在大肠杆菌K99(中国QH株)菌毛形成亚单位基因(fanC)的克隆和测序中发现IS1 插入序列

目的 构建大肠杆菌K99菌毛表达载体。方法 通过逐级亚克隆的方法克隆k99菌毛形成亚单位基因（fanC），并进行序列测定和fanC亚单位的抗原决定簇模拟，以确定突变部位。结果 克隆了包括K99菌毛形成亚单位基因（fanC）和部分fanD基因在内的一段基因，该基因与国外报道相比明显偏大。序列测定表明：在fanC尾部多出 24bp，在fanC与 fanD，的结合部有一 800bp左右的新基因。经与基因库中大肠杆菌基因作同源性比较发现，该新基因为768bp的插入序列IS1。其他基因为K99自身fonC与fanD片段之间的过渡基因。IS1的插入未改变fanD，基因的阅读框架，只是使fanD片段末端增加了 8个氨基酸。fanC亚单位的抗原决定簇模拟结果显示，K99 fanC片段具有一个非常突出的亲水区域，根据经验它应是抗原决定簇所在的部位。结论 首次发现大肠杆菌K99基因中含有插入序列IS1。

Discovery of Inserted Sequence IS1 During Cloning and Sequencing of E.coli K99(Chinese QH Strain) Pilus Forming Subunit Gene(fanC)

Abstract] Objective To construct E.coli K99 pilus expression vector.Methods E. coli K99 pi-lus forming subunit gene(fanC) was cloned by a series of subcloning method and sequenced. The mutation site was determined by the epitope mimesis of fanC subunit. Results A gene fragment containing fanC and a part of fanD genes was cloned. The fragment was significantly longer than that reported abroad. Sequencing showed a new gene fragment at the binding region of fanC and fanD. Compared with the E. coli gene in Genebank, the new fragment was identified as the inserted sequence IS1 with a length of 768bp. No obvious change was observed in the reading frame of fanD gene after IS1 was inserted. However,8 amino acids were added at the end of fanC fragment. The epitope mimesis of fanC subunit showed an obvious hydrophilic region in K99 fane C fragment. According to the previous experiences, the region was judged as the site in which epitope was located. Conclusion Inserted sequence IS1 was observed in E. coli K99 gene for the first time.