含人IGF-1基因杆状病毒表达载体在sf-9细胞中表达.pdfVIP

业生物技术学报 Journal of Agricultural Biotechnology 2005,13 (4): 452~455 ·研究论文· 人I 基因重组杆状病毒表达载体的构建及在sf9 细胞中的表达* 丽翠 王雅梅 闫豫东 张静宜 司 杨 王 民 祁雅慧** (首都医科大学实验中心，北京 100054) ： ，采用 ， 摘要 以pcDNA3.1/ 为模板 PCR 方法对 基因进行扩增 R 和 酶切回收含 基因的DNA 片 ，与pFastHTA 连接，获得重组载体pFast/ ；将其转化大肠杆菌( )DH 10Bac 感受态细胞，经卡那霉素、四 环素、庆大霉素及蓝白斑筛选得到重组杆状病毒载体pBacmid-Fa- ，PCR 鉴定得到单一 条带。将该病毒载体转染 sf9 细胞，进行病毒重组，Western-blot 检测 表达, 并用MTT 法检测促内皮细胞生长活性。结果成功获得了重组病毒， ， Western-blot 检测出现1 条约 13.0 kD 的带 MTT 法检测 促细胞生长率为24.4%。 关键词：人 基因；重组杆状病毒载体；sf9 细胞 Construction of Recombinant Baculovirus Expressive Vector and Its Expression in sf9 Cells SUN Li-Cui WANG Ya-Mei YAN Yu-Dong ZHANG Jing-Yi SI Yang WANG Min QI Ya-Hui** gene was amplified using PCR and cloned into pFastHTA with the R and sites. The constructed pFast/ was transformed into DH 10Bac. After screening with kanamycin, tetracycline, gentamicin and X-gal/IPTG, the recombinant baculovirus expressive vector pBacmid-Fa- was obtained. Then the sf9 cells were transfected us- ing the recombinant pBacmid-Fa- . After packing in the sf9 cells, recombinant viruses were obtained, which could be observed under electron microscope . A band of protein of about 13.0 kD was identified using Western-blot, which demonstrated that the was successfully expressed in the sf9 cells. The cell growth promotion rate was 24.4% measured using MTT method. human gene; recombinant baculovirus vector; sf9 cells 人胰