中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (2): 91-93.

• 论著 • 上一篇    下一篇

表达人乳头瘤病毒6b和11型E6/E7基因的小鼠肿瘤细胞株的建立

赵可佳1, 程浩1, 陈民利2, 丁志山2, 耿礼义3, 方永明3   

  1. 1. 浙江大学医学院附属邵逸夫医院皮肤科 杭州 310016;
    2. 浙江省中医学院实验动物中心 杭州 310016;
    3. 浙江大学医学院附属第二医院肿瘤研究所 杭州 310016
  • 收稿日期:2003-03-09 出版日期:2004-02-15 发布日期:2004-02-15
  • 基金资助:
    国家自然科学基金资助(30271190);浙江省自然科学基金资助(491030-N20239)

Establishment of Murine Tumor Cell Line Expressing HPV types 6b and 11 E6/E7 Genes

ZHAO Ke-jia1, CHENG Hao1, CHEN Min-li2, DING Zhi-shan2, GENG Li-yi3, FANG Yong-ming3   

  1. Department of Dermatology and Venereology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, China
  • Received:2003-03-09 Online:2004-02-15 Published:2004-02-15

PDF (PC)

209

赞

摘要： 目的 构建含有人乳头瘤病毒(HPV)6b和11型E6/E7基因的表达质粒和建立这些基因的转染细胞株。方法 分别将HPV6bE6、HPV6bE7、HPV11E6和HPV11E7克隆于含绿色荧光蛋白基因的真核表达载体pcDNA3.1,经脂质体介导转染B16细胞,以G418筛选阳性克隆,荧光显微镜观察,RT-PCR鉴定mRNA转录。结果 通过酶切鉴定和基因测序证实,构建质粒中目的基因片段序列完全正确。转染后荧光显微镜下可观测到B16细胞发出绿色荧光。以G418筛选的抗性细胞克隆提取RNA并经RT-PCR扩增出与目的基因大小一致的基因片段。结论 建立的小鼠肿瘤细胞B16表达HPV6bE6、HPV6bE7、HPV11E6和HPV11E7基因。提示转染的细胞株可移植于小鼠,利于体内研究这些基因的生物学作用和免疫学机制。

关键词: 乳头状瘤病毒,人, 基因表达, 质粒, 转染

Abstract: Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

Key words: Papillomavirus, human, Gene expression, Plasmids, Transfection