关键词: 脱γ羧基凝血酶原, Ｎｉ柱纯化, 原核表达, 蛋白免疫印迹

Abstract: To achieve efficient expression of des-γ-carboxyprothrombin (DCP) in a prokaryotic system, this paper refers to the CDS sequence of DCP gene published in GenBank (accession number LX095963). Based on a correct amino acid sequence of the DCP protein, the codon of the DCP gene is optimized. The CDS sequence gene is synthesized and fused into pET28a(+) plasmid to construct pET28a(+)/DCP recombinant plasmid. Then, the plasmid is transferred into the receptor state BL21(DE3) for induction expression, and the DCP protein is purified by a nickel column to conduct purity measurement, concentration determination and western blot analysis of the recombinant protein DCP. The results show that the recombinant plasmid has a double enzyme cut band at 1 875 bp, which is sequenced to match its optimized gene sequence. The highest expression of the DCP protein is obtained when the recombinant bacteria are induced at an IPTG final concentration of 0.8 mmol/L for 6 hours at 25 ℃. After nickel column purification, the recombinant protein DCP has a high purity, the concentration of which reaches 1.04 mg/mL. As expected, there is a clear protein band at 72 kD. It shows that the recombinant protein DCP is successfully expressed in E. coli system, which provides reagent reference for the subsequent study of DCP detection methods.