禽致病性大肠杆菌P型菌毛papA基因克隆与基因分型分析

Cloning and genotyping of papA gene of type P pili of avian pathogenic Escherichia coli

为克隆禽致病性大肠杆菌P型菌毛papA基因,确定其基因分型并分析其变异性,从GenBank数据库中查找大肠杆菌P型菌毛papA基因,并运用Snap Gene 3.2.1软件设计合成3对引物。其中,第1对引物为扩增不同基因分型通用引物,预计扩增片段大小为281 bp;第2对和第3对引物是为了扩增完整papA基因并分析有无变异而设计的,预计扩增片段大小分别为522 bp和943 bp。以禽大肠杆菌HJ2、TK3菌株的基因组为模板进行PCR扩增;将所有扩增片段回收、酶切,并分别与pTG19-T载体进行连接,转化进入DH5α受体菌,提取获得含扩增片段的重组质粒,测序并进行序列分析。结果表明,用引物1从HJ2、TK3菌株的基因组中均扩增获得接近281 bp大小的片段,用引物2则只从HJ2菌株基因组中扩增获得接近522 bp大小的片段,而未能从TK3菌株基因组中扩增出片段;用引物3从TK3菌株基因组中扩增出接近943 bp大小的片段,而未能从HJ2菌株基因组中扩增出产物。经过基因碱基序列分析比对,完整的papA基因则位于943 bp的片段之中,确定HJ2和TK3两个菌株P型菌毛papA基因均属基因11型,但发现二者papA基因存在一定的碱基差异。PapA蛋白氨基酸序列比对发现,同属于基因11型的papA基因编码的PapA蛋白氨基酸序列也存在位点差异。

In order to clone the papA gene of type P pili of avian pathogenic Escherichia coli, determine its genotyping and analyze its variability, the papA gene of Escherichia coli P-type pili was searched from the GenBank database, and three pairs of primers were designed and synthesized using Snap Gene 3.2.1 software. Among them, the first pair of primers was a universal primer for amplifying different genotypes, and the expected amplified fragment size was 281 bp. The second and third pairs of primers were designed to amplify the complete papA gene and analyze the variation, and the expected amplified fragments were 522 bp and 943 bp, respectively. The genes of avian Escherichia coli HJ2 and TK3 strains were used as templates for PCR amplification. All amplified fragments were recovered, digested and connected with the pTG19-T vector, respectively, and transformed into DH5α receptor bacteria. Recombinant plasmids containing amplified fragments were extracted, sequenced and analyzed. The results showed that a fragment of 281 bp was amplified from the genome of HJ2 and TK3 strains by primer 1, and a fragment of 522 bp was amplified from the genome of HJ2 strain by primer 2 but not from the genome of TK3 strain. A fragment of 943 bp was amplified from the genome of TK3 strain by primer 3 but not from the genome of HJ2 strain. After sequencing analysis, the complete papA gene was located in the fragment of 943 bp. It was confirmed that the papA genes of P-type pili of the two strains belonged to genotype 11, but there were some base differences in their papA genes. Amino acid sequence alignment of PapA protein showed that there were site differences in the amino acid sequence of PapA protein encoded by papA gene, which belonged to genotype 11.