猪水肿病大肠杆菌志贺样毒素Ⅱ型突变体新基因的分子特征

Characteristics of A Novel Shiga-like Toxin Ⅱ Variant Gene in Escherichia coli from Swine With Edema Disease

从仔猪水肿病样品中分离的一株大肠杆菌菌株NP96 2 1,抽提染色体DNA ,构建NP96 2 1的总DNA文库 ,经菌落原位杂交筛选、限制酶酶切和Southern印迹等分析 ,获得含有志贺样毒素基因的重组质粒 ,核苷酸序列分析表明 ,该基因的A亚基与SLT IIes的同源性为 97 6 %～ 98 1% ,与SLT IIc、SLT IId及EHECO15 7:H7产生的SLT II的A亚基之间的同源性分别为 93 1%、93 0 %和 92 9% ;B亚基与SLT IIes的同源性为 99 6 2 %～ 10 0 % ,与SLT IIc、SLT IId及SLT II的同源性分别为 81 5 %、81 1%和 81 5 % ;与 2 9种已知序列的SLT II、SLT I及志贺毒素STX进行聚类分析 ,结果证实大肠杆菌NP96 2 1菌株中的志贺样毒素基因属于一种新的SLT IIe亚型。SLT IIe NP96 2 1的A亚基与其它SLT IIe之间存在 7～ 9个氨基酸的差异 ,B亚基的氨基酸序列完全相同 ,与A亚基毒力活性密切相关的氨基酸分别为Thr4、Glu16 7、Arg170和Arg176。

E. coli strain NP9621 was isolated from the feces of a swine with edema disease in Guizhou province. The fragment containing the Shiga-like toxin gene in NP9621 was cloned from the genome DNA library of NP9621. The recombinant plasmid pEK36 was subjected to nucleotide sequence analysis. It contained the whole genes of Shiga-like toxin Ⅱ in the plasmid pEK36. The subunit A gene of SLTII in NP9621 presented 97.6-98.1% identity of nucleotide sequence with the published SLT-IIe A and its identity of nucleotide sequences with SLT-IIc、SLT-IId and SLT-II/O157:H7 was only about 93% or so. The subunit B gene of SLTII of NP9621 showed a single nucleotide difference from the nucleotide sequence of the published SLT-IIe B at position 187 but revealed 81% identity with subunit B genes of SLT-IIc、SLT-IId or SLT-II from the E. coli O157:H7. The phylogenetic analysis of the nucleotide sequence homology of Shiga-like toxin from NP9621 compared with 29 those of SLT-II, SLT-I and Shiga toxin genes showed that the Shiga-like toxin from NP9621 belonged to the group of SLT-IIes. Therefore the Shiga-like toxin from NP9621 was designated SLT-IIe/NP9621. The deduced amino acid sequence of the subunit B of SLT-IIe/NP9621 was completely identical to those of SLT-IIes submitted in the Genbank. There were seven to nine amino acids different from the other SLT-IIes in subunit A. The amino acids of Glu167, Arg170 and Arg176 were conserved in the subunit A of the SLT-IIe/NP9621, which were crucial to the catalytic activity of the subunit A. The threonine at position 4 in the amino acid sequence of the SLT-IIe/NP9621 might result in the virulence of the SLT-IIe/NP9621 less than the toxins contained Met4. In conclusion, SLT-IIe/NP9621 cloned from the chromosome of E. coli was a novel gene type of SLT-IIe that resulted in edema disease of Guizhou province.