摘要
目的 :以绿色荧光蛋白 (GFP)基因为报告基因 ,将GFP基因克隆入pUC1 8建立大通量的筛选载体。方法 :用PCR方法扩增目的DNA片段 ,碱裂解法小量制备质粒DNA ,用限制性内切酶消化质粒DNA ,用低熔点琼脂糖凝胶分离和纯化大片段DNA ,GFP基因与 pUC1 8连接 ,用氯化钙法转化感受态细胞 ,DNA序列测定 ,琼脂糖凝胶电泳 ,变性聚丙烯酰胺凝胶电泳。结果 :pUC1 8 atg GFP载体意外表达GFP蛋白 ,新合成的上游引物与原下游引物扩增GFP基因 ,克隆入 pUC1 8构建 pUC1 8 GFP载体 ,通过PCR、酶切鉴定和DNA序列分析 ,GFP基因的读码框架与原设计方案完全一致 ,该载体自身无GFP表达。结论 :筛选载体内的GFP基因与克隆基因的融合基因可表达一种融合蛋白 ,在琼脂平板上含有该融合基因的克隆在紫外线激发下可发绿色荧光 ,因此 ,可表达基因能被挑选出来。
Aim: To construct a screening vector. Methods: Polymerase chain reaction(PCR), alkaline lysis miniprep of plasmid DNA, digestion of DNA with restriction endonucleases, isolation and purification of large DNA fragments using low melting temperature agarose gels, ligation of GFP gene and pUC18 transformation using calcium chloride, DNA sequence analysis, agarose gel electrophoresis, and denaturing polyacrylamide gel electrophoresis were performed. Results: Newly synthesized upper primer and original down primer amplified the GFP gene. After cloning and identification, GFP gene sequence and reading framework were confirmed correctly, but pUC18 GFP did not express GFP. Conclusions: A fusion gene of GFP and cloned gene inserted into the pUC18 GFP screening vector can produce a fusion protein, and the clone containing the fusion gene on the plate can emit the green fluorescence under the UV light.Thus, expressiable genes in the screening vector can be selected
出处
《郑州大学学报（医学版）》
CAS
北大核心
2002年第5期621-624,共4页
Journal of Zhengzhou University(Medical Sciences)
