一种带有组氨酸标签的新型表达载体的构建及应用

Construction and Application of a New Type Expression Vector with a Histidine Tag

目的:利用基因重组技术,对已有载体pSE380进行改造,获得具有组氨酸标签的新型表达载体。方法:以pSE380载体为出发材料,通过一步反向PCR技术在多克隆位点处添加一个六聚组氨酸纯化标签,并用该载体对xdh外源基因进行克隆表达及纯化验证。结果:测序结果显示该标签已成功插入,xdh基因克隆表达及纯化表明,镍柱亲和层析完全可以将重组的表达蛋白纯化,并达到电泳纯级别,融合标签并没有影响的该重组酶的功能。结论:通过一步反向PCR技术成功地获得了改造pSE380载体,该技术为载体的相关改造提供了一种方便可行的方法。

Objective：A new type of histidine tag expression vector was obtained on the basis of vector pSE380 through gene recombination technology.Method：Vector pSE380 was modified and a 6×His Tag was inserted into this vector using one-step-reverse PCR.Then demonstration test of dowing,expression and purification of xdh gene was carried out using the modified vector.Result： DNA sequencing revealed that 6×His Tag was accurately inserted into the expression vector,and demonstration test showed that the modified vector was constructed successfully,showed this Tag was constructed successfully through sequence analysis and xdh gene cloning and expression.Recombinant XDH expressed by this modified vector could be purified homogeneous simply by Ni-nitrilotriacetate affinity chromatography,and this tag had no influence on the characteristic of recombinant enzyme.Conclusion：A modified expression vector was obtained successfully by one-step-reverse PCR,and a convenient and practical method of vector modification was formed.