致肾盂肾炎大肠杆菌粘附素papG基因克隆及序列分析

Cloning and sequencing of adhesin gene papG derived from uropathogenic Escherichia coli

目的 克隆F13型致肾盂肾炎大肠杆菌 (UPEC) 132株的粘附素基因papG并作序列分析。 方法 根据Ⅰ型papG(papGJ96 )和Ⅱ型 papG(papGIA2 )基因序列设计 3条引物 ,PG2和PG3分别为下游 3’端引物 ,PG1为两型papG上游 5’端共用引物 ,并在各引物 5’端加入限制性内切酶位点。以UPEC132染色体DNA为模板进行PCR扩增 ,将扩增产物克隆入质粒载体 ,筛选阳性重组质粒作序列分析。结果 UPEC132株以Ⅰ型 papG引物扩增时为阴性结果 ,以Ⅱ型 papG引物扩增时可获得约 110 0bp的DNA片段。扩增产物经克隆获得阳性重组质粒 pGC39和pGC10 3,对其序列分析显示 papG132编码337个氨基酸 ,与 papGJ96的同源性仅为 4 5 % ,而与 papGIA2的同源性为 98.6 %。与papGIA2比较 ,papG132核苷酸序列 +3位缺失 1个三联体密码 ,并在特异性受体结合域 +49位、+16 0位和PapD蛋白亚单位作用区 +2 72位、+314位存在错义突变 ,此外还存在 7处同义突变。结论UPEC132株的P菌毛不同于相同血清型的UPECJ96株 ,前者为F13型papA与Ⅱ型papG组合 ,后者为F13型 papA与Ⅰ型papG组合。Ⅱ型PapG粘附力较强 ,具有重要的临床意义。

Aim To clone and sequence adhesin gene papG obtained from uropathogenic E.coli strain 132 (UPEC132).Methods papG of UPEC132 was amplified by PCR with primer pairs specific for two classes of papG with one of them for UPECJ96 whose F13 serotype of P pili contained classⅠpapG and the other for UPECIA2 characterized by F11 serotype of P pili including classⅡpapG.The recombinants constructed by the insertion of the amplified fragment from UPEC132 into the plasmid pUC18 were then identified and subjected to DNA sequencing.Results A single predicted fragment of about 1100 bp from UPEC132 was identified with the primer specific for classⅡpapG but not the primer specific for classⅠpapG.Sequence analysis of the recombinant plasmids(pGC39 and pGC103) carrying such a product of papG of UPEC132 (PapG132) showed that a 377 amino acid long polypeptide could be translated.UPEC132 and J96 shared only 45% homology of deduced amino acid sequence, but the homology of deduced amino acid sequence between UPEC132 and IA2 was 98.6%.There were four missense mutations in the PapG of UPEC132 with two of them involved in the receptor binding domain and the other two in the PapD PapG interaction domain, as compared to IA2.Seven synonymous mutations were also shown, as well as a deleted codon at +3 site occurred, causing one amino acid residue shorter in PapG132 protein than in PapGIA2 equivalent. Conclusion The PapG adhesin from UPEC132 differs from extensively from the corresponding protein from J96, although both strains expressed the same serotype of P pili.Thus, the structure of fimbriae of UPEC132 consists of class II PapG variant in conjunction with the F13 PapA protein.