大肠杆菌O23 O-抗原基因簇序列的破译及UDP-N-乙酰葡萄糖-C4异构酶的鉴定

Molecular characterization of O-antigen gene cluster of Escherichia coli O23 reference strain and identification of UDP-N-acetylglucosamine 4-epimerase

采用鸟枪法破译大肠杆菌O23标准株的O-抗原基因簇序列,并用生物信息学的方法进行了基因注释和分析;采用基因缺失和互补的方法鉴定了O23的UDP-GlcNAc C4异构酶(Gne);用同源建模的方法构建了O23 Gne的高级结构并对其活性位点进行了分析;分析了不同血清型大肠杆菌O-抗原基因簇中gne基因的多样性;根据O23O-抗原基因簇中的特异基因筛选出了可用于大肠杆菌O23快速检测的特异DNA序列。

Lipopolysaccharide （LPS） is one of the major components of the outer membrane of gram-negative bacteria. It is an amphipathic molecule composing of lipid A, a core oligosaccharide and an O-specific antigen. O-antigen, which is a repeat unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface. Genes involved in O-antigen biosynthesis are generally found to be clustered between the housekeeping genes galF and gnd on the chromosome of E. coll. E. coli O23 is one of the enterotoxigenic E. coli causing pediatric diarrhea in the developing world. The O-antigen gene cluster of E. coli O23 type strain was amplified by long-range PCR using primers based on galF and gnd and then sequenced. Except for galF and gnd, seven open reading frames were identified and assigned functions on the basis of their similarity to those from available databases. The seven genes include a UDP-N- acetylglucosamine 4-epimerase gene （gne）, the O-antigen polymerase gene （wzy）, the O-antigen transferase gene （wzx） and four glycosyltransferase genes （orf2, orf4, orf5, orf6）. The UDP-N-acetylglucosamine 4-epimerase （Gne） was identified by mutation and complementation complement tests. The structure of Gne was predicted by the homology modeling method, and the active sites were also analyzed. The phylogenetic and structural analysis showed that the Gne derived from the common ancestor with E. coli O23 Gne were UDP-GlcNAc/UDP-GalNAc epimerases. The specific DNA used for rapid molecular genotyping for E. coli O23 was also identified.