禽致病性大肠杆菌Ⅰ型菌毛fimH基因缺失突变株的构建 及部分生物学特性的分析

Construction and characterization of type I fimbriae fimH deletion mutant from avian pathogenic Escherichia coli

摘要:

基于禽大肠杆菌Ⅰ型菌毛黏附素fimH 基因的已知序列, 利用λ噬菌体的Red重组系统构建禽致病性大肠杆菌国内分离株A2(血清型O2:K89)Ⅰ型菌毛黏附素fimH 基因缺失突变株A2△fimH::Cat, 在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20(温度敏感性)以去除上述缺失突变株中抗性基因标志, 结合PCR扩增和测序结果, 证明fimH 基因缺失株A2△fimH 的正确构建。通过fimH 基因互补试验使A2△fimH 缺失突变株恢复了与野生株具有相同的凝集活性。红细胞和酵母细胞凝集试验结果表明, 野生株呈现良好的凝集效果, 并能被0.5%甘露糖完全抑制, 而A2△fimH 缺失突变株未呈现任何凝集现象。体外生长试验结果表明,在同样的培养条件下, A2△fimH 缺失突变株生长周期的各个阶段都要稍慢于野生株。禽致病性大肠杆菌国内分离株Ⅰ型菌毛黏附素fimH 基因缺失突变株成功构建, 为进一步深入研究禽大肠杆菌Ⅰ型菌毛与机体相互作用的分子机制, 肠道外感染的致病机理及对国内禽大肠杆菌病的防控策略奠定了一定基础。

关键词:Red重组系统; Ⅰ型菌毛; fimH 基因; 生物学特性

Abstract:

The wild type avian pathogenic Escherichia coli (APEC) isolate A2 (Serotype O2:K89) was selected as the prototype of the (APEC ) TypeⅠFimbriae. Based on the original sequences of TypeⅠFimbriae operon gene clusters in the GenBank,we generated PCR products by using primers with the homologies extension of fimH gene to be deleted and template plasmid pKD3 carrying selectable antibiotic chloramphenicol resistance(cat) gene that is flanked by FRT(FLP recognition target)sites. By using the PCR products, the A2 △fimH::Cat deletion mutant from A2 isolate was constructed by λRed-mediated recombination system in the flanking homologies. After selection, the resistance gene located in the A2△fimH::Cat mutant was eliminated in the second recombination, by using a helper plasmid pCP20, a tempera-ture-sensitive one encoding and expressing the FLP recombinase, which acts on the directly repeated FRT sites flanking the resistance gene. The A2△fimH mutant obtained was further confirmed by fimH PCR amplification and sequencing. The A2△fimH mutant with the deletion of fimH adhesin in the fim gene cluster lost the ability of agglutination reaction with both guinea pig erythrocytes and yeast cells. The A2△fimH deletion mutant restored the agglutination ability of both binding guinea pig erythrocytes and yeast cells when transfected the compatible recombinant plasmid pBR322-fimH with fimH insert in the fimH complementation assay. Very similar to the wild type A2 isolate, The obtained binding activity from the A2△fimH mutant in the complementation assay was completely inhibited when pretreated with 0.5% mannose solution. Compared with the wild type isolate, the A2△fimH mutant grew slowly during all stages of growth. This work provides the basis for us to study the molecular pathogenesis mechanisms of interaction between the APEC TypeⅠ Fim-briae and susceptible host cells, extra-intestinal infection, prevention and control of the APEC-caused disease.

Key words:λRed recombination system; avian Escherichia coli type Ⅰ fimbriae (APEC); fimH mutant; biological characteristics