一种简便有效的枯草杆菌质粒克隆方法

A Rapid and Effective Method for B.subtilis Plasmid Cloning

目的 在用枯草杆菌质粒克隆并表达白喉毒素突变型DT del14 8时 ,出现A70 0T突变 ,导致Ile2 0 9Leu氨基酸取代 ,为纠正这一突变 ,设计一种简便有效的枯草杆菌质粒克隆方法。方法 首先对含I2 0 9L突变的载体PSM6 0 4 DT del14 8 I2 0 9LDNA进行计算机限制内切酶谱分析 ,以位于PflMⅠ及HindⅢ间的唯一切点的限制内切酶KpnⅠ消化 ,然后去磷酸化 ,热灭活相应酶 ,加入不含I2 0 9L突变的大肠杆菌质粒PET15b DT del14 8,再以PflMI、HindⅢ消化 ,浓缩后进行连接、转化、筛选克隆 ,最后DNA序列分析。结果 经限制内切酶消化及序列分析 ,所有转化子均含目的基因片段。载体DNA仅需 30 0ng ,转化效率达 4 5× 10 2 转化子 / μg ;结论 方法简便、有效、快捷 ,成本低、成功率高 。

Objective A700T mutation was found in cloning and expressing diphtheria toxin (DT) gene mutant DT del148, which led to amino acid substitution of Ile209Leu. A simple and effective method for B. subtilis plasmid cloning was developed to correct this mutation.Methods All restriction sites in sequence of PSM604-DT del148 I209L was fully mapped by computer, and then digested with Kpn Ⅰ, whose sole site located between cloning sites pfl MⅠ and Hin dⅢ. The products were dephosphated, heat inactivated and co digested with E.Coli plasmid PET15bD T del148 that contained no I209L mutation. Concentration of digested DNA, ligation, transformation and clone screening were followed, and the selected clones were finally sequenced to clearify if mutation was corrected.Results All transformants were found to have the expected DNA fragment without I209L mutation testified by restriction mapping and DNA sequencing. The present method only needed 300 ng vector DNA and resulted in high transformation efficiency of 4 5×10 2 transformants per microgram vector DNA.Conclusion The presented method was simple, rapid, effective with low cost but high through out, and could be extensively used in plasmid cloning.