霍乱弧菌甘露醇-1-磷酸脱氢酶基因mtlD的克隆表达

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。

Cloning and expression of mtlD encoding mannitol-1-phosphate dehydrogenase of Vibrio cholerae

Objective To clone and express the mtlD gene encoding mannitol-1-phosphate dehydrogenase from Vibrio cholerae mannitol specific PTS operon. Methods PCR was conducted to amplify the mtlD gene with the chromosome of N16961 as template. The PCR product was cloned as NdeⅠ-XhoⅠ fragment into the expression vector pET-30a. The mtlD expression was induced with 0.1 mmol/L IPTG, the cell lysis supernatant treated with sonication was purified with Ni-NTA affinity column. The enzyme activity was tested with chromometry assay. Results mtlD was successfully cloned into pET-30a and expressed in the E.coli host cell BL21. The purified MtlD protein containing 6His-tag at its carboxyl terminus showed specific enzyme activity. Conclusion Mannitol-1-phosphate dehydrogenase of V. cholerae was functionally expressed in E. coli as a His-tagged recombinant protein and purified to homogeneity, which severed as the basis for further function study.