人乳头瘤病毒16型E7抗原表达模型的构建  

Construction of HPV Type 16 E7 Antigen Expressing Model

作　　者：李辉[1] 伍欣星[1] 

机构地区：[1]武汉大学医学院病毒学研究所分子生物学研究室,武汉430071

出　　处：《武汉大学学报（医学版）》2004年第1期7-11,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助 ( 3 0 1710 42 )

摘　　要：目的 :构建pEGFP HPV1 6E7表达载体 ,并观察其在肝癌细胞中的瞬时和稳定表达及小鼠肝癌细胞皮下成瘤情况 ,为建立表达HPV1 6E7的实体瘤动物模型奠定基础。方法 :应用基因重组技术 ,构建增强绿色荧光蛋白(EGFP)与HPV1 6E7的融合表达载体 ,经限制性内切酶酶切鉴定和PCR分析 ,用脂质体转染技术将其转入小鼠肝癌细胞 ,2 4h后RT PCR检测HPV1 6E7mRNA的生成 ,荧光显微镜观察EGFP HPV1 6E7融合蛋白的表达 ,将转染细胞接种小鼠皮下 ,观察成瘤及成瘤后融合蛋白的表达情况。结果 :酶切鉴定和PCR分析证实重组质粒中插入目的基因片段及载体DNA大小、方向和插入位点均正确 ,在转染的小鼠肝癌细胞中检测到HPV1 6E7mRNA的生成和绿色荧光蛋白的表达 ,接种的转染细胞在小鼠皮下可成瘤且可检测到HPV1 6E7mRNA的生成。结论 :pEGFP HPV1 6E7表达载体便于观察转染细胞中EGFP HPV1 6E7融合蛋白的表达情况 ,小鼠肝癌细胞接种小鼠皮下可成瘤 ,成瘤后可检测到HPV1Objective: To do basic research for construction of animal tumor model in which EGFP HPV16E7 was expressed,to construct pEGFP HPV16E7 expression vector and to detect its transient expression in transfected liver cancer cell and its stably expression in the formed tumor. Methods: Recombinant expression vector was constructed by techniques of gene recombination and screening,and identified by restriction and PCR. Then the recombinant was transfected into mouse liver cancer cell by techniques of gene transfection and expression were detected by RT PCR and fluoroscopy. After that,transfected cells were inoculated into subcutaneous of mouse and the forming tumor and expression of HPV16E7 protein after tumor was formed was observed. Results: Identification of pEGFP HPV16E7 by enzyme digestion and PCR showed that the length,inserted location and direction of the target gene which was inserted into the recombinant was correct and the expression of EGFP in transfected cell was observed. And inoculated cells could form tumor in vivo obviously and HPV16E7 protein could express in the cells stably. Conclusion: The pEGFP HPV16E7 expression vector makes it easy to assess the expression of EGFP HPV16E7 fusion protein and to sift the transfected cells. And transfected cells could form tumor in the body of animal and express HPV16E7 protein stably.

关 键 词：人乳头瘤病毒 表达载体 基因转染 HPV16E7 动物模型 基因重组 抗原 

分 类 号：R373.9[医药卫生—病原生物学] R394[医药卫生—基础医学]