脱-γ-羧基凝血酶原蛋白的原核表达与鉴定  

Prokaryotic expression and identification of des-γ-carboxyprothrombin protein

作　　者：龚吕鸿 徐丽 刘雅楠 吴胜昔 许东 秦萍 韦广苗 曾鹏 GONG Lyuhong;XU Li;LIU Yanan;WU Shengxi;XU Dong;QIN Ping;WEI Guangmiao;ZENG Peng(School of Pharmacy and Bioengineering,Chongqing University of Technology,Chongqing 400054,China;The Seventh People’s Hospital of Chongqing,Chongqing 400054,China)

机构地区：[1]重庆理工大学药学与生物工程学院,重庆400054 [2]重庆市第七人民医院,重庆400054

出　　处：《重庆理工大学学报（自然科学）》2023年第5期331-336,共6页Journal of Chongqing University of Technology：Natural Science

基　　金：重庆市教委科学技术研究项目(KJZD-K201901101);重庆理工大学研究生创新基金项目(gzlcx20222093);大学生创新创业训练计划项目(2022CX204,2022CX205)。

摘　　要：为实现脱-γ-羧基凝血酶原(des-γ-carboxy-prothrombin,DCP)在原核系统中高效表达,参考GenBank中发布的DCP基因CDS序列(LX095963),在保证DCP蛋白氨基酸序列正确的基础上,优化该基因的密码子;化学合成CDS序列基因,并将其融合至pET28a(+)质粒中,构建pET28a(+)/DCP重组质粒;将该质粒转化至感受态BL21(DE3)中进行诱导表达,利用Ni柱纯化DCP蛋白,进行DCP重组蛋白纯度检测、浓度测定及蛋白印迹分析。结果表明:重组质粒在1875 bp处的双酶切条带,经测序后与已优化的基因序列完全切合。重组菌的DCP蛋白表达最高时,诱导条件为25℃、IPTG终浓度为0.8 mmol/L、时间6 h;经Ni柱纯化后的DCP重组蛋白纯度较高,浓度达1.04 mg/mL;在72 kD处有明显的蛋白条带,切合预期。在大肠杆菌系统中成功表达出DCP蛋白,为后续DCP检测方法研究提供试剂参考。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 1875 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.

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 

分 类 号：R392-33[医药卫生—免疫学]