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血友病B是凝血IX因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,构建含人凝血IX因子基因(hFIX,2.8kb)植物双元表达载体p35s2300∷gus∷noster,用农杆菌介导法转化烟草“百日红”,通过PCR和Southernblot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RTPCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性。为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。
Hemophilia B is a bleeding disorder caused by a lack of coagulation factor IX (hFIX), which is efficacious in the treatment of blood transfusions and hFIX concentrates, but with high cost of treatment and potential safety issues resulting in safe, inexpensive human coagulation IX Factors for the treatment of hemophilia B is of great significance. Plant systems expressing foreign proteins have advantages in terms of production costs and safety. For this purpose, a plant binary expression vector p35s2300 :: host :: noster containing the human coagulation factor VIII gene (hFIX, 2.8kb) was constructed and Agrobacterium tumefaciens-mediated transformation of tobacco “Bairihong” was carried out. Four strains were confirmed by PCR and Southern blot analysis The independent transgenic plants showed 1 to 4 copies of hFIX in the genome of transgenic tobacco. The results of RTPCR and ELISA showed that hFIX was successfully expressed at transcription and translation level. The expression level of hFIX in transgenic tobacco leaves was 2.5-8.8 ng / g · FW, and is immunologically active. It provides the necessary preparation for the follow-up study of expressing hFIX by plant systems and also provides some theoretical and experimental references for the research of expressing other medicinal proteins by plant systems.