肺炎链球菌体内启动子诱捕文库的构建与初步分析

Construction of Promoter-trap Library Screening In Vivo-Induced Gene in Streptococcus pneumoniae

构建肺炎链球菌体内启动子诱捕文库（promoter-trap library），用于筛选肺炎链球菌体内诱导的基因。以穿梭质粒pEVP3为骨架，将无启动子的galU基因作为体内报告基因定向克隆到pEVP3上，与无启动子的体外报告基因lacZ基因融合，构建用于筛选体内诱导基因的载体pEVP3-galU；再把肺炎链球菌基因组DNA的随机酶切片段（200-500bp）克隆到此载体galU基因上游的BglⅡ位点，构建了启动子诱捕文库，并转化肺炎链球菌galU缺陷菌株，获得相应的菌株库。文库大约覆盖基因组全长的5倍，插入率达到90％以上，保持了较高的复杂性。对得到的约450000个肺炎链球菌转化子进行体内、外初步分析，那些从小鼠体内分离出的细菌，并在X-gal平板上为白色的菌落表明galU报告基因上游含仅在体内表达的启动子，提示所构建的启动子诱捕文库可用于筛选肺炎链球菌的体内诱导基因。

To identify in vivo-induced gene in Streptococcus pneumoniae （S. pn）through a novel in vivo expression technology（IVET）, a large promoter-trap library using galU and lacZ as the reporters was constructed. Based on the suicide vector pEVP3, a new vector pEVP3-galU was constructed with promoterless galU gene as an in vivo reporter. Firstly, promoterless galU gene was directly cloned into pEVP3 fusing with promoterless lacZ gene （an in vitro reporter）. Then the random pieces of S. pn chromosomal DNA （200-500bp）, obtained by partial Sau3AI restriction digestion, were subcloned into the Bgl Ⅱ site of pEVP3-galU. Upon introduction of the ligated plasmid library into E. coli DH5cα by transformation, about 70 000 recombinants were recovered. Considering insert DNA orientation and insert size, this represents 5 coverage of the 2.2Mb S. pn genome; 90% of these clones had 250- to 500-bp inserts. Thus, the library retained maximal complexity. Transformation by this plasmid library yield 450 000 S. pn transformants. The library was used to infect animals in intraperitoneal model. Those strains survived in vivo while exhibiting a white colony phenotype on TSA agar containing X-gal would indicate that the DNA fragment upstream of the galU reporter contained an in vivo-induced promoter. The promoter-trap library is suitable for screening in vivo-induced gene of S. pn.