胸膜肺炎放线杆菌apxIIC^-/kan^(r+)基因缺失减毒株的构建  

Construction of the Gene Deleted Attenuated Mutant Strain apxIIC^-/kan^(r+) of Actinobacillus pleuropneumoniae

作　　者：李建[1] 曹三杰[2] 文心田[2] 黄小波[2] 都启晶[2] 张明[2] 余慧[2] 

机构地区：[1]中国兽医药品监察所,北京100081 [2]四川农业大学,四川雅安625014

出　　处：《中国兽药杂志》2012年第3期5-9,共5页Chinese Journal of Veterinary Drug

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0555-9)

摘　　要：重组转移载体pBSKA通过电转化导入亲本菌胸膜肺炎放线杆菌血清7型(APP-7)WF83株,电转化后的产物涂布于TSB/Kan平板,2 d获得突变株。卡那霉素抗性实验证实突变株有卡那霉素抗性;NAD依赖性实验证实突变株依赖NAD生长;PCR鉴定证实了卡那霉素抗性基因置换了apxIIC基因,并证实突变株中无pBSKA质粒的存在;溶血活性实验证实突变株完全失去了溶血活性;细胞毒性实验证实突变株的细胞毒性完全丧失;对小鼠的安全性实验证实突变株的毒力显著减弱,突变株对小鼠是安全的;遗传稳定性实验证实,突变株在体外连续传30代和在体内传10代均不会发生卡那霉素抗性的丢失。结果表明实验成功构建了基因缺失减毒株,为进一步以此突变株作为基因工程弱毒活疫苗株开展研究奠定了一定的基础。The recombinant transfer vector pBSKA was electroporated into parent strain Actinobacillus pleuropneumoniae serovar 7 （APP -7 ） strain WF83. Product of the electroporation was plated onto TSB agar containing kanamyeine （Kan）. After 2 days the recombinant strains were selected. Resistance of kanamycine experiment confirmed that mutant strain can counteract kanamycine. Dependence experiment of NAD confirmed that mutant strain needed NAD in growth. Identification of PCR confirmed that complete apxIIC gene was substitute for kanamycine resistance gene and there was no presence of pBSKA. Hemolytic experiment confirmed that mutant strain had no ability of haemolysis. Cytotoxicity test confirmed that mutant strain had no cytotoxieity. Safety experiment of injected mice confirmed that eytotoxicity and haemolysis of mutant was attenuated significantly so that mutant was safe to mice. Experiment of genetic stability confirmed that kanamycine resistance of mutant was stable 30 successive generations in vitro and 10 generations in vivo. All of the above - mentioned tests indicated that the gene deleted attenuated strain was constructed successfully, which provided certain basis for further genetic live vaccine research with mutant strain.

关 键 词：胸膜肺炎放线杆菌 重组转移载体 电转化 突变株 基因工程弱毒活疫苗 

分 类 号：S858.28[农业科学—临床兽医学]