卡介苗基因组的克隆

Cloning of Bacillus Calmette-Guerin Genomic DNA

以质粒pUC19为载体克隆了卡介苗(即卡介菌,BCG)DNA.Sau3Al部分酶解BCG DNA,通过琼脂糖电泳分离2～9Kb DNA片段.BamHl酶切质粒pUC19,碱性磷酸酶去磷酸化.2～9kb DNA片段与pUC19载体用T4连接酶连接转化大肠杆菌HB101,在含氨苄青霉素(Amp)、5-溴-4-氯-3-吲哚-β-D-半乳糖苷的LB平板上获得抗Amp白色转化子.随机挑选12个转化子快速提取重组子、电泳检查结果:其中8个分子量扩大.选择5个分子量扩大重组子经BamHl酶切,电泳分析表明DNA插入片段约在2～7kb之间.

Bacillus Calmette-Guerin (BCG) genomic DNA in Escherichia coli was cloned by cloning sau3A1 cleaved BCG DNA fragments 2-9 kb into BamHl-digested. pUC19 vector. The white ampicillin (Amp) resistance colonies were obtained in plated Luria-Bertani medium containing Amp and 5-bromo-4-chloro-3-indolyl-β-D-galactoside. 12 transforments chosen randomly were compared in size of the plasmid by rapid preparation of plasmid. A-mong 12 clonies tested, the length of these DNA molecules increased. The test results of 5 recombinants showed that the size of the inserted DNA fragment was 2-7 kb.