构建胸膜肺炎放线杆菌apxIC基因插入突变株

Construction of an apxIC Mutant of Actinobacillus pleuropneumoniae

目的:构建胸膜肺炎放线杆菌(APP)apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法:根据apxI核酸序列(U05042)设计1对引物,用于自APP血清10型参考菌株(D13039)基因组DNA中扩增apxIC基因及其上下游约2.8kb的基因片段,经克隆测序后在apxIC基因下游XhoⅠ酶切位点处插入约0.9kb的氯霉素(Chl)抗性基因表达盒,构建用于转化的转移载体pUIC-Chlr,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果:在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株(D13039C-Chlr);利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论:利用电转化和同源重组技术构建成功APPapxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。

Objective： In order to analyze biological characters of the Apx I toxin, an apxlC mutant Actinobacillus pleuropneumoniae was constructed by insertional inactivation. Methods： A 2.8 kb fragment containing the apxIC gene was amplified from the genomic DNA of A.pleuropneumoniae serovar 10 reference strain（D13039） by PCR. Based on cloning and sequence, the transfer plasmid pUIC-Chl^r was constructed by inserting a chloramphenicol resistance gene cassette into the Xho I site of the apxIC gene. The transfer plasmid was introduced into the electrocompetent A.pleuropneumoniae serovar 10（D13039） for homologous recombination by electroporation. Results： The mutant strain, termed D13039C-Chl^r, was obtained by selection on the culture containing chloramphenicol. The chloramphenicol resistance gene in the genome of the mutant strain was identified by PCR and Southern blot. Conclusion： The apxIC mutant strain of A.pleuropneumoniae was constructed successfully by insertional inactivation, and the mutant strain may be developed as a live attenuated vaccine candidate of A.pleuropneumoniae.