B群脑膜炎球菌lpxL2基因敲除突变株的构建及初步鉴定

 应用基因敲除技术的方法和原理,通过PCR扩增B群脑膜炎球菌lpxL2基因及载体pGBK T7上的Kan抗性片段,lpxL2片段与puc-18载体连接得到重组质粒msb-puc,以重组质粒msb-puc为基础,分别通过反向PCR和酶切2种方法构建lpxL2基因中间片段的缺失,并在缺失位点连入Kan抗性表达盒,从而得到重组质粒mpK,mpK转化B群脑膜炎球菌,并用PCR的方法对转化子进行初步筛选鉴定,初步确定突变株1株.本研究通过基因敲除MenB中LPS合成途径相关基因lpxL2的方法,降低LPS毒性,为B群脑膜炎球菌OMV疫苗的研发做了铺垫.

Construction and primary identification of meningococcal serogroup B mutant strain that knocked out gene LpxL2

To construct lpxL2 deletion mutants of meningococcal serogroup B,the lpxL2 gene and Kan resistance gene was amplified from wild-type N.meningitidis serogroup B strain 29325 and plasmid pGBK T7 respectively.The lpxL2 gene was cloned into plasmid puc-18 to constitute plasmid msb-puc.Based on msb-puc,inverse PCR and restriciton enzyme digestion were performed respectively to delete some internal fragments,the resulting products were digested and ligated with the Kan resistance gene,yielding recombiant plasmid ...