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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.
Using the method and principle of gene knockout technique, the lpxL2 gene of group B meningococcus and the Kan resistance fragment of vector pGBK T7 were amplified by PCR. The lpxL2 fragment was ligated with the vector puc-18 to obtain the recombinant plasmid msb-puc, msb-puc as the basis, respectively, by reverse PCR and enzyme digestion two methods to construct lpxL2 gene fragment deletion, and in the deletion site connected Kan resistance expression cassette, resulting in recombinant plasmid mpK, mpK transformed into group B meningitis Cocci and preliminary PCR screening of transformants preliminary identified mutant 1. In this study, gene knockout Men L in the LPS synthesis pathway related gene lpxL2 method to reduce the toxicity of LPS group B meningococcal OMV vaccine R & D done pave the way.