hGM-CSF基因穿梭表达载体的构建及其在鱼腥藻7120中的克隆  

Cloning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene in Anabaena sp.PCC7120

作　　者：魏兰珍[1] 金锐[1] 马为民[2] 施定基[1] 甘人宝[3] 王全喜[1] 

机构地区：[1]上海师范大学生命与环境科学学院,上海200234 [2]中国科学院上海生命科学研究院植物生理生态研究所中国科学院研究生院,上海200032 [3]中国科学院上海生命科学院生物化学研究所,上海200031

出　　处：《植物研究》2005年第4期436-440,共5页Bulletin of Botanical Research

摘　　要：人粒—巨噬细胞集落刺激因子(hGM-CSF)作为一种造血生长因子,能够刺激T细胞和巨噬细胞增殖、成熟和分化,具有极其重要的免疫调解功能。本研究运用PCR方法,从质粒pAG-MT-8中克隆该基因,并在其5′端添加有利于在蓝藻细胞中高效表达的SD序列,然后插入到表达载体(pRL-439)强启动子PpsbA的下游,进一步与穿梭表达载体pDC-08相连构建成穿梭表达载体pDC-GM。利用三亲接合转移方法将该穿梭表达载体(pDC-GM)转入丝状鱼腥藻7120,通过相应抗生素筛选后得到能稳定遗传的转基因藻。以该转基因藻的基因组DNA为模板进行PCR检测,结果表明hGM-CSF基因已转入鱼腥藻7120。这是首次尝试把蓝藻作为制备重组hGM-CSF的新宿主,具有潜在的经济价值和社会效益。Human granulocyte-macrophage colony stimulating factor （hGM-CSF） is one of a family glycoprotein cytokines that have potent effects in stimulating proliferation, maturation and function of hematopoietic cells. The gene encoding hGM-CSF was PCR amplified from the plasmid pAGMT-8 and modified by addition SD sequence to promote prokaryotic expression. The resultant hGM-CSF was inserted into downstream of the strong promoter, PpsbA of expression vector pRL-439, then ligated with pDC-08 to produce the shuttle expression vector, pDC-GM. The resulting pDC-GM was transferred into the filamentous, heterocystour cyanobacterium, Anabaena PCC7120, by the tri-parental conjugation transfer method. When cells were cultivated without antibiotics for many generations, the transgenic cyanobacteria remained resistant to neomycin. PCR amplification of wild type and transgenic Anabaena cells with primers P1 and P2 revealed no band in wild type and plasmid free cells, whereas transgenic cells yielded a about 390 bp band. This is the first report of expression hGM-CSF in cyanobacteria cells.

关 键 词：基因穿梭表达载体 鱼腥藻7120 基因克隆 人粒-巨噬细胞集落刺激因子 造血生长因子 

分 类 号：Q949.2[生物学—植物学] Q785