Objective: To construct recombinant prokaryotic expression vector to express the important NIDO Domain protein and further purify and identify the NIDO protein. Methods: The NIDO gene was subcloned into pMD19-T vector for DNA sequencing analysis. Plasmid extraction,purification,enzyme,connection,competent cell preparation and transformation technology were performed to construct and identify the prokaryotic expression vector pGEX-4T-1-NIDO. The protein NIDO-GST was expressed in E.coli-BL21 as a fusion protein induced by IPTG. The expression products were isolated,and the target proteins of supernatants and inclusion precipitation were detected by 12%SDS-PAGE. After denaturation and renaturation of inclusion body,the expression products were purified by GST-affinity chromatography. The purity of NIDO-GST fusion protein was identified by 12% SDS-PAGE and the Mass-Spectrographic analysis. Results: NIDO coding region was cloned into pMD19-T vector and the sequence was confirmed. Prokaryotic expression system of pGEX-4T-1-NIDO was constructed successfully,and were induced in E.coli BL21,to obtain high expression of N-terminal with GST tag sequence of the recombinant fusion protein(NIDO-GST) expression(30%). Target protein in bacterial precipitation was in the form of inclusion body mostly after Ultrasonic crack bacteria. After denaturation and renaturation of the inclusion body and further GST affinity chromatography purification,the purity of protein NIDO-GST was more than 80% and confirmed by mass spectrum identification. Conclusion: The purified fusion protein NIDO-GST was constructed successfully by the prokaryotic expression and affinity chromatography.