鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC^i的克隆、鉴定  

Cloning and Identification of H一1i Gene(fliC￣i) of Salmonella Typhimurium

作　　者：蔡学忠[1] 焦新安[1] 张如宽[1] 刘秀梵[1] 

机构地区：[1]扬州大学农学院

出　　处：《农业生物技术学报》1995年第3期71-76,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金;江苏省青年科技基金

摘　　要：以提纯的鼠伤寒沙门氏菌８７０５染色体ＤＮＡ为材料，经ＥｃｏＲＩ消化，过柱（Ｓｅｐｈａｒ－ｃｙｌＳ一４００），得到大于４００ｂｐ的酶切片段；然后随机克隆到质粒ｎＧＥＭ一３Ｚｆ（一）中，转化大肠杆菌ＬＣ２ａ（ｈａｇ－，ｒｅｃＡ－）；在氨苄青霉素平板上共得到６０ｌ３个转化子，从中筛选出１个有动力的克隆，小量制备质粒ＤＮＡ，经酶切电泳鉴定，该克隆的外源片段大小为１５．３ｋｂ，将其命名为ｐＧＩ４０ｌ５。动力和动力抑制试验、Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交试验证明ｎＧＩ４０ｌ５中载有鼠伤寒沙门氏菌Ｉ相鞭毛蛋白基因ｆｌｉＣｉ；利用其ＢａｍＨＩ和ＳａＩＩ位点切除与鞭毛蛋白表达无关的序列，构建亚克隆质粒ｐＧＩ４０ｌ５８８，使得ｆｌｉＣｉ定位于更小的区域─—３．８ｋｂ的ＢａｍＨＩ／ＳａｌＩ插入片段上。Purified Salmonella typhimurium 8705 chromosomal DNA was digested with EcoR I.DNAfragments above 400 bp were obtained by Sepharcyl S一400 chrornatography and cloned into vector pGEM一3Zf(一),then transformed into host cell LC2a(hag- is recA-).One out of 6013 transformants was foundto be motile and this clone was named pGI4Dl5.Miniprep proved that pGI40l5 contained an inserted frag-ment about l5.3kb. Motility/inhibition teSts as well as Southern blot hvbridization showed that pGI40l5bear the H一1i gene of S,typhimurirm. With the aid of BamH I and Sal I site,sequences unrelated to flag-ellin in the cloned DNA fragment was removed,and a 3.8 kb fragment containing fliCi gene was sub-cloned.

关 键 词：鼠伤寒沙门氏菌 I相鞭毛 蛋白基因 克隆 

分 类 号：S852.43[农业科学—基础兽医学]