绿色荧光蛋白(GFP)基因的克隆与表达.doc

FILENAME 绿色荧光蛋白（GFP）基因的克隆与表达 PAGE PAGE I 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） 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A600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 ,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 TOC \o 1-3 \h \z \u 1.前言： 1 1.1绿色荧光蛋白（green fluorescent protein，GFP） 1 1.1.1 GFP研究背景 1 1.1.2 GFP研究应用 2 1.2基因的克隆与表达 3 2.实验试剂及实验仪器： 5 2.1实验试剂与材料： 6 2.2实验仪器： 7 3.实验方法 7 3.1质粒提取方法: 7 3.2琼脂糖凝胶电泳及回收: 9 3.3酶切及连接： 11 3.4E. coLi DH5α或E. coLi BL21感受态