以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

Construction and Analysis of a Promoter-trap Library of Streptococcus pneumoniae Which Employed gfp as a Reporter Gene

摘要:

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP, 将肺炎链球菌基因组DNA的随机酶切片段(200 bp~800 bp)克隆到该质粒gfp基因上游的多克隆位点, 得到约58000个含有肺炎链球菌基因组DNA随机酶切片段的重组子, 提取质粒即为质粒库, 该库大约覆盖肺炎链球菌基因组全长的5倍, 插入率达到90%以上, 且有较强的随机性, 质量较高。将该质粒库转化入肺炎链球菌TIGR4菌株, 带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后, 利用质粒的抗生素抗性基因筛选出重组菌株, 从而构建出相应的菌株库, 共获得包含约500000个肺炎链球菌转化子的菌株库, 经体内、外实验表明, 其包含插入了S. pn体内、外表达基因片段的细菌, 可以报告特定条件下的基因表达, 并可通过流式细胞仪识别、分选。该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础。

Abstract:

Firstly a suicide plasmid pEVP3-SDGFP which employed gfp as a reporter gene was constructed. DNA fragments of S. pneumoniae TIGR4 were cloned upstream of the promoterless green fluorescence protein (gfp) gene in pEVP3-SDGFP and a plasmid library which includes 58000 recombinants was constructed. Considering insert DNA orientation and insert size, this library represents 5 coverages of the 2.2 Mb S. pneumoniae genome. 90% of these clones had DNA fragments of S. pneumoniae and the library is random. Then this plasmid library was transformed into TIGR4. Through recombination, the plasmid DNA which includes the random DNA fragments was placed behind the homologous sequence of the genomic DNA of S. pneumoniae. The recombinants were screened according to the antibiotic gene in plasmid, and the S. pneumoniae library was obtained. This library includes 500000 S. pneumoniae transformants. Analysed by fluorescence microscope and flow cytometry, this S. pneumoniae library contains both the in vivo-induced gene fragments and in vitro-expressing fragments which can be reported by GFP. So this promoter-trap library can be used in analyzing the in vivo-induced gene of S. pneumoniae by differential fluorescence induction.