禽病原性大肠杆菌aes-31基因突变株的构建和致病性鉴定

【目的】通过禽病原性大肠杆菌(APEC)aes-31突变株的构建和动物实验来初步鉴定此基因片段对E058株的毒力影响。【方法】对在芯片杂交试验中筛选到的APEC E058株体外表达差异基因片段aes-31(来自SSH方法筛选到的E058株特异片段),用SSH引物从重组质粒中扩增出目的片段,克隆到pGEM-Teasy Vector中,然后用SphⅠ和SpeⅠ从中切下此片段,将之克隆到pMEG-375自杀性载体中,构建自杀性重组质粒pMEG375-aes-31,将突变载体转化到受体菌中,再和APEC E058株进行固相杂交,根据同源重组原理,筛选出基因突变株E058(Δaes-31)。【结果】E058株和突变株的LD50没有明显差别,突变株对35日龄SPF鸡的致死率高于E058株;两者接种鸡6 h内,E058(Δaes-31)突变株在各内脏器官和血液中的细菌数和E058株差异均不显著;接种鸡24 h后,E058(Δaes-31)突变株在脾脏和肺中的细菌数显著大于E058株,差异显著,E058(Δaes-31)突变株在心脏、肝脏和血液中细菌数显著大于E058株,差异极显著;接种鸡48 h后,E058(Δaes-31)突变株在心脏、肝脏和脾脏中的细菌数比E058株多,差异极显著,而肺脏和血液中的细菌数均无明显差异;48 h后突变株所引起的感染鸡的大肠杆菌病变比亲本株稍严重。【结论】以上数据表明aes-31有可能与E058株毒力的负调控有关。

Construction and pathogenic identification of aes-31 gene mutant of Avian Pathogenic Escherichia coli strain E058

Abstract: [Objective] To find the primary function of aes-31 fragment through construction of defined mutation of Avian Pathogenic Escherichia coli strain E058 and animal experiments. [Methods] The fragment of aes-31 was generated by PCR and cloned into pGEM-T-easy vector. A resultant suicide vector containing the aes-31 fragment named pMEG375-aes-31 was constructed and transformed to a receptor strain SM10. Then recombinant strain SM10 was hybridized with E058 strain in solid state. Mutant derivatives of strain E058 were generated by homologous recombination and were named E058(?aes-31). The 50% lethal dose (LD50) of E058 and E058(?aes-31) in commercial day-old chickens experimentally inoculated via intratrachea were 104.3CFU and 103.5CFU, respectively. The same way was used to inoculate with 108 CFU to obtain the pathogenic ability of E058 and E058(?aes-31) in 35-days-old SPF chickens. In the chicken challenge model, the mutant was tested to determine the individual function for virulence and persistence in 2-week-old SPF chicks. [Results] The pathogenicity test for E058 strain and E058(?aes-31) strain showed that the mutant had a higher mortality (75%) to 35-day-old specific pathogen-free (SPF) chicks than that of E058 (62.5%). In the chicken challenge model, there was no obviously CFUs difference in blood and lung in chicks of E058 group and E058(?aes-31) group 6 hours after inoculation. After 24 hours there was obvious CFUs difference in heart, liver, spleen, lung and blood in chicks of E058 group and E058(?aes-31) group. After 48 hours, there was also obvious CFUs difference in heart, liver and spleen in chicks of E058 group and E058(?aes-31) group E058(?aes-31) had a trend of increasing virulence in chicks. [Conclusion] Aes-31 might be associated with negative regulatory gene for E058 virulence and its actual function needed further study.