B群脑膜炎球菌lpxL2基因敲除突变株的构建及初步鉴定  

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

作　　者：李晓晶[1] 姬秋彦[1] 肖红剑[1] 彭正华[1] 罗娜[1] 杨增福[1] 杨槐[1] 李健峰[1] 李智华[1] 徐维明[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《云南大学学报（自然科学版）》2011年第6期710-715,共6页Journal of Yunnan University(Natural Sciences Edition)

基　　金：云南省自然科学基金资助项目(2009ZC168M)

摘　　要：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.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 mpK which was used to transform into wild - type strain 29325. Antibiotic - resistant transformants were screened by using PCR, and a mutant strain was primary identified. The LPS biosynthesis gene lpxL2 mutants can reduce toxicity by means of knocking out gene lpxL2, which established serogroup B. approach for the development of OMV vaccine against Nm serogroup B.

关 键 词：脑膜炎奈瑟氏菌 外膜囊泡 基因敲除 脂多糖 

分 类 号：R392.11[医药卫生—免疫学] Q782[医药卫生—基础医学]