中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (5): 347-349.

• 研究报道 • 上一篇    下一篇

人乳头瘤病毒6bL1双顺反子载体的构建及其在哺乳动物细胞内的表达

邓列华1,殷董2,胡云峰1,田静3,计雄飞4,范洪涛5,郭秀枝6,林泽7,赵永铿8   

  1. 1. 广州市暨南大学附属第一医院皮肤科
    2. 陕西省人民医院皮肤科
    3. 广州市天河区慢性病防治中心皮肤性病科
    4. 福建医科大学附属第一医院
    5. Medical department of johnson，Radnor PA USA
    6. 广州暨南大学附属第一医院皮肤科
    7. 广州暨南大学医学院附院皮肤科
    8.
  • 收稿日期:2010-06-21 修回日期:2010-08-10 出版日期:2011-05-15 发布日期:2011-05-03
  • 通讯作者: 邓列华 E-mail:liehuadeng@126.com
  • 基金资助:

    广东省自然科学基金项目

Construction of a bicistronic expression vector containing human papillomavirus （HPV） type 6b L1 gene and its expression in mammalian cells

  • Received:2010-06-21 Revised:2010-08-10 Online:2011-05-15 Published:2011-05-03
  • Contact: DENG Lie-hua E-mail:liehuadeng@126.com

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摘要：

目的 构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因（HPV6b L1）的双顺反子表达载体，并使其在哺乳动物细胞内表达，以期建立含有HPV6b L1的细胞模型。方法 表达质粒pEGFP-HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接，酶切鉴定，挑选阳性克隆进行测序。重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞（NIH3T3），荧光显微镜下观察EGFP蛋白的表达，RT-PCR检测HPV6b L1 mRNA的生成。结果 成功构建含HPV6b L1的重组质粒pIRES2-HPV6bL1-EGFP。重组体成功转染进NIH3T3细胞，并用G418筛选。同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达。进一步进行RT-PCR，检测到HPV6b L1 mRNA的生成。结论 成功构建携带HPV6b L1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞。经荧光倒置显微镜观察及RT-PCR方法检测证明HPV6b L1在NIH3T3细胞内成功表达。

关键词: 基因表达

Abstract:

Objective To construct a bicistronic expression vector containing HPV type 6b L1 gene, to express the recombinant vector in mammalian cells, and to establish a cell strain stably expressing HPV6b L1 gene. Methods After endonuclease digestion and purification, the gene fragment of HPV6b L1 was cloned into the eukaryotic expression vector pIRES2-enhanced green fluorescent protein （EGFP）. The identification of the recombinant was realized via endonuclease digestion and sequence analysis. Then, the recombinant plasmid pIRES2-HPV6bL1-EGFP was transfected into NIH3T3 （a mouse embryonic fibroblast cell line） cells. Subsequently, the expression of EGFP was observed by fluorescent inverted microscopy, and HPV6b L1 mRNA expression by reverse transcription （RT）-PCR. Results The recombinant plasmid pIRES2-HPV6bL1-EGFP was successfully constructed, transfected into NIH3T3 cells, and selected by G418. The expression of EGFP was seen under an inverted fluorescence microscoy. RT-PCR proved the expression of HPV6b L1 mRNA in transfected cells. Conclusions The recombinant plasmid pIRES2-HPV6bL1-EGFP was successfully constructed and transfected into NIH3T3 cells. Inverted fluorescent microscopy and RT-PCR confirmed the successful expression of HPV6b L1 in NIH3T3 cells.

Key words: Gene expression