CAJ | 학술논문

目的构建自杀性质粒载体,对布鲁杆菌M16株pgm基因进行精确突变,并对得到的pgm基因突变活菌苗株进行鉴定.方法在puc19质粒载体上构建正向筛选标记--蔗糖敏感基因和反向筛选标记--卡那霉素抗性基因融合序列;用卡那霉素抗性基因融合序列对布鲁杆菌pgm基因进行修饰(插入突变),完成pucS1.6K自杀性质粒载体的构建;通过电转化获得布鲁杆菌M16株pgm基因突变菌株;应用PCR方法对pgm基因突变菌株进行鉴定.结果鉴定结果显示,布鲁杆菌M16株pgm基因在卡那霉素抗性基因插入后失活,突变后的布鲁杆菌M16株pgm基因DNA片段长度约为3525 bp,与预期的相符,布鲁杆菌M16株pgm基因突变菌株构建成功.结论构建的自杀性质粒载体能成功对布鲁杆菌进行精确毒力基因突变,为获得布鲁杆菌突变株提供了一个有效的技术平台,也为新型减毒活疫苗的研制奠定了基础.
목적 구건자살성질립재체,대포로간균M16주pgm기인진행정학돌변,병대득도적pgm기인돌변활균묘주진행감정.방법 재puc19질립재체상구건정향사선표기--자당민감기인화반향사선표기--잡나매소항성기인융합서렬;용잡나매소항성기인융합서렬대포로간균pgm기인진행수식(삽입돌변),완성pucS1.6K자살성질립재체적구건;통과전전화획득포로간균M16주pgm기인돌변균주;응용PCR방법대pgm기인돌변균주진행감정.결과 감정결과현시,포로간균M16주pgm기인재잡나매소항성기인삽입후실활,돌변후적포로간균M16주pgm기인DNA편단장도약위3525 bp,여예기적상부,포로간균M16주pgm기인돌변균주구건성공.결론 구건적자살성질립재체능성공대포로간균진행정학독력기인돌변,위획득포로간균돌변주제공료일개유효적기술평태,야위신형감독활역묘적연제전정료기출.
Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines.