新疆O139群霍乱弧菌93006菌株基因组文库的构建

Genomic library construction of O139 Vribrio cholerae(93006) from Xinjiang

目的 :构建我国新疆分离O1 39群霍乱弧菌菌株 (930 0 6菌株 )的基因组文库 ,为研究我国O1 39群霍乱弧菌的来源及其基因组特征提供资料。方法 :制备霍乱弧菌高相对分子质量的基因组DNA ,用Sau3AI酶将基因组DNA部分酶切成平均大小为 32～ 4 0kb的片段 ,将部分酶切并经碱性磷酸酶 (CIAP)处理的DNA片段与经XbaI、CIAP、BamHI处理的粘粒载体SuperCos1的载体臂相连接 ,连接产物经体外包装并转染大肠杆菌XL Blue1MR ,经氨苄抗性筛选得到阳性克隆 ,构建霍乱弧菌的基因组文库。结果 :该文库共获得了 1 2 0 0个单克隆 ,平均插入片段的大小为 32kb ,空载率为 0 .0 5 % ,相当于 9个库容量 ,从本文库中筛选到任一霍乱弧菌的DNA单拷贝序列的机率为99.98% ,该文库能承受 7次反复冻溶。结论 :成功构建了我国新疆分离O1 39群霍乱弧菌菌株 930 0 6的基因组文库 ,为进一步克隆霍乱弧菌的基因和研究我国O1

Aim:To construct the genomic library of the O139 Vibrio cholerae(93006) from Xinjiang. Methods: The genomic DNA of Vibrio cholerae was extracted by phenol chloroform. 30～40 kb DNA fragments were prepared by partial digestion with Sau3AI,and treated with CIAP. The fragments were ligated to XbaI, CIAP and BamHI digested SuperCos1, packaged in vitro with the package extract, then infected E.coli XL Blue1 MR. Transformants were selected on LB agar containing ampicillin. Results: The obtained library consisted of 1 200 clones with average insertion size of 32 kb. About 90 percent of the clones contained the inserted DNA. The capacity of this library was about 9 times of the equivalent of Vibrio cholerae genome. The clones of this library could survive after more than 7 times freeze thaw. Conclusions:Genomic library of O139 Vibrio cholerae has been successfully constructed. It is very useful for further gene cloning of Vibrio cholerae 93006 and studying of the origin of Vibrio cholerae CV39.