绿色荧光蛋白(GFP)基因的克隆与表达.docxVIP

绿色荧光蛋白(GFP)基因的克隆与表达.pdf 分子生物学综合实验论文 题  目: 绿色荧光蛋白(GFP)的基因克隆及表达 中国·黄石 2010 年 12 月 绿色荧光蛋白（GFP）基因的克隆与表达 胡丽丹 （湖北师范学院生命科学院 0803 班 湖北 黄石 43500） 绿色荧光蛋白(GFP)基因的克隆与表达.pdf 摘要：绿色荧光蛋白（GFP）是一类存在于包括水母、水螅和珊瑚等腔肠动物 体内的生物发光蛋白。用碱裂解法提取的的质粒 pEGFP-N3 和 pET-28α 经过 BamH I 和 Not I 双酶切连接后，得到 pET-28α-pEGFP-N3 重组质粒。取部分的 重组质粒做酶切实验，验证重组质粒的存在性，剩下的重组质粒导入表达菌 E. coLi BL-21 大肠杆菌感受态细胞中。重组有 GFP 基因的 E. coLi BL-21，在含有 1 μL/mL 的卡纳霉素的 LB 培养基上培养。当 A  600  达到 0.7 时，用终浓度为 0.8 mM 的异丙基硫代-β-D-半乳糖苷（IPTG）诱导培养 3 小时，离心得到下层绿 蓝色沉淀物即可。 关键词：绿色荧光蛋白 GFP  基因的克隆  荧光蛋白  水母 CLoning and Expression of Green FLuorescent Protein Gene HU Li-Dan ( Class three Grade eight, College of Life Sciences department, Hubei Normal university ,Huangshi 435000) Abstract：Green fluorescent protein（GFP）,one kind of bioluminenscent proteins I 绿色荧光蛋白(GFP)基因的克隆与表达.pdf ,has been found existing in the internal of Coelenterates,such as fellyfish,polyp and coral pEGFP-N3 and pET-28α was harvested by sodium dodecylsulfate(SDS) alkaline process extraction and Agarose Gel Electrophoresis.After treating the two targeted plasmids with BamH I and Not I, the recombinant plasmid,namely pET-28α- pEGFP-N3, can be retrieved by furthermore Agarose Gel Electrophoresis. Taking slight recombinant plasmid proves that recombinant plasmid does exist by dienzyme cutting(BamH I and Not I).The recombinant plasmid lefting would be transported to E. coLi BL-21 the competent cells. E. coLi BL-21 containing recombinant GFP gene was grown overnight in LB solid medium containing kanamycin (Final concentration: 1 μL/mL). When absorbance at 600 nm value was 0.7, isopropyL β-D-thiogalactopyranoside was added to 0.8 mM and the incubation was continued an addition 3 h. Key words: Green fluorescent protein Fellyfish Genetic Cloning Fluorescin 目录 1. 前 言 ： ...............................................................................................1 1.1 绿 色 荧 光 蛋 白 （ GREEN FLUORESCENT PROTEIN ， GFP ） ......................1 1.1.1 GFP