摘要
应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达。酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达。构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达。由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对HPV16E7分子生物学特性、致瘤机理及APC提呈等的研究。为建立表达HPV16E7的实体瘤动物模型奠定了基础。
In order to make bases for construction of animal tumor model which expresses EGFP-HPV16E7, recombinant expression plasmid was constructed by techniques of gene recombination after screening and identifing by restriction and PCR. Then the recombinant was transfected into mouse liver cancer cell by techniques of gene transfection and detected expression by fluoroscopy, pEGFP-HPV16 E7 was identified by enzyme digestion and PCR. The resent showed that the length, inserted location and direction of the target gene was correct and the expression of EGFP in transfected cell was observed. Because of it's pEGFP-HPV16E7 expression plasmid that makes it easy to assess the expression of EGFP- HPV16E7 fusion protein and to sift the transfected cells.
出处
《中国病毒学》
CSCD
2003年第4期344-347,共4页
Virologica Sinica
基金
湖北省自然科学基金(97J077)
关键词
人乳头瘤病毒
表达载体
基因转染
真核表达
Human papillomavirus
Eukaryotic expression
Expression vector
e7 gene
