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目的 克隆福氏 2a志贺菌 30 1株染色体编码与代谢相关的酶类基因 ,分析结构特点并进行同源性比较 ,以期部分阐明其与大肠杆菌生化特性相似的分子生物学基础。方法 鸟枪法随机克隆福氏 2a志贺菌染色体DNA ,经探针杂交和末端核苷酸序列测定筛选出带有磷酸烯醇丙铜酸盐合成酶 (pps)完整基因的阳性克隆 ,利用exonucleaseⅢ制备嵌套缺失体亚克隆 ,采用双脱氧链末端终止法测定核苷酸序列。结果 福氏 2a志贺菌 30 1株pps基因 (全长 2 4 74bp)与大肠杆菌pps基因核苷酸及氨基酸序列的同源性分别为 98.8%和 97.6 %。编码区上游和下游存在较多变异。福氏 2a志贺菌 30 1株pps基因与GenBank中其它菌株pps基因的同源性均低于 5 5 %。结论 福氏 2a志贺菌pps基因核苷酸序列与大肠杆菌pps基因核酸序列高度同源
Objective To clone and express the enzyme genes related to metabolism of 30 1 Chromosome of Shigella flexneri 2a, analyze the structural features and compare homology, in order to clarify the molecular biology basis similar to the biochemical characteristics of Escherichia coli. Methods The chromosomal DNA of Shigella flexneri 2a was randomly cloned by shotgun method. The positive clones carrying the complete gene of phosphoenolpyruvate synthase (pps) were screened by probe hybridization and terminal nucleotide sequencing. The positive clones were prepared by exonuclease Ⅲ Nested subclones were nested, and the nucleotide sequence was determined by the dideoxy chain termination method. Results The homology of the pps gene of P. pastoris 2a and pps gene was 98.8% and 97.6%, respectively. There are many variations upstream and downstream of the coding region. The homology of 30 1 pps gene of Shigella flexneri 2a with other strains of pps gene in GenBank was lower than 55%. Conclusion The nucleotide sequence of pps gene of Shigella flexneri 2a is highly homologous to the pps gene of Escherichia coli