鸡白痢沙门氏菌与鸡伤寒沙门氏菌基因组消减文库的构建

为了解鸡白痢沙门氏菌与鸡伤寒沙门氏菌的基因组差别，筛选鸡白痢沙门氏菌特有的序列，利用SSH对鸡白痢沙门氏菌C79-13（实验方）与鸡伤寒沙门氏菌Sg9（驱动方）基因组进行了比较。选择四碱基内切酶Rsa I将C79-13与Sg9基因组DNA酶切,酶切的C79-13基因组DNA分成两份，分别与接头1和接头2R连接，然后进行两轮杂交和两轮PCR ,得到消减混合物, 将其与PCR?2.1克隆载体连接，转化感受态E.coli DH5α建立消减文库。结果共获得400个阳性克隆；筛选240个克隆制备质粒进行PCR检测，均扩增出100～2000 bp大小的片段。差异DNA消减文库的构建为进一步筛选、克隆鸡白痢沙门氏菌特异的未知新基因、建立分子鉴别新体系奠定了基础。

Construction of a Subtracted DNA Library between Salmonella Pullorum and Salmonella Gallinarum

To understand the genomic differences between Salmonella pullorum and Salmonella gallinarum and to screen Salmonella pullorum-specific sequences that could serve as diagnostic markers, SSH technique was used to compare the genomic differences between S. pullorum C79-13 (tester) and S.gallinarum Sg9 (driver). The tester and driver genomic DNA were digested with Rsa I to give a range of blunt-ended fragments between 100bp and 2000bp. The digested tester DNA was then subdivided into two portions, which were ligated with adaptor 1 and adaptor 2R respectively. Twice hybridizations and two times PCR were performed. The mixture of subtracted DNA fragments were cloned into PCR?2.1 cloning vector and transformed into E.coli DH5α competent cell to construct the subtracted DNA library.The results are as follows: 400 positive clones were obtained; Analysis of 240 clones with PCR shows that all plasmids in the clones contain about 100~2000 bp inserts．The constructed DNA subtractive library lays foundation for screening and cloning relational genes of Salmonella pullorum, and further establishing new system for molecular differentiation .