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The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene andto express it in E. Coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction (PCR) from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. Coli strain TG I. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KGNanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. Coli strain TG I and was transformed into BL21(DE3) for expression. After induction by isopropyl-β-D-thiogalactoside (IPTG) at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. Coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.