摘要
胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失。信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法。通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株,再以萘啶酸抗性菌株为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coliCC118λpir或S17-1λpir为供体菌,在或不在E.coliDH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过抗性筛选、PCR和Southern杂交鉴定转座突变株。结果表明:体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株,该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关。在APP与E.coli接合实验中,两亲本接合比三亲本接合操作更简单,效率也较高;APP不同菌株在接合和转座效率上存在很大差异,血清1型菌株高于血清3型菌株,3型标准菌株高于地方分离株JL03-R。本研究为APPSTM突变体库的构建与毒力基因的鉴定奠定了基础。
Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis（STM） is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains ofAPP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 2pir or S17-1 2pir containing mini-Tnl0 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5ct （pRK2073）. We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP.
出处
《微生物学报》
CAS
CSCD
北大核心
2008年第1期73-79,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金(30530590
30771599)
教育部新世纪优秀人才支持计划(NCET-04-0740)~~
关键词
胸膜肺炎放线杆菌
萘啶酸抗性
信号标签突变
细菌接合
Actinobacillus pleuropneumoniae
Nalidixic acid resistance
signature-tagged mutagenesis
mating
