The human epidermal growth factor （hEGF） is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro . A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.
人表皮生长因子（hEGF）基因在蓝藻中的表达
戴? 1,2 施定基1* 张卉1,2 钟晖3 冉亮1 彭国宏4 甘人宝5 陈素娟3 连慕兰2
（1. 中国科学院植物研究所，北京100093；2. 北京师范大学生命科学学院，北京100875；3. 解放军301 医院东亚免疫研究所，
北京100853；4. 中国科学院海洋研究所，青岛266071; 5. 中国科学院生物化学研究所，上海200031）
摘要：人表皮生长因子(hEGF)是由53个氨基酸组成的蛋白,在临床上内服与外敷可促进内外表皮细胞的生长.将人工合成的hEGF基因连接到质粒pRL-489上,位于启动子psbA下游.验证连接成功后,用三亲接合转移方法将载体pRL-hEGF导入聚球藻Synechococcus sp. PCC 7002和鱼腥藻Anabaena sp. PCC 7120.由于pRL-hEGF没有能在单细胞蓝藻中自主复制的复制子,通过筛选,hEGF在聚球藻7002中是整合到蓝藻染色体上进行表达的.用PCR扩增的方法在两种转基因藻中均检测到hEGF基因的存在.放射免疫分析证明,hEGF基因在两种转基因藻中均得到了表达.而且,在聚球藻7002中是采用分泌形式将表达产物分泌到培养液中.
关键词： 人表皮生长因子；鱼腥藻Anabaena sp.PCC7120；聚球藻Synechococcus.sp.PCC7002；载体；转基因蓝藻；三亲接合转移
全 文 ：