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[目的]克隆表达和纯化野生型及3种缺失突变型人PDIP38谷胱甘肽S转移酶(glutathion S trans-ferase,GST)融合蛋白。[方法]采用常规PCR扩增法得到野生型及3种缺失突变型人PDIP38的cDNA;4种cD-NA与GST融合载体pGEX-4T-1重组并转化大肠杆菌DH5α;采用BamH Ⅰ和Xho Ⅰ双酶切鉴定插入的DNA序列;采用谷胱甘肽-sepharose 4B亲和层析柱纯化重组蛋白。[结果]野生型和3种缺失突变型人PDIP38 GST融合蛋白在DH5α中高效表达;经谷胱甘肽-sepharose 4B亲和层析柱一步纯化后,4种蛋白的纯度均达到85％以上。[结论]pGEX-4T-1 GST蛋白融合载体可高效表达人PDIP38蛋白;用谷胱甘肽-sepharose 4B亲和层析柱一步纯化法可简单、快速地得到高纯度的蛋白。
[Objective] The research aimed to clone and express the wild-type and three mutant human PDIP38 glutathione S trans-ferase (GST) fusion proteins. [Method] The cDNA of wild-type and three mutant human PDIP38 was obtained by conventional PCR amplification. Four kinds of cD-NA and GST fusion vector pGEX-4T-1 were recombined and transformed into E. coli DH5α. BamH Ⅰ and Xho Ⅰ Double digestion to identify the inserted DNA sequence; purified recombinant protein using glutathione-sepharose 4B affinity column. [Result] The wild-type and three mutant human PDIP38 GST fusion proteins were highly expressed in DH5α. After purification by glutathione-sepharose 4B affinity chromatography, the purity of the four proteins reached more than 85%. [Conclusion] The pGEX-4T-1 GST protein fusion vector can efficiently express human PDIP38 protein. The high purity protein can be obtained simply and rapidly by the one-step purification method with glutathione-sepharose 4B affinity chromatography column.