论文部分内容阅读
目的克隆表达霍乱弧菌甘露醇特异性磷酸烯醇式丙酮酸依赖的磷酸转移酶系统操纵子中的甘露醇-1-磷酸脱氢酶基因mtlD。方法以测序株N16961染色体为模板,聚合酶链反应(polymerase chain reaction,PCR)扩增mtlD基因,扩增产物经NdeⅠ和XhoⅠ酶切后克隆入表达载体pET-30a。0.1 mmol/L IPTG诱导表达,超声裂解上清经Ni柱亲和层析纯化,比色法测定酶活性。结果 mtlD基因成功克隆入表达载体pET-30a,在宿主菌BL21中诱导表达,利用Ni亲和层析柱获得较高纯度的羧基端带His标签的MtlD蛋白,并具有酶活性。结论表达纯化了有活性的霍乱弧菌甘露醇-1-磷酸脱氢酶,为进一步的功能研究打下了基础。
Objective To clone the mannitol-1-phosphate dehydrogenase gene mtlD in the operator of the Vibrio cholerae mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system. Methods The mtlD gene was amplified by polymerase chain reaction (PCR) using the sequence of N16961 as a template. The amplified product was digested with Nde Ⅰ and Xho Ⅰ and cloned into the expression vector pET-30a. The recombinant plasmid was induced by 0.1 mmol / L IPTG. The supernatant was purified by Ni-affinity chromatography and the enzyme activity was determined by colorimetric assay. Results The mtlD gene was successfully cloned into the expression vector pET-30a and expressed in the host strain BL21. The high affinity His-tagged MtlD protein was obtained by Ni affinity chromatography and showed high activity. Conclusion Expression and purification of active V. cholerae mannitol-1-phosphate dehydrogenase laid the foundation for further functional studies.