An approach to identify the etiological agent of Crohn's disease by molecular cloning technique

克罗恩病是一种病因不明的慢性炎症性肠病。本研究旨在通过分子技术寻找克罗恩病可能的病因。从克罗恩病患者的结肠组织中构建了cDNA文库。用另一位克罗恩病患者的血清筛选了120*10^6个λ噬菌体克隆。免疫筛选选择了20个噬菌体克隆，内含150-1000个bp-cDNA插入。然而，核苷酸测序显示，20个阳性插入片段中有12个与保存在GenBank和欧洲分子生物学实验室（EMBL）数据库中的任何病毒或细菌基因序列没有同源性。由于麻疹病毒最近被报道为克罗恩病的病原，我们试图用PCR（聚合酶链反应）扩增病毒基因组的一部分，引物特异性编码核衣壳、基质、融合蛋白和血凝素的基因。用引物对核衣壳基因进行PCR，在所有标本中均观察到与预期分子大小一致的单个条带，而其他引物对均未扩增出片段。然而，测序结果显示，这些片段与麻疹病毒核衣壳基因或存放在GenBank和EMBL数据库中的任何其他已知序列没有同源性。从这些结果，我们得出结论，没有传染因子，特别是麻疹病毒，直接参与克罗恩病的发病机制。

Crohn's disease is a chronic inflammatory bowel disease with unknown etiology. This study was undertaken to seek by molecular techniques the possible etiological agents of Crohn's disease. A cDNA library was constructed from the affected colonic tissue of a Crohn's disease patient. A total of 120*10^6 of lambda phage clones were screened with the serum taken from another patient with Crohn's disease. The immunoscreening selected 20 phage clones containing 150-1000 bp-cDNA inserts. Nucleotide sequencing revealed, however, that 12 of 20 such positive inserts exhibited no homology with any of viral or bacterial gene sequences deposited in the GenBank and European Molecular Biology Laboratory (EMBL) databases.Because measles virus has recently been reported as an etiological agent of Crohn's disease, we attempted to amplify portions of the viral genome by using the PCR (polymerase chain reaction) with primers specific to the genes encoding nucleocapsid, matrix, fusion protein and hemagglutinin.Single distinct bands with and expected molecular size were observed in all specimens by PCR with primers for the nucleocapsid gene, while no fragment was amplified by any of the other primer pairs. Sequencing these fragments revealed, however, no homology with the nucleocapsid gene of measles virus or any other known sequences deposited in the GenBank and EMBL databases.From these results, we conclude that no infectious agent, measles virus in particular, is directly involved in the pathogenesis of Crohn's disease.