大肠杆菌F18菌毛E亚单位基因克隆与序列分析

Cloning and Identification of the Minor Subunit fedE Gene of the Fimbriae F18 of Escherichia coli

根据已发表的F18ab菌毛E亚单位的基因序列(fedE/ab),设计1对引物,利用PCR技术从10株大肠杆菌(F107/86、YCED1、YCED2、C、D、E、12F、2134P、8199、8813)中分别扩增到1个片段,并克隆至pGEM-T载体,获得重组质粒TF107E、TYCED1E、TYCED2E、TCE、TDE、TEE、T12FE、T8813E、T8199E、T2134PE。琼脂糖凝胶电泳、序列测定及分析表明,这10个片段大小均为516 bp。通过与已发表的fedE/ab序列进行比较,发现这10个菌株的fedE序列高度同源,其中F107/86、YCED2、C、D、E、8813、8199、2134P的fedE序列完全相同,只有YCED1株在第45位碱基处存在1个G→A转换、12F株在第316位碱基处存在1个G→转换。这说明F18ab和F18ac菌毛的fedE基因相同,fedE是F18菌毛的保守性亚单位。

A pair of primers were designed and synthesized according to fedE of fimbriae F18ab (fedE/ab), and the coordinate subunit genes were amplificated from ten different Escherichia coli strains, F107/86, YCED1, YCED2, C, D, E, 12F, 2134P, 8199 and 8813. Thereinto, F107/86 is display fimbriae F18ab, whereas 2134P, 8199 and 8813 are all display fimbriae F18ac. All the PCR products were cloned into pGEM-T vector respectively. Color screening, PCR and restriction endonucleases analysis were used to identify the recombinant plasmids, and ten recombinant plasmids, TF107E, TYCED1E, TYCED2E, TCE, TDE, TEE, T12FE, T8813E, T8199E and T2134PE were obtained. The ten recombinant plasmids sequences were analyzed and compared with the published sequences of fedE/ab. The data of sequences for the ten genes showed that they were all in accord with the sequences of fedE/ab, 516 bp. Although the strain YCED1 has a variance of adenine to guanine at the 45th nucleotide site, and the strain 12F has variance of adenine to guanine at the 316th nucleotide site. All the data confirmed that the fedE is conservative gene of fimbriae F18, whether it is F18ab or F18ac.