CD特异性产物。这些结果表明M.PTB没有发挥作用。为了检查作为食物抗原的酵母是否与 CD 的病因有关，使用识别酿酒酵母、白色念珠菌和总状毛霉的每种引物进行了巢式 PCR，CD 和总状毛霉的检出率没有差异。此外，我们之前的研究表明，CD 和对照之间的血清抗体水平没有差异，这表明酵母可能在其中发挥次要作用。 CD 的病原学也使用巢式 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 中不发挥病原学作用。无CD

项目成果

A Munakata, Y Haga: "Crohn's disease as aspects of infectious disease." Bio Clinica. 12(5). 18-21 (1997)

A Munakata、Y Haga：“克罗恩病是传染病的一个方面。”

芳賀 陽一: "Absence of measles viral genomic sequence in intestinal tissues from Crohn's disease by nested polymerase chain reaction" Gut. 38. 211-215 (1996)

Yoichi Haga：“通过巢式聚合酶链式反应检测克罗恩病肠道组织中麻疹病毒基因组序列的缺失”Gut，38. 211-215 (1996)。

棟方 昭博: "炎症性腸疾患の感染症的側面" Bio Clinica. 12. 18-21 (1997)

Akihiro Munakata：“炎症性肠病的感染方面”Bio Clinica 12. 18-21 (1997)。

芳賀 陽一: "Nested Reverse Transcription Polymerase Chain Reactionによる炎症性腸疾患腸管中の真菌遺伝子の研究" The Japanese Journal of Parenteral and Enteral Nutrition. 18. 83-85 (1996)

Yoichi Haga：“通过巢式逆转录聚合酶链反应研究炎症性肠病肠道中的真菌基因”《日本肠外和肠内营养杂志》18. 83-85 (1996)。

Y Haga, O Funakoshi, H Nakajima, H Saito, Y Murata, A Munakata, K Kuroe, Y Yoshida: "A study of viral genomic sequence in intestinal tissues from Crohn's disease by nested reverse transcription polymerase chain reaction." M Omata, ed.Gene Transfection and

Y Haga、O Funakoshi、H Nakajima、H Saito、Y Murata、A Munakata、K Kuroe、Y Yoshida：“通过巢式逆转录聚合酶链反应研究克罗恩病肠道组织中的病毒基因组序列。”