肺炎链球菌荚膜缺陷菌株的构建及功能研究.doc

肺炎链球菌荚膜缺陷菌株的构建及功能研究
(作者:___________单位: ___________邮编: ___________)
作者:孟江萍,尹一兵,袁军,张雪梅,蓝锴,陈淑惠,王虹,涂植光
【Abstract】AIM:To construct galU-deletion mutant of us pneumoniae and get the capsule deficient strain, which can be used in al functional genome research. METHODS: LFHPCR was introduced to generate a gene disruption construct consisting of Emr cassette with long flanking homology regions to the target gene. The us pneumoniae was then transformed directly with this PCR product. The galUdeletion mutant was obtained on the TSA agar containing erythromycin and identified by PCR. The deficient strain (5×106 CFU) was applied to infect BALB/c mice to determine the virulence. RESULTS: GalU gene was pletely by erm cassette. The mutant had low virulence to BALB/c mice. CONCLUSION: The galUdeletion mutant can be used to screen in vivopromoter in us pneumoniae because it is incapable of synthesizing capsular haride and hence loses virulence. The target gene can be pletely by LFHPCR product, so this method is simple, rapid and effective for functional genome research of us pneumoniae.
【Keywords】 us pneumoniae;harides ,bacterial;mutation;polymerase chain reaction
【摘要】目的: galU基因是肺炎链球菌荚膜合成的关键基因,构建该基因的缺失突变体,获得肺炎链球菌荚膜缺陷菌株,研究其生物学功能,并为肺炎链球菌功能基因组研究提供实验菌株和技术平台. 方法: 采用长臂同源多聚酶链式反应(LFHPCR)方法制备中间为红霉素耐药基因(erm),两侧为galU基因上、下游同源序列的连接片段,并将此片段转化入肺炎链球菌,,: PCR结果显示ga