| 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,2d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。遗传稳定性试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。这表明成功构建基因缺失减毒株,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。 |
| 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 |
| DOI： |
| 投稿时间：2011-11-17修订日期：2011-11-24 |
| 基金项目:教育部长江学者和创新团队发展计划资助项目(IRT0555-9)。 |
|
| Construction of the Gene Deleted Attenuated Mutant Strain apxIIC-/kanr of Actinobacillus Pleuropneumoniae |
| Li Jian |
| (China Institute of Veterinary Drugs Control;Sichuan Agricultural University) |
| Abstract: |
| 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 kanamycine(Kan).After 2 days,the recombinant strains were selected.Resistance of kanmycine experiment confirmed that mutant can counteract kanamycine.Dependence of NAD experiment confirmed that mutant need NAD in growth.Identification of PCR confirmed that complete apxIIC gene was substitute with kanamycine resistance gene and there was no presence of pBSKA.Experiment of haemolysis confirmed that mutant had no ability of haemolysis. Cytotoxicity test confirmed that mutant had no cytotoxicity.Safty of injected mice experiment confirmed that cytotoxicity and haemolysis of mutant was attenuated significantly so that mutant is safy to mice.Experiment of genetic stability conformed that kanamycine resistance of mutant is stable in 30 vitro successive generations and 10 vivo generations.All of the above indicated that the gene deleted attenuated strain was constructed successfully,which provided a certain basis for further researching genetic live vaccine with the mutant strain. |
| Key words: Actinobacillus pleuropneumoniae Recombinant transfer vector Electroporate Mutant strain Genetic engineering live vaccine |
