卫氏并殖吸虫基因文库的构建——Ⅰ重组克隆的方法

CONSTRUCTION OF GENOMIC DNA LIBRARY OF PARAGONIMUS WESTERMANI 1 COMPARASION OF RECOMBINANT CLONING METHODS

以SDS-酚-乙醇沉淀法提取卫氏并殖吸虫成虫基因组DNA,分别用Sau 3A和Eco RI核酸限制性内切酶酶切,电泳回收0.4—4Kb大小的DNA片段,与相应的大肠杆菌质粒载体pBR^(322)或pUR^(222)重组连接,转染大肠杆菌7118。结果显示,pUR^(222)质粒经碱性磷酸脂酶处理后与DNA片段以1/4(W/W)比例重组连接,获得的重组克隆最多,并且可以利用菌落的色泽不同,从麦康凯培养基平板上直接挑选出重组克隆。是利用质粒载体构建基因文库重组克隆的较好方法。

We isolated the genomic DNA of Paragonimus westermani using SDS-phenolalcohol precipitation method. Genomic DNA was digested with restrictive endonucleases Sau 3A or Eco RI. the fragments of size 0. 4-4 Kb were harvested and randomly ligated into linearized vea-tors plasmid pBR322 or pUR222 respectively, using T4 DNA ligase. The recombinant plasmids were transformed into E. coli strain 7118. We found it is best to recombine the vector and the fragment in the ratio of 1: 4 (w/w) . Phosphatased linearized plasmid pUR222 could prevent it from self-circling and raise the recombinant efficiency to 90. 8%, and be easily screened by the colour of bacteria colonies on MacConkey lactose indicator agar plates. It is a better way to construct the genomic DNA library to select DNA probe.