关键词: 人源, DCF1, 基因, PTD, TAT

Abstract: In order to further study the role of human DCF1 gene, the target fragment DCF1-TAT was amplified from plasmid pcDNA3.1- DCF1-TAT by PCR, and then cloned into a prokaryotic expression vector pET32a to construct the recombinant plasmid pET32a-DCF1-TAT,which was then transformed into Escherichia coli Rosetta(DE3)cells to express the fusion protein and induced to express by isopropyl-β-D-thiogalactoside(IPTG), and meanwhile the expression condition was optimized. The inclusion body was dissolved by urea after the optimization of dissolution condition, purified by Ni ion affinity chromatography, and renatured by dialysis. SDS-polyaerylamide gel electrophoresis(SDS- PAGE)and Western blot analysis were used to detect the fusion protein. The results indicated that the recombinant expression vector pET32a- DCF1-TAT was constructed and expressed in Rosetta successfully. The fusion protein existed in the form of inclusion body, and the target protein was obtained with high purity through the dissolution of urea and purification of affinity chromatography. SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.

Key words: Humanized, DCF1 gene, PTD, TAT