增强型青色荧光蛋白慢病毒表达载体的构建  

Construction of Enhanced Cyan Fluorscent Protein Lentiviral Vector

作　　者：曹鸿国[1,2] 卢智刚[2] 罗敏[2] 袁克湖[2] 邓宏魁[2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]北京大学深圳研究生院,广东深圳518055

出　　处：《生物技术通讯》2008年第6期836-839,共4页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2001CB510106)

摘　　要：目的:通过多点突变构建增强型青色荧光蛋白(ECFP)慢病毒表达载体。方法与结果:根据增强型绿色荧光蛋白(EGFP)和ECFP基因序列的差异设计3对引物,以pLentiLox3.7-EGFP为模板进行分段PCR扩增,再以分段PCR扩增产物为模板扩增出突变的ECFP基因片段,将其与载体连接,得到ECFP慢病毒表达载体pLentiLox3.7-ECFP,测序结果证实经过多点突变扩增的ECFP片段基因序列完全正确;磷酸钙介导pLentiLox3.7-ECFP在293T细胞中表达,48h后在荧光显微镜下观察到青色荧光蛋白。结论:通过多点突变的方法得到了ECFP慢病毒表达载体。Objective： To construct enhanced cyan fluorescence protein（ECFP） lentiviral vector using the gene multipoint mutation. Methods ＆ Results： Three pairs of mutation premiers were designed and synthesized according to the difference between enhanced green fluorescent protein（EGFP） and ECFP DNA sequence. Three different DNA fragments were ampli- fied with pLentiLox3.7-EGFP as the DNA template. Consequently, the DNA fragment with the length of ECFP was ampli- fied using the three different DNA fragments as DNA templates, and it was confirmed by DNA sequence analysis. ECFP gene fragment and plasmid vector pLentiLox were ligated to be the pLentiLox3.7-ECFP. Finally, the expression of ECFP could be detected in 293T cells after 48 h of reconstructed lentiviral vector pLentiLox3.7-ECFP introduced into 293T cells mediated by calcium phosphate. Conclusion： Lentiviral vector pLentiLox3.7-ECFP was reconstructed with EGFP gene as the template by muhipoint mutation.

关 键 词：慢病毒载体 绿色荧光蛋白 青色荧光蛋白 点突变 

分 类 号：Q78[生物学—分子生物学]