禽病原性大肠杆菌温度敏感性血凝素(Tsh)突变株的构建

Construction of defined mutations of avian pathogenic Escherichia coli strain

运用基因重组方法将庆大霉素抗性基因(GM)连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下目的片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APECE037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株。E037和E037(Δtsh)株的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明,E037(Δtsh)株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有了明显降低。

Gentamycin-resistant gene （GM） was ligated with the flanking fragments of tsh gene amplified by PCR,and then the product was transferred into MCS of pUC18 via DNA recombination techniques. The resulting plasmid was named pUC18-tshFRGM. The fragment of interest was digested and subcloned into suicide vector pMEG375 to generate the plasmid pMEG375-tshFRGM. The plasmid was transformed into APEC E037 used as recipient strain containing tsh gene. ΔTsh-deleted E037 mutant strain was screened by homologous recombination. The animal infection results revealed that the pathogenicity of E037 （Δtsh） strain reduced. Successful construction of Δtsh-deleted E037 mutant strain has an important value and significance for elucidating the function of tsh gene and the role of tsh gene playing in the pathogenicity. The 50% lethal dose （LD50） of E037 and E037 （Δtsh） in commercial day-old chickens experimentally inoculated via intratrachea were 105.6 CFU and 109.0 CFU,respectively. In the chicken challenge model,the mutants were tested to determine the individual role of this system for virulence and persistence in chickens.