禽病原性大肠杆菌aes-31基因突变株的构建和致病性鉴定  

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

作　　者：宦海霞[1,2] 张科[3] 陈祥[2] 高崧[2] 刘秀梵[2] 

机构地区：[1]淮阴师范学院生命科学学院,淮安223300 [2]扬州大学,农业部畜禽传染病学重点开放实验室,扬州225009 [3]淮安出入境检验检疫局,淮安223001

出　　处：《微生物学报》2010年第12期1681-1685,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30972196,30771460,30471281);国家“863计划”(2003AA222141);江苏省高校自然科学研究项目(09KJB230002)~~

摘　　要：【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。[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 experimentally inoculated via intratrachea were 104.3 CFU and 103.5 CFU,respectively.The same way was used to inoculateE058 strain in solid state.Mutant derivatives of strain E058 were generated by homologous recombination and were named E058（Δaes-31）.The 50% lethal dose（LD50） of E058 and E058（Δaes-31） in commercial day-old chickens with 108 CFU to obtain the pathogenic ability of E058 and E058（Δaes-31） in 35-days-old SPF chickens.In the chickenchallenge 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（Δaes-31） strain showed that the mutant had ahigher 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（Δaes-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（Δaes-31） group.After 48 hours,there was also obvious CFUs difference in heart,liverand spleen in chicks of E058 group and E058（Δaes-31） group E058（Δaes-31） had a trend of increasing virulence inchicks.[Conclusion]Aes-31 might be associated with negative regulatory gene for E058 virulence and its actual function needed further study.

关 键 词：禽病原性大肠杆菌E058株 aes-31基因片段 突变株 致病性 

分 类 号：S852.61[农业科学—基础兽医学]