鼠李糖乳杆菌LS8基因组文库的构建

Construction of Genomic Library of Lactobacillus rhamnosus LS-8

为通过构建基因组文库方法,探究鼠李糖乳杆菌LS8菌株的细菌素编码基因,采用质粒消除方法证明LS8菌株细菌素编码基因位于基因组DNA。在此基础上,提取LS8菌株基因组DNA,采用同尾酶Sau3A1和Bam H1分别酶切基因组DNA,回收250~1 000 bp的片段,然后采用T4 DNA连接酶将回收片段与已酶切并去磷酸化后的载体（p Bluescript SK）进行连接,采用Ca Cl2处理法转化大肠杆菌（TOP10）。通过蓝白斑和氨苄抗性筛选,得到阳性克隆1 480个,采用菌液PCR对克隆进行检测。将测序并拼接后所得101条序列在NCBI上比对,发现5个可能的细菌素编码基因。分别设计引物对上述序列进行PCR扩增和测序,为细菌素基因的异源表达奠定基础。

Genomic DNA library was constructed to study the possible bacteriocin encoding genes that may existed in Lactobacillus rhamnosus LS-8. Firstly, it was proved that bacteriocin encoding gene of LS8 was existed in genomic DNA by plasmid curing. Thereafter, the genomic DNA of LS8 was extracted and digested with Sau3A1 and Bam H1,after which 250 bp-1 000 bp DNA fragments were recovered and linked with dephosphorylated p Bluescript SK vector by T4 ligase. The recombinant plamids were transformed into Top10 and recombinants were screened by the blue-white screening and resistance of ampicillin, by which 1 480 clones were selected and verified by PCR. 101 DNA fragments were sequenced and five possible bacteriocin encoding genes were found according to blast search on NCBI, after which five genes were amplified by PCR.