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生物学技术寻找克罗恩病的可能病因。从克罗恩病患者的受影响的结肠组织构建cDNA文库。用取自另一名克罗恩病患者的血清筛选了总共120*10^6个λ噬菌体克隆。免疫筛选筛选出20个含有150-1000 bp cDNA插入片段的噬菌体克隆。然而，核苷酸测序显示，20个这样的阳性插入物中有12个与GenBank和欧洲分子生物学实验室（EMBL）数据库中保存的任何病毒或细菌基因序列都没有同源性。我们尝试用PCR扩增病毒基因组的一部分（聚合酶链反应），用对编码核衣壳，基质，用核衣壳基因的引物通过PCR在所有标本中观察到具有和预期分子大小的单个不同条带，而任何其他引物对均未扩增出片段。然而，这些片段的测序显示，没有同源性与麻疹病毒的核衣壳基因或任何其他已知的序列存放在GenBank和EMBL databases.From这些结果，我们得出结论，没有感染性因子，特别是麻疹病毒，是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.