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目的 对致肾盂肾炎大肠杆菌 (UPEC) 132株的papA基因进行克隆和序列分析。方法根据F13型papA基因序列设计引物 ,并在二引物 5′端加上限制性内切酶EcoRⅠ和BamHⅠ的酶切位点。采用此引物扩增 132菌株p菌毛粘附基因群的papA基因 ,扩增产物克隆入质粒 ,选取阳性重组质粒进行核苷酸序列分析。结果 UPEC132株经PCR法扩增获得约 70 0bp的papA片段 ,经克隆获得阳性重组质粒pCT2 0 ,对其进行序列分析显示papA编码 184个氨基酸多肽 ,与UPECJ96株papA相比较同源性达 98%以上 ,于 +4 3位和 +73位的错义突变对该菌毛的免疫学特性没有影响 ,保守的信号肽序列 16位出现同义突变 , 3位缺失三联体密码使 1, 3位切割三联体由ANN变为ANV。结论 pa pA信号肽序列变异可能影响其正确的切割 ,致使 132株 (Mr为 16 6× 10 3)和J96株 (Mr为 19 6× 10 3)p菌毛蛋白的相对分子质量不同。
Objective To clone and sequence the papA gene of 132 strains of pyogenic pyogenic E. coli (UPEC). Methods Primers were designed according to the type F13 papA gene sequence, and restriction endonucleases EcoRI and BamHI were added to the 5 ’end of the two primers. This primer was used to amplify the papA gene of 132 strains of p pilus adhesion gene. The amplified product was cloned into the plasmid and the positive recombinant plasmids were selected for nucleotide sequence analysis. Results The UPA132 strain was amplified by PCR to obtain a 70 bp fragment of papA. The recombinant plasmid pCT20 was obtained by cloning. Sequence analysis showed that papA encoded a 184 amino acid polypeptide with a homology of 98% with papA of UPECJ96. Above, the missense mutations at positions +43 and +73 have no effect on the immunological properties of the pili, and the conservative signal peptide sequence has a synonymous mutation at position 16 and a deletion of the triplet codon leads to cleavage at position 1 and 3 Triplet changed from ANN to ANV. Conclusion The variation of pa pA signal peptide sequence may affect its correct cleavage, resulting in different relative molecular mass of 132 strains (Mr 16 6 × 10 3) and J96 strain (Mr 19 6 × 10 3).