瘤胃中小韦荣球菌H2基因文库的构建

Construction of genomic DNA library for Veillonella parvula H2 in rumen

为了构建小韦荣球菌H2基因的文库,研究提取小韦荣球菌H2总基因组,采用机械剪切方法获得DNA随机片段,将获得的DNA片段与SmaⅠ酶切质粒pUC18体外连接并电转化至大肠杆菌DH5α,在含有IPTG、X-gal和氨苄西林的LB琼脂平板上用蓝白斑法筛选含重组质粒的大肠杆菌,并进行扩增分析。结果表明:重组质粒经鉴定分析证实包含小韦荣球菌H2的3～4 kb插入片段,文库滴度为1×105pfu/mL;根据公式N=ln(1-p)/(1-f)证明99%基因组包含在文库中。

The experiment was conducted to construct the genomic library of VeiUonellaparvula H2. The total DNA was extracted and sheared by Hydroshear machine and the acquired DNA fragments was ligated to vector pUC18 which was digested with the restriction enzyme Sma Ⅰ and transferred into the E. coli DH5α DNA selected and proliferated on Luria - Bertani （LB） plates containing ampieillin, 5 - bromo -4 - chloro - 3 - indoyl - 3 - galae - （ X - gal ） and isopropy - 13 - D - thiogala ctoside（IPTG） and proliferated. The recombinant plasmid was analyzed and the presence of insert 3 -4 kb DNA fragment was detected by eleetrophoresis mapping. The titer of the library is 1 × 10^5 pfu/mL according to the formula N = ln（ 1 -p）/（ 1 -f）, the genomic library consisits of 99% genome of Veillonella parvula.