CAJ | 학술논문

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apxlIC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。
중조전이재체pBSKA통과전전화도입친본균흉막폐염방선간균혈청7형（APP-7）WF83주，전전화후적산물도포우TSB／Kan평판，2d획득돌변주。잡나매소항성실험증실돌변주유잡나매소항성；NAD의뢰성실험증실돌변주의뢰NAD생장；PCR감정증실료잡나매소항성기인치환료apxlIC기인，병증실돌변주중무pBSKA질립적존재；용혈활성실험증실돌변주완전실거료용혈활성；세포독성실험증실돌변주적세포독성완전상실；대소서적안전성실험증실돌변주적독력현저감약，돌변주대소서시안전적；유전은정성실험증실，돌변주재체외련속전30대화재체내전10대균불회발생잡나매소항성적주실。결과표명실험성공구건료기인결실감독주，위진일보이차돌변주작위기인공정약독활역묘주개전연구전정료일정적기출。
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.