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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。
Objective To clone the adhesin gene papG from 132 strains of F13 pyogenic E. coli (UPEC) and analyze the sequence. Methods According to the sequence of papGJ96 and papGIA2, three primers were designed. PG2 and PG3 were the downstream 3 ’primers and PG1 was the common primer of upstream 5’ of papG. ’End of the restriction enzyme site. UPEC132 chromosomal DNA was used as a template for PCR amplification. The amplified product was cloned into a plasmid vector and the positive recombinant plasmids were screened for sequence analysis. Results UPEC132 strain was negative when it was amplified by type Ⅰ papG primer. About 110 bp DNA fragment was obtained by amplification with type Ⅱ papG primer. The amplified products were cloned and the positive recombinant plasmids pGC39 and pGC103 were obtained. Sequence analysis showed that the deduced amino acid sequence of papG132 encoded 337 amino acids, which shared 45% homology with papGJ96 and 98.6% with papGIA2. Compared with papGIA2, the deduced amino acid sequence of the papG132 nucleotide sequence was deleted at +3, +49, +160, +272 and +314 positions of the PapD protein subunit There are missense mutations, in addition there are seven synonymous mutations. Conclusion The P pilus of UPEC132 strain is different from the UPECJ96 strain of the same serotype. The former is a combination of F13 type papA and type Ⅱ papG, the latter is F13 type papA and type Ⅰ papG. Type Ⅱ PapG strong adhesion, has important clinical significance.